Cytokeratin pattern of clinically intact and pathologically changed oral mucosa

Cytokeratin pattern of clinically intact and pathologically changed 0ral mucosa

J/irgen Schulz 1, Thomas Ermich 2, Michael Kasper 3, Gerd Raabe 4, Dieter Schumann 2 qnstitute of Biochemistry, Humboldt University, Berlin, 2Department of Maxillo-Facial and Plastic Surgery, Friedrich Schiller University, Jena, 3Department of Pathology, District Hospital G6rlitz, and 4Institute of Pathological Anatomy, Friedrich Schiller University, Jena, Germany

J. Schulz, T. Ermich, M. Kasper, G. Raabe, D. Schumann: Cytokeratin pattern of

clinically intact and pathologically changed oral mucosa. Int. J. Oral Maxillofac. Surg. 1992; 21: 35-39. Abstract. The various cytokeratin polypeptides in oral epithelia are expressed in dependence on site and formation of a stratum corneum. Certain cytokeratins o c c u r permanently and others occasionally, in fibrous hyperplasia and Lichen ruber planus, patterns of cytokeratins did not deviate significantly from normal. In some but not all cases of squamous cell carcinoma and leukoplakia studied, marked aberrations of pattern were characterized by (i) appearance of cytokeratin No. 19, (ii) somewhat more frequent occurrence of cytokeratins Nos. 8 and 18, (iii) proteolytic modifications of cytokeratins, and (iv) partial loss of a few sitespecific cytokeratins. The aberrations m a y be taken as additional diagnostic criteria for differentiation between non-aggressive and potentially aggressive leukoplakic lesion, even if they are not correlated with the conventional histological grading of dysplasia.

There is a continuing search for more specific means to differentiate between aggressive and non-aggressive lesions, benign and precancerous alterations o f mucosa, and virulence of carcinoma u' 25, 3o. For this reason, the behaviour of cytoskeletal intermediate filaments (IF) and, particularly, their different polypeptides have been biochemically and immunochemically analysed 5, 17, 22, 31 The I F can be subdivided into 5 classes15, 23 and, as a rule, are specifically expressed in the various cells of different tissues. Epithelial cells always contain a certain n u m b e r of proteins of the I F class named cytokeratins (CK numbers 1 to 19) 2°. The cytokeratin pattern (CKP) is site-specifiC, 20,26, 27, but shows inter-individual changes and pathologically caused shifts J, 3, is, 19,20,21, 27,28, 31, 32 Moreover, co-expressions of several I F proteins have been found in cells under normal as well as pathological conditions12, 17, 29 This study was conducted with a view to finding whether the C K P in cases of oral precancerous lesions and squamous cell carcinomas could provide information for differential diagnosis and c o u l d be helpful in early detection of malignant alterations. Proper understanding of the various types of C K P in

normal oral epithelia was a prerequisite for this study. It is also important to be aware of possible alterations of the C K P in hyperkeratotic lesions, such as Lichen ruber planus and fibrous hyperplasia.

Material and methods Tissue samples The biopsy specimens were excised, under clinical indications from 58 patients suffering from fibrous hyperplasia (14 cases), Lichen ruber planus (10), leukoplakia (20) and squamous cell carcinoma (14). Twenty-two samples of apparently unsuspicious "normal" oral mucosa were obtained in the course of dental extractions, among them l0 biopsies from free mucosa and 12 from keratinized* gingiva. The age of the patients ranged from 17 to 72 years (43 males and 37 females). The directly adjacent regions of each biopsy specimen were biochemically and histologically examinedT'8.

Biochemical techniques The CK were isolated from epithelia as described by FRANKEet al. 9, and were submitted * The term "keratinized" might be confnsing in the context of this paper. In conformity with habitual usage in medicine it will be used to define cornifying epithelium which forms a stratum corneum despite the fact that every epithelial cell synthesizes keratins.

Key words: carcinoma; cytokeratins; immunoblotting; immunohistology; leukoplakia; Lichen ruber planus; SDS electrophoresis. Accepted for publication 3 September 1991

to SDS polyacrylamide gel electrophoresis which separated the mixture of all the 19 CK into 16 distinct protein bands (Fig. 1). CK Nos. 6/11, 7/13 and 14/15 formed one band each on account of identical molecular weights. For specific staining of CK by means of "Western blotting ''L°, the polypeptides were blotted out of the polyacrylamide gel onto nitrocellulose and were incubated either with the broad-spectrum anti-cytokeratin rabbit antiserum AKS 426 or the mouse monoclonal antibody A53-B/A2 which reacted only with CK No. 19~3'~4 The antibody binding to CK was visualized by using a second antibody to rabbit or mouse immunoglobulins, respectively, conjugated with horse radish peroxidase 7' 8, i0

Histological techniques Pathohistological diagnosis were performed by conventional techniques on paraffin-embedded formalin-fixed tissue slices stained with hematoxylin and eosin. In the immunohistological studies, cryosections (4 ~tm) were stained by means of the indirect immunoperoxidase method, as described elsewhere 6, 7, 8, 12. 13, f4 using the monoclonal antibodies TROMA 1 (directed to CK No. 8), 2 RKSE 60 (directed to CK No. 10, both of them kindly provided by Prof. F. C. S. Ramaekers, Nijmegen, The Netherlands) and V9 (antivimentin, Boehringer Mannheim, Germany).

36

S c h u l z et al.

CK No. 1

14/15

of epithelia, as had been described also by OUHAYOUNet al. 24 (Fig. 4). CK No. 8. not found in keratinized gingiva by other authors 4' 24, 32, has been electrophoretically as well as immunohistochemically recorded (Fig. 5). In conformity with CLAUSEN et al. 4, we did not identify CK No. 13 in intact oral mucosa.

Mr 68

52.5-'~~-_ / F., 50

17

46¢Q

18

45

19

40 j Cytokeratin pattern in oral mucosa with benign alteration

1

2

3

4

5

6

T

8

Fig. 1. SDS polyacrylamide gel electrophoresis of cytokeratins isolated from various epithelial

sources. Lanes 1 and 2: leukoplakia of free oral mucosa; lanes 3 and 4: oral squamous cell carcinoma, Grade III; lane 5: lingual mucosa; lane 6: mucosa of hard palate; lane 7: epidermis of skin; lane 8: buccal mucosa.

FREQUENCY OF OCCURRENCE (%)

Cytokeratins in oral leukoplakia and carcinoma

lOO

75

. . . . . . . . . . .

50

25

o 6/11 7 / 1 3

8

12

14/15

16

(42)

lg

CYTOKERATIN No. OR (Mr)

Fig. 2. Normal and leukoplakic patterns of cytokeratins on sites of free oral mucosa. Frequencies of occurrence of distinct cytokeratins are given in percentage of the total number (n) of patients investigated. []: apparently unsuspicious non-keratinized free normal mucosa (n = 10); I1: leukoplakic lesions of free mucosa (n= 14).

Results Normal oral pattern of cytokeratins

In SDS electrophoretic CKP of clinically intact normal human oral mucosa, CK of permanent occurrence should be distinguished from those of occasional presence (Figs. 2 and 3). CK Nos. 5, 6/11, and 14/15 were solidly expressed across the entire oral mucosa, with No. 1 in all regions forming a stratum corneum, Nos. 3 and 16 in the kera-

Hyper-orthokeratotic or hyper-parakeratotic epithelia of fibrous hyperplasias yielded CKP which were similar to the CKP in intact keratinized mucosa, which in these cases had been attributable to mechanical irritation. In Lichen ruber planus, shifts of CKP were also located within the normal range of variability of apparently unsuspicious oral mucosa (for details see 8).

tinized parts of the hard palate and gingiva, and No. 8 in keratinized gingiva as well as in free non-keratinized mucosa. CK Nos. 16 and 18 were of variable occurrence as minor components in non-keratinized mucosa, and Nos. 2, 4, 17, and 18 in keratinized mucosa. Higher variability of CKP has been observed in mucosa of the tongue 7 (not shown). CK No. 10 was immunohistochemically observed only in superficial layers

CKP in leukoplakia showed significant, partially quantitative and also qualitative aberrations from the normal sitespecific pattern, relating, above all, to CK Nos, 1-3, 8, and 19. The aberrations were found in leukoplakic lesions in both areas of mucosa, that is those of keratinization and no keratinization under normal conditions (Figs. 2 and 3). However, we could not ascertain any correlation between the pathohistologically diagnosed degree of dysplasia or inflammation and the appearance of distinct CK, as described by L6NINa et al. is. CK No. 1 was recognized in leukoplakic specimens of 15 patients, although cornification had been observed as an obligate histological feature in all the 20 eases analysed. The identity of the 40kD-protein with the CK No. 19 was examined by means of the Western blotting technique using the monoclonal antibody A53-B/A2. In 2 cases, this antibody decorated not only the 40kD-protein but also an additional 38kD-polypeptide (Fig. 6). It may represent a proteolytically shortened CK No. 19. In 2 other cases of leukoplakia, the 40 kD-protein did not react with the monoclonal anti-CK19 antibody, but did so with the broad-spectrum anticytokeratin antiserum AKS 42 and, therefore, may be regarded as a proteolytic product of higher molecular CK. In those cases in which CK No. 19 had

Cytokeratin patterns o f oral mucosa

37

F R E Q U E N C Y OF O C C U R R E N C E (%) 100

76

OK No.

Mr

18 --

40 38

~k

60

25 0

I

1

2

3

4-

5

6111 7113

9

10

12

14/15

16

17

18

Fig,. 6. Western blot of cytokeratin No. 19 and its proteolytically shortened form. Cytokeratins isolated from oral specimen with leukoplakic dysplasia were separated by SDS electrophoresis and transferred onto nitrocellulose where they were immunochemically stained with CK No. 19-specific monoclonal antibody A53-B/A2S'~4; note that both a 40kD-protein and a 38kD-protein were decorated by the antibody•

......

(42)

19

C Y T O K E R A T I N No. OR (Mr)

Fig. 3. Normal and leukoplakic patterns of cytokeratins on sites of gingiva and hard palate. Frequencies of occurrence are given as in Fig. 2. Nt: apparently unsuspicious keratinized normal mucosa of gingiva plus hard palate (n-12); note that CK No. 8 was found only in gingiva (*, n - 8 ) ; B: leukoplakic lesions on sites of normally keratinized mucosa (n-6). been biochemically identified, the immunohistological investigations resulted in positively as well as negatively reacting dysplasia cells of the suprabasal layers of epithelia after staining with the monoclonal anti-CK19 antibody. A 42kD-protein, not listed in the cytokeratin catalogue 2°, was recorded for the first time from 3 of the leukoplakic specimens. It b o u n d the polyclonal broad-spectrum anticytokeratin antibodies, when checked by means of Western blotting. Hence, it may as well be proteolytically derived from larger CK. The predominance of the C K Nos. 5 6/11, 14/15 and 16, as established in

of surgery. Further, C K No. 1 was not detectable from all cornifying carcinomas and was also electrophoretically recorded from one of the undifferentiated carcinomas which had been histologically classified as non-cornifying. Highly frequent occurrence of C K Nos. 18 and 19 was the most striking finding from oral squamous cell carcin o m a (Fig. 7). Six of the specimens were immunohistochemically analysed, using the m o n o c l o n a l anti-CK19 antibody

most samples of squamous cell carcinoma, suggested the oral origin of the malignomas (Fig. 7). However, these C K were hardly demonstrable even in 3 cases of highly undifferentiated carcinomas (2 cases of degree III and 1 of degree II). The loss of tissue specific C K P correlated with poor prognosis. The 3 patients died within 6 months

t3

A

CK No. I 3 5 611'1 8 14,/15 16

Mr

•

•

63 ----~ 6s ~ i 48 ~

ii

;

ii

711!i! ii

ii :i$ ]JJ

!

')i=

•:

.

Fig. 4. Immunohistological demonstration of cytokeratin No. 10 within suprabasal layers of keratinized intact gingiva. A cryosection of the gingival specimen was stained with the CK No. 10-specific antibody RKSE 60 by means of the indirect inmauno-peroxidase method 12A3( × 150).

.;'

,

-

Fig. 5. Evidence of cytokeratin No. 8 in keratinized intact gingiva. (A) S I S electrophoresis of CK isolated from keratinized intact gingiva; arrow indicating CK No. 8; (B) Cryosection of region adjacent to gingival specimen, stained with the CK No. 8-specific antibody TROMA 12 by means of the indirect immuno-peroxidase method~Z.~3;note that colour of origin has changed in photographic black/white reproduction; so, brownish cytoplasmatic staining of CK No. 8 appears grey, whereas nuclei countei~stained with hematoxylin appear black. ( x 105)

38

S c h u l z et al.

latter finding did not confirm the assumption ~9,2~that the origin of an undifferentiated carcinoma can be safely identified by its CKP. A n d lastly, C K Nos. 1 and 3 failed to be secure indicators of cornification 5' 7, 20, 22, 27, if mucosa was pathologically changed. The results, on principle, correspond to the findings about changes o f C K P in malignantly transformed epithelia of various orgins ~' 3, 16, 17, 19, 22, 28, 31

F R E Q U E N C Y O F O C C U R R E N C E (%) 100

75

......

50

25

0 1

2

3

4

5

6/11 7 / 1 3

8

g

10

12

14115

16

17

18

(42)

19

C Y T O K E R A T I N NO. OR ( M r )

Fig. 7. Cytokeratin pattern of oral squamous cell carcinomas. Frequencies of occurrence are given as in Fig. 2. [] : Cornified carcinomas (n = 7); • : non-cornified carcinomas (n = 7).

broad-spectrum anticytokeratin antiserum in the blotting experiments, and again, they might be considered as proteolytic products of CK. When antibody V9 was used in immunohistological staining, co-expression of I F protein vimentin was not observed in any of the carcinoma cells or in the leukoplakic epithelia investigated.

Discussion

Fig. 8. Immunohistological demonstration of cytokeratin No. 19 in oral squamous cell carcinoma (Grade II). A cryosection of the carcinoma specimen was stained with the CK No. 19-specific antibody A53-B/A2 by means of the indirect immuno-peroxidase method~2-~4; note staining of carcinoma cell strains within stroma under a region of normal epithelium (margin cut) which partially contains also CK No. 19 in basal keratinocytes ( × 105)

A53-B/A2. All t u m o u r cells reacted positively (Fig. 8). The 42kD-protein was shown in 6 carcinoma specimens and its immunochemical analysis gave the same results as mentioned above. We also found in carcinomas proteins of the cytoskeleton which in the isolation procedure behaved like cytokeratins but had molecular masses < 40 k D (Fig. 1). They also reacted positively with the

Two basic patterns of C K have been found in the oral cavity, i.e. C K P o f keratinized and o f non-keratinized mucosa. Certain C K are permanently expressed and characterize the site-specificity of C K P depending on the type of mucosa. Moreover, additional C K occur as minor components with varying frequency. The essential aspects of these findings corresponded to those described by other authors 4'24'32, except for C K Nos. 8 and 13, as mentioned above. In benign changes of oral mucosa, such as f b r o u s hyperplasia and Lichen ruber planus, the expression of C K did not exhibit any deviation in significant excess of normal variability. However, in some cases of squamous cell carcin o m a as well as o f leukoplakia of oral mucosa, marked aberrations from normal C K P were observed. They were characterized by (i) appearance of C K No. 19, (ii) the somewhat more frequent occurrence of C K Nos. 8 and 18, (iii) proteolytic modifications of CK, and (iv) partial loss of site-specific C K R The

The above results, at present, are hard to evaluate in respect to their relevance to differential diagnosis of leukoplakia, since aberrations of C K P in leukoplakia were not found in all cases and did not correlate with the traditional histological grading of dysplasia. By comparison with benign changes and with carcinomas of oral mucosa, the aberrations of C K P in some of the leukoplakic lesions m a y be regarded to follow a precancerous tendency. The above aberrations of CKP, consequently, should be viewed as additional criteria of diagnostic differentiation between non-aggressive and potentially aggressive leukoplakic lesions. Thus, immunohistological examination of C K Nos. 19, 18, 8 and 1 seems to be promising1,16,17, 22, 28. On the other hand, whether the aberrations of C K P are unambiguously indicative of malignancy or of a pre-malignant state, seems to be as uncertain for a diagnosis as is any single parameter. Better understanding of C K P shifts during epithelial cell transformation and t u m o u r progression may be expected when the molecular regulatory mechanisms of the expression of C K have been explored. References

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