Epithelial dysplasia (ED) grading systems are criticized for low reliability. The effects of diagnostic tests or stains on reducing examiner variability in grading ED of the upper aerodigestive tract have not been investigated. The study aim was to examine the effectiveness of cytokeratin 19 (K19) immunostain on enhancing inter and intraexaminer reliability of ED grading and to reiterate the relation of K19 positivity with epithelial keratinization. The study sample consisted of 122 paraffin blocks that fulfilled the inclusion criteria. Each paraffin block had three sections cut: one immunostained for K19 and two for hematoxylin and eosin stain (H&E). Each examiner graded the study sample in six rounds; three using H&E stain only and three using paired K19–H&E stains. The study examiners were three American-Board certified practicing oral pathologists. The results were analyzed using Krippendorff’s alpha, ROC curve, Chi square test and binary logistic regression. Upon the use of paired K19–H&E stains the results showed that the intraexaminer reliability coefficients of grading were improved from 0.70, 0.69, 0.78 to 0.73, 0.88, 0.91 for examiners 1, 2, and 3 respectively. Reliability coefficients for inter-examiners improved from 0.55 to 0.73 (Krippendorff alpha). The accuracy of identifying the diseased cases (high-grade dysplasia) increased from 0.82 to 0.94 (ROC curve). Binary logistic regression revealed that K19 positivity is negatively associated with hyperkeratinization of surface epithelium (P = 0.001). To conclude, for grading non-keratinized epithelial dysplastic lesions of the upper aerodigestive tract, paired K19–H&E stains proved to reduce inter and intra-examiner variability by highlighting the extension of dysplastic epithelial cells within epithelial thickness, thus identifying the involved epithelial third and assigning a more reliable and better reproducible grade.
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Harrison LB, Sessions RB, Hong WK. Head and neck cancer: a multidisciplinary approach. Philadelphia: Lippincott Williams & Wilkins; 2009. p. 24.
van Hulst AM, Kroon W, van der Linden ES, Nagtzaam L, Ottenhof SR, Wegner I, et al. Grade of dysplasia and malignant transformation in adults with premalignant laryngeal lesions. Head Neck. 2016;38(S1):E2284–90.
Reibel J. Prognosis of oral pre-malignant lesions: significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med. 2003;14(1):47–62.
Cowan C, Gregg T, Napier S, McKenna S, Kee F. Potentially malignant oral lesions in Northern Ireland: a 20-year population-based perspective of malignant transformation. Oral Dis. 2001;7(1):18–24.
Sadiq H, Gupta P, Singh N, Thakar S, Prabhakar I, Thakral J. Various grading systems of the oral epithelial dysplasia: a review. Int J Adv Health Sci. 2015;1:20–6.
Villa A, Woo SB. Leukoplakia—a diagnostic and management algorithm. J Oral Maxillofac Surg. 2017;75(4):723–34.
Reibel JGN, Hille J. Oral potentially malignant disorders and oral epithelial dysplasia. World Health Organization classification of tumors: head and neck tumors. Lyon: IARC Press; 2017. p. 435.
Sharma N, Hosmani JV, Tiwari V. Epithelial dysplasia: different grading system and its applications. J Int Oral Health. 2010;2(1):1–8.
Fleskens S, Slootweg P. Grading systems in head and neck dysplasia: their prognostic value, weaknesses and utility. Head Neck Oncol. 2009;1(1):11.
Speight PM. Update on oral epithelial dysplasia and progression to cancer. Head Neck Pathol. 2007;1(1):61–6.
Kujan O, Oliver RJ, Khattab A, Roberts SA, Thakker N, Sloan P. Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol. 2006;42(10):987–93.
Mahajan MC, Hazarey V. An assessment of oral epithelial dysplasia using criteria of Smith & Pindborg Grading System’ & Ljubljana Grading System’ in oral precancerous lesions. J Oral Maxillofac Pathol. 2004;8(2):73.
Scully C, Sudbø J, Speight P. Progress in determining the malignant potential of oral lesions. J Oral Pathol Med. 2003;32(5):251–6.
Gonzalez-Moles MA. Comment on: Küffer and Lombardi “Premalignant lesions of the oral mucosa. A discussion about the place of intraepithelial neoplasia”. Oral Oncol. 2002;38(8):809–10.
Kuffer J, Lombardi S. Reconsideration oral risk lesions. Oral Dis. 2002;38:302–7.
Manchanda A, Shetty D-C. Reproducibility of grading systems in oral epithelial dysplasia. Medicina oral, Patologia oral y Cirugia bucal. 2012;17(6):e935.
Gale NPB. Epithelial precursor lesions. In: Barnes LEJ, Reichart P, Sindransky D, editors. World Health Organization classification of tumours pathology and genetics of head and neck tumours. Lyon: IARC; 2005. p. 177–9.
Nankivell P, Williams H, Matthews P, Suortamo S, Snead D, McConkey C, et al. The binary oral dysplasia grading system: validity testing and suggested improvement. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115(1):87–94.
Gnepp D. Diagnostic surgical pathology of the head and neck. 2nd ed. Saunders: Elsevier Health Sciences; 2009. p. 647.
Gale N, Zidar N, Poljak M, Cardesa A. Current views and perspectives on classification of squamous intraepithelial lesions of the head and neck. Head Neck Pathol. 2014;8(1):16–23.
Izumo T. Oral premalignant lesions: from the pathological viewpoint. Int J Clin Oncol. 2011;16(1):15–26.
Kujan O, Khattab A, Oliver RJ, Roberts SA, Thakker N, Sloan P. Why oral histopathology suffers inter-observer variability on grading oral epithelial dysplasia: an attempt to understand the sources of variation. Oral Oncol. 2007;43(3):224–31.
Trask DK, Band V, Zajchowski DA, Yaswen P, Suh T, Sager R. Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells. Proc Natl Acad Sci. 1990;87(6):2319–23.
Safadi RA, Musleh AS, Al-Khateeb TH, Hamasha AA-H. Analysis of immunohistochemical expression of k19 in oral epithelial dysplasia and oral squamous cell carcinoma using color deconvolution-image analysis method. Head Neck Pathol. 2010;4(4):282–9.
Yoshida K, Sato K, Tonogi M, Tanaka Y, Yamane G-Y, Katakura AC. Expression of cytokeratin 14 and 19 in process of oral carcinogenesis. Bull Tokyo Dent Coll. 2015;56(2):105–11.
Kobayashi T, Maruyama S, Cheng J, Ida-Yonemochi H, Yagi M, Takagi R, et al. Histopathological varieties of oral carcinoma in situ: diagnosis aided by immunohistochemistry dealing with the second basal cell layer as the proliferating center of oral mucosal epithelia. Pathol Int. 2010;60(3):156–66.
Su L, Morgan PR, Lane EB. Keratin 14 and 19 expression in normal, dysplastic and malignant oral epithelia. A study using in situ hybridization and immunohistochemistry. J Oral Pathol Med. 1996;25(6):293–301.
Lindberg K, Rheinwald JG. Suprabasal 40 kd keratin (K19) expression as an immunohistologic marker of premalignancy in oral epithelium. Am J Pathol. 1989;134(1):89.
Jacques CM, Pereira AL, Maia V, Cuzzi T, Ramos-e-Silva M. Expression of cytokeratins 10, 13, 14 and 19 in oral lichen planus. J Oral Sci. 2009;51(3):355–65.
Antoine J-Y, Villaneau J, Lefeuvre A, editors. Weighted Krippendorff’s alpha is a more reliable metrics for multi-coders ordinal annotations: experimental studies on emotion, opinion and coreference annotation. In: EACL 2014; 2014.
Krippendorff K. Reliability in content analysis. Hum Commun Res. 2004;30(3):411–33.
Gale N, Blagus R, El-Mofty SK, Helliwell T, Prasad ML, Sandison A, et al. Evaluation of a new grading system for laryngeal squamous intraepithelial lesions—a proposed unified classification. Histopathology. 2014;65(4):456–64.
Tilakaratne W, Sherriff M, Morgan P, Odell E. Grading oral epithelial dysplasia: analysis of individual features. J Oral Pathol Med. 2011;40(7):533–40.
Fleskens SA, Bergshoeff VE, Voogd AC, van Velthuysen M-LF, Bot FJ, Speel E-JM, et al. Interobserver variability of laryngeal mucosal premalignant lesions: a histopathological evaluation. Mod Pathol. 2011;24(7):892.
Mallia RJ, Subhash N, Mathews A, Kumar R, Thomas SS, Sebastian P, et al. Clinical grading of oral mucosa by curve-fitting of corrected autofluorescence using diffuse reflectance spectra. Head Neck. 2010;32(6):763–79.
Mittal KR, Demopoulos RI, Goswami S. Patterns of keratin 19 expression in normal, metaplastic, condylomatous, atrophic, dysplastic, and malignant cervical squamous epithelium. Am J Clin Pathol. 1992;98(4):419–23.
Carletta J. Assessing agreement on classification tasks: the kappa statistic. Comput Linguist. 1996;22(2):249–54.
Bland JM, Altman DG. Statistics notes: Cronbach’s alpha. BMJ. 1997;314(7080):572.
Tavakol M, Mohagheghi MA, Dennick R. Assessing the skills of surgical residents using simulation. J Surg Edu. 2008;65(2):77–83.
Streiner DL. Starting at the beginning: an introduction to coefficient alpha and internal consistency. J Pers Assess. 2003;80(1):99–103.
Dooley K. Questionnaire programming language, version 1 (software package). Washington, DC: US General Accounting Office; 1988.
Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33(1):159–74.
Bosman FT. Dysplasia classification: pathology in disgrace? J Pathol. 2001;194(2):143–4.
Speight PM, Abram TJ, Floriano PN, James R, Vick J, Thornhill MH, et al. Interobserver agreement in dysplasia grading: toward an enhanced gold standard for clinical pathology trials. Oral Surg Oral Med Oral Pathol Oral Radiol. 2015;120(4):474–82.
Lasisi F, Mouchli A, Riddell R, Goldblum JR, Cummings OW, Ulbright TM, et al. Agreement in interpreting villous elements and dysplasia in adenomas less than one centimetre in size. Dig Liver Dis. 2013;45(12):1049–55.
Barnes L. Diseases of the larynx, hypopharynx, and trachea. Surgical pathology of the head and neck. 3rd ed. Boca Raton: CRC Press; 2008. p. 117–208.
Gale N, Michaels L, Luzar B, Poljak M, Zidar N, Fischinger J, et al. Current review on squamous intraepithelial lesions of the larynx. Histopathology. 2009;54(6):639–56.
Wenig BM. Squamous cell carcinoma of the upper aerodigestive tract: precursors and problematic variants. Mod Pathol. 2002;15(3):229–54.
Zur Hausen H. Papillomavirus infections—a major cause of human cancers. Biochim Biophys Acta. 1996;1288(2):F55–78.
Mehanna HM, Rattay T, Smith J, McConkey CC. Treatment and follow-up of oral dysplasia—a systematic review and meta-analysis. Head Neck. 2009;31(12):1600–9.
Boy SC. Leukoplakia and erythroplakia of the oral mucosa—a brief overview. SADJ. 2012;67(10):558–60.
Amagasa T, Yamashiro M, Uzawa N. Oral premalignant lesions: from a clinical perspective. Int J Clin Oncol. 2011;16(1):5–14.
McLaren KM, Burnett RA, Goodlad JR, Howatson SR, Lang S, Lee FD, et al. Consistency of histopathological reporting of laryngeal dysplasia. The Scottish Pathology Consistency Group. Histopathology. 2000;37(5):460–3.
Coltrera MD, Zarbo RJ, Sakr WA, Gown AM. Markers for dysplasia of the upper aerodigestive tract. Suprabasal expression of PCNA, p53, and CK19 in alcohol-fixed, embedded tissue. Am J Pathol. 1992;141(4):817–25.
The research is funded by King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, Ministry of National Guard, Riyadh, Saudi Arabia. Grant Number: RC 17/100. Special thanks to Sarah A. AlJundi, Medical Technologist, Department of Pathology and Laboratory Medicine at King Abduaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, KSA for her valuable technical support.
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The Research was approved by the IRB committee of King Abdullah International Medical Research Center; reference number IRBC/1154/17.
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Safadi, R.A., AlRomaizan, A., Alshagroud, R.S. et al. Cytokeratin 19 Immunostain Reduces Variability in Grading Epithelial Dysplasia of the Non-Keratinized Upper Aerodigestive Tract Mucosa. Head and Neck Pathol 14, 183–191 (2020). https://doi.org/10.1007/s12105-019-01038-w