Diagnostic Value of CK7 Immunostaining Patterns in Cervical Intraepithelial Neoplasia Running title: CK7 value in CIN

Diagnostic Value of CK7 Immunostaining Patterns in Cervical Intraepithelial Neoplasia Running title: CK7 value in CIN | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diagnostic Value of CK7 Immunostaining Patterns in Cervical Intraepithelial Neoplasia Running title: CK7 value in CIN Elham Omidi, Farzaneh Nayeri, Mohammad Javad Tarrahi, Maryam Soltan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7579340/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Accurate classification of cervical intraepithelial neoplasia (CIN) is essential for the appropriate treatment strategy and monitoring disease progression. While morphological evaluation remains the standard, interobserver variability and grading ambiguity—particularly in CIN2—necessitate additional biomarkers. This study evaluates CK7 immunostaining patterns and their association with CIN grade, Ki67 and p16 expression, and colposcopy findings. Methods: In a retrospective cross-sectional study, 106 histologically confirmed CIN cases (CIN1–CIN3) were assessed for CK7 expression patterns (negative, patchy, gradient, full-thickness). Ki67 and p16 scores (0–3) were obtained from prior pathology reports. Chi-square analysis was used to evaluate associations between biomarker expression and CIN grade. Results: CK7 expression was significantly associated with CIN grade (p < 0.001), with full-thickness staining observed in 68.8% of CIN3 cases and 0% of CIN1. High Ki67 and p16 scores (≥ 2) were also significantly associated with higher-grade CIN (p 0.001), p16 (p = 0.002), and abnormal colposcopic findings (p = 0.004). No significant association was found with past medical history of having CIN (p = 0.088). Conclusions: CK7 expression patterns reflect the severity of CIN and correlate with proliferation (Ki67) and HPV-driven transformation (p16). Full-thickness and gradient CK7 staining, when combined with Ki67 and p16, increase diagnostic accuracy and may inform clinical decision-making, particularly in morphologically ambiguous cases or settings with limited access to molecular testing. CK7 cervical intraepithelial neoplasia Ki-67 Antigen P16 immunohistochemistry Figures Figure 1 Figure 2 Introduction Cervical cancer remains a significant global health concern, ranking as the fourth most common cancer among women and the second leading cause of cancer-related mortality in low-income countries [ 1 , 2 ]. The majority of cases are preceded by premalignant changes known as cervical intraepithelial neoplasia (CIN), which are histologically classified into grades 1 to 3 (CIN1–CIN3) based on the extent of epithelial dysplasia [ 3 , 4 ]. Accurate identification and grading of these lesions are critical, as CIN1 lesions often regress spontaneously, whereas CIN2 and CIN3 carry a substantially higher risk of progression to invasive carcinoma [ 5 – 7 ]. Despite the use of standardized histological criteria, CIN grading—especially CIN2—remains challenging due to considerable interobserver variability and overlapping morphological features [ 4 , 8 ]. As a result, immunohistochemical (IHC) markers such as p16^INK4a^, KI-67 antigen, and cytokeratin 7 (CK7) have been increasingly employed to enhance diagnostic accuracy and reproducibility [ 9 – 11 ]. CK7 is a low-molecular-weight intermediate filament protein typically expressed in glandular epithelium, particularly at the squamocolumnar junction of the cervix—the most vulnerable area for HPV-mediated oncogenic transformation [ 5 , 12 ]. Studies have shown that CK7 expression increases with lesion severity and may assist in identifying CIN lesions with higher malignant potential [ 6 , 13 ]. However, most existing studies have been conducted in Western or East Asian populations, with limited data from Middle Eastern or Iranian cohorts. This gap highlights the urgent need for context-specific research to better understand regional variations in CIN grading and CK7 expression, particularly in Middle Eastern populations. This study aims to characterize CK7 IHC staining patterns—categorized as negative, patchy, gradient, and full-thickness—in histologically confirmed CIN lesions (CIN1–CIN3) and to examine their associations with two widely used proliferation and surrogate HPV biomarkers, Ki67 and p16. By investigating a regional Iranian population, this study also provides context-specific data that may aid in standardizing interpretation protocols and refining risk stratification strategies in settings with limited access to molecular HPV testing. Materials and Methods Study Design and Setting This retrospective cross-sectional study was conducted at the Department of Pathology, Beheshti Hospital, affiliated with Isfahan University of Medical Sciences, between 2021 and 2023. Archived cervical biopsy and cone specimens with histopathologically confirmed squamous intraepithelial lesions (SIL) were retrieved for immunohistochemical (IHC) analysis. Inclusion and Exclusion Criteria All available samples diagnosed as CIN1, CIN2, or CIN3 during the study period were included using a sequential sampling method. Cases were excluded if patients had a history of immunosuppressive medication, organ transplantation, or current pregnancy. Sample Size A total of 106 eligible cases were included. The estimated sample size was based on a presumed prevalence of CK7 negativity in CIN1 (~ 80%), with a 5% margin of error and 95% confidence level. Data Collection Patient demographics, including age and clinical history, were obtained from hospital records. Colposcopic findings were categorized as normal or abnormal. Past history (PMH) of having CIN was also recorded. Histopathological and Immunohistochemical Evaluation All hematoxylin and eosin (H&E)-stained slides were reviewed to confirm diagnosis and CIN grade. IHC staining for CK7 was performed on formalin-fixed paraffin-embedded (FFPE) sections using a recombinant mouse monoclonal antibody (Long Island Antibody, China). Positive and negative controls were included in each staining batch. Standard protocols were applied, including deparaffinization, antigen retrieval, endogenous peroxidase blocking, primary antibody incubation, and DAB visualization. CK7 staining was assessed for cytoplasmic and membranous positivity and categorized into four patterns (Fig. 1 ): Negative: 40% of epithelial cells stained in contiguous groups of ≥ 5 cells Ki67 and P16 scores were extracted from prior pathology reports and scored from 0 to 3, based on the extent of epithelial involvement. Details of the scoring system are provided in Table 1 . Table 1 Descriptive Distribution of IHC Staining Patterns (CK7, Ki67, P16) and age by CIN Grade CIN IHC Marker CIN1 (n = 69) CIN2 (n = 21) CIN3 (n = 16) CK7 negative 53(76.8%) 5(23.8%) 1(6.3%) patchy 11(15.9%) 0(0%) 1(6.3%) gradient 5(7.2%) 9(42.9%) 3(18.8%) Full thickness 0(0%) 7(33.3%) 11(68.8%) Ki67 Score0 4(26.7%) 2(15.4%) 0(0.0%) Score1 11(73.3%) 3(23.1%) 0(0.0%) Score2 0(0.0%) 4(30.8%) 1(16.7%) Score3 0(0.0%) 4(30.8%) 5(83.3%) P16 Score0 15(100.0%) 5(38.5%) 0(0.0%) Score1 0(0.0%) 1(7.7%) 1(16.7%) Score2 0(0.0%) 4(30.8%) 0(0.0%) Score3 0(0.0%) 3(23.1%) 5(83.3%) Mean Age 40.06(8.76) 32.81(5.42) 36.88(6.71) Statistical Analysis All data were analyzed using SPSS version 22. Categorical variables were presented as frequencies and percentages. Chi-square tests were used to assess associations between CIN grade and biomarker expression (CK7, Ki67, P16). A p-value < 0.05 was considered statistically significant. To facilitate a more precise analysis, the expression of CK7 was categorized into limited (negative and patchy) versus extensive (including gradient and full-thickness staining). Additionally, Ki67 and p16 data were classified into low expression (scores of 0 or 1) versus high expression (scores of 2 or 3). Results Patient Characteristics A total of 106 cases were included: 69 (65.1%) were diagnosed as CIN1, 21 (19.8%) as CIN2, and 16 (15.1%) as CIN3. The mean age of patients was 38.1 years (SD: 8.4; minimum: 22–maximum: 62 years). Detailed demographic and clinical characteristics are summarized in Table 1 . CK7 Immunohistochemistry Patterns Across CIN Grades The distribution of CK7 staining patterns showed a significant association with CIN grade (p < 0.001). CIN1 lesions predominantly exhibited negative (76.8%) and patchy (15.9%) staining, with no full-thickness positivity. In contrast, 33.3% of CIN2 and 68.8% of CIN3 lesions showed full-thickness staining. Gradient staining was also more frequent in higher grades. These results are detailed in Table 1 and Fig. 2 . When CK7 expression was dichotomized into limited (negative + patchy) versus extensive (gradient + full-thickness), 7.2%% of CIN1 cases showed extensive expression, while 76.2% of CIN2 and 87.5% of CIN3 exhibited extensive staining (p < 0.001). (Table 2 ) Table 2 Distribution of IHC Staining Patterns (CK7, K67, P16) by CIN Grade CIN IHC Marker CIN1 CIN2 CIN3 P-value CK7 Limited 64 (92.8%) 5(23.8%) 2(12.5%) < 0.001 extensive 5(7.2%) 16(76.2%) 14(87.5%) Ki67 LOW 15 (100.0%) 5 (38.5%) 0(0.0%) < 0.001 HIGH 0(0.0%) 8(61.5%) 6(100.0%) P16 LOW 15(100.0%) 6(46.2%) 1(16.7%) < 0.001 HIGH 0(0.0%) 7(53.8%) 5(83.3%) Abbreviations: CIN, Cervical Intraepithelial Neoplasia; CK7, Cytokeratin 7; P16, p16INK4a. Ki67 and P16 Expression Patterns Among the 34 cases with Ki67 data, high expression (score 2 or 3) was significantly more frequent in CIN2 (61.5%) and CIN3 (100%) compared to CIN1 (0%) (p < 0.001). Similarly, non of CIN1 cases (0%) showed P16 score 2 or 3, while 53.8% of CIN2 and 83.3% of CIN3 had scores 2 or 3 (p < 0.001). Full distribution of Ki67 and P16 scores across CIN grades is shown in Table 2 . Clinical Characteristics of Patients Across CIN Grades Colposcopy findings were available for 105 patients, with abnormal findings seen in 42.0% of CIN1, 71.4% of CIN2, and 100% of CIN3 cases (p < 0.001). (Table 3 ) Table 3 Clinical Characteristics of Patients by CIN Grade CIN grade Clinical data CIN1 CIN2 CIN3 P-value colposcopy Normal 40 (58.0%) 6 (28.6%) 0 (0%) < 0.001 abnormal 29 (42.0%) 15 (71.4%) 15 (100%) Past medical history of CIN Present 6 (8.7%) 3 (14.3%) 4 (25.0%) 0.191 Absent 63 (91.3%) 18 (85.7%) 12 (75.0%) Mean Age (SD) 40.06(8.76) 32.81(5.42) 36.88(6.71) 0.001 Abbreviations: CIN, Cervical Intraepithelial Neoplasia; PMH, past medical history of CIN There was statistically significant difference in mean age among the three groups (p < 0.001). According to Tukey’s post hoc test, this difference was specifically attributable to comparison between CIN1 and CIN2 mean age. (Table 3 ) No significant relationship was found between CIN grade and past medical history of previous CIN (p = 0.191). (Table 3 ). Association Between CK7 and Other Variables CK7 expression patterns were significantly associated with Ki67 (p < 0.001) and P16 scores (p = 0.002). High Ki67 expression (scores 2 or 3) occurred more frequently in the CK7 extensive pattern (68.4%), while high P16 expression (scores 2 or 3) was also more prevalent in this pattern (57.9%). Full-thickness CK7 staining frequently co-occurred with high Ki67 and P16 expression. (Table 4 ) Table 4 Distribution of IHC Staining Patterns (Ki67, P16) by CK7 Grade CK7 grade OTHER markers grade Limited Extensive P-value Ki67 Low 14(93.3%) 6(31.6%) < 0.001 High 1(6.7%) 13(68.4%) P16 Low 14(93.3%) 8(42.1%) 0.002 High 1(6.7%) 11(57.9%) Additionally, CK7 pattern was associated with colposcopic findings (p = 0.004), with extensive CK7 positivity seen in 76.5% of cases with abnormal colposcopy. No significant relationship was found between CK7 expression and History of CIN (p = 0.088). (Table 5 ) Table 5 Clinical Characteristics of Patients by CK7 Grade CK7 grade Clinical data Limited Extensive P-value Colposcopy Normal 38(53.5%) 8(23.5%) 0.004 Abnormal 33(46.5%) 26(76.5%) Past medical history of CIN Present 6(8.5%) 7(20.0%) 0.088 Absent 65(91.5%) 28(80.0%) Discussion The findings of this study confirm a strong association between CK7 immunoreactivity patterns and the histological grade of cervical intraepithelial neoplasia (CIN). A progressive shift from negative or patchy CK7 expression in CIN1 to full-thickness staining in CIN3 was clearly observed, consistent with previous studies suggesting CK7 as a marker of neoplastic transformation at the squamocolumnar junction [ 5 , 6 , 9 , 10 ]. CK7 expression has been proposed to reflect the involvement of reserve cells at the squamocolumnar junction, a region vulnerable to HPV-mediated carcinogenesis [ 5 ]. Our results, which showed increased CK7 expression along with higher CIN grades, are in line with those of Lee et al., who demonstrated increased CK7 and CK19 expression in CIN3 and cervical carcinoma [ 6 ]. Paquette et al. also emphasized the predictive value of CK7 in low-grade squamous intraepithelial lesions (LSIL), with CK7-positive LSILs more likely to progress to high-grade disease [ 7 ]. A key observation in our study was the co-occurrence of full-thickness CK7 staining with high Ki67 and p16 scores in most CIN2 and CIN3 lesions. This reinforces the idea of a coordinated upregulation of these biomarkers in dysplastic epithelium. Previous reports have established the diagnostic and prognostic utility of Ki67 and p16 in distinguishing high-grade lesions and in predicting biological behavior [ 11 – 13 ]. Similarly, Huang et al. found that the combination of CK7 and p16 provided superior predictive accuracy in identifying LSILs at risk of progression [ 8 ]. Our study’s results align with those of Samyuktha et al., who showed a significant correlation between CK7 intensity and CIN grade [ 9 ]. Furthermore, Umphress et al. proposed the complementary use of CK7 with p16 to resolve diagnostic uncertainty in CIN2, a notoriously ambiguous category [ 11 ]. Despite these strengths, certain limitations must be acknowledged. First, the retrospective design restricts our ability to assess temporal progression or establish causality. Second, the absence of follow-up data precludes direct assessment of lesion outcomes. Third, immunohistochemical data for Ki67 and p16 were not available for all cases, which may have introduced a degree of selection or reporting bias. These limitations are shared by many previous studies in the field [ 3 , 4 , 7 ]. In terms of generalizability, our study was limited to a single-center sample within a defined geographic region, which may restrict the external applicability of findings to more diverse populations. Nonetheless, the consistency of our results with multiple international studies enhances confidence in the biological validity of CK7 as a marker of CIN severity [ 6 – 9 ]. From a clinical standpoint, the addition of CK7 staining to routine diagnostic panels could improve the stratification of patients, especially in cases with morphologically ambiguous findings. In resource-limited settings where HPV genotyping or molecular profiling is not feasible, combining CK7 with p16 and Ki67 may offer a cost-effective alternative to enhance diagnostic accuracy and guide patient management [ 1 , 13 ]. Conclusion In conclusion, our findings support the growing body of evidence indicating that CK7, particularly in full-thickness and gradient patterns, is a valuable immunohistochemical marker in CIN. When used alongside p16 and Ki67, CK7 not only improves diagnostic precision but may also provide insight into lesion biology and progression risk. Future prospective studies with long-term clinical follow-up are recommended to validate these findings and explore their utility in clinical decision-making. Declarations Ethical Approval and Consent to Participate This study was approved by the Research Ethics Committee of the School of Medicine, Isfahan University of Medical Sciences (Approval ID: IR.MUI.MED.REC.1403.150, Approval date: 15 July 2024). The study was conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments. The requirement for informed consent was waived by the Ethics Committee, as this was a retrospective study conducted on archived pathology slides and blocks, with no identifiable patient data included. Consent for Publication - Not applicable. This manuscript does not contain any person’s data in any form (including individual details, images, or videos) Financial Support: No financial support. Availability of data and materials: All data supporting the findings of this study are available within the paper. Competing interests: I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper Authors' contributions E O was contributed in literature search, data acquisition, data analysis, manuscript preparation, manuscript editing, and manuscript review F N was contributed in literature search, data acquisition, data analysis, manuscript preparation, manuscript editing, and manuscript review M J T was contributed in data acquisition and data analysis M S was contributed in literature search, manuscript preparation, manuscript editing, and manuscript review Acknowledgements: Not applicable Authors' information Elham Omidi, Assistant Professor of Pathology, Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Farzaneh Nayeri, Resident of Pathology, Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Mohammadjavad Tarrahi: Professor of Biostatistics and Epidemiology, Department of Health Sciences, Isfahan University of Medical Sciences, Isfahan, Iran Maryam Soltan, Assistant Professor of Pathology, Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran References Lin S, Gao K, Gu S, Chen Y, Li S, Wang Y, et al. 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07:11:54","extension":"html","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93649,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7579340/v1/22d40c20e84a74b3cab49347.html"},{"id":95894779,"identity":"1dc84eca-94a3-4612-9d2a-1aed846954db","added_by":"auto","created_at":"2025-11-14 07:09:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":496585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCK7 staining, a) Full staining, b) Gradient staining, c) Negative staining and d) Patchy staining\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7579340/v1/6c481cee233ef9a54de10198.png"},{"id":95894781,"identity":"da0fe3f0-0bdb-404e-b231-f00c71a07065","added_by":"auto","created_at":"2025-11-14 07:09:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of CK7 Staining Patterns Across CIN Grades\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7579340/v1/159ebd69925b9963c269c4bf.png"},{"id":97896448,"identity":"b487ac81-081d-4670-a79a-e7c0b4062050","added_by":"auto","created_at":"2025-12-10 15:36:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1180731,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7579340/v1/079d5319-9b96-4684-bfda-5cee8a0b466c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diagnostic Value of CK7 Immunostaining Patterns in Cervical Intraepithelial Neoplasia Running title: CK7 value in CIN","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical cancer remains a significant global health concern, ranking as the fourth most common cancer among women and the second leading cause of cancer-related mortality in low-income countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The majority of cases are preceded by premalignant changes known as cervical intraepithelial neoplasia (CIN), which are histologically classified into grades 1 to 3 (CIN1\u0026ndash;CIN3) based on the extent of epithelial dysplasia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Accurate identification and grading of these lesions are critical, as CIN1 lesions often regress spontaneously, whereas CIN2 and CIN3 carry a substantially higher risk of progression to invasive carcinoma [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the use of standardized histological criteria, CIN grading\u0026mdash;especially CIN2\u0026mdash;remains challenging due to considerable interobserver variability and overlapping morphological features [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a result, immunohistochemical (IHC) markers such as p16^INK4a^, KI-67 antigen, and cytokeratin 7 (CK7) have been increasingly employed to enhance diagnostic accuracy and reproducibility [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCK7 is a low-molecular-weight intermediate filament protein typically expressed in glandular epithelium, particularly at the squamocolumnar junction of the cervix\u0026mdash;the most vulnerable area for HPV-mediated oncogenic transformation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies have shown that CK7 expression increases with lesion severity and may assist in identifying CIN lesions with higher malignant potential [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, most existing studies have been conducted in Western or East Asian populations, with limited data from Middle Eastern or Iranian cohorts. This gap highlights the urgent need for context-specific research to better understand regional variations in CIN grading and CK7 expression, particularly in Middle Eastern populations.\u003c/p\u003e\u003cp\u003eThis study aims to characterize CK7 IHC staining patterns\u0026mdash;categorized as negative, patchy, gradient, and full-thickness\u0026mdash;in histologically confirmed CIN lesions (CIN1\u0026ndash;CIN3) and to examine their associations with two widely used proliferation and surrogate HPV biomarkers, Ki67 and p16. By investigating a regional Iranian population, this study also provides context-specific data that may aid in standardizing interpretation protocols and refining risk stratification strategies in settings with limited access to molecular HPV testing.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eStudy Design and Setting\u003c/p\u003e\u003cp\u003eThis retrospective cross-sectional study was conducted at the Department of Pathology, Beheshti Hospital, affiliated with Isfahan University of Medical Sciences, between 2021 and 2023. Archived cervical biopsy and cone specimens with histopathologically confirmed squamous intraepithelial lesions (SIL) were retrieved for immunohistochemical (IHC) analysis.\u003c/p\u003e\u003cp\u003eInclusion and Exclusion Criteria\u003c/p\u003e\u003cp\u003eAll available samples diagnosed as CIN1, CIN2, or CIN3 during the study period were included using a sequential sampling method. Cases were excluded if patients had a history of immunosuppressive medication, organ transplantation, or current pregnancy.\u003c/p\u003e\u003cp\u003eSample Size\u003c/p\u003e\u003cp\u003eA total of 106 eligible cases were included. The estimated sample size was based on a presumed prevalence of CK7 negativity in CIN1 (~\u0026thinsp;80%), with a 5% margin of error and 95% confidence level.\u003c/p\u003e\u003cp\u003eData Collection\u003c/p\u003e\u003cp\u003ePatient demographics, including age and clinical history, were obtained from hospital records. Colposcopic findings were categorized as normal or abnormal. Past history (PMH) of having CIN was also recorded.\u003c/p\u003e\u003cp\u003eHistopathological and Immunohistochemical Evaluation\u003c/p\u003e\u003cp\u003eAll hematoxylin and eosin (H\u0026amp;E)-stained slides were reviewed to confirm diagnosis and CIN grade. IHC staining for CK7 was performed on formalin-fixed paraffin-embedded (FFPE) sections using a recombinant mouse monoclonal antibody (Long Island Antibody, China). Positive and negative controls were included in each staining batch. Standard protocols were applied, including deparaffinization, antigen retrieval, endogenous peroxidase blocking, primary antibody incubation, and DAB visualization.\u003c/p\u003e\u003cp\u003eCK7 staining was assessed for cytoplasmic and membranous positivity and categorized into four patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eNegative: \u0026lt;1% of epithelial cells stained\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePatchy: 1\u0026ndash;40% of cells stained in isolated clusters\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGradient: progressive increase from superficial to basal layers\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eFull-thickness: \u0026gt;40% of epithelial cells stained in contiguous groups of \u0026ge;\u0026thinsp;5 cells\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eKi67 and P16 scores were extracted from prior pathology reports and scored from 0 to 3, based on the extent of epithelial involvement. Details of the scoring system are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescriptive Distribution of IHC Staining Patterns (CK7, Ki67, P16) and age by CIN Grade\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCIN\u003c/p\u003e\u003cp\u003eIHC Marker\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCIN1\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;69)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCIN2\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;21)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCIN3\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eCK7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53(76.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5(23.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1(6.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epatchy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11(15.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0(0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1(6.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egradient\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5(7.2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9(42.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3(18.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFull thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7(33.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11(68.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eKi67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4(26.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2(15.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11(73.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3(23.1%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4(30.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1(16.7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4(30.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5(83.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eP16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15(100.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5(38.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1(7.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1(16.7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4(30.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScore3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3(23.1%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5(83.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eMean Age\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.06(8.76)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.81(5.42)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36.88(6.71)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using SPSS version 22. Categorical variables were presented as frequencies and percentages. Chi-square tests were used to assess associations between CIN grade and biomarker expression (CK7, Ki67, P16). A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003cp\u003eTo facilitate a more precise analysis, the expression of CK7 was categorized into limited (negative and patchy) versus extensive (including gradient and full-thickness staining). Additionally, Ki67 and p16 data were classified into low expression (scores of 0 or 1) versus high expression (scores of 2 or 3).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePatient Characteristics\u003c/p\u003e\u003cp\u003eA total of 106 cases were included: 69 (65.1%) were diagnosed as CIN1, 21 (19.8%) as CIN2, and 16 (15.1%) as CIN3. The mean age of patients was 38.1 years (SD: 8.4; minimum: 22\u0026ndash;maximum: 62 years). Detailed demographic and clinical characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eCK7 Immunohistochemistry Patterns Across CIN Grades\u003c/p\u003e\u003cp\u003eThe distribution of CK7 staining patterns showed a significant association with CIN grade (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). CIN1 lesions predominantly exhibited negative (76.8%) and patchy (15.9%) staining, with no full-thickness positivity. In contrast, 33.3% of CIN2 and 68.8% of CIN3 lesions showed full-thickness staining. Gradient staining was also more frequent in higher grades. These results are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eWhen CK7 expression was dichotomized into limited (negative\u0026thinsp;+\u0026thinsp;patchy) versus extensive (gradient\u0026thinsp;+\u0026thinsp;full-thickness), 7.2%% of CIN1 cases showed extensive expression, while 76.2% of CIN2 and 87.5% of CIN3 exhibited extensive staining (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of IHC Staining Patterns (CK7, K67, P16) by CIN Grade\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCIN\u003c/p\u003e\u003cp\u003eIHC Marker\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCIN1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCIN2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCIN3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCK7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLimited\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64 (92.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5(23.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2(12.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eextensive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5(7.2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16(76.2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14(87.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eKi67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLOW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15 (100.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 (38.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHIGH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8(61.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6(100.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLOW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15(100.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6(46.2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1(16.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHIGH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7(53.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5(83.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eAbbreviations: CIN, Cervical Intraepithelial Neoplasia; CK7, Cytokeratin 7; P16, p16INK4a.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eKi67 and P16 Expression Patterns\u003c/p\u003e\u003cp\u003eAmong the 34 cases with Ki67 data, high expression (score 2 or 3) was significantly more frequent in CIN2 (61.5%) and CIN3 (100%) compared to CIN1 (0%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eSimilarly, non of CIN1 cases (0%) showed P16 score 2 or 3, while 53.8% of CIN2 and 83.3% of CIN3 had scores 2 or 3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Full distribution of Ki67 and P16 scores across CIN grades is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eClinical Characteristics of Patients Across CIN Grades\u003c/p\u003e\u003cp\u003eColposcopy findings were available for 105 patients, with abnormal findings seen in 42.0% of CIN1, 71.4% of CIN2, and 100% of CIN3 cases (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical Characteristics of Patients by CIN Grade\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCIN grade\u003c/p\u003e\u003cp\u003eClinical data\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCIN1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCIN2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCIN3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ecolposcopy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40 (58.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6 (28.6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 (0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eabnormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29 (42.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15 (71.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePast medical history of CIN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 (8.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (14.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4 (25.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.191\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63 (91.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18 (85.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12 (75.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eMean Age (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.06(8.76)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.81(5.42)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36.88(6.71)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eAbbreviations: CIN, Cervical Intraepithelial Neoplasia; PMH, past medical history of CIN\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThere was statistically significant difference in mean age among the three groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). According to Tukey\u0026rsquo;s post hoc test, this difference was specifically attributable to comparison between CIN1 and CIN2 mean age. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eNo significant relationship was found between CIN grade and past medical history of previous CIN (p\u0026thinsp;=\u0026thinsp;0.191). (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAssociation Between CK7 and Other Variables\u003c/p\u003e\u003cp\u003eCK7 expression patterns were significantly associated with Ki67 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and P16 scores (p\u0026thinsp;=\u0026thinsp;0.002). High Ki67 expression (scores 2 or 3) occurred more frequently in the CK7 extensive pattern (68.4%), while high P16 expression (scores 2 or 3) was also more prevalent in this pattern (57.9%). Full-thickness CK7 staining frequently co-occurred with high Ki67 and P16 expression. (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of IHC Staining Patterns (Ki67, P16) by CK7 Grade\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCK7 grade\u003c/p\u003e\u003cp\u003eOTHER markers grade\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLimited\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExtensive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eKi67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14(93.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6(31.6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1(6.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13(68.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14(93.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8(42.1%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1(6.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11(57.9%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAdditionally, CK7 pattern was associated with colposcopic findings (p\u0026thinsp;=\u0026thinsp;0.004), with extensive CK7 positivity seen in 76.5% of cases with abnormal colposcopy. No significant relationship was found between CK7 expression and History of CIN (p\u0026thinsp;=\u0026thinsp;0.088). (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical Characteristics of Patients by CK7 Grade\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCK7 grade\u003c/p\u003e\u003cp\u003eClinical data\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLimited\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExtensive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eColposcopy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38(53.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8(23.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbnormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33(46.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26(76.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePast medical history of CIN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6(8.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7(20.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.088\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65(91.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28(80.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings of this study confirm a strong association between CK7 immunoreactivity patterns and the histological grade of cervical intraepithelial neoplasia (CIN). A progressive shift from negative or patchy CK7 expression in CIN1 to full-thickness staining in CIN3 was clearly observed, consistent with previous studies suggesting CK7 as a marker of neoplastic transformation at the squamocolumnar junction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCK7 expression has been proposed to reflect the involvement of reserve cells at the squamocolumnar junction, a region vulnerable to HPV-mediated carcinogenesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Our results, which showed increased CK7 expression along with higher CIN grades, are in line with those of Lee et al., who demonstrated increased CK7 and CK19 expression in CIN3 and cervical carcinoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Paquette et al. also emphasized the predictive value of CK7 in low-grade squamous intraepithelial lesions (LSIL), with CK7-positive LSILs more likely to progress to high-grade disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA key observation in our study was the co-occurrence of full-thickness CK7 staining with high Ki67 and p16 scores in most CIN2 and CIN3 lesions. This reinforces the idea of a coordinated upregulation of these biomarkers in dysplastic epithelium. Previous reports have established the diagnostic and prognostic utility of Ki67 and p16 in distinguishing high-grade lesions and in predicting biological behavior [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, Huang et al. found that the combination of CK7 and p16 provided superior predictive accuracy in identifying LSILs at risk of progression [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur study\u0026rsquo;s results align with those of Samyuktha et al., who showed a significant correlation between CK7 intensity and CIN grade [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, Umphress et al. proposed the complementary use of CK7 with p16 to resolve diagnostic uncertainty in CIN2, a notoriously ambiguous category [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite these strengths, certain limitations must be acknowledged. First, the retrospective design restricts our ability to assess temporal progression or establish causality. Second, the absence of follow-up data precludes direct assessment of lesion outcomes. Third, immunohistochemical data for Ki67 and p16 were not available for all cases, which may have introduced a degree of selection or reporting bias. These limitations are shared by many previous studies in the field [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn terms of generalizability, our study was limited to a single-center sample within a defined geographic region, which may restrict the external applicability of findings to more diverse populations. Nonetheless, the consistency of our results with multiple international studies enhances confidence in the biological validity of CK7 as a marker of CIN severity [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFrom a clinical standpoint, the addition of CK7 staining to routine diagnostic panels could improve the stratification of patients, especially in cases with morphologically ambiguous findings. In resource-limited settings where HPV genotyping or molecular profiling is not feasible, combining CK7 with p16 and Ki67 may offer a cost-effective alternative to enhance diagnostic accuracy and guide patient management [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings support the growing body of evidence indicating that CK7, particularly in full-thickness and gradient patterns, is a valuable immunohistochemical marker in CIN. When used alongside p16 and Ki67, CK7 not only improves diagnostic precision but may also provide insight into lesion biology and progression risk. Future prospective studies with long-term clinical follow-up are recommended to validate these findings and explore their utility in clinical decision-making.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan lang=\"EN-GB\"\u003eEthical Approval and Consent to Participate\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Research Ethics Committee of the School of Medicine, Isfahan University of Medical Sciences (Approval ID: IR.MUI.MED.REC.1403.150, Approval date: 15 July 2024). The study was conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments. The requirement for informed consent was waived by the Ethics Committee, as this was a retrospective study conducted on archived pathology slides and blocks, with no identifiable patient data included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp;Not applicable. This manuscript does not contain any person\u0026rsquo;s data in any form (including individual details, images, or videos)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial Support:\u0026nbsp;\u003c/strong\u003eNo financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan lang=\"EN-GB\"\u003eCompeting interests:\u003c/span\u003e\u003c/strong\u003e\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE O\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003ewas contributed in literature search, data acquisition, data analysis, manuscript preparation, manuscript editing, and manuscript review\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eF N\u0026nbsp;was contributed in literature search, data acquisition, data analysis, manuscript preparation, manuscript editing, and manuscript review\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan lang=\"EN-GB\"\u003eM J T was contributed in data acquisition and data analysis\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eM S\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003ewas contributed in literature search, manuscript preparation, manuscript editing, and manuscript review\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003e\u003cspan lang=\"EN-GB\"\u003eNot applicable\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElham Omidi, Assistant Professor of Pathology, Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran\u003c/p\u003e\n\u003cp\u003eFarzaneh Nayeri, Resident of Pathology, Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran\u003c/p\u003e\n\u003cp\u003eMohammadjavad Tarrahi: Professor of Biostatistics and Epidemiology, Department of Health Sciences, Isfahan University of Medical Sciences, Isfahan, Iran\u003c/p\u003e\n\u003cp\u003eMaryam Soltan, Assistant Professor of Pathology, Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLin S, Gao K, Gu S, Chen Y, Li S, Wang Y, et al. Worldwide trends in cervical cancer incidence and mortality, with predictions for the next 15 years. Cancer. 2021;127(21):4030\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoeneman MM, Kruitwagen RFP, Nijman HW, Slangen BFM, Van Gorp T, Kruse AJ. Natural history of high-grade cervical intraepithelial neoplasia: a review of prognostic biomarkers. Expert Rev Mol Diagn. 2015;15(4):527\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoopik DL, Bentley HA, Eijgenraam MN, van der Aa MA, van der Velden J, Ebisch RMF, et al. The natural history of cervical intraepithelial neoplasia grades 1, 2, and 3: a systematic review and meta-analysis. J Low Genit Tract Dis. 2021;25(3):221\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang EJ, Quick MC, Hanamornroongruang S et al. Microanatomy of the cervical and anorectal squamocolumnar junctions: a proposed model for anatomical differences in HPV-related cancer risk. Modern Pathology. 2015;28(7):994\u0026ndash;1000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/modpathol.2015.54\u003c/span\u003e\u003cspan address=\"10.1038/modpathol.2015.54\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 25839700.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerfs M, Soong TR, Delvenne P, Crum CP. Deciphering the multifactorial susceptibility of mucosal junction cells to HPV infection and related carcinogenesis. Viruses. 2017;9(4):85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee H, Lee H, Cho YK. Cytokeratin7 and cytokeratin19 expression in high-grade cervical intraepithelial neoplasm and squamous cell carcinoma and their possible association in cervical carcinogenesis. Diagn Pathol. 2017;12(1):18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaquette C, Mills AM, Stoler MH. Predictive value of cytokeratin 7 immunohistochemistry in cervical low-grade squamous intraepithelial lesion as a marker for risk of progression to a high-grade lesion. Am J Surg Pathol. 2016;40(2):236\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang EC, Tomic MM, Hanamornroongruang S, Crum CP. p16^INK4a^ and cytokeratin 7 immunostaining in predicting HSIL outcome for low-grade squamous intraepithelial lesions: a case series, literature review and commentary. Mod Pathol. 2016;29(12):1501\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSamyuktha A, Shetty P, Mysorekar V. A study of cytokeratin-7 expression and clinicopathological correlation in cervical dysplasia and carcinoma. Asian Pac J Cancer Prev. 2022;23(3):885\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan der Marel J, van Baars R, Hesselink AT, Melchers WJ, Massuger LF, Quint WG, et al. The role of cytokeratin 7 and cytokeratin 19 in the development of high-grade cervical intraepithelial neoplasia. Am J Surg Pathol. 2014;38(4):470\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUmphress B, Sanchez B, Paintal A, Nayar R, Maniar KP. Utility of CK7 versus p16 as a prognostic biomarker in CIN 2. Am J Surg Pathol. 2018;42(4):479\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoussef M, Canete-Portillo S, Li Z, Wang HL. Patterns of cytokeratin 7 expression in cervical squamous intraepithelial lesions: a potential marker for lesion progression. Am J Clin Pathol. 2020;154(Suppl 1):S37\u0026ndash;S.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanthiya K, Khunnarong J, Tangjitgamol S, Puripat N, Tanvanich S. Expression of the p16 and Ki67 in cervical squamous intraepithelial lesions and cancer. Asian Pac J Cancer Prev. 2016;17(7):3201\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CK7, cervical intraepithelial neoplasia, Ki-67 Antigen, P16, immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-7579340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7579340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eAccurate classification of cervical intraepithelial neoplasia (CIN) is essential for the appropriate treatment strategy and monitoring disease progression. While morphological evaluation remains the standard, interobserver variability and grading ambiguity\u0026mdash;particularly in CIN2\u0026mdash;necessitate additional biomarkers. This study evaluates CK7 immunostaining patterns and their association with CIN grade, Ki67 and p16 expression, and colposcopy findings.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eIn a retrospective cross-sectional study, 106 histologically confirmed CIN cases (CIN1\u0026ndash;CIN3) were assessed for CK7 expression patterns (negative, patchy, gradient, full-thickness). Ki67 and p16 scores (0\u0026ndash;3) were obtained from prior pathology reports. Chi-square analysis was used to evaluate associations between biomarker expression and CIN grade.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eCK7 expression was significantly associated with CIN grade (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with full-thickness staining observed in 68.8% of CIN3 cases and 0% of CIN1. High Ki67 and p16 scores (\u0026ge;\u0026thinsp;2) were also significantly associated with higher-grade CIN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). CK7 pattern correlated strongly with Ki67 (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), p16 (p\u0026thinsp;=\u0026thinsp;0.002), and abnormal colposcopic findings (p\u0026thinsp;=\u0026thinsp;0.004). No significant association was found with past medical history of having CIN (p\u0026thinsp;=\u0026thinsp;0.088).\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e\u003cp\u003eCK7 expression patterns reflect the severity of CIN and correlate with proliferation (Ki67) and HPV-driven transformation (p16). Full-thickness and gradient CK7 staining, when combined with Ki67 and p16, increase diagnostic accuracy and may inform clinical decision-making, particularly in morphologically ambiguous cases or settings with limited access to molecular testing.\u003c/p\u003e","manuscriptTitle":"Diagnostic Value of CK7 Immunostaining Patterns in Cervical Intraepithelial Neoplasia Running title: CK7 value in CIN","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 07:09:39","doi":"10.21203/rs.3.rs-7579340/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6dfc8152-4e0b-48a2-b078-0d074661d90a","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-09T19:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 07:09:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7579340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7579340","identity":"rs-7579340","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}