EXPRESSION OF CALRETININ AND CYTOKERATIN19 IN RADICULAR CYST,

DENTIGEROUS CYST, ODONTOGENIC KERATOCYST AND AMELOBLASTOMA - AN

IMMUNOHISTOCHEMICAL STUDY

Abstract:

Background: Calretinin is a protein associated with cellular

differentiation and proliferation and is an inhibitor of

apoptosis. Calretinin is known to be a distinctive and specific

immunohistochemical marker for ameloblastoma. Cytokeratin is a

protein associated with odontogenic epithelial cell and

ameloblasts being epithelial derivative also expresses some

cytokeratin. This study was done to ascertain if calretinin and

cytokeratin could be responsible for the differences between

aggressiveness of certain odontogenic cysts and tumors. Aims &

Objectives: To evaluate the expression of calretinin and

cytokeratin 19 (CK19) in odontogenic cysts and

ameloblastoma. Materials and Methods: Sixty samples of formalin

fixed paraffin embedded tissue specimens (15 radicular cysts, 15

odontogenic keratocysts (OKC), 15 dentigerous cysts and 15

ameloblastoma) were evaluated for the expression of CK19 and

calretinin using immunohistochemistry. Results: Calretinin showed

positive expression in all cases of ameloblastoma. Among the

samples of OKC, 13% of the cases showed positivity. All the cases

of radicular and dentigerous cysts were completely negative for

calretinin. CK19 was negative in all cases of radicular cyst and

OKC, whereas among the dentigerous cyst 2 cases showed mild

expression and one case showed moderate staining intensity for CK

19. Only one case of ameloblastoma showed moderate staining for

CK19. Conclusion: The results of this study showed that calretinin

can be a immunohistochemical marker for neoplastic ameloblastic

epithelium and the difference of expression among the lesions is

probably due to their diverse histopathological characteristics

and their developmental origin. CK19 a marker of simple

epithelia and its absence could probably be due to absence

of CK 19 epitope, superimposition of other cytokeratins or

masking of the epitopes.

Key Words: Calretinin, Cytokeratin19, Immunohistochemistry,

Odontogenic lesions.

Introduction:

The odontogenic lesions originate from odontogenic epithelium or

ectomesenchyme with varying degrees of inductive tissue

interaction.1 The most commonly occurring odontogenic cysts are

radicular, dentigerous and odontogenic keratocyst (OKC). 2,3 The

biological behaviour of few OKCs is as aggressive as benign

neoplasm such as ameloblastoma. OKC is now designated by World

Health Organization (WHO) as a Keratocystic odontogenic tumor

(KCOT). The clinically aggressive behaviour is a result of the

properties of lining epithelial cells and connective tissue

capsule.4 Calretinin, a calcium binding protein with molecular

weight of 29kda, is a member of the large family EF-hand proteins.

This protein is expressed primarily in certain subtypes of neurons

in the retina and in the central and peripheral nervous system.

Its precise behaviour is still unknown but possible roles are

calcium buffer, calcium sensor and regulator of apoptosis .5

Studies in rats have demonstrated that calretinin is expressed in

neural element of tooth pulp, periodontal ligament, viscerosensory

nerve fibres of oral tissues and also during odontogenesis in

molar tooth germs. Various studies have shown positivity of

calretinin in ameloblastoma as well as in aggressive areas of

OKC.6

Cytokeratin belonging to intermediate family protein that are

divided into two subfamilies based on their charges,

immunoreactivity and amino acid sequence: acidic proteins

with low molecular weight and basic proteins with high

molecular weight.7 The expression pattern of intermediate

filaments has been investigated in normal and neoplastic

human cells including oral epithelial cells, odontogenic

epithelia, tumors and cysts. These investigators

hypothesize that intermediate filaments expression

patterns are characteristic for each kind of cells.8

Cytokeratin 19 is the smallest known acidic type of

cytokeratin, having molecular mass of 40kD. It is not

paired in epithelial cells. It is expressed in most simple

epithelia, in various ductal epithelia, intestinal epithelia,

gastric foveolar epithelium, and in the mesothelium. It is

expressed in all kinds of odontogenic epithelial cells,

developing tooth germs and in neoplastic epithelial cells

in some odontogenic tumors. It is also detected in cell

rests of Malassez, cell rests of Serrae and lining of

odontogenic cysts. 9Various other studies have demonstrated

the presence of cytokeratin in human oral embryonic

tissues.10 Fukumashi et al has demonstrated the expression of CK19

in odontogenic epithelium to discuss histogenesis of epithelium in

tumors like ameloblastoma.11

Materials and Methods:

The study was conducted in the Department of Oral and

Maxillofacial Pathology, Ragas Dental College and Hospital,

Chennai and was approved by Institutional Review Board. The study

material comprised of 60 formalin fixed, paraffin embedded

specimens taken from archival tissue blocks. Clinically,

radiographically and histologically confirmed cases of dentigerous

cyst (group I, n=15), odontogenic keratocyst (group II, n=15),

radicular cyst (group III, n=15) and ameloblastoma (group IV,

n=15) were included in the study.

Immunohistochemistry Procedure:

5µ sections from paraffin embedded blocks were made and

transferred onto the amino propyl epoxy saline (APES) coated

slides. The slides with tissue sections were treated with three

changes of xylene to remove paraffin wax, followed by descending

grades of alcohol and rehydration with water. The slides were

then transferred to citrate buffer and autoclaved for antigen

retrieval at 15 lbs pressure for 15 minutes. The slides were

allowed to cool and washed in phosphate buffer solution (PBS) (pH

7.2) for 5 minutes. Peroxidase and protein blocking were done

using 3% hydrogen peroxide and protein block reagent respectively

for 10 minutes each. The slides were incubated with primary

antibodies (Concentrated, Rabbit Polyclonal Anticalretinin

antibody; Biogenex super sensitivity detection system) at room

temperature for overnight. Anti Human Cytokeratin 19

antibody (concentrated mouse monoclonal antibody;

Biogenex super sensitive detection system) was added to

the tissues and incubated for 2 hours at room

temperature. The secondary antibody used was an enhancer

followed by streptavidin solution for 20 minutes from the

secondary polymer kit (Biogenex). A drop of freshly prepared DAB

(3' Diaminobenzidine Tetrahydrochloride - a substrate chromogen)

was added. Three changes of PBS washes were performed after every

step during the immunostaining procedure. The sections were

counter stained with hematoxylin and mounted with DPX®.

Criteria for evaluation of staining:

Labeling index (LI) was calculated by dividing the number of

positive cells by the total number of cells counted in the slide

and expressed as percentage. A minimum of thousand cells was

counted for each slide. The cytoplasmic staining intensity was

evaluated and graded as mild (+), moderate (++) and intense (+++)

as described by Cheung et al . Mild staining is denoted by light

brown color, moderate by brown color and intense by dark brown

color. The cells which did not take up any brown stain were

considered negative.

Statistical analysis:

Data entry and statistical analysis was done using SPSS TM

software (version 10.05). Chi-square test was done to compare

tissue localization of stain, nature of stain, intensity of stain

and the percentage of cells stained among the study groups. p

value less than 0.05 was considered to be statistically

significant.

Results:

Table 1: Staining intensity of calretinin among the study groups:

Staining

intensity Dentigerous OKC Radicular

Ameloblasto

ma p-

Cyst cyst value

n % n % n % n %

No stain (-) 15 100 13 86.6 15 100 0 0

0.000

*

Mild stain

(+) 0 0 1 6.6 0 0 10 66.6

Moderate

stai

n 0 0 1 6.6 0 0 4 26.6

(++)

Intense

stai

n 0 0 0 0 0 0 1 6.6

(+++)

Staining Intensity of CK19 in Study groups

Staining Intensity Dentigerous

cyst

OKC

Radicular cyst p-value

n % N % n %

0.169

No stain ( - ) 12 80 15 100 15 100

Mild stain (+) 2 13.3 0 0 0 0

Moderate stain (++) 1 6.6 0 0 0 0

Intense stain(+++) 0 0 0 0 0 0

Among the odontogenic cysts, all the cases of dentigerous

Staining

Intensity

Ameloblastoma

N %

No stain (-) 14 93.3

Mild stain (+) 0 0

Moderate stain (+

+)

1 6.6

Intense (+++) 0 0

cysts and radicular cysts were negative for calretinin stain,

whereas only 2 cases (6.7%) of OKC exhibited positive

staining. Of the 2 positive cases of OKC, one showed mild

stain and the other exhibited moderate staining intensity.

All the cases of ameloblastoma expressed positive calretinin

stain with 66.6% (n=10) showing mild staining, 26.6% (n= 4)

moderate staining and 6.6% (n=1) intense staining. When the

staining intensity of OKC and ameloblastoma was compared, a

significantly higher intensity of calretinin staining was

noted in ameloblastoma. (p=0.004). Mean labelling index of

the study group which had stained positive for calretinin was

calculated as 1.28 ± 3.39 for Group II (OKC) and 19.21± 13.22

for Group IV (Ameloblastoma).

Among the odontogenic cysts, all the cases of radicular and

OKC were negative for CK 19, whereas among the dentigerous

cyst 2 cases showed mild staining (13.3%) and 1 case (6.6%)

was moderately stained. Among the cases of ameloblastoma only

one case (6.6%) showed moderate staining for CK19. No

significant difference was noted in the staining intensity

between odontogenic cysts and ameloblastoma.

Discussion:

Odontogenic tumors originate from successional and accessional

dental lamina and differentiate into various entities. The

mechanisms that trigger proliferation of the odontogenic

epithelial rests are unknown, but various sub cellular and

developmentally related factors may be responsible for their

differentiation.12 Ameloblastoma is the most frequently

encountered tumor arising from enamel organ.1,11 Among the

cysts, keratocystic odontogenic tumor deserves a special

consideration because of its destructive behaviour, in a

lesser degree when compared to ameloblastoma.4 There are

clinical and radiographic similarities between ameloblastoma

and OKC which may also be reflected at the histologic level if

the tissue sample is small and if neoplastic epithelium

displays reactive changes induced by inflammation. Although

the histology presentation of both OKC and ameloblastoma are

different, both represent an aberration in some stage of

odontogenesis.11

Calretinin is a calcium binding protein of EF hand family and

has a role as a calcium buffer, sensor and also been

demonstrated as an antiapoptotic factor .13,14 In odontogenesis

calretinin was expressed focally in dental lamina, outer

enamel epithelium, stellate reticulum and stratum intermedium

at different stages. In contrast it was intensely expressed in

the inner enamel epithelium and presecretory ameloblasts. This

distribution suggests a possible role of this protein in

enamel formation. 15 Calretinin is known to be a distinctive

and specific immunohistochemical marker for ameloblastoma.16

The expression of calretinin in our studies were in accordance

with earlier studies by Devilliers, Sundaragiri, Chitra and

desliva17,18,19,20 in which ameloblastomas showed 100% positive

staining. None of these studies showed staining in odontogenic

keratocyst except for studies done by desilva20, in which 40 %

of OKC were positive for calretinin. In our study, 13% of OKC

cases showed positive staining for calretinin. The

localization of the protein in ameloblastoma was intense in

areas of stellate reticulum like epithelium, whereas in

peripheral areas its expression was weak.17,21 In OKC the

positive cells were seen in the basal and parabasal layers of

the liningepithelium.22 These findings suggest that the role of

calretinin in ameloblastoma could be a calcium ferry in the

path of transition towards enamel matrix, thus making

calretinin a definitive marker for neoplastic ameloblastic

epithelium.22The location of positivity (the stellate reticulum

like epithelium was strongly reactive for calretinin) explains

the role of calretinin as a antiapoptotic proliferating

protein in the outer layer and apoptotic differentiating

factor in the stellate reticulum like areas.25 In OKC the

positivity in suprabasal layers similar to other markers like

PCNA and p53 could point to increased activity of calcium

binding protein and abnormal cell cycle control.22

In our study, 80% of dentigerous cysts did not

show any staining for CK 19, while 3.3% cases showed

mild staining and 6.6% cases showed moderate staining

intensity. Stoll et al reported that, 48.3% of

dentigerous cyst showed positive staining of CK 19 in

the superficial layers. In our study, staining was not

detected for cytokeratin 19 in any of the layers in the

epithelial lining of OKC. Recent studies done by Stoll

et al on odontogenic keratocysts showed a complete

negative staining. 25In contrast, the study conducted by

Mathews et al showed cytokeratin 19 expression in the

epithelial linings of the odontogenic keratocysts

irrespective of their level within the epithelium

and/or differentiation.

Cytokeratin19 is a marker of simple epithelia and

is an obligatory constituent of all normal epithelium,

junctional epithelium and odontogenic epithelium.

Although OKC is lined by odontogenic epithelium, all

the cases in our study showed negative staining for CK

19. A reason for negative staining could be due to the

superimposition of the cornification markers like

cytokeratin1 and 10 as suggested by Morgan et al.26 Shear

et al suggested that there could be change in the

pattern of cytokeratin profiles in odontogenic

epithelial cells during odontogenesis and also when

quiescent cells proliferate in certain pathological

situations like odontogenic cysts and tumors 27. Hence,

negative staining could also be attributed to the

epithelial characteristics, modification or absence of

epitope.

In our study, none of the cases of radicular cysts

showed any staining characteristics for CK 19. However,

48.3% of the radicular cysts showed positive

immunoreactivity in the suprabasal layers in study

conducted by Stoll et al. In our study, 6.6% of

ameloblastoma showed positive staining characteristics

for CK 19, while 93.3% of cases did not show

immunoexpression. In the study conducted by Fukumashi

et al, all the cases of follicular and desmoplastic

ameloblastomas showed positivity for CK 19, whereas

95% of plexiform and 67% of granular ameloblastomas

exhibited positive staining. Kumamoto et al conducted a

study on all histological types of ameloblastoma and

found decreased expression in keratinizing cells of

acanthomatous type suggesting the dedifferentiation

from odontogenic epithelial characteristics. The

reasons for the absence of staining in our study could

be due to the dedifferentiation of cells in

ameloblastoma which is similar to the results of above

mentioned authors.1 Further, keratin profile of an

epithelium is linked to several factors such as

differentiation, proliferation and histogenesis. The

biological rules of keratin expression are yet to be

defined fully and often there may be subtle differences

between keratin at the molecular levels that may be

reflected as absence of immunoreactivity with

antibodies that have generated to known epitopes. 23

In conclusion, the results of the current study show that

calretinin was shown to be a specific immunohistochemical

marker for neoplastic ameloblastic epithelium and the

discrepancy among the selected odontogenic lesions (higher

immunoreactivity for ameloblastoma, less reactivity for OKC

and non reactivity for dentigerous and radicular cysts) is

probably interconnected with diverse clinical and

histopathological characteristics. The variable expression of

calretinin in ameloblastoma and OKC may reveal a role of this

protein as a second messenger in the control of abnormal cell

cycle proliferation and also indicates that higher the

expression of the protein the least differentiated will be

odontogenic epithelium and hence the calretinin expression in

stronger in ameloblastoma and weaker in OKC. Cytokeratin 19

is a marker of simple epithelia and its absence could

probably be due to absence of cytokeratin 19 epitope,

superimposition of other cytokeratins or masking of the

epitopes due to fixation needs to be investigated.

References:

1. Reichart PA, Philipsen HP. Odontogenic tumors and allied

lesions

2. Shear M. Cysts of the oral region, Fourth edition

3. Browne RM. Pathogenesis of odontogenic cysts: a review.

Journal of Oral Pathology 1975; 4: 31-46.

4. Shear M. The aggressive nature of the odontogenic

keratocyst: is it a benign cystic neoplasm? Part1.Clinical and

early experimental evidence of aggressive behaviour. Oral

Oncology 2002; 38:219-226.

5. Rogers J, Khan M, Ellis J. Calretinin and other CaBPs

in the nervous system. Adv Exp Me d Biol. 1990; 269:195-203.

6. Altini M, Coleman H, Doglioni C, Favia G and Maioran

E. Calretinin expression in ameloblastomas.

Histopathology 2000; 37: 27-32.

7. Lu D-P, Tatemoto Y, Kimura T. Expression of

cytokeratins 8, 13 and 18 and their mRNA in

epithelial linings of radicular cysts: implication

for the same CK profiles as nasal columnar epithelium

in squamous epithelial lining. Oral

Diseases2002;8:30-36.

8. HarveyL, Arnold B. Molecular cell biology. New York

2000:Pg19.6.

9. Shruthi DK et al. Cytokeratin 14 and cytokeratin 18

expressions in reduced enamel epithelium and dentigerous

cyst: Possible role in oncofetal transformation and

histogenesis- of follicular type of adenomatoid odontogenic

tumor ; J Oral Maxillofac Pathol. 2014;18: 365–371.

10. Sawant SS et al. Cytokeratin expression in human fetal

tongue and buccal mucosa. J. Biosci.2000;25:235–242.

11. Fukumashi et al. Cytokeratins expression of constituting

cells in ameloblastoma. Bulletin of Tokyo dental college

2002:43;13-21.

12. Mosqueda-Taylor A. New findings and controversies in

odontogenic tumors. Med Oral Pato l Oral Cir Bucal. 2008:13;

555-558.

13. Alaeddini M ,Etemad-Moghadam S. Comparative expression of

calretinin in selected odontogenic tumors: a possible

relationship to histogenesis Histopathology 2008:52; 299-304.

14.Gotzos V, Schwaller B, Hetzel N, Bustos-Castillo H, Celio

MR. “Expression of the calcium binding protein calretinin in

WiDr cells and its correlation to their cell cycle,” Exp Cell

Res 1992: 202; 292–302.

15. Piattelli A, Fioroni M, Iezzi G, Rubini C. Calretinin

expression in odontogenic cysts.

J Endod. 2003: 29; 394-396.

16. Altini M, Coleman H, Doglioni C, Favia G and Maioran

M ;Calretinin expression in ameloblastomas. Histopathology

2000:37; 27-32.

17. DeVilliers P, Liu H, Suggs C,et al. Calretinin

expression in the differential diagnosis of human

ameloblastoma and keratocystic odontogenic tumor. Am J Surg

Pathol.2008:32; 256-260.

18. Anandani C, Metgud R, Singh K. Calretinin as a Diagnostic

Adjunct for Ameloblastoma. Patholog Res Int 2014:1-7.

19. Sundaragiri SK, Chawda J, Gill S, Odedra S, Parmar G.

Calretinin Expression in Unicystic Ameloblastoma: An Aid in

Differential Diagnosis J Biosci 2010:52;164-169.

20. D’Silva S, Sumathi MK, Balaji N, Shetty NKN, Pramod

KM, Cheeramelil J. Evaluation of Calretinin expression in

Ameloblastoma and Non-Neoplastic Odontogenic Cysts – An

immunohistochemical study. J Int Oral Health. 2013; 5(6): 42–

48.

21. Coleman H, Altini M, Ali H, Doglioni C, Favia G,

Maiorano E. Use of Calretinin in the differential diagnosis of

unicystic ameloblastoma. Histopathology 2001: 38; 312-331.

22. Main D.M.G. Epithelial jaw cysts:10 years of the WHO

classification. Journal of Oral Pathology 1985:14; 1-7.

22. Mistry D, Altini M, Coleman HG, Ali H, Maiorano E.

The spatial and temporal expression of calretinin in

developing rat molars (Rattus norvegicus). Arch Oral Biol.

2001:46 (10); 973- 981.

23. Sandra F, Nakamura N, Mitsuyasu T, Shirasuchi Y, Ohoshi M.

Two relatively distinct patterns of ameloblastoma: an

antiapoptotic proliferating site in the outer layer

(periphery), and a pro apoptotic differentiating site in the

inner layer (centre). Histopathology 2001:39; 93-98.

24. Gupta.B, Ponniah. The pattern of odontogenic

tumors in government teaching hospital in the southern

Indian state of tamil nadu. Oral Surg Oral Med Oral

Pathol Oral Radiol Endod 2010:110;e32-9.

25. Stoll C, Stollenwerk C et al. Cytokeratin

expression patterns for distinction of odontogenic

keratocysts from dentigerous and radicular cysts. J

Oral Pathol Med 2005:34; 558-64.

26. Morgan PR, Shirlaw PJ et al. Potential applications

of anti- keratin antibodies in oral diagnosis. J Oral

Pathol 1987:16; 212-222.