J Clin Pathol 1989;42:736-739

Expression of cytokeratin in the epithelium ofdentigerous cysts and odontogenic keratocysts: an aid todiagnosis

A W MAcDONALD, A FLETCHER

From the Department ofPathology, University ofLeeds, and the Department ofHistopathology, LeicesterRoyal Infirmary

SUMMARY Sections of tissue embedded in paraffin wax from 18 selected odontogenic cysts were

studied both histologically and immunohistochemically with antibodies to cytokeratins using theindirect peroxidase technique. The cysts were divided on a clinical and histological basis into twoequal groups comprising dentigerous cysts and odontogenic keratocysts. It was possible todifferentiate the two cyst types in every case by the pattern of staining using the monoclonal antibodyLP34 for cytokeratins of intermediate molecular weight. The monoclonal antibody CAM 5 2 forcytokeratins of low molecular weight was not discriminatory. Such a clear distinction may prove

useful diagnostically in distinguishing between two cysts of similar appearance but very differentbehaviour.

Dentigerous cysts and odontogenic keratocysts areboth thought to arise from the odontogenic epith-elium-the former arising from the enamel organusually after the crown of the tooth has formed, thelatter from the dental lamina or primordial odon-togenic epithelium. Consequently odontogenickeratocysts are often associated with a site where thereis absence of a tooth from the series. Despite having asimilar odontogenic origin clinical behaviour differs,particularly the rate of recurrence. A dentigerous cystshould not recur after adequate excision and may evenbe treated by marsupialisation. The keratocyst,however, has a strong tendency to recur, the rate ofrecurrence being as high as 60%.'-3

Ideally, the diagnosis is best made preoperatively sothat the surgical procedure can be tailored accordin-gly. Clinical and radiological assessment is obviouslyof considerable importance but may sometimes bequite misleading.4

Consequently, much work has centred on theodontogenic keratocysts in an attempt to find areliable marker. Soluble protein estimation, if below48 g/l, would tend to indicate a keratocyst.' Morerecent work has centred on the qualitative assessmentof "specific" cyst contents6 7; these techniques, usefulas they may be, do not, however, replace histologicalexamination of the cyst after excision, which is stillrequired for definitive diagnosis.

Accepted for publication 2 February 1989

Both cyst types are lined by a stratified squamousepithelium. Subtle differences may be seen on routineexamination of slides stained with haematoxylin andeosin which may enable those with experience todistinguish these cysts, but misinterpretation caneasily occur. A further diagnostic aid would be useful.

Material and methods

Eighteen odontogenic cysts were selected from the filesof the Leicester Royal Infirmary between 1981 and1987. Inflammatory cysts such as radicular andresidual cysts were excluded from the study along withany other cyst showing a high degree of inflammation.Initial diagnosis was made according to the criteriashown in table 1, which includes clinical, radiological,and histological variables, based on the WHO his-tological typing of odontogenic cysts.8

Sections were cut from the original blocks,rehydrated, and treated with 01% trypsin in 0-12%calcium chloride, adjusted to pH 7-8, at 37°C for 20minutes. Endogenous peroxidase activity was blockedusing H202 in methanol for 30 minutes. The sectionswere washed in water and phosphate buffered saline(PBS) before being treated with a 1/20 dilution ofnormal rabbit serum for 10 minutes.The sections were then drained and treated with the

antisera at appropriate dilution (LP 34 (CKI, Dak-opatts) 1/200, CAM 5 2 (Buton Dickenson) 1/25) andincubated overnight at room temperature. After wash-

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Expression ofcytokeratin in dentigerous cysts and odontogenic keratocysts

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Fig 1 LP34 staining ofepithelium ofan uninflamed dentigerous cyst showingfull thickness staining.

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Fig 2 LP34 staining ofepithelium ofan uninflamed odontogenic keratocyst showing absence ofstaining ofthe basal layer.

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738Table 1 Criteria used to segregate odontogenic cysts

Clinical Dentigerous Odontogenic keratocysts

Associated with the Not intimately related tocrown of an unerupted a tooth, often in place oftooth a missing tooth

Radiological Unilocular around Unilocular/multilocularcrown of tooth

Protein* Greater than 48 g/l Less than 48 g/l solublesoluble protein protein

Histology Stratified squamous Stratified squamousepithelium three to four epitheliumcells thick Prominent basalNo basal palisading palisadingMay be mucous cells or Rete ridges generallyciliated cells absent but occasionalKeratinisation may budding of basal layersoccur Epithelial infoldingsCell vacuolation rare commonwhen present usually Parakeratosis common,keratinising orthokeratosis lessFibrous stroma common

Cell vacuolation commonStroma tends to be loose

*In no case was there evidence of this having been undertaken inany of the present cases.

ing with PBS twice for 15 minutes each, the sectionswere stained with 5 mg diaminobenzidine in 10 mlTris-HCI, 10 ml H202 for five minutes and thenwashed and counterstained with Mayer's hematoxylinfor 90 seconds.

Results

Table 2 shows that staining with the antibody CAM5-2 was very weak or absent altogether. When presentit was seen in the basal stratum of odontogenic

Table 2 Results ofstaining intensity

LP34 CAM s52CaseNo Cyst Basal Mid Superficial Basal Superficial

I OKC 0 + + + + +/- 02 OKC 0 + + 0 03 OKC 0 + + + + + +/- 04 OKC 0 + + + + + + 0 05 OKC 0* + + + + + 0 06 OKC 0 +++ +++ 0 07 OKC 0 + + + + + 0 08 OKC 0* + + + ++ +/- 09 OKC 0 +++ +++ +/- 010 Dent. +++ ++ +++ 011 Dent. +++ +++ +++ 0 +/-12 Dent. +++ +++ +++ 0 013 Dent. +++ +++ +++ 0 014 Dent. +++ +++ +++ 0 015 Dent. + + ++ + +++ 0 +patchy16 Dent. +±+±+ +±+4+ +-+4+ 0 0

17 Dent. +++ +++ +++ 0 +/-18 Dent. +++ +++ +++ 0 0

The intensity of staining was assessed semiquantatively and scoredas below: 0 = None; + = weak; + + = moderate;+ + + = strong.0* = generally no staining but weak.

MacDonald, Fletcherkeratocysts and in the superficial stratum of den-tigerous cysts.LP34 showed two patterns of staining, one where

the basal layer was strongly positive, usuallyassociated with stronger staining of more superficiallevels, and one where the basal layer was negative orfocally very weak, associated with less strong stainingof mid and superficial layers. The former patternoccurred in dentigerous cysts (fig 1); the latter inondontogenic keratocysts (fig 2). Two odontogenickeratocysts showed weak focal staining (case 5 andcase 8) in areas which were directly adjacent to chronicinflammatory cellular infiltration within the connec-tive tissue stroma. Similar foci of mild inflammationwere evident in the connective tissue surroundingsome of the dentigerous cysts. The staining pattern inthe epithelium of these cysts remained unchangedbetween these mildly inflamed foci and uninflamedareas. This finding would suggest that the basalstaining of dentigerous cysts using LP34 was aproperty of the epithelial differentiation rather than asecondary effect of inflammation, unlike the basalstaining seen in odontogenic keratocysts which onlyappeared adjacent to inflammation.

Discussion

Although dentigerous cysts and odontogenickeratocysts both arise from odontogenic epitheliumand may have a similar histological appearance theirbehaviour in terms of recurrence is sufficientlydifferent to warrant different treatment and follow up.These types of cyst are relatively uncommon and theexperience of the individual pathologist may belimited, so any additional diagnostic methods are to bewelcomed. Much current research has been directed atfeatures specific to the odontogenic keratocystsincluding keratocyst antigen9 and enzyme histo-chemistry of the epithelium'"'2 in an effort to aiddiagnosis. Recent work has been directed towards theintermediate filament keratin proteins within the cyto-plasm of odontogenic keratocysts which have beenidentified and catalogued.'3 A similar catalogue ofcytokeratins from other odontogenic cysts may high-light differences in expression ofcytokeratins allowinga given epithelium or epithelial cell to be characterisedby the specific pattern of its cytokeratin components. 14The results of our study suggest that there are, indeed,differences between the expression of cytokeratincomponents between dentigerous cysts and odon-togenic keratocysts that can be exploited diagnos-tically.Thus far the emphasis in research has concentrated

on the epithelium itself, with only occasional passingreference to the underlying connective tissue stroma.One feature among the histological differences bet-

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Expression of cytokeratin in dentigerous cysts and odontogenic keratocysts 739ween dentigerous cysts and odontogenic keratocyststhat has been cited many times is the stroma, which inthe case of the latter is described as delicate, loose, andrich in mucopolysaccharide. The interaction betweenan epithelium and its stroma is complex and not wellunderstood, but observations from this study showthat focal inflammation within the stroma of odon-togenic keratocysts affects the local expression ofcytokeratins. This observation, in addition to otherobservations that odontogenic keratocyst epithelium,when transplanted into nude mice, only retains itstypical appearance histologically if supported by itsown connective tissue stroma,'5 suggest that thedifferentiation of the epithelium is not independent ofthe stroma.The results of this study show that odontogenic

keratocysts and dentigerous cysts, in the absence ofclinically important inflammation, may be reliablydistinguished on the basis of staining ofthe basal layerof the cyst lining using the commonly availablemonoclonal antibody LP34. Similarly, the techniquedescribed may have limitations in distinguishing bet-ween cysts in which there is considerable inflammatorychange, a situation where histological distinction isparticularly difficult.

We thank Mr R Cullen and staff of the specialhistology laboratory, Leicester Royal Infirmary fortheir kind assistance and Mrs Lynne Bailey for typingthe manuscript.

References

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2 Ahlfors E, Larsson A, Sjogen S. The odontogenic keratocyst: a

benign cystic tumour? J Oral Maxillofac Surg 1984;42:10-9.3 Zachariades N, Papanicolaou S, Triantafyllou D. Odontogenic

kertatocysts: review of the literature and report of sixteen cases.J Oral Maxillofac Surg 1985;43:177-82.

4 Soskolne WA, Shear M. Observations on the pathogenesis ofprimordial cysts. Br Dent J 1967;123:321-6.

5 Kramer IRH, Toller PA. The use of exfoliative cytology andprotein estimations in pre-operative diagnosis of odontogenickeratocysts. Int J Oral Surg 1973;2:143-51.

6 Kunsela P, Hormia M, Trompo H, Ylipaavalniemi P. Demonstra-tion and partial characterisation of a novel soluble antigenpresent in keratocysts. Oncodev Biol Med 1982;3:383-90.

7 Kunsela P, Ylipaavalniemi P, Thesleff l. The relationship betweenthe keratocyst antigen (KCA) and keratin. J Oral Pathol1986;15:287-91.

8 Pindberg JJ, Kramer IRH, Torloni H. Histological typing ofondotogenic tumours,jaw cysts and allied lesions. InternationalHistological Classification of Tumours. No S Geneva: WHO1971.

9 Douglas CWI, Craig GT. Recognition of protein apparentlyspecific to odontogenic keratocyst fluids. J Clin Pathol1986;39:1 108-15.

10 Kobayasi T, Hansen J. Ultrastuctural studies ofodontogenic cystsIII. Acid phosphatases. Acta Morphol Neerl Scand 1970;8:63-78.

11 Magnusson BC. Odontogenic keratocysts: a clinical and his-tological study with special reference to enzyme histochemistry.J Oral Pathol 1978;7:8-18.

12 Stenman G, Magnusson B, Lennartsson B, Juber-Ode M. In vitrogrowth characteristics of human odontogenic keratocysts anddentigerous cysts. J Oral Pathol 1986;15:143-5.

13 Shuler CF, Schiver BJ. Identification of intermediate filamentkeratin proteins in parakeratinised odontogenic keratocysts.Oral Surg Oral Med Oral Pathol 1987;64:439-4.

14 Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. Thecatalogue of human cytokeratins: patterns of expression innormal epithelium, tumours and cultured cells. Cell 1982;31:11-24.

15 Vedtofte OP, Holmstrup P, Dabelsteen E. Human odontogenickeratocyst transplants in nude mice. Scand J Dent Res1982;90:306-14.

Requests for reprints to: Dr A W MacDonald, Departmentof Pathology, University of Leeds, Leeds LS2 9JT, England.

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