Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease

The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. Objectives: To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Material and Methods: Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Results: Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk. Key words:Oral lichenoid disease, oral lichen planus, oral lichenoid lesion, oral carcinogenesis, EGFR.


Material and Methods
We ha�e studied 44 biopsies obtained �rom patients clinicall� and histopatholo�icall� dia�nosed with OL� in the Oral Medicine and Oral and Maxillo�acial Pa-tholo�� �nit (�ental Clinic Ser�ice o� the �ni�ersit� o� Basque Countr�/EH�)� durin� the period o� Januar� 2006 to June 2008. Out of 44 patients, 30 (68.2%) were females and 14 (31.8%) males, with a mean age of 56.4 �ears (ran�e 31-82 �ears). Clinical and histopatholo�ical data were collected usin� a protocol based on previous studies (2,3,12). Briefly, the clinical data like sex� a�e� t�pe and site o� the lesions were collected. Re�ardin� histopatholo�ical �ea-tures� presence or absence o� the main epithelial and inflammatory infiltrate characteristics were recorded and �raded in mild� moderate or se�ere in each case. Cases were clinically classified in: Group C1 with 26 (59.1%) cases with only papular reticular lesions, and Group C2 with 18 (40.9%) cases with papular reticular and other lesions such as atrophic� erosi�e� and ulcera-ti�e and/or plaque lesions. As inclusion criterion� none o� the patients should ha�e been recei�in� treatment �or OL� at the time o� the stud� or pre�ious to dia�nosis. The mean �ollow up time was 43.5 months (ran�e 20-78 months)� period in which no mali�nant trans�ormation was obser�ed in an� o� the cases.
-Histolo�� and Immunohistochemistr� The cases were classified histopathologically following the dia�nostic criteria proposed b� �an der Meij and van der Waal (2) in an "histologically typical of OLP" (Group HT) with 23 (52.3%) cases, and "histologically compatible with OLP" (Group HC) with 21 (47.7%) cases. Briefly, HT cases had to show: (1) well-defined superficial "band like" predominantly lymphocytic chronic inflammatory infiltrate, (2) epithelial basal cell de�eneration� and (3) absence o� d�splasia; and HC cases did not show either one or none o� the characteristics described �or HT cases. Cases with epithelial d�splasia were excluded �rom the stud�. Samples o� oral mucosa without epithelial and/or inflammatory alterations and OSCC were emplo�ed as controls. All cases and controls underwent an hematox�lin and eosin standard his-tolo�ical procedure as well as con�entional immunohistochemical anal�sis. For immunohistochemical analysis, 4ųm paraffin sections were treated with a citrate bu��er solution at 100°C �or 2 minutes as anti�en retrie�al and incubated usin� the No�olink Pol�mer �etection Kit (No�o-castra®� New Castle �pon T�ne� �K) �ollowin� the manu�acturer´s instructions. All cases were incubated with the primar� prediluted monoclonal antibod� anti-EGFR protein (clon 31G7� Z�med Labs®� Camarillo CA� �SA.) �or 1 hour at room temperature. The anti-bod� emplo�ed has been tested pre�iousl� in other studies (18�22). For �isualization� sections were colored with the substrate/chromo�en 3�3= -diaminobenzidine (�AB) usin� the Pol�mer �etection Kit (No�ocastra ® � New Castle �pon T�ne� �K) showin� a �isible brown precipitate at the anti�en site. Based on pre�ious studies (23)� and considerin� the EGFR expression in controls� a semiquantitati�e anal�sis o� the expression percent-a�e and the expression pattern o� epithelial cells was per�ormed� usin� the So�t Ima�e S�stem Cell A so�tware (Ol�mpus ® � Munster� German�). Immunohistochemical analysis was performed randomly in 5 fields b� 3 obser�ers (�C� JCC and JMA)� independentl� and without knowled�e o� clinical data� usin� a li�ht optic microscope Ol�mpus ® BX41 20x objecti�e and 10x ocular. A consensus a�reement was reached �or an� �i�en sample when discrepancies existed amon� obser�ers �or an� �i�en sample. The epithelial EGFR cell expression was assessed as follows: mild expression when <20% of epithelial cells were positi�e� moderate expression when ≥20% but <40% of epithelial cell were positive and severe expression when ≥40% of cells were positive. For statistical anal�sis� the �ariables were dichotomized in "low EGFR expression" when the expression was mild or moderate, and "overexpression of EGFR" when it was se�ere. The expression pattern was assessed in: membrane pattern (Mm)� c�toplasmatic pattern (Ct) and mixed membrane-c�toplasmatic pattern (Mm-Ct)� and the expression intensit� in mild� moderate and se-�ere. For statistical anal�sis the cellular expression was dichotomized in mild and moderate-se�ere intensit�. The stud� was appro�ed b� the Ethics� In�esti�ation and Teachin� Committee (CEISH) o� the �ni�ersit� o� the Basque Countr�/EH�. The data underwent a descrip-ti�e and comparati�e statistical anal�sis with X2 Pear-son method and Fisher exact test� usin� the statistical so�tware SPSS (Version 15.0� SPSS Inc.� Chica�o� IL).

Results
As expected� �i�en the location o� proli�eratin� cells� the epithelial expression o� EGFR in controls o� nonaffected oral mucosa was confined to basal and parabasal cell la�ers with an Mm expression and scarce Ct expression pattern. In contrast� OSCC controls showed an intense EGFR expression in the peripher� and the center o� neoplastic nests� with a mixed Mm-Ct se�ere expression pattern (Fi�. 1). All cases showed EGFR expression in basal and parabasal epithelial cells. Thirty-nine cases (88.6%) showed EGFR expression in the spinous la�er and onl� in 5 (11.4%) cases reached the superficial layers. EGFR overexpression was recognized in 35 (79.5%) cases and low expression in 9 (20.5%) (Fig. 1). We think that chronic inflammation mediators might be the main reason of this hi�h number o� cases with EGFR o�erexpression� rather than a hi�her mali�nant risk. All cases showed an Mm expression pattern, but in 31 (70.5%), we also obser�ed a Ct pattern� which was mild and moderatesevere in 13 (41.9%) and 18 (58.1%) cases respectively. This was an interesting finding because main EGFR expression pattern in non-a��ected oral mucosa controls was Mm rather than Ct� which in turn was intense in OSCC controls. We did not find significant differences in EGFR expression between the clinical and histolo�ical �roups (p>0.05) ( Table 1)� su��estin� that the expression o� EGFR in OL� subt�pes does not seem to be completel� related with the se�erit� o� the clinical lesions� in other words� epithelial inte�rit� (ulcers and/or erosions) did not affect significantly EGFR expression. Out of the cases with EGFR overexpression, 17 (38.6%) cases showed a moderate-se�ere Ct expression pattern (Fi�. 1), 6 (40%) and 11 (84.6%) cases from HT and HC groups respectively showing a significant difference (p=0.016) ( Table  1). This finding suggests, that when specific histological dia�nostic criteria (2) are applied there are di��erences in the cellular localization o� EGFR in OL� subt�pes� regardless of other histological features like inflammatory infiltrate intensity and/or epithelial thickness or inte�rit�. When we anal�zed the histopatholo�ical �eatures o� cases with EGFR o�erexpression� we onl� obser�ed significant differences regarding the keratinization type, since 19 (54.3%) of these cases showed parakeratosis compared with only 1 (11.1%) case without EGFR o�erexpression (p=0.027) ( Table 2). To this respect� we thought that is difficult to consider this associa-tion� since parakeratosis is �requentl� obser�ed in OLP and OLL samples. Howe�er� it mi�ht be reasonable to observe modifications in the epithelial lining features

Discussion
OLD includes different chronic inflammatory processes like OLP and OLL� characterized clinicall� b� the presence o� white lineal papular lesions in the oral mucosa (1). The mali�nant potential o� these processes is controversial and sometimes difficult to analyze due to the lack of defined and uniform diagnostic criteria emplo�ed in each stud� (7-10). Se�eral �ears a�o� �an der Meij and �an der Waal (2) proposed a clinical and histolo�ical dia�nostic criteria to di��erentiate t�pical cases o� OLP �rom those that were onl� compatible called OLL. These authors (2) demonstrated that when these criteria were applied� onl� cases dia�nosed as OLL showed an e�ident risk �or mali�nant trans�ormation (7�8). Althou�h a clear clinical and histopatholo�ical di��erentiation o� OLP and OLL is critical� an immunohistochemical anal�sis o� these processes would give valuable information about their molecular profiles and their possible mali�nant risk. The EGFR protein is a biomarker o� earl� carcino�enesis (24�25)� o�erexpressed in se�eral oral premali�nant diseases (13�14�16). Moreo�er� EGFR has an s�ner�ic participation with other carcino�enesis biomarkers like c�cloox��enase-2 (COX-2)� inducible nitric oxide s�nthase (iNOS) (26)(27)(28). iNOS is associated with EGFR throu�h the stimulation o� STAT-3 (Si�nal Transducer and Acti�ator o� Transcription-3) and with COX-2 throu�h the s�nthesis o� prosta�landin (PGE2) (16). The upre�ulation o� STAT-3 and COX-2 expression maintain directl� and/or indirectl� the EGFR intracellular path-wa�s� due to acti�ation o� other proteins in�ol�ed in this process� enhancin� oral carcino�enesis in OPML such as OLP and OLL (16�24). To this respect� pre�iousl� we ha�e demonstrated the o�erexpression o� COX-2 in these same samples (27). These results ma� support the s�ner�� between EGFR and COX-2 and hi�hli�hts the possible pro�nostic implications o� this molecular interaction in OLP and OLL. Our stud� has demonstrated that EGFR is o�erexpressed in a �reat percenta�e o� OL� cases� showin� no di�-�erences between the clinical and histolo�ical �roups. These findings may suggest that the clinical type of lesions does not always reflect the molecular features o� OL� processes� in other words� the presence o� an erosive or ulcerative lesion "clinically more aggressive and strikin�"� not necessaril� implies an o�erexpression o� EGFR� and in the same wa�� the presence o� reticular white lesion "clinically less aggressive or striking", does not necessaril� impl� a low EGFR expression. It  is worth mentioning that these findings were also ob-ser�ed in a pre�ious stud� (27) when we anal�ze the COX-2 expression in these same cases. The histolo�ical HT and HC groups showed no significant differences in the EGFR o�erexpression� su��estin� that the presence or absence o� a particular histolo�ical �eature like an intense inflammatory infiltrate and/or extensive epithelial basal cell de�eneration or thickness it is not related with EGFR expression in OLP and OLL. These data indicate the existence o� other molecular mechanisms implicated in EGFR expression in OL� subt�pes. �ue to the lack o� clinical and histolo�ical separation in similar studies (20�21)� the comparison o� our results is difficult. However, our results contrast with those obtained b� Ebrahimi et al. (20) where� althou�h the� do not mention the t�pe o� clinical lesion o� their cases� the� obser�ed a low EGFR expression in OLP samples compared with controls. These authors (20) su��est that hi�h p53 expression in OLP or the presence o� mutated EGFR protein (EGFR �III) ma� explain the low EGFR expression in OLP. p53 protein is one o� se�eral control mechanisms o� EGFR expression (24�25)� thus its o�erexpression ma� explain EGFR low expression. Howe�er� se�eral studies (13�17�29) ha�e pointed out that EGFR mutations in OLP and other OPML is an uncommon e�ent. Moreo�er� se�eral studies (16�25�29) ha�e demonstrated that the expression o� EGFR �III is mainl� obser�ed in late oral carcino�enesis sta�es and alwa�s accompanied with the expression o� the wild t�pe EGFR protein. In this sense� the antibod� used in this stud� reco�nizes both wild and mutated EGFR protein, which makes it difficult to ensure the main type of EFGR protein detected in our stud�. Howe�er� based on pre�ious studies (13�17�29) and considerin� the low per-centa�e o� cases with expression o� EGFR �III in OL� and other OPML� we assume that the main protein detected in our stud� is the wild t�pe EGFR protein. Similar to our results� Kuma�ai et al. (21) obser�ed EGFR o�erexpression in all OLP samples� and additionall� the� obser�ed o�erexpression o� EGFR li�ands (am-phire�uline� epire�uline � HB-EGF [Heparin-bindin�-EGF like �rowth �actor]) and decreased expression o� the li�ands EGF and TGF-alpha. Supported b� their re-sults� these authors (21) su��est that EGFR o�erexpression in OLP ma� contribute to the carcino�enesis o� this disorder. These authors (21) ha�e onl� included reticular lesions� there�ore the comparison with our results is not totall� �alid in this sense. Howe�er� we did not obser�e significant differences in EGFR expression among the Group C1 (papular-reticular) cases. Most o� our samples showed a mixed Mm-Ct EGFR expression pattern which is in concordance with other studies (13,21). However, we observed significant differences between the histolo�ical �roups� since hi�h per-centa�e o� HC cases showed a Ct moderate-se�ere ex-pression pattern compared with HT cases. Other studies (20�21) that anal�ze the EGFR expression in OLP make no reference to this finding. It is worth to note that this Ct expression pattern was not present in controls o� non-a��ected oral mucosa but was intense in OSCC controls. To this respect� Muller et al. (18) ha�e linked pre�iousl� the Ct EGFR expression pattern with mali�nant potential in patients with HNC. These authors (18) obser�ed that Ct EGFR expression pattern in HNC cell lines was associated with nodal metastasis and increased resistance to t�rosine-kinase inhibitors. Other studies (13)� ha�e demonstrated in samples o� normal oral mucosa adjacent to HNC� that the localization o� Ct EGFR expression and the li�and TGF-alpha was the same� and �urthermore� the intensit� and extension o� this expression increased proportionall� to the se�erit� o� the lesion. Similarl�� Srini�asan et al. (17) ha�e obser�ed that Ct EGFR expression in OPML proportionall� increased with the se�erit� o� the epithelial d�splasia. These authors su��est (17) that the Ct EGFR expression could be due to the internalization o� the receptor once it has been acti�ated b� extracellular li�ands� or alternati�el�� due to it being transport to its final location after being s�nthesized. I� the presence o� an increased Ct EGFR expression in HT or OLL cases is associated or ma� contribute to a �reater mali�nant potential as was demonstrated pre�iousl� b� �an der Meij et al. (7�8) is di�ficult to ascertain with these results, but may be worth considerin�.
We ha�e demonstrated that EGFR o�erexpression was not associated with an� particular clinical or histolo�ical �eature� su��estin� that di��erent and/or more complex molecular mechanisms are in�ol�ed in this process. The main cause o� EFGR o�erexpression in OL� samples remains unknown. Howe�er� some authors (22) ha�e �ound that EGFR o�erexpression in other OPML with mali�nant trans�ormation was associated with an increased EGFR �en cop� number� which can also be a possibilit� that need to be in�esti�ated in OL� subt�pes. Considerin� the low rate o� mali�nant trans�ormation of OLP (<1%) (9,11), it is obvious that the high percent-a�e o� cases with EGFR o�erexpression herein obser�ed will not su��er a mali�nant trans�ormation� since se�eral �enetic alterations would be necessar� to make that possible. Ne�ertheless� the EGFR and COX-2 o�erexpression pre�iousl� demonstrated (27) in these samples� ma� enhance the carcino�enic process in those �eneti-call� susceptible cases. There�ore� these molecular alterations can �i�e us �aluable in�ormation about intracellular pathwa�s altered in OL� subt�pes like OLP and OLL. So� with all the pre�ious back�round� we can propose two possible scenarios: the first that HC subtypes lesions with EGFR o�erexpression and increased Ct expression pattern ma� ha�e a di��erent biolo�ical be-ha�ior �rom those HT subt�pes lesions� which mi�ht be e457 possibl� associated with a hi�her mali�nant potential; the second scenario� ma� be that these di��erences in the EGFR expression pattern� could be the result o� di�ferent molecular profiles involved directly or indirectly in EGFR immunoexpression in OLP and OLL� which is probabl� determined b� etiolo�ical �actors o� each process and ma� not be related with mali�nant potential. Howe�er� re�ardless the pro�nostic implication o� these immunohistochemical di��erences� we ha�e demonstrated that althou�h the clinical and histopatholo�ical �eatures are �er� similar in all OL� processes� there are molecular di��erences in each OL� subt�pe. We did not observe significant association between EGFR expression and an� particular histolo�ical �ea-ture� except �or keratinization t�pe� since more than 50% of the cases with EGFR overexpression showed parakeratosis. This finding has not been reported previ-ousl� b� other authors (20�21). We do not know exactl� the meaning of this finding, or even if it has clinical rele�ance. Howe�er� considerin� that parakeratosis is a common finding in OLD samples it is very difficult to e�aluate this association. Ne�ertheless� what we do consider rele�ant is the lack o� association o� EGFR o�erexpression with the presence and/or intensit� o� some particular features from the epithelial lining and inflammatory infiltrate. Inflammatory intensity and epithelial thickness are some o� the �eatures classicall� associated with the acti�it� o� OL� lesions. We belie�e that these findings may have relevance in those cases with mild histolo�ical acti�it� at dia�nosis and then de�elop ma-li�nant trans�ormation in a short period o� time. We can conclude that there is a hi�h EGFR o�erexpression in OL� subt�pes� which increase their susceptibil-it� to the EGFR stimulation e��ects like cell proli�era-tion� di��erentiation� apoptosis inhibition� an�io�enesis� mi�ration and cellular in�asion. Neither the clinical t�pe nor particular histolo�ical �eatures were associated with EGFR overexpression. Cases considered as "his-tolo�icall� compatible" (HC) or OLL show a �reater c�toplasmic EGFR expression� su��estin� biolo�ical di��erences with HT cases. The lon� term �ollow up o� these OL� patients will �i�e us more consistent in�ormation about the significance of EGFR expression and mali�nant potential in these processes. Howe�er� �urther studies are needed to ascertain the exact pro�nostic �alue o� EGFR expression in di��erent t�pes o� OPML like OLP and OLL.