Immunohistochemical Study of Differential Expressions of Cytokeratin-13, -14, - 17 and p53 in Epithelial Dysplasia and Carcinoma of the Tongue

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1 Original Yasuo Okada et al.: Immunohistochemical Expressions of Cytokeratin and p53 in Tongue ucosal Lesions Journal of Hard Tissue Biology 19[2] (2010) p The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODENJHTBFF, ISSN Immunohistochemical Study of Differential Expressions of Cytokeratin13, 14, 17 and p53 in Epithelial Dysplasia and Carcinoma of the Tongue Yasuo Okada and ichiko oride Department of Pathology, The Nippon Dental University, School of Life Dentistry at Niigata, 18 Hamauracho, Chuoku, Niigata City, Niigata , Japan. (Accepted for publication, July 25, 2010) Abstract: In cancers of the oral cavity, high histological malignancy grade is associated with higher risk of cervical lymph nodes, lung and bone metastases, which have great impact on the prognosis. Furthermore, early detection of these cancers is desirable so that treatment can aim at conserving oral functions and improving prognosis. It is known that several percent of leukoplakia that develop in the tongue become cancerous. Leukoplakia is a clinical diagnostic term, and histopathologically most cases show hyperkeratosis with epithelial dysplasia or hyperkeratosis without epithelial dysplasia. On the other hand, difficulties in histopathological diagnosis of severe epithelial dysplasia and carcinoma in situ are often encountered. In recent years, immunohistochemical staining of cytokeratins (CK), p53 and Ki67 for precancerous lesions and squamous cell carcinoma (SCC) of the oral cavity has been reported. However, few reports have applied a combination of several immunohistochemical stainings to diagnosis. In the present study, with the objective to achieve a higher precision in histopathological diagnosis, we conducted and compared the immunohistochemical stainings of CK13, CK17, CK14 and p53 in 10 cases each of hyperkeratosis without epithelial dysplasia, hyperkeratosis with epithelial dysplasia, carcinoma in situ, and SCC in the tongue. The following results were obtained. CK13 was positive in all 10 cases of hyperkeratosis without epithelial dysplasia, and negative in all cases of hyperkeratosis with epithelial dysplasia, carcinoma in situ, and SCC. CK17 was negative in all 10 cases of hyperkeratosis without epithelial dysplasia, and was positive in all 10 cases of hyperkeratosis with epithelial dysplasia as well as 10 cases of carcinoma in situ and 9 of 10 cases of SCC. CK14 was negative or positive only in the basal cell layer or a few rows of cells in spinous layer adjacent to the basal cell layer in cases of hyperkeratosis without epithelial dysplasia, but was positive in all the epithelial layers in all 10 cases of hyperkeratosis with epithelial dysplasia, and also positive in the tumor in all 10 cases each of carcinoma in situ and SCC. For p53, positive reaction was observed in some cells of the basal cell layer in 2 of 10 cases of hyperkeratosis without epithelial dysplasia, while high positive rates were found in hyperkeratosis with epithelial dysplasia (10 of 10 cases) and SCC (9 of 10 cases). On the other hand, only 5 of 10 cases of carcinoma in situ were positive for p53. In conclusion, although the histopathological diagnosis of epithelial dysplasia and carcinoma in situ is based on hematoxylineosin stained sections, immunostaining and evaluation of CK13, CK17, CK14 and p53 are useful as an adjunct to histopathological diagnosis. Key words: Tongue, Epithelial dysplasia, Carcinoma, Cytokeratin, p53 Introduction reports have indicated that several percents of leukoplakia undergo In cancers of the oral cavity, high histopathological malignancy malignant changes 7,8). Leukoplakia is a clinical diagnostic term, grade is associated with increased risks of metastases to cervical and histopathologically most cases of leukoplakia show lymph nodes, lung and bone, which greatly impact the prognosis 1 hyperkeratosis with epithelial dysplasia or hyperkeratosis without 6). oreover, early detection of these cancers is desirable so that epithelial dysplasia. Histopathological diagnoses of severe treatment can aim at conservation of oral functions and epithelial dysplasia and carcinoma in situ occurring on the tongue improvement of prognosis. An accurate and early diagnosis is are often difficult. In addition, differentiation between epithelial therefore desirable. Epithelial dysplasia occurring on the tongue dysplasia and carcinoma in situ is extremely important to is most commonly presented and diagnosed as leukoplakia, and understand the malignant transformation process of epithelial Correspondence to: Yasuo Okada, DDS, PhD. Department of Pathology, dysplasia. In recent years, immunohistochemical studies of The Nippon Dental University, School of Life Dentistry at Niigata, 18 cytokeratin (CK), p53 and Ki67 in precancerous lesions and Hamauracho, Chuoku, Niigata City, Niigata , Japan. Tel: , Fax: yokada@ngt.ndu.ac.jp squamous cell carcinoma (SCC) of the oral cavity have been 123

2 J.Hard Tissue Biology Vol. 19(2):123130, 2010 reported 913). However, the application of a combination of several immunohistochemical stains to diagnosis is rarely reported. Table 1. Expression of CK13, CK17, CK14 and p53 in hyperkeratosis without epithelial dysplasia of the tongue With the objective to improve the precision of histopathological diagnosis, we conducted and evaluated immunohistochemical Case No. Gender Age CK13 CK17 CK14 p53 stainings of CK13, CK17, CK14 and p53 not only in cases of hyperkeratosis with epithelial dysplasia and carcinoma in situ, but also in cases of hyperkeratosis without epithelial dysplasia and SCC occurring in the tongue F F Patients and ethods Patients Among patients who underwent biopsy or resection of lesions located at the lateral edge of tongue, 10 each of the most recent primary cases diagnosed histopathologically as hyperkeratosis F F without epithelial dysplasia, hyperkeratosis with epithelial Staining intensity:, intermediate; +, weak;, negative. dysplasia, carcinoma in situ, and SCC (40 cases in total) were CK13: Positive reaction was observed from the cornified layer to studied. The male to female ratios and age ranges (mean age) were, respectively, 6 : 4 and 3779 (62.4) years in hyperkeratosis without epithelial dysplasia cases, 5 : 5 and 2483 (62.3) years in hyperkeratosis with epithelial dysplasia cases, 8 : 2 and 2680 spinous layer. CK14: Positive reaction was observed in the basal cell layer and a few rows of cells in the spinous layer adjacent to the basal cell layer. p53: Positive reaction was observed in some cells of the basal cell layer. (64.4) years in carcinoma in situ cases, and 6 : 4 and 5986 (74.3) years in SCC cases. For carcinoma in situ and SCC cases, the initial biopsy specimens were evaluated. Table 2. Expression of CK13, CK17, CK14 and p53 in hyperkeratosis with epithelial dysplasia of the tongue Procedures In each case, immunohistochemical studies for CK13 (DE K13, Dako, Glostrup, Denmark), CK14 (LL002, BioGenex, San Ramon, CA, USA), CK17 (E3, Dako) and p53 (DO7, Dako) were conducted. Immunohistochemical staining of formalinfixed, paraffinembedded sections was done using an Envision kit (Dako). After blocking endogenous peroxidase by treating the sections for 30 min with methanol containing 0.3% H 2 O 2, pretreatment in immunosaver (Nissin E, Tokyo, Japan) was performed for antigen retrieval. The sections were incubated with the primary antibodies (working dilution 1/100 for all antibodies) for CK13, CK14, CK17 and p53 overnight at 4, followed by incubation with polymerperoxidase antimouse immunoglobulin for 30 min at room temperature. Color was developed with 3,3' diaminobenzidine. The slides were observed under a light microscope by two investigators and the differences in staining were evaluated. For hyperkeratosis without epithelial dysplasia and hyperkeratosis with epithelial dysplasia, staining reactions in the cornified layer, granular layer, spinous layer, and basal cell layer were examined. For carcinoma in situ, staining reactions at sites equivalent to the four abovementioned layers were observed. For SCC, the staining reactions from the superficial part to the most advanced part of the tumor were observed. This study was approved by the Ethics Committee of The Nippon Dental University, Life Dentistry at Niigata. Case No. Gender Age CK13 CK17 CK14 p F F F F F 60 + Staining intensity: +, strong;, intermediate; +, weak;, negative. CK17: Positive reaction was observed from the cornified layer to spinous layer. CK14: Positive reaction was observed in all the epithelial layers. p53: Positive reaction was observed in some cells of the basal cell layer. Results For hyperkeratosis without epithelial dysplasia, CK13positive reaction was observed from the cornified layer to spinous layer in all 10 cases, while the basal cell layer was negative. CK17 was negative in all 10 cases. CK14 was positive in 7 of 10 cases, but positive staining was limited to the basal cell layer and a few rows of cells in the spinous layer adjacent to the basal cell layer. Immunostaining of p53 was positive in some cells of the basal cell layer in 2 of 10 cases (Table 1, Fig. 1AE). 124

3 Yasuo Okada et al.: Immunohistochemical Expressions of Cytokeratin and p53 in Tongue ucosal Lesions Figure 1. Histopathological and immunohistochemical findings in the tongue lesions. (AE) A typical case of hyperkeratosis without epithelial dysplasia of the tongue. (A) HE stained section of the initial biopsy showing hyperkeratosis and thickening of the spinous layer, but no epithelial dysplasia. (B) Staining of CK13 showing positive reaction from the cornified layer to spinous layer but negative in basal cell layer. (C) Staining of CK17 showing negative finding in all epithelial layers. (D) Staining of CK14 showing positive reactions in the basal cell layer and a few rows of cells in the spinous layer adjacent to the basal cell layer. (E) Staining of p53 showing negative finding in all epithelial layers. (FJ) A typical case of hyperkeratosis with epithelial dysplasia of the tongue. (F) HE stained section of the initial biopsy showing hyperkeratosis and thickening of the spinous layer, as well as moderate epithelial dysplasia. (G) Staining of CK13 showing negative finding in the majority of the lesion but sparse positive reaction in a small portion. (H) Staining of CK17 showing positive reaction from the cornified layer to spinous layer. (I) Staining of CK14 showing positive reaction in all epithelial layers. (J) Staining of p53 showing positive reaction in the basal cell layer. (Contenued on the following page) 125

4 J.Hard Tissue Biology Vol. 19(2):123130, 2010 Table 3. Expression of CK13, CK17, CK14 and p53 in carcinoma Table 4. Expression of CK13, CK17, CK14 and p53 in SCC of the in situ of the tongue tongue Case No. Gender Age CK13 CK17 CK14 p F F Staining intensity: +, strong;, intermediate; +, weak;, negative. CK17 and CK14: Positive reaction was observed in the lesions. p53: Positive reaction was observed in cells equivalent to the basal cell layer or from the basal cell layer to spinous layer. Case No. Gender Age CK13 CK17 CK14 p F F F F 65 + Staining intensity: +, strong;, intermediate; +, weak;, negative. CK17 and CK14: Positive reaction was observed in the tumor. p53: Positive reaction was observed in some cells at the margin of the tumor nest in 6 cases. For hyperkeratosis with epithelial dysplasia, CK13 was negative in the lesions of all 10 cases, but positive reaction was observed sparingly in a part of the spinous layer. CK17 was positive in all 10 cases, but the basal cell layer was negative in 4 cases. Strong CK14 positive reaction was observed in all 10 cases. While p53positive cells were observed in all 10 cases, the positive reaction was limited to some cells of the basal cell layer in 5 cases and in some cells from the basal cell layer to spinous layer in the remaining 5 cases (Table 2, Fig. 1FJ). For carcinoma in situ, CK13 was negative in the lesions of all 10 cases. CK17 was positive in the lesions of all 10 cases, but the staining intensity was weak in 6 cases. CK14 was strongly positive in the lesions of all 10 cases. Positive reaction for p53 was observed in cells equivalent to the basal cell layer or from the basal cell layer to spinous layer in only 5 of 10 cases (Table 3, Fig. 1KO). In addition, at the cancer transition zone, formation of an oblique line (or front) was observed in the hematoxylineosin (HE) stained section. With this line as border, the tumor portion showed CK13 negative, and CK17 and CK14positive findings; while the nontumor portion was CK13positive, CK17negative, and CK14 positive confined to the basal cell layer and a few rows of cells in the spinous layer adjacent to the basal cell layer (Fig. 2). For SCC, CK13 was negative in the tumor tissue of all 10 cases. CK17 was positive in 9 of 10 cases, and was mostly negative but sparingly positive in the remaining case. All 10 cases showed strong positive reaction for CK14. Immunostaining of p53 was positive in 9 of 10 cases, 6 of which showed positive reaction in some cells at the margin of the tumor nest (Table 4, Fig. 1PT). Discussion Cancers of the oral cavity with high histopathological malignancy grade are associated with increased risks of metastases to cervical lymph nodes, lung and bone, which greatly affect the prognosis 16). oreover, early detection of these cancers is desirable so that treatment can aim at conservation of oral functions and improvement of prognosis. On the other hand, difficulties are (KO) A typical case of carcinoma in situ of the tongue. (K) HE stained section of the initial biopsy showing no tumor invasion of the lamina propria, confirming cancer in situ. (L) Staining of CK13 showing negative finding in the lesion. () Staining of CK17 showing positive reaction in the lesion. (N) Staining of CK14 showing positive reaction in the lesion. (O) Staining of p53 showing positive reaction from the spinous layer to basal cell layer. (PT) A typical case of SCC of tongue. (P) HE stained section of the initial biopsy shows well differentiated SCC. (Q) Staining of CK13 showing negative finding in the tumor. (R) Staining of CK17 showing positive reaction in the tumor. (S) Staining of CK14 showing positive reaction in the tumor. (T) Staining of p53 showing positive reaction in some cells at the margin of the tumor nest. 126

5 Yasuo Okada et al.: Immunohistochemical Expressions of Cytokeratin and p53 in Tongue ucosal Lesions Figure 2. A case of carcinoma in situ of the tongue. (A) Oblique line (front) formation is observed (arrows). With this zone as border, malignant transformation occurs from the right to the left. (B) Serial section of (A) immunohistochemically stained for CK13: With the zone corresponding to the oblique line (HE staining) as border, negative finding is observed on the tumor side and positive reaction on the nontumor side. (C) Serial section of (A) immunohistochemically stained for CK17: With the zone corresponding to the oblique line as border, positive reaction is observed on the tumor side and negative finding on the nontumor side. (D) Serial section of (A) immunohistochemically stained for CK14: With the zone corresponding to the oblique line as border, positive reaction is observed on the tumor side and negative finding in a portion of the nontumor side. often encountered in histopathological diagnosis of severe epithelial dysplasia and carcinoma in situ. ost cases of epithelial dysplasia are diagnosed clinically presented as leukoplakia. Previous reports indicated that several percents of leukoplakia cases undergo malignant transformation 7,8). Hansen et al 8) reported that a particular form of proliferative verrucous leukoplakia may undergo transformation from leukoplakia to verrucous hyperplasia, verrucous carcinoma, papillary SCC, and then less differentiated SCC. The WHO classification of 1997 classified epithelial dysplasia as a precancerous lesion and listed 13 items of cellular atypia and structural atypia as diagnostic criteria for epithelial dysplasia 14). The 2005 WHO classification included squamous intraepithelial neoplasia (SIN) 15) and currently much research is being conducted in this field. We consider SIN not as a squamous intraepithelial neoplasm but as a squamous intraepithelial neoplastic lesion. In other words, SIN is not yet a true neoplasm but a lesion that is susceptible to malignant transformation; SIN2 more susceptible than SIN1, and SIN3 more so than SIN2, representing a classification based on working prediction. Therefore, differentiation between epithelial dysplasia and carcinoma in situ is important. In addition, basic research on the possibility of malignant transformation of SIN is desirable, and refined diagnostic methods are needed for this kind of research. With this background, we evaluated and compared immunohistochemical stainings of CK13, CK17, CK14 and p53 in hyperkeratosis without epithelial dysplasia, hyperkeratosis with epithelial dysplasia, carcinoma in situ and SCC arising in the tongue. Cytokeratin belongs to the intermediate sized filaments of the cytoskeleton. Over 20 subtypes of cytokeratin are known and they express differentially in the normal epithelium and various epithelial tumors 1618). Acidic keratins such as CK13, CK14, CK15, CK16, CK17 and CK19 as well as basic keratins including CK4, CK5 and CK6 have been identified on the lingual mucosal epithelium 16) and the expressions of these subtypes vary in different layers of the epithelium. CK13 evaluated in the present study is a high molecular weight (51 kd) keratin expressed in the parabasal cell layer of noncornified stratified squamous epithelium. CK17 is a low molecular weight (46 kd) keratin, and CK14 is a high molecular weight (50 kd) keratin, both of which are expressed in the basal cell layer. Immunohistochemical expression of the cancer suppressor gene p53 reflects gene mutation and is being used as a 127

6 J.Hard Tissue Biology Vol. 19(2):123130, 2010 marker for malignant transformation and prognosis 19). In the present study, CK13 was positive in all 10 cases of hyperkeratosis without epithelial dysplasia, and was expressed from the spinous layer to cornified layer but not in basal cell layer. These findings are consistent with the report of Ohta et al 13). On the other hand, CK13 was negative in all cases of hyperkeratosis with epithelial dysplasia, carcinoma in situ and SCC. These findings suggest that this cytokeratin may be used in the diagnosis of the presence or absence of epithelial dysplasia, or cancer. CK17 was negative in all 10 cases of hyperkeratosis without epithelial dysplasia. On the other hand, CK17 was positive in all cases of hyperkeratosis with epithelial dysplasia, either in all layers or from the spinous layer to cornified layer; was positive in all Histopathological diagnosis has to be conducted bearing in mind that p53 may be negative even in carcinoma in situ. In summary, although HE stained sections are fundamentally used for the histopathological diagnosis of hyperkeratosis with epithelial dysplasia and carcinoma in situ and for the evaluation of the presence or absence of tumor cells at the stump of resected tumor tissues, immunohistochemical studies of CK13, CK17 and CK14 that indicate abnormal differentiation of epithelium as well as the cancer suppressor gene p53 is useful as an adjunct to the histopathological diagnoses of hyperkeratosis without epithelial dysplasia, hyperkeratosis with epithelial dysplasia, carcinoma in situ and SCC. Based on the present results, further studies on the classification and malignant transformation of SIN are required. cases of carcinoma in situ although staining intensities varied; and was positive in 9 of 10 cases of SCC. These results are consistent with the report of Wei et al 12). This cytokeratin may also be used in the diagnosis of the presence or absence of epithelial Acknowledgement We would like to thank all staff members at the Department of Pathology for their assistance in this research. dysplasia, or cancer. CK14 was negative in 3 of 10 cases of hyperkeratosis without References epithelial dysplasia, and positive in the remaining 7 cases but confined only to the basal cell layer or a few rows of cells in the spinous layer close to the basal cell layer. On the other hand, CK14 1. Yamamoto E, iyakawa A and Kohama G. ode of invasion and lymph node metastasis in squamous cell carcinoma of the oral cavity. Head Neck Surg 6: , 1984 was positive in all the epithelial layers in all 10 cases of hyperkeratosis with epithelial dysplasia, and also positive in the tumor in all 10 cases each of carcinoma in situ and SCC. These findings are consistent with the report of Hamano et al 10). In the 2. Anneroth G, Batsakis J and Luna. Review of the literature and a recommended system of malignancy grading in oral squamous cell carcinomas. Scand J Dent Res 95: , 1987 case that the staining intensity of CK17 is somewhat weak, CK14 may be used as an auxiliary finding in the diagnosis of the presence or absence of epithelial dysplasia, or cancer. The differential staining properties of CK13, CK17 and CK14 demonstrated in the present study are consistent with the genetic 3. Okada Y, ataga I, Katagiri and Ishii K. An analysis of cervical lymph nodes metastasis in oral squamous cell carcinoma: Relationship between grade of histological malignancy and lymph nodes metastasis. Int J Oral axillofac Surg 32: , 2003 analysis by Ohkura et al 20), and are important findings for epithelial 4. Woolgar JA. The topography of cervical lymph node dysplasia, carcinoma in situ and SCC of the tongue. However, it metastases revisited: the histological findings in 526 sides should be noted that the staining reaction of CK13, CK17 and of neck dissection from 439 previously untreated patients. CK14 per se does not indicate the presence or absence of neoplasm. Therefore it is important not to diagnose the presence or absence Int J Oral axillofac Surg 36: , 2007 of neoplasm based on the immunohistochemical staining property of any of the cytokeratins. Recognizing the oblique line (front) is useful during histopathological diagnosis. 5. Yasui A, Okada Y, ataga I and Katagiri. An analysis of distant metastases in oral squamous cell carcinoma. J Hard Tissue Biol 19(1): 2732, 2010 Positive reaction of p53 was observed in some cells of the basal cell layer in 2 of 10 cases of hyperkeratosis without epithelial dysplasia. On the other hand, p53 was positive in all 10 cases of hyperkeratosis with epithelial dysplasia, and in 9 of 10 cases of 6. Okada Y. Relationships of cervical lymph node metastasis to histopathological malignancy grade, tumor angiogenesis, and lymphatic invasion in tongue cancer. Odontology 98: , 2010 SCC, showing high rates of expression consistent with previous reports 21,22). Contrary to expectation, p53 was positive in only 5 of 10 cases of carcinoma in situ. This finding is consistent or 7. Silverman S Jr, Gorsky and Lozada F. Oral leukoplakia and malignant transformation: a followup study of 257 patients. Cancer 53: , 1984 similar to a previous report that p53 expression rate is higher in moderate or severe epithelial dysplasia than in carcinoma in situ or well differentiated SCC 21), and a study showing higher p53 8. Hansen LS, Olson JA and Silverman S Jr. Proliferative verrucous leukoplakia. Oral Surg Oral ed Oral Pathol 60: , 1985 expression rate in epithelial dysplasia than in early cancer 22). 9. Ogden GR, Chrisholm D, Adi and Lane EB. Cytokeratin 128

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