JOURNAL OF PATHOLOGY, VOL. 178: 146-150 (1996)

EXPRESSION OF MARKERS OF DIFFERENTIATION IN NORMAL BRONCHIAL EPITHELIUM AND

BRONCHIAL DYSPLASIA NEIL PENDLETON*, GLEN R . D I X O N ~ , JOHN A. GREEN$ AND MICHAEL w. MYSICOW?

*Clatterbridge Cuncer Research Trust, J. K. Douglus Luborutories, Clutterbridge Hospital, Merseyside L63 45 Y, U. K., t Drpurttnent of Pathology, Broudgrrrn Hospitul, Liverpool L14 I HX, U. K. I. $ Clatterbridge Centre ,for Oncology,

Clutterbridge Hospital, Merseyside L63 4JY, U. K.

SUMMARY Bronchial epithelial dysplasia is a non-invasive cellular change often associated with physical or chemical injury and considered a

pre-neoplastic lesion in the formation of lung cancer. A series of 39 bronchial dysplasias associated with both neoplastic and non-neoplastic lesions were assessed for expression of markers of differentiation by immunocytochemistry and compared with samples of normal bronchial epithelium. The normal bronchial epithelium studied expressed cytokeratins (CKs) 4, 6, 7, 8, 18, and 19 in all cases; CK 13 in 13 cases; and peanut agglutinin (PNA) in seven cases. Involucrin, CK 10, and CK 14 were not observed in the normal bronchial samples. In the dysplastic bronchial biopsies, epithelial staining was observed with epithelial CKs 7,8, 18, and 19 in all cases; CK 13 was seen in 26 cases; CK 14 in 13 cases; CK 6 in I I cases: and CK 10 in five cases. In 13 cases of dysplasia, only simple epithelial antigens were identified. lnvolucrin expression was observed in 17 dysplastic biopsies and PNA in 12. By Fisher's exact test, a significant association between non-severe histological grade of dysplasia and CK 6 expression (P= 0.018) was found. Comparison of the results using the same analysis showed significant correlations between the loss of CK 6 expression (P<O.OOl) and the expression of CK 14 (P=0-008) and involucrin (P=0.0018) with bronchial dysplasia. These data show that the pattern of differentiation antigen expression in bronchial dysplasia is significantly different from that of the normal bronchial epithelium, but the phenotypic heterogeneity of these lesions is similar to that of bronchial carcinomas.

KEY WORDS-bronchial epithelium; dysplasia; cytokeratins; involucrin; peanut agglutinin

INTRODUCTION

Bronchial epithelial dysplasia is a non-invasive cellu- lar change often associated with physical or chemical injury.',' In the case of severe dysplasia or carcinoma in sifu, there is limited evidence of progression to frank neoplasia.' However, little is understood about the behaviour of bronchial epithelial dysplasia or its role in the development of the heterogeneous group of carcino- mas found in man. Previous studies have identified molecular and genetic changes in bronchial dysplasia, such as mutations in the p53 gene,435 an increase in the proliferative fraction of dysplastic cells,6 and changes in the subepithelial extracellular m a t r i ~ . ~ The expression of cytokeratins* and involucrin9 and reactivity to peanut agglutinin lectin'" are known to be associated with particular patterns of epithelial differentiation. This report examines these factors in a series of bronchial dysplasias, in comparison with normal bronchial epit he1 ium.

MATERIALS AlVD METHODS

Thirty-nine formalin-fixed, paraffin-embedded bronchial biopsies of dysplastic epithelium were retrieved from the archives of the Histopathology

Addressee for correspondence: Dr N. Pendleton, University Hospital of South Manchester, Nell Lane, Manchester M20 2LR, U.K.

CCC 0022-3417/96/020146-05 (c> 1996 by John Wiley & Sons, Ltd.

Department of Broadgreen Hospital for comparison with 17 biopsy samples taken from pulmonary re- section specimens (I4 for bronchial carcinoma, two for bronchiectatic disease, and one for a bronchial carcinoid) and immediately frozen in iso-pentane cooled to - 165°C for 2 niin and stored in liquid nitrogen at - 196°C. Serial sections were then cut, in the case of archival material, at 4 p m with a Reichert 2030 rotary microtome; floated out on water in a bath heated to 48°C; and then mounted on slides and dried at 37°C overnight. In the case of frozen material, the tissue was embedded in OCT compound and cut at 5 p m using a rotary microtome in a Bright OTF cryostat set to a temperature of - 24°C and collected onto a glass slide coated in chrome gelatin solution for section adhesion. Sections were then either dewaxed through a xylene and alcohol gradient with paraffin-embedded material, or fixed in cold acetone at 4°C for 10 min.

The morphology of biopsy specimens was examined independently by two histopathologists using routine haematoxylin and eosin-stained tissue sections. Sequential sections were examined for continuity of the epithelium to be studied. In the case of the dysplastic biopsies, sections were graded as described in a previous report.6 In each case, the epithelium was graded histologically by the level containing undifferentiated, non-stratified cells with pleomorphic nuclei, a high nuclear-cytoplasmic ratio, and mitoses. In mild dys- plasia, these changes are limited to the lower-third of the epithelium; in moderate dysplasia, beyond the

Received 8 July 1994 Accepted 26 June 1995

DIFFERENTIATION MARKERS IN BRONCHIAL DYSPLASIA 147

Table I-Antisera used for immunolocalization of antigens studied in normal and dysplastic bronchus

Antiserum Antigen Source

6 B10 Anti-K6

RCK105 Anti-K7 (f) M20 RKSE60 Anti-K10 (f) 1 C7 Ks 13.1 (f) CKB 1

RGE53

Ks 19.1 BA 17 (f) v9 Peanut agglutinin

BT 600

LL002 (f)

Ks-B17.2 (f)

(Arachis hypogaea)

CK 4 CK 6

CK 7 CK 7 CK 8 CK 10 CK 10 CK 13 CK 13 CK 14 CK 14 CK 18 CK 18 CK 19 CK 19

Vimentin Beta-D-gal

(1-3)-~-galNAc Involucrin

Biogenesis Professor E. Fuchs,

U.S.A. Biogenesis Sigma Sigma Biogenesis Biogenex Biogenesis ICN Sigma Biogenex Biogenesis Sigma ICN Dako Dako Sigma

Biomedical Technologies

CK=Cytokeratin with Moll classification number. An antiserum followed by (f) indicates that this antiserum was used

on formalin-fixed, paraffin-embedded tissue sections. Those without (f) were used on snap-frozen, acetone-fixed tissue sections with the exception of antisera anti-K6, M20, V9, peanut agglutinin, and BT600, which were used on both tissue section types.

lower-third but not into the upper-third; and in severe dysplasia, the changes occupy more than two-thirds, or the full thickness of the epithelium.

In all the immunocytochemical experiments, the avidin-biotin-peroxidase method was used;" antigen localization was visualized by 3,3-diaminobenzidene tetrahydrochloride; and nuclei were counterstained with haematoxylin. Only cytoplasmic staining was recorded as positive. The antibodies used are listed in Table I and included both commercially available and donated antisera to cytokeratin peptides, vimentin, involucrin, and peanut agglutinin. All immunocyto- chemical experiments included the appropriate positive and negative controls.

Immunocytochemically stained sections were all reviewed by two independent assessors, one of whom is a consultant histopathologist. Both the presence of antigen and its distribution were recorded. Following this, inter-observer agreement was evaluated using the kappa statistic and 95 per cent confidence internals (CIS). l 2 The significance of antigen presence and absence was assessed using either a two-tailed chi square or Fisher's exact test as appr~pr i a t e . ' ~

RESULTS

The inter-observer agreement of the two independent assessors was determined to be high, with a kappa statistic of 0.94 (95 per cent CI 0.90-0.98) for normal

bronchial epithelium and 0.8 (95 per cent CI 0.75-0.85) for the dysplastic bronchial epithelium.

The 17 cases of normal bronchial epithelium exam- ined all expressed cytokeratins (CKs) 4, 6, 7, 8, 18, and 19. Thirteen biopsies expressed CK 13 and no demon- stration of CK 10 or CK 14 was noted. CKs 6, 8, and 19 were found in both basal and columnar cells, whereas CKs 4, 7, 13, and 18 were found only in the columnar cells. The expression of CKs 4 and 13 was focal in distribution throughout the columnar cell layer. Focal columnar labelling with anti-vimentin and peanut agglutinin (PNA) lectin was observed. No difference in antigen expression was found in the normal bronchial epithelium from lungs resected for malignant disease as compared with non-malignant disease.

Limited clinical information was available for the cases of bronchial dysplasia, but the donor patients were of a mean age of 66 years (53-81 years) and all were cigarette-smokers. Twenty-nine of the cases were associ- ated with a diagnosis of lung cancer and there was histological confirmation of this in 19 cases. Of the remaining cases, five were associated with benign pathol- ogy (three pneumonidabscess; one lipoma; one asthma) and five were without other pulmonary pathology.

CKs 7 and 19 were detected in all dysplastic samples, and in the 19 samples where there was sufficient material present, simple CKs 8 and 18 were visualized. Supra- basal and focal cellular distribution of CK 13 was seen in 26 cases, CK 14 in 13 cases, CK 6 in 11 cases, and CK 10 in 5 cases. Some basal cell cytokeratin expression was observed in 8 cases. An example of CK localization in dysplastic bronchial epithelium is shown in Fig. 1. Focal involucrin expression was observed in 17 biopsies and PNA binding lectin in 12. In all the cases positive for involucrin and five of those positive for PNA binding lectin, the staining distribution was suprabasal. In the remaining seven PNA binding lectin-positive cases, five exhibited both basal and suprabasal staining, and two basal cell staining only. Vimentin was demonstrated in the stroma of all biopsies, but focal basal and suprabasal epithelial staining was observed in five cases. CK 6 was found to be associated with a non-severe histological grade (PO.018) (see Fig. 2). There were no associations demonstrated between clinical or histopathological data and other antigens expressed.

Between the normal and dysplastic biopsy groups, significant associations were found between the loss of CK 6 peptide in bronchial dysplasia (P<O.OOl) and the presence of CK 14 (P=O.OOS) and involucrin (P=O.O18) in bronchial dysplasia, compared with normal bronchial epithelium (see Fig. 3).

DISCUSSION

This study has shown that CK 6 is expressed in both basal and suprabasal normal bronchial epithelial cells and that the presence of this cytokeratin peptide correlates significantly with the less atypical degrees of bronchial dysplasia. Suprabasal expression of involucrin and CK 14 was also significantly associated with the dysplastic phenotype.

148 N. PENDLETON ET AL.

Fig. I-(a) Immunolocalization o f cytokeratin 13 in a case of severe bronchial dysplasia. Cytoplasmic staining is noted in the epithelium. (b) lmmunolocalization of cytokeratin 13 in a case o f severe bronchial dysplasia. Negative case

100 In Q) cn m u Q) > cn .- .- c 8 50 Q) cn m C Q)

Q)

U

2 n

0

Mildhnoderate dysplasia number of samples=l2

a Severe dysplasia number of samples=27

CK=cytokeratin, INV=involucrin, PNA=peanut agglutinin

Significant differences indicated by p value 1 CK6 CK7 CK8 CK18 CKlSCKl3CKlOCK14 INV PNA P=O.O18 Antigen detected

Fig. 2 Comparison of the differentiation antigen expression of mild/moderate and severe bronchial dysplasia

Normal Bronchus number of

k% Bronchial Dysplasia number of

samples= 17

samples=39

CK=cytokeratin, INV=involucrin, PNA=peanut agglutinin

Significant differences indicated by p value

CK6'CK7 CK8 CK18 CK19CK13CKlO CK14 INV PNA pco.001 Antigen detected P=o.ooBP=o.018

Fig 3-Comparison of normal and dysplastic bronchial epithelial diRerentiation antigen expression

Although there is considerable circumstantial evi- linking progression of bronchial dysplasia to lung carcinoma. As there is a marked morphological alter- ation in dysplastic bronchial epithelium to a stratified, rather than a pseudo-stratified pattern, this study was

dence that squamous cell metaplasia and carcinoma in sittr' are linked to the histogenesis of lung carcinoma, no direct evidence exists. There is equally no direct evidence

DIFFERENTIATION MARKERS IN BRONCHIAL DYSPLASIA 149

designed to examine whether the expression of differentiation-associated antigens would fit an ordered pattern similar to normal stratified epithelia, or be more heterogeneous as in epithelial malignancies, including lung cancer.

Cytokeratin polypeptide-containing intermediate filaments are the structural unit of epithelial cells and their expression corresponds to patterns of epithelial differentiation.* There are a total of 20 cytokeratin polypeptides, subdivided into two equal families based on a combination of biochemical and molecular bio- logical properties. I4, l5 However, their expression can also be divided into cytokeratins expressed in simple or glandular epithelium (CKs 7 , 8 , 18, and 20), cytokeratins expressed in stratified epithelium (CKs 1, 2, 3, 4, 5 , 6, 9, 10, 11, 12, 13, 14, 15, 16, and 17), and CK 19, which is found in both.

Previous studies have allowed a meta-analysis of the expression of cytokeratins in bronchial e p i t h e l i ~ r n . ' ~ ~ ~ ' As in our study, CKs 7, 8, 18, and 19 have been demonstrated in the basal cells, and CKs 4, 7, 8, 13, 18, and 19 in the columnar CK 14, not demon- strated in our study, has been visualized focally in the basal cell layer of bronchial epitheli~m,'~. '~ while CKs 17 and 20 have been shown also in the columnar cell layer.l5>l9 Using biochemical analysis, CKs 5 and 6 are known to be part of the intermediate filament profile of bronchial epithelium, but until this report the basal and suprabasal location of CK 6 expression had not been described.16 As in our series, vimentin has been demon- strated in the columnar cell layer by other group^.'^ In lung tumours, cytokeratin expression commonly follows the pattern of differentiation, adenocarcinomas showing a simple cytokeratin profile and squamous cell carcino- mas a complex profile of both simple and stratification ~ytokeratins.~,' 5-17,19 Small cell carcinomas vary in the ability to express cytokeratin and other less common cell types show variable cytokeratin profiles. 17,18 Other inter- mediate filament peptides, including ~ i m e n t i n , ' ~ , ' ~ have been demonstrated in lung tumours.

Involucrin, an envelope protein, and the glycoprotein binding the lectin, PNA, are associated with stratifica- tion of epithelial cells and thus have been most exten- sively studied in skin.'.'' However, PNA staining of rat tracheobronchial epithelium has shown a basal cell layer distribution.20 Involucrin expression has been examined in normal bronchus and bronchial metaplasia;21 normal bronchial epithelium was entirely negative and bronchial metaplasia was strongly positive. In lung neoplasms, involucrin expression was found almost exclusively in squamous cell carcinomas.21

Although there have been a number of published studies presenting the behaviour and presence of cellular differentiation antigens in squamous cell metaplasia induced in animal r n ~ d e l s , ~ ' ~ ~ ~ - ~ ~ there has been only one detailed analysis of the molecular characteristics of squamous cell metaplasia in samples of human tracheo- bronchial e p i t h e l i ~ m . ~ ~ In this work, there was a clear pattern of increased expression of stratification- associated cytokeratin peptides (CKs 4, 6, 13, 15 and 16) orientated to a suprabasal distribution, with a reduction in glandular or simple epithelial cytokeratin expression

(CKs 7, 8, 18, and 20). This would be entirely in keeping with an alteration of a pseudo-stratified to a stratified- columnar epithelium, with or without cornification. In our study, a high proportion of dysplastic epithelial biopsies showed stratification-associated antigens such as CKs 6 and 13 in the normal bronchial epithelium. The results also show a significant association between the severity of bronchial dysplasia and the absence of CK 6, which may indicate that biological or molecular differ- ences exist with different degrees of atypia in these lesions. Involucrin and CK 10 were also detected; although not expressed in the normal pseudo-stratified columnar epithelium of the bronchus, they may simply reflect stratified-cornified differentiation of these dys- plastic samples. However, a more surprising result is that over a third of the dysplastic biopsies examined expressed only simple epithelial cytokeratin profiles and all biopsies expressed simple CK 7, usually only observed in epithelia exhibiting glandular differentia- tion.' Similarly, a suprabasal distribution of CK 14, regarded as a marker of basal cell phenotype, is unusual and unexplained.26

The comparison of results from the snap-frozen, acetone-fixed normal bronchial epithelium and formalin-fixed, paraffin-embedded bronchial dysplasias shows significant differences already discussed. It is possible that differences in the immunocytochemical demonstration of cytokeratin antigens depend on the antisera and type of fixation used. Snap-frozen mate- rial is accepted as optimal for cytokeratin localization, but to obtain sufficient cases of bronchial dysplasia, archival formalin-fixed, paraffin-embedded tissue was essential. In this study, therefore, antisera were chosen appropriate to the fixation method. The antigen pro- files of the normal and dysplastic epithelium could reflect differences in fixation methods. We would have expected this to demonstrate less antigen localization in archival material, as in the case of CK 6. However, CK 14 and involucrin showed significantly higher percentages of positive cases in the formalin-fixed dysplasia sections.

In conclusion, our results indicate that the pattern of differentiation-associated antigen expression in bronchial epithelial dysplasia is significantly different from that of the normal bronchus. The pattern of antigen expression, however, does not correlate well with the morphological changes in dysplasia. The expression of the simple, non-stratification-associated epithelial cytokeratin peptide alone in some dysplastic lesions is unusual in a stratified epithelial structure. This may indicate that unlike squamous metaplasia of the bronchus, bronchial dysplasias are a heterogeneous group of lesions from which the whole spectrum of lung carcinomas could potentially develop.

ACKNOWLEDGEMENTS

This work was supported by the Clatterbridge Cancer Research Trust. Antiserum to cytokeratin 6 polypeptide was kindly donated by Professor E. Fuchs, Harvard Medical Institute, U.S.A.

150 N. PENDLETON ET AL.

REFERENCES I . Auerbach 0, Stout AP, Hammond EC, Garfinkel L. Change in bronchial

epithelium in relation to cigarette smoking and in relation to lung cancer. N Engl J Med 1961; 265: 253-257.

2 . Trump BF, McDowell EM, Glavin F, rt ul. The respiratory epithelium (111). Histogenesis of epidermoid metaplasia and carcinoma in-situ in the human. J Nut/ Cancer In.rr 1978; 61: 587-606.

3. Mason MK, Jordan JW. Outcome of carcinoma in-silu and early invasive carcinoma of the bronchus. Thorax 1982; 37: 453456.

4. Nuorva K, Soini Y , Kame1 D, et al. Concurrent p53 expression in bronchial dysplasias and squamous cell lung cancer. A m J Pathol 1992; 142: 725-732.

5. Sundaresan V, Ganly P, Haselton P, et ul. p53 and chromosome 3 abnormalities, characteristic of malignant lung tumours, are detectable in preinvasive lesions of the bi-onchus. Oncogene 1992; 7 1989-1997.

6. Pendleton N, Dixon GR, Burnett HE, Occleston NL, Myskow MW, Green JA. Expression of proliferating cell nuclear antigen (PCNA) in dysplasia of the bronchial epithelium. J Puthol 1993; 1 7 0 169-172.

7. Fisseler-Eckhoff A, Preberg M, Voss B, Muller KM. Extracellular matrix in prencoplastic lesions and early canccr of the lung. Puthol Res Pruct 1990; 1 8 6 9 5 101.

8. Moll R, Franke WW, Schiller DL. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumours and cultured cells. Cell 1982; 31: I 1 24.

9. Banks-Schegel SP, Green H. lnvolucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia. J Cell Biol 1980; 9 0 732.

10. Watt FM, Keeble S, Fisher C, Hudson DL, Codd J, Salisbury JR. Onset of expression of peanut lectin-binding glycoproteins is correlated with stratifi- cation of keratinocytes during human epidermal development m vivo and in i'irro. J Cell Sci 1989; 94: 355-359.

1 I . Hsu SM, Raine L, Franger H. Use of avidin-biotin-peroxidase complex (ABC) arid unlabelled antibody (PAP) procedures. J Histocliem Cytochern 1981; 2 9 571-579.

12. Altman RG (ed ). Some common problems in medical research. In: Practical Statistics in Medical Research. London: Chapman and Hall, 1992; 403409.

13. Altman RG (ed.). Comparing groups-ategorical data. In: Practical Sta- tistics in Medical Research. London: Chapman and Hall, 1992: 253-257.

14. Sun TT, Eichner R, Schcrmer A, Cooper D, Nelson WG, Weiss RA. Classification, expression and possible mechanisms of evolution of mamma- lian epithelial keratins: a unifying model. In: Levine A, Topp G, Van de

Woude D, Watson JD, eds. The Cancer Cell. Vol. I . Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 3984; 169-176.

15. Moll R, Schiller DL, Franke WW. Cytokeratin 20 in human carcinomas: a new histodiagnostic marker detected by monoclonal antibodies. A m J Pathol 1992,140 4 2 7 4 7 .

16. Blobel GA, Moll R, Franke WW, Vogt-Moykopf I . Cytokeratins in normal lung and lung carcinomas: adenocarcinomas, squamous cell carcinomas and cultured cell lines. Virchows Arch B /Cell Puthol] 1984; 4 5 407429.

17. Broers JLV, Ramaekers FCS, Rot MK, el al. Cytokeratins in different typcs of human lung cancer as monitored by chain-specific monoclonal anti- bodies. Cancer Res 1988; 4 8 3221 -3229.

18. Elias AD, Cohen BF, Bernal SD. Keratin subtypes of small cell lung cancer. Cancer Res 1988; 4 8 2724-2729.

19. Wetzels RHW, Schaafsma HE, Leigh IM, et ul. Laminin and type V11 collagen distribution in different types of lung carcinoma: correlation with expression of keratins 14, 16, 17 and 18. HistopathoLogy 1992, 2 0 295-303.

20. Schimizu T, Nettesheim P, Mahler JF, Randel SH. Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: quantitative studies with Grifonia .sirnplicifoliu I isolectin B,. J Histochon Cytochrm 1991; 3 9 7-14.

21. Said JW, Nash G, Sassoon AF, Shintaku P, Banks-Schlegel S. lnvolucrin in lung tumours. A specific marker for squamous differentiation. Lab I m m 1983; 4 9 563-568.

22. Reznik-Schuller H. Sequential morphological alterations in the bronchial epithelium of Syrian Golden hamsters during N-nitrosomorpholine-induced pulmonary tumorigenesis. Am J Pathol 1977; 8 9 59-66.

23. Jetten AM, George MA, Smits HL, Volberg TM. Keratin 13 expression is linked to squamous differentiation in the rabbit tracheal epithelial cells and down-regulated by retinoic acid. Exp Cell Res 1989: 182: 622 634.

24. Rutten AA, Bruyntjes JP, Rameakers FC. Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium. An immunohistomorphological study using monoclonal antibodies to keratins. Virchowas Arch B /Cell Pathol] 1988; 5 6 111-117.

25. Leube RE, Ruustad TJ. Squamous cell metaplasia in the human lung: molecular characteristics of epithelial stratification. Virchows Arch B (Cell Puthol] 1991; 61: 227-253.

26. Purkis PE, Steel JB, MacKenzie IC, Nathrath WBJ, Leigh IM, Lane EB. Antibody markers of basal cells in complex epithelia. J Cell Sci 1990; 97: 39-50.