CD44 variant 6 (CD44v6) expression as a progression marker in benign, premalignant and malignant oral epithelial tissues

Int. J. Oral Maxillofac. Surg. 1997; 26:443 446 Printed in Denmark. All rights reserved

Copyright 9 Munksgaard 1997 lntemadonalJaumal of

Oral& Max ofacial Surgery ISSN 0901-5027

CD44 variant 6 (CD44v6). expression as a progression marker in benign, premalignant and malignant oral epithelial tissues

Rumana Bahar, Masanori Kunishi, Yoshiaki Kayada, Koji Yoshiga Department of Oral and Maxillofacial Surgery 1, Hiroshima University School of Dentistry, Hiroshima, Japan

R. Bahar, M. Kunishi, Y.. Kayada, K. Yoshiga: CD44 variant 6 (CD44v6) expression as a progression marker in benign, premalignant and malignant oral epithelial tissues. Int. J. Oral Maxillofac. Surg. 1997; 26: 443-446. 9 Munksgaard, 1997 Abstract. The change in the expression pattern of CD44 variant 6 (CD44v6) protein in benign, premalignant, and malignant (SCC) oral epithelial lesions was studied immunohistochemically and compared with the pattern in normal mucosa in order to examine whether this gene can serve as a progression marker in patients with SCC. The principal finding is that CD44v6 expression was clearly downregulated in most cases of severe premalignant lesions as well as in most of the SCCs. The staining pattern and intensity varied according to the degree of dysplasia and to the degree of differentiation of the SCCs. Premalignant severe epithelial dysplasia cases with early features of invasion, not yet developed into SCC, showed distinctly downregulated expression of CD44v6 protein whereas hyperplastic and benign epithelial lesions (papilloma) expressed positive staining patterns comparable to those of the normal counterparts. The authors conclude that alteration in CD44v6 may occur as an early event in primary oral SCC development, as well as in premalignant severe epithelial dysplasia. It can thus, be used as a molecular progression marker when screening for oral cancer.

Some benign epithelial lesions such as dysplasia (leukoplakia, erythroplakia) and lichen planus, have the potential to undergo malignant transformation and become squamous cell carcinomas (SCCs). There is no easily available method for clinically assessing the progression of these conditions towards malignancy. In preneoplastic lesions and SCC of the oral mucosa, a correlation has already been established between degree of dysplasia and potential malignancy. Recently, a number of genetic markers, most notably the p53 tumour suppressor gene, have seen wide-

spread use in clinical screening of cancers and premalignant lesions for possible cellular proliferation or progression. For primary oral SCCs, treatment varies according to the progression and aggressiveness of the tumour. The pathological evaluations of early morphological changes in epithelia are the basis for identifying and characterizing the tumour cells that possess advanced metastatic potential, which may be regarded as the first step in the progression of a primary tumour to a metastasizing tumour, a fact that is particularly important in clinical cancer

Key words: CD44v6; oral epithelium; dysplasia; squamous cell carcinoma. Accepted for publication 28 May 1997

screening. In keeping with this trend we investigated CD44 gene products, where early molecular changes occur before invasion or metastasis takes place. CD44 protein is a widely distributed transmembrane glycoprotein that functions as a receptor for the extracellular matrix glycan, hyaluronan 1,2,9,16,17,2~ and plays an important role in cellular adhesion, lymphocyte homing and activation and tumour metastasis. Recently, many researchers have reported both altered expressions of standard CD44 (CD44s) and variant forms of CD44 (CD44v) appearing in a wide range of

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developing epithelial tumours, including gastrointestinal, renal, and pulmonary carcinogenesis. M a n y epithelia carry splice variants of CD44, including the epithelial cells of h u m a n skin, oral, and intestinal mucosa s'6. A m o n g the different forms of this protein, the 'variant 6' (CD44v6) is said to be involved in t u m o u r metastasis 1~ which is normally expressed by oral epithelium 6'18. Yet it is not known how cancer cells make use of these splice variants of CD44 in order to achieve metastatic behaviour, although it has been shown that multiple genetic factors are involved in regulating the expression of this cellular adhesion molecule during the progression of the disease 12,14,21 To our knowledge there is no published information on CD44v6 expression in oral t u m o u r development. The recent availability of different exon-specific monoclonal antibodies against CD44 variants has enhanced the ease a n d accuracy of immunohistochemical analysis. In this study we, therefore, performed immunohistochemical analysis to compare CD44v6 gene expression in benign, premalignant and malignant oral epithelium.

Material and methods The samples were collected from patients of the Department of Oral and Maxillofacial Surgery 1, Hffbshima University School of Dentistry, who presented with epithelial hy-

Fig. 1. CD44v6 expression pattern in normal oral epithelium. Cells in basal and spinous layer show strong membrane staining of CD44v6 on immunohistochemistry (magnification • 100).

Table 1. CD44v6 expression in benign, premalignant and malignant oral epithelium Positive expression observed Type of lesion Hyperplasia (n= 10) Papilloma (n= 8) Dysplasia (n= 19) SCC (n=38)

+ + +ve

+ +ve

1 2

9 6 4

Downregulation of expression +ve

Focally

Severely

2 27

6 9

7 2

-ve

+ve=weakly positive, + +ve=strongly positive, + + +ve-very strongly positive, -ve=negative.

perplasia, dysplasia of various degrees and benign (papilloma) or malignant primary oral epithelial tumours. Cancer patients were subjected to a routine follow up after treatment, and close observation was recommended for patients with severe dysplasia (premalignant), who were considered to be at risk. Ten cases of epithelial hyperplasia, 8 papillomas, 19 dysplasias and 38 cases of primary SCC were studied. Ten normal oral mucosa samples (as controls) were collected from patients who had no tumours. All cases included in the study were randomly selected. On histological analysis, the degree of dysplasia varied from mild to moderate, to severe. Tumour stages were determined using the histopathologic results and the TNM classification system (UICC, 1987). The immunohistochemical studies were performed on formalin-fixed, routinely processed paraffin-embedded samples, and antigen detection was carried out by the avidinbiotin peroxidase technique with the Histofine | SAB-PO(M) kit (Nichirei Corp., Tokyo, Japan). Tissues were sliced in 5-micron sections, placed on albumin-coated glass slides and prewarmed overnight before immunostaining. Briefly, prior to immunohistochemistry, the sections were dewaxed by xylene and subjected to an antigen retrieval procedure by microwave oven treatment for 5 rain in citrate buffer solution. Afterwards slides were treated by 1% H202 in methanol to inactivate the internal peroxidase. To block nonspecific binding, sections were incubated with 10% rabbit blocking serum for 30 min. Next, they were incubated with the primary antibody antihuman CD44v6 mouse monoclonal antibody (clone 2F10, R & D Systems Europe Ltd., Abingdon, UK), diluted at 1:2000 for 1 h in a humid chamber. Slides were washed in PBS and incubated for another hour with the secondary antibody (antimouse rabbit immunoglobulins), then complexed with avidinbiotin-peroxidase complex for 30 min and treated with DAB (3-3'-diaminobenzidine tetrahydrochloride) for colour expression. All incubations were done at room temperature. The sections were counterstained with hematoxylene. Only dysplasia samples were stained immunohistochemically for p53 expression using antihuman p53 mouse monoclonal antibody (clone BP 53-12, YLEM s.r.1.), di-

luted at 1:50. This antibody detects both wild and mutant types of p53 protein in formalinfixed paraffin-embedded samples. The immunohistochemical procedure for the dysplasia samples was the same as for the other samples above, with the exception of the omission of microwave treatment.

Results Immunohistochemistry

CD44v6 expression pattern in normal mucosa: In n o r m a l mucosa the expression of CD44v6 was detected as membranous protein localized on the surface of the epithelial cells. Both basal and spinous layer epithelial cells expressed strong positive staining of CD44v6, which then gradually faded into the negative staining of the cells of the superficial keratin layer (Fig. 1).

Determination of staining intensity: The staining pattern observed in the spinous layer was used as a standard and scored as positive with two pluses ( + +ve). In comparison to this pattern, weaker staining patterns were scored as weakly positive with one plus (+ve), and very strong staining patterns scored as strongest staining with three pluses ( + + + v e ) , while no staining at all was scored as negative staining (-ve).

CD44v6 expression in study samples (Table 1): The CD44v6 expression pattern in hyperplastic mucosa remained nearly identical to that of n o r m a l mucosa, while the thickened epithelial layer stained positively for CD44v6, with the staining intensity varying from moderate to strong. The papillomas showed a positive staining pattern throughout with a very strong expression in two cases (Fig. 2), as well as both membranous and cytoplasmic localization of CD44v6. In case of dysplasia, 15 of 19 cases showed a tendency toward downregulation of expression and this tendency was correlated to the degree of dysplasia (Table 2). In severe dysplasia, overall downregulation of CD44v6 expression was found in wide areas

CD44v6 expression in oral premalignant lesions

throughout all sections (Fig. 3); however, focal areas with downregulated expression of the CD44v6 in the basal layer (Fig. 4A) were also observed in moderate to severe degree dysplasias. Highly pleomorphic cells showed downregulation to almost negative expression (Fig. 4B), whereas less pleomorphic or normal cells expressed CD44v6 positively in the same section. Four cases of mild dysplasia showed normal positive expression patterns of CD44v6. In primary SCCs of the oral cavity, tumour tissues also showed downregulated expression in all cases to a vari-

Fig. 2. Very strong immunopositivity detected in cells of benign papilloma (magnification • 100).

Table 2. Correlation between CD44v6 downregulation in dysplastic epithelium and clinicopathologic findings

Degree of dysplasia

Status of CD44v6 expression*

Mild to Moderate

Positive expression (4)

All -ve

Slight downregulation (2)

All - v e

Moderate to Severe

Focal downregulation (6)

+ve (2)

Operated for oral SCC (2).

Severe downregulation (7)

+ve (6)

Precancerous and/or carcinoma in situ (4).

p53 expression*

Clinicopathologic findings*

+ve=positive, -ve=negative. *Number of cases indicated within parentheses.

able degree. Among these, 27 cases were classified as weakly positive (having slight downregulation); 9 showed focal areas of downregulated expression, and 2 expressed severe, overall downregulation of CD44v6 expression (Table 1). We were unable to establish any significant correlation between CD44v6 expression status and differentiation of SCCs on the basis of these 38 cases, but we did observe that the tendency for CD44v6 downregulation was more pronounced in the poorly differentiated than in the well or moderately differentiated carcinomas. p53 expression: Positive immunoreactivity was detected in 42% of dysplasia cases. Eight of the total 19 cases were immunopositive for p53, and histologically all cases showed signs of moderate to severe dysplasia. The staining pattern was nuclear, although the staining intensity varied. A significant relationship existed between the degree of dysplasia and the presence of p53 positive cells (Table 2). Discussion

As stated earlier, the purpose of our study was to evaluate the efficacy of

Fig. 3. Severe downregulation of CD44v6 over large areas, a case of severe dysplasia (magnification x 100).

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Fig. 4. A) In severe dysplasia, focal downregulation of CD44v6 expression in cells of basal layer. B) High-power magnification of same case showing weak cellular expression and partial loss of expression (arrow) of CD44v6. (A) magnification x 100; B) magnification •

CD44v6 gene expression as a progression marker in clinical cancer screening. Changes in CD44v6 expression have been correlated with poor prognosis in several human malignancies. Elevated CD44v6 expression has been observed in benign and malignant colorectal epithelium II, carcinoma of the vulva 24, primary brain tumours 7, cutaneous lymphoma 4 and certain types of gastric adenocarcinoma 3. There is increasing evidence that the expression of CD44 variants is related to the invasive and metastatic potential of tumour cells. Fox et al. 5,6 demonstrated upregulated expression of CD44v6 in breast carcinoma as well as downregulated expression of this variant in invasive breast tumour tissue. Other investigators have reported, however, that epithelium expressing CD44v6, under normal conditions, tended toward downregulation during turnout progression and metastatic spread. This tendency has been reported in human tumours that originated from squamous epithelium of skin 22, endometrium 8, urothelium 13, laryngeal mucosa 23 and oral mucosa 15. In this study, we analysed the expression of CD44v6 in oral premalig-

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n a n t a n d m a l i g n a n t epithelium a n d f o u n d t h a t the C D 4 4 v 6 expression altered from its n o r m a l positive p a t t e r n to a downregulated state with progression o f the neoplastic process from severe dysplasia toward SCC. This correlation between degree o f dysplasia a n d C D 4 4 v 6 d o w n r e g u l a t i o n m i g h t reflect the fact of early cellular change f r o m n o r m a l cell-cell a n d cell-matrix interaction towards a bizarre, p a t h o physiological heterotypic cell surface a d h e s i o n property, which m i g h t be cont r i b u t o r y to the cells to achieve invasiveness w h e n they b e c o m e fully malignant. The overexpression o f p53 t u m o u r suppressor protein in the positive cases m a y f u r t h e r reflect a g r o w t h deregulation, a d d i n g proliferation advantages. As shown in Table 2, d o w n r e g u l a t i o n of C D 4 4 v 6 a m o n g dysplasia samples is m u c h m o r e p r o n o u n c e d in cases presenting with either a h i s t o r y o f previous oral SCC or presenting with c a r c i n o m a in situ. These findings suggest t h a t b o t h the loss of control of cell proliferation implied by overexpression o f p53 a n d the altered cellular a d h e s i o n implied by the g r a d u a l loss o f C D 4 4 v 6 molecules, m a y be early events in the d e v e l o p m e n t o f m a l i g n a n t t u m o u r in the oral cavity. T h e e x a m i n a t i o n s o f b e n i g n hyperplasia a n d p a p i l l o m a revealed a n increased expression o f C D 4 4 v 6 in comp a r i s o n to n o r m a l epithelium. Two cases of particularly elevated C D 4 4 v 6 expression with cytoplasmic deposition suggest t h a t b e n i g n epithelial cells may utilize this v a r i a n t of C D 4 4 to m a i n t a i n the histoarchitecture by epithelial mesenchymal interactions. A t present, it is difficult to explain the significance o f over a n d downregulated expression o f C D 4 4 v 6 in benign, premalignant, a n d m a l i g n a n t epithelium, a l t h o u g h this v a r i a n t o f C D 4 4 has previously been associated with t u r n o u t progression 12'19. These p r i o r a n d present results indicate t h a t v a r i a n t 6 of C D 4 4 is expressed differentially by the epithelia o f different o r g a n s a n d m a y play different roles in t u m o u r development. Thus, the relation between C D 4 4 v 6 expression a n d disease prognosis m a y d e p e n d u p o n tissue specificity. D o w n r e g u l a t i o n o f CD44v6, however, occurs in oral cancer a n d also in severe p r e m a l i g n a n t epithelial dysplasias.

Acknowledgment. We thank Dr Mutsumi Miyauchi (Department of Oral Pathology) for

her kind cooperation in preparing the illustrations.

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Address: Dr Rumana Bahar Department of Oral and Maxilh?facial Surgery 1 Hiroshima University School of Dentistry Minami ku, Kasumi 1-2-3 Hiroshima City 734 Japan