Vol. 120 No. 2 August 2015

Paradental cyst is an inclusion cyst of the junctional/ sulcular epithelium of the gingiva: histopathologic and immunohistochemical confirmation for its pathogenesis Satoshi Maruyama, DDS, PhD,a Manabu Yamazaki, DDS, PhD,b Tatsuya Abé, DDS,c Hamzah Babkair, DDS,d Jun Cheng, MD, PhD,e and Takashi Saku, DDS, PhDf Niigata University Hospital and Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Objective. The aim of this study was to characterize the histologic and immunohistochemical profiles of paradental cyst- lining epithelia to clarify its histopathogenesis. Study Design. Ten surgical specimens of paradental cysts were examined for clinical profiles and to determine the histopathologic characteristics of the lining epithelia. Immunohistochemical profiles for keratin (K) subtypes, as well as for perlecan, UEA-I lectin binding, and proliferating cell nuclear antigen (PCNA), were determined and compared. Results. The paradental cyst was clinically characterized by its occurrence in young adults (mean age, 36.8 years; male, 42.8, female 27.8). Eight of the 10 cases arose in the retromolar area. The cyst wall was basically granulation tissue that was attached to the periodontal ligament space. Thin irregular anastomosing epithelial cords lined the cyst walls of immature granulation tissue with vascular dilation and hemorrhage. The intercellular space of the lining epithelia was widened with inflammatory cell infiltrates. Immunohistochemically, the lining was positive for K13, K14, K17, K19, UEA-I binding, and perlecan, suggesting its junctional/sulcular epithelial character. Conclusion. The results showed that the paradental cyst was lined by epithelial cells with characteristics of the junctional/ sulcular epithelium. The cyst can thus be considered as a kind of inclusion cyst arising in the periodontal pocket, most frequently of the mandibular third molars of young adults. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;120:227-237)

The disease entity of the paradental cyst, also known as inflammatory pathogenesis of the paradental cyst.4 the inflammatory paradental cyst, inflammatory collat- Even after reviewing 342 cases in the literature, eral cyst, and mandibular infected buccal cyst, was Philipsen et al. concluded that reduced enamel established as 1 of 2 types of inflammatory jaw cysts in epithelium, epithelial rests of Malassez, or remnants the second version of the World Health Organization of the dental lamina are the possible sources of cyst- (WHO) classification in 1992.1 The cyst has also been lining epithelia and that the histologic features are referred to as Craig’s cyst in acknowledgment of the indistinguishable from those of the inflammatory work of Craig, who examined a series of 49 cases radicular cyst.5 Thus, it remains unclear whether the and helped clarify the cyst’s clinicopathologic differential diagnosis of the paradental cyst is characteristics2; Craig’s findings were similar to those histopathologically possible or what the origin of the previously reported by Main, who had analyzed 8 lining epithelium of the paradental cyst is. cases.3 Subsequent reports have confirmed the Jaw cysts are one of the most common diseases encountered in routine oral pathology services. How- ever, their differential diagnoses at the histopathologic aLecturer, Oral Pathology Section, Department of Surgical Pathology, level are not always easy because the epithelial linings Niigata University Hospital. of the cyst wall are easily modified by inflammation, bAssistant Professor, Division of Oral Pathology, Niigata University even though the diagnosis of cystic jaw lesions is made Graduate School of Medical and Dental Sciences. c by considering clinical and radiographic information Resident, Oral Pathology Section, Department of Surgical Pathology, along with histopathologic specimens. Histopathologic Niigata University Hospital; Graduate Student, Division of Oral Pa- thology, Niigata University Graduate School of Medical and Dental differential diagnosis is especially important when Sciences. dGraduate Student, Division of Oral Pathology, Niigata University Graduate School of Medical and Dental Sciences. eAssociate Professor, Division of Oral Pathology, Niigata University Statement of Clinical Relevance Graduate School of Medical and Dental Sciences. fProfessor, Division of Oral Pathology, Niigata University Graduate This study proposes a possible histopathogenesis of School of Medical and Dental Sciences and Oral Pathology Section, the paradental cyst based on histopathologic and Department of Surgical Pathology, Niigata University Hospital. Received for publication Dec 12, 2014; returned for revision Mar 27, immunohistochemical investigations. The cyst is a 2015; accepted for publication Apr 1, 2015. kind of inclusion cyst arising in the periodontal Ó 2015 Elsevier Inc. All rights reserved. pocket, most frequently of the mandibular third 2212-4403/$ - see front matter molars of young adults. http://dx.doi.org/10.1016/j.oooo.2015.04.001

227 ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 228 Maruyama et al. August 2015 neoplastic lesions such as cystic ameloblastoma or specimens were then routinely processed and keratocystic odontogenic tumor (KCOT) are clinically embedded in paraffin. In addition, 20 biopsy samples suspected. from epulides, which contained junctional/sulcular To resolve this challenging issue, we have introduced epithelia in the inner surface, and 10 cases each of the several combinations of immunohistochemistry for other five major cystic jaw lesions such as unicystic epithelial linings in our diagnostic services, since we ameloblastoma, KCOT, dentigerous cysts, lateral have found that hematoxylin and eosin (H&E)-stained periodontal cyst, and radicular cyst were selected for sections occasionally do not work in the differential control experiments. Serial sections cut at 4 mm from diagnoses of inflamed cystic jaw lesions6-11 or of paraffin blocks were used for H&E and immunohis- odontogenic tumors with cystic changes.12-15 So far, we tochemical staining. The experimental protocol for have defined immunohistochemical profiles specificto analyzing surgical material was reviewed and the six most common cystic lesions: unicystic amelo- approved by the Ethical Board of the Niigata Uni- blastoma, KCOT, dentigerous cyst, lateral periodontal versity Graduate School of Medical and Dental Sci- cyst, radicular cyst,6,7 and nasopalatine duct cyst.11 ences (Oral Life Science). However, we have not included the paradental cyst as an object for differential diagnosis because we have Clinical findings considered that its occurrence was much rarer than In addition to clinical data (patient age and gender and that of the 6 cystic lesions mentioned above and cyst location) of paradental cyst cases that were docu- because the diagnosis of the paradental cyst is mented in patients’ clinical records, the relationship apparently easier due to its characteristic location. At with the affected tooth and the condition of associated the same time, however, we have occasionally teeth (vitality and periodontal disease) were carefully encountered paradental cyst cases showing some determined in every case by analyzing simple radio- characteristic histopathologic features that have not graphic and computed tomography (CT) images in been well documented in the literature. Furthermore, comparison with the documented clinical records and we have come to realize that the histopathogenesis of by referring to the conventional criteria for paradental paradental cysts still remains poorly understood. cyst.4,5 Thus, we wanted to apply our immunohistochemical method for better understanding the histopathogenesis of paradental cysts. Histopathology The purpose of this study was to analyze the Tissue sections from the 10 cases of paradental cyst clinicopathologic and histologic characteristics of 10 were reviewed histopathologically, and their charac- paradental cyst cases documented in our hospital to teristic features were analyzed in terms of lining determine the relationship between the cyst-lining epithelia, blood vasculature, and inflammatory cells epithelia and the gingival junctional/sulcular epithe- within the cyst walls. lium. This investigation was possible because we had recently determined the immunohistochemical profiles Antibodies of the junctional/sulcular epithelium.16 Mouse monoclonal antibodies against human keratin 10 (K10, DE-K10, immunoglobulin [Ig] G1), K13 (DE- K13, IgG ), K17 (E3, IgG ), K19 (RCK108, IgG ), MATERIALS AND METHODS 2a 2b 1 and proliferating cell nuclear antigen (PCNA) Patients and specimens (PC10, IgG ) were purchased from Dako (Glostrup, Surgical specimens of 10 cases of paradental cysts 2a Denmark). A monoclonal antibody against K14 (CK- were collected for the present study from the surgical B1, IgM) was obtained from Sigma Chemical Co., (St pathology files of the Division of Oral Pathology, Louis, MO). Rabbit polyclonal antibodies against the Niigata University Graduate School of Medical and core protein of perlecan were raised in rabbits, as Dental Sciences, during a 15-year period from 2000 to described elsewhere.17 2014. During the same period, a total of 2329 jaw cyst cases were documented. The 10 paradental cyst cases were typical cases, diagnosed on the basis of not only Immunohistochemistry histopathologic findings but also clinical and radio- Immunohistochemical staining was performed by using logic findings. The surgical samples were fixed in the ChemMate Envision system (Dako).7 For K13, K14, 10% formalin and decalcified with Planck-Rychlo’s and K19, sections were autoclaved in citric acid buffer solution, which contained 8.5% hydrochloric acid and (pH 6.0) at 121C for 10 minutes. For K17, sections 5% formic acid, for up to 48 hours when cyst walls were autoclaved in 0.01 M Tris buffer (pH 9.0) were surgically removed along with teeth. The containing 0.001 mol/L ethylenediaminetetra-acetic acid OOOO ORIGINAL ARTICLE Volume 120, Number 2 Maruyama et al. 229

Table I. Clinical summary of 10 cases of paradental cyst investigated in the present study Age (y) Associated Case # and sex Location Tooth site Symptoms tooth pulp Treatment 1. 17 M Mandible Second molar, distal No, bone resorption found Vital Extirpation (no third molar) during dental treatment for other teeth 2. 27 M Mandible Third molar, distal Repeated pain Vital Extirpation with tooth 3. 22 F Mandible Third molar, distal Acute pain Vital Extirpation with tooth 4. 28 F Mandible Third molar, distal Continuous pain Vital Extirpation with tooth 5. 29 F Mandible Third molar, distal Repeated gingival swelling Vital Extirpation with tooth 6. 32 F Mandible Third molar, distal Food impaction, pain Vital Extirpation 7. 47 M Mandible Third molar, distal No, found during dental Vital Extirpation with tooth treatment 8. 45 M Mandible First and second premolar, Periodontitis Vital Extirpation buccal 9. 56 M Maxilla Third molar, palatal No, found during dental Vital Extirpation with tooth treatment for multiple caries with periapical lesions and for periodontitis 10. 65 M Mandible Lateral incisor, distal Repeated pain, gingival Vital Extirpation swelling, periodontitis

(EDTA) at 121 C for 10 minutes. After the pretreat- RESULTS ment, the sections were rinsed in phosphate-buffered Age and gender saline (PBS) containing 0.5% milk protein (Morinaga The age and gender distribution in the 10 cases of Milk Industry Co. Ltd., Tokyo, Japan) and 0.05% paradental cysts are summarized in Table I. Those cases Triton X-100 (T-PBS) and treated with 0.3% accounted for 0.4% of the 2329 of jaw cysts hydrogen peroxide in methanol for 30 minutes at documented in the file during the same period. They room temperature to block endogenous peroxidase were ranked sixth among jaw cysts, of which activities. After rinsing in T-PBS, the sections were radicular cysts were the most common (1136 cases, incubated with 5% milk protein in T-PBS for 1 hour 48.8%), followed by dentigerous cysts (328, 14.1%), at room temperature to block nonspecific protein- KCOTs (159, 6.8%), nasopalatine duct cysts (61, binding sites. They were then incubated overnight at 2.6%), and lateral periodontal cysts (29, 1.2%). The 4C with the primary antibodies diluted in T-PBS. mean age of the patients was 36.8 years (42.8, male; After incubation, the sections were rinsed in T-PBS 27.8, female) with ages ranging from 17 to 65 years. and incubated with the secondary antibodies, which There were 6 male (60%) and 4 female (40%) were conjugated with peroxidase-labeled dextran patients. The data indicated that paradental cysts polymers, for 1 hour at room temperature. After occur in young adults but not in children, and with no rinsing with T-PBS, they were treated with 0.02% obvious gender predilection. 3,3’-diaminobenzimine in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.005% hydrogen peroxide to visualize the reaction products. Finally, the sections were Tooth association counterstained with hematoxylin. For control studies Of the 10 paradental cysts, 8 (80%) were associated on antibodies, the primary antibodies were replaced with the third molars or with second molars which with preimmune mouse IgG subclasses (Dako). For lacked a distally neighboring third molar; 9 (90%) were Ulex Europaeus agglutinin I lectin (UEA-I) binding mandibular, and 1 (10%) was maxillary. One of the 8 immunohistochemistry, sections were stained using cysts was located on the mesial side of the third molar, the lectin-antilectin method.6 UEA-I and rabbit poly- while the others were on the distal side (see Table I). clonal antibodies against UEA-I were obtained from Radiographically, they were identified as unilocular E-Y Laboratories Inc. (San Mateo, CA). The lectin radiolucencies next to tooth roots without any tooth concentration was 1 mg/mL. Staining results were root resorption. Most showed ovoid shapes, with evaluated as positive when staining intensities of swelling toward their distal ends and looked different epithelial cells were evident from those of connective from simple periodontal pockets (Figure 1, case #7). tissue areas and their positive areas occupied recog- Their longest diameters ranged from 8 to 18 mm, nizable extents of the epithelial zone. with a mean of 11 mm on panoramic radiographs. In ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 230 Maruyama et al. August 2015

Fig. 1. Radiographic views of a paradental cyst arising in the distal side of the mandibular third molar of a 45-year-old man (case #7). (A) Panoramic X-ray, (B) computed tomography (CT) image, (C) Three-dimensional (3D) reconstruction of CT images. The patient had no obvious periodontitis (A). A paradental cyst typically arose on the distal side of the mandibular third molar which was vital, although the third molar did not erupt completely but obliquely inclined against the distal side of the second molar (A, B). The reconstructed 3D image demonstrated that the occlusal surface of the third molar was exposed from the bone and that there was some bone resorption from the surface to deeper zones of the retromolar triangle area (C).

addition, younger patients (cases #1-7; ages 17-47 the background of inflammatory cell infiltrates years) did not suffer from periodontitis but from focal (Figures 2F and G). The congestive status of blood pericoronitis around their third molar teeth, whereas vascular channels seemed to result in hemorrhage at older patients (cases #8-10; ages 45-65 years) had surgery (Figure 2G). In 2 of the 10 specimens, some extensive periodontitis, which might have caused the connection was identified (Figure 2D, arrow) between deepening of the periodontal pockets. All of the teeth the cyst linings (Figure 2D, open asterisk) and the associated with the cysts were determined to be vital junctional/sulcular epithelia (Figure 2D, closed by electric pulp testing (see Table I). asterisk) forming the bottom of the periodontal pockets (Figure 2D, double circle), although there was no direct continuity between the periodontal Histopathology pockets and the cystic lumina. The paradental cysts were located along tooth roots The lining epithelia in anastomosing cords were char- within the range from the enamel-cementum junction acterized by their wide intercellular space (Figure 3A). (case #4, Figure 2A; rectangle area indicated by dashed Occasionally, microcystic changes with eosinophilic line, a high-resolution version of this slide for use with (Figure 3B, open asterisk)orpale-coloredfluid contents the Virtual Microscope is available as eSlide: (Figure 3B, arrows) as well as round-shaped hyaline VM00565) to the apical ends (case #2, Figure 2B; bodies (Figure 3B, closed asterisk) were observed within rectangle, eSlide: VM00579). The cyst wall was the lining epithelia. Blood capillaries and venules were composed of granulation tissue, measuring 1 to 3 mm prominently dilated with red blood cell (RBC) retention in thickness on H&E-stained sections. In case #3, the between the anastomosing epithelial cords (Figure 3C). cyst wall granulation tissue extended to the Those RBCs tended to coagulate with each other, to bifurcation zone of tooth roots (Figure 2C; rectangle, be hemolyzed within the vascular channels, or to eSlide: VM00572). The cystic lumen was not smooth be extravasated over the epithelial compartment surfaced but was indented, with immature granulation (Figure 3D). However, there were no obvious tissue protrusions (Figures 2D to 2G). The cyst wall hemosideroses in the tissue specimens investigated, granulation tissue showed edematous changes, but which indicated that the hemorrhagic changes were there was no definite layering of granulation tissues in related to surgical intervention. the different organizing processes, which is In 2 cases, the round-shaped hyaline bodies were characteristic of the walls of radicular cysts, the other observed in close association with deeply eosinophilic major type of inflammatory cysts. The inner surface epithelial cells (Figure 3E). Some of those deeply of the cystic lumina was lined by extensively stained cells contained pyknotic or fragmented nuclei anastomosed cords of stratified squamous epithelial or even lacked nuclei, and they were occasionally cells without keratinizing tendencies (Figures 2Eto indistinguishable from round-shaped eosinophilic 2G). The epithelial layer was overlapped by the hyaline bodies, some of which were composed of immature granulation tissue layer, in which vascular granular materials or contained calcified centers channels were prominently dilated with congestion in (Figure 3F). OOOO ORIGINAL ARTICLE Volume 120, Number 2 Maruyama et al. 231

Fig. 2. Histopathologic variations of paradental cysts arising in the periodontal space of mandibular third molars. (A) Low power view of case #4, rectangle area indicated by dashed lines (a high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM00565). (B) Low power view of case #2, rectangle area (a high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM00579). (C) Low power view of case #3, rectangle area (a high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM00572). (D, E) High-power view of the cyst wall (A), case #4.; (F) High-power view of (B), case #2. (G) High-power view of (C), case #3; hematoxylin and eosin (H&E) stain; A- C, 8, D-G, 25. Paradental cysts are located along tooth roots from the enamel-cementum junction (A) to the lower half of the roots (B). When tooth roots were bifurcated, granulation tissue, which consisted of cyst walls, extended to the periodontal space underneath the bifurcation (C). The inner surface of the cyst wall appeared basophilic due to dense infiltration of inflammatory cells and lining epithelial layers (A-C). D, A transitional zone (arrow) between cyst lining epithelium (✩open asterisk, cystic lumen) and the junctional/sulcular epithelium (+closed asterisk) at the bottom of the periodontal pockets (0double circle). Cordlike anastomosing of lining epithelial cells intervened by immature granulation tissue with dense infiltration of chronic inflammatory cells and vascular dilation (E). In areas with severe inflammatory infiltrates, lining epithelia disappeared (F). Hemorrhagic ten- dencies were prominent, and the cystic lumen occasionally pooled bloody contents (G).

Immunohistochemistry for lining epithelia found in the surface part, but their appearance was not The 10 cases showed basically the same staining pro- stable (not shown). There were no positive reactions files. Among the keratin subtypes, K13 (Figure 4A), for the other K subtypes (not shown). Perlecan was K14 (Figure 4B), and K19 (Figure 4C) were characteristically localized mainly on the cell border, demonstrated in almost the whole layer of the showing vesicular patterns in nearly the whole linings. K17-positive (þ) cells were occasionally epithelial layer (Figure 4D). UEA-I binding was ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 232 Maruyama et al. August 2015

Fig. 3. Histopathology of the cyst wall of paradental cyst. Higher power views of Figure 2E (A) and Figure 2F (B-D); hyaline bodies in lining epithelia (E-F). H&E stain, (A, B) 40, (C-E) 160, (F) 300. In immature granulation tissue separated by the anastomosed cords of lining epithelia, blood capillaries showed marked congestion with vascular dilation. The intercellular space was characteristically widened in the epithelial compartment (A), which occasionally showed microcystic changes due to retention of tissue fluid, bloody contents, or some foreign body-derived materials (B, arrows). Foreign bodies with macrophage reactions were also found in the epithelial compartment (B, +closed asterisk) or in the cystic lumen (B, ✩open asterisk). At higher magnification, red cells congested within the blood capillary space were coagulated or hemolyzed (C). Extravasated red cells were coagulated within the epithelial compartments or in their intercellular spaces (D). Eosinophilic, round-shaped mate- rials were occasionally formed within the epithelial compartment (E). They were composed of granular materials or circumscribed with condensed rims; some contained nuclear traces or calcified materials in the center (F).

mainly shown on the cell border of the upper half of epithelia of the dentigerous cyst, lateral periodontal the epithelial layer (Figure 4E). PCNAþ cells were cyst, and radicular cyst (data not shown), while it scarcely observed (Figure 4F). We also investigated the was not obviously demonstrated in unicystic immunohistochemical profiles for K14 in the ameloblastoma and KCOT (data not shown). The dentigerous cyst, which is included in the differential immunohistochemical profiles of paradental cyst diagnosis with the paradental cyst, unicystic linings are summarized in Table II, where the profiles ameloblastoma, KCOT, lateral periodontal cyst, and of the linings of the other 5 major jaw cysts are compared. radicular cyst, whose immunohistochemical modes for Hyaline bodies within the anastomosed epithelial K10, K13, K17, K19, UEA-I binding, perlecan, and processes (see Figures 3E and 3F) were PCNA have been documented.7 As a result, K14 was immunohistochemically positive for K13 (Figure 5A), sporadically positive in the surface layer of the lining K14 (Figure 5B), K19 (Figure 5C), and faintly for OOOO ORIGINAL ARTICLE Volume 120, Number 2 Maruyama et al. 233

Fig. 4. Immunohistochemistry of lining epithelia of paradental cyst. Immunoperoxidase stain for keratin (K) 13 (A), K14 (B), K19 (C), perlecan (D), Ulex Europaeus agglutinin I lectin (UEA-I) binding (E), and proliferating cell nuclear antigen (PCNA) (F), hematoxylin counterstain, (A-C) 60, (D-F) 150. Anastomosing cords of the lining epithelia showed positive reactions for K13 (A), K14 (B), and K19 (C), although their staining intensities and extents differed by case. At higher magnification, the cell border of the lining epithelial cells were positive for perlecan (D) and UEA-I binding (E). There was no definite PCNA labeling among the epithelial cells (F).

Table II. Immunohistochemical profiles of lining epithelia compared five major jaw cysts with the paradental cyst Lateral Unicystic Keratocystic Dentigerous periodontal Radicular Paradental Junctional Antigens ameloblastoma* odontogenic tumor* cyst* cyst* cyst* cyst epithelium Keratin 10 þþ Keratin 13 þþþþþþþ Keratin 14 /z /z y y y þþ Keratin 17 þþþþy y Keratin 19 þþþþþþþ UEA-I þþþþþþ Perlecan þþþþþ PCNA þþþ UEA-I, Ulex Europaeus agglutinin I lectin; PCNA, proliferating cell nuclear antigen. *Modified from reference 7. yonly surface. zonly basal.

K17 (Figure 5D). The results indicated that the (Figure 6D). K14 (Figure 6E), and K19 (Figure 6F) round-shaped hyaline bodies were derived from the were more extensively localized in the epithelial lining epithelial cells. There were occasionally eosino- layer, although their extents varied from case to case. philic conglomerates of round-shaped calcified mate- In cases with severe inflammation, K17 tended to be rials within fibrous tissues, which did not look like positive only in the surface zone (Figure 6G). epithelial components (Figure 5E). However, immunohistochemically, those round-shaped materials DISCUSSION were positive for keratin subtypes (K13; Figure 5F). The present study reconfirmed the clinical characteris- Junctional/sulcular epithelia covering the inner tics of the paradental cyst: it mostly arises in the peri- surface of epulis samples showed irregular-shaped rete odontal space of the mandibular third molar of young ridges that were anastomosed with each other in the adults. Histopathologically, the cyst wall is granulation background of acute and chronic inflammation. tissue, and its inner surface is lined by an irregular- Compared with the outer surface epithelia, their shaped squamous epithelial layer, which indicates its intercellular spaces were more enhanced (Figure 6A). inflammatory origin. Based on its immunohistochem- Immunohistochemically, those cell borders were ical profiles, we were also able to demonstrate that the positively labeled for perlecan (Figure 6B) as well as nature of the lining epithelium resembles that of the for UEA-I (Figure 6C), and junctional/sulcular junctional/sulcular epithelium of the gingiva. Thus, our epithelial cells in the upper zone were positive for K13 present definition of the paradental cyst is that of an ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 234 Maruyama et al. August 2015

Fig. 5. Immunohistochemistry of hyaline bodies in lining epithelia and calcified bodies in cyst walls of paradental cyst (serial sections of Figure 3E). Immunoperoxidase stains for keratin (K)13 (A, F), K14 (B), K19 (C), and K17 (D), hematoxylin counterstain, and (E) H&E stain. 180. Eosinophilic round-shaped materials within the epithelial compartment (Figure 3E) were immunohistochemically positive for K13 (A), K14 (B), K19 (C) together with surrounding epithelial cells. K17 was faintly positive in some of them (D). Within the connective tissue compartment of the cyst wall, there were conglomerates of round-shaped calcified materials (E), which were immunohistochemically positive for K13 (F), indicating that they were derived from lining epithelial cells. inflammatory cyst generated by inclusion of the In addition to the histologic features already docu- gingival sulcular or junctional epithelia of adjacent vital mented, the present study adds the following charac- teeth. Paradental cysts most often arise in the distal teristic findings: (1) anastomosed epithelial cords of aspect of the third molar of younger adults between 20 the cyst lining, which have a wide intercellular space; and 30 years old but also arise around teeth affected by (2) vascular dilation with congestion or hemorrhage, periodontitis in older men. The cyst wall, attached to which seems to be generated by mechanical stress the upper part of tooth roots, is composed of fibrous during surgery; (3) retention of tissue fluid containing granulation tissue lined with nonkeratinous epithelium foreign bodies within the epithelial compartment with reticularly anastomosed rete ridges, between or along the inner surface of the cyst wall; and (4) which are dilated and congestive blood capillaries. association between the junctional epithelium and the Immunohistochemically, the lining epithelium shows cyst-lining epithelium. Surgical intervention was the same profile as the junctional/sulcular epithelium16: considered as a cause of hemorrhage because RBCs Both are positive for K13, K14, K17, K19, UEA-I were intact, although some were hemolytic and not binding, and perlecan. associated with hemosiderosis. The anastomosing OOOO ORIGINAL ARTICLE Volume 120, Number 2 Maruyama et al. 235

Fig. 6. Immunohistochemistry of junctional/sulcular epithelia of the gingiva (epulis). (A) H&E stain and immunoperoxidase stain for perlecan (B), UEA-I binding (C), K13 (D), K14 (E), K19 (F), and K17 (G), hematoxylin counterstain, (A) 110, (D-G) 140. Junctional/sulcular epithelia included in epulis samples were usually infiltrated with acute and chronic inflammatory cells (A). Epithelial cell borders were immunohistochemically labeled for perlecan (B) and UEA-I binding (C). The epithelial cells were positive for K13 (D), K14 (E), K19 (F), and faintly for K17 (G), although their staining intensities and extents varied from area to area.

epithelial cords may cause mechanical stress to blood cysts indicates that it is a reactive lesion as opposed to vessels between the cords, which may result in a cir- a neoplastic one. culatory disturbance with a tendency toward hemor- A summary of the immunohistochemical profiles of rhage. Their characteristic wide intercellular space lining epithelia of major jaw cysts7 is provided in must be one of the most important tissue architectural Table II. The pathogenesis of the paradental cyst, features of the junctional/sulcular epithelium, which including the origin of its lining, has remained allows the intercellular passage of leukocytes as well unclarified.4,5 However, we now consider it possible as tissue fluids, including gingival crevicular fluid to objectively evaluate the differentiation degrees of the (GCF).18 Such a characteristic tissue architecture lining epithelia with the aid of combined immunohis- should be an infrastructure of microcyst formation tochemistry in order to speculate on their origins.7,33,34 within the lining epithelium. The presence of foreign Judging from the immunohistochemical profiles bodies also suggests the continuity between cystic (Table II), the paradental cyst can be regarded as an lumina and periodontal pockets in the process of independent cyst entity in which the lining either paradental cyst formation. shows the junctional/sulcular-epithelial differentiation Immunohistochemically, the lining epithelium is or keeps the junctional/sulcular-epithelial character. positive for K13, K14, and K19, perlecan, and UEA-I The unstable and faint expression of K17 indicates binding. The results indicate its junctional/sulcular that the paradental cyst differs from pure odontogenic epithelium characteristics. The keratin expression lesions such as ameloblastoma or KCOT. Among profiles for the junctional/sulcular epithelium or the those jaw cyst types, K14 was not demonstrated in periodontal pocket epithelium have been documented the whole epithelial layer, but their stainings were in the literature.16,19-25 We have chosen K14 and K19 occasional and sporadic, which was different from as the junctional/sulcular epithelium markers in our those in the junctional/sulcular epithelium or the diagnostic services because these 2 keratin subtypes paradental cyst. The presence of perlecan indicates are most stably localized in the whole layer of the junctional/sulcular-epithelial characteristics, in which junctional epithelium in formalin-fixed surgical human cellular migration is allowed, as seen in the sulcular specimens.16,19 As to the inter-epithelial-cellular deposit or normal junctional epithelium.16 Those combined of perlecan, we have reported its significance as one of immunohistochemical profiles, summarized in the constituents of the intraepithelial stroma,8,12-16,26 Table II, indicate the resemblance between junctional/ which functions as a growth factor reservoir for cellular sulcular epithelia and paradental cyst linings. growth27-29 in such tissue circumstances without blood Philipsen et al. stated that histologic features are circulation as inside epithelial compartments.30-32 The indistinguishable from those of the inflammatory, peri- absence of UEA-I binding has been shown to be specific apical (radicular) cyst.5 However, it is now possible to to ameloblastoma,6 and thus its presence in paradental distinguish these 2 cysts by immunohistochemistry. ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 236 Maruyama et al. August 2015

They also listed 3 candidates for the origin of lining: The authors thank Dr. Yutaka Terao at Niigata Station Dental reduced enamel epithelium, cell rests of Malassez, and Clinic for providing them with radiographic imaging data. remnants of the dental lamina, which are stimulated by inflammation in the pathogenic processes of paradental cysts.5 Epithelial rests of Malassez have been REFERENCES considered to be the source for radicular cysts.1,4 1. Kramer IRH, Pindborg JJ, Shear M. Nasopalatine duct (incisive Because they are the only epithelial component within canal) cyst. In: Rich G, Lee KW, van der Waal I, eds. World Health Organization International Histological Classification of the periodontal space between the tooth root and the Tumours: Histological Typing of Odontogenic Tumours. 2nd ed. alveolar bone of erupted teeth, there is no doubt about the Berlin, Germany: Springer-Verlag; 1992:39. role of epithelial rests of Malassez in the histopatho- 2. Craig GT. The paradental cyst. A specificinflammatory odonto- genesis of radicular cysts. However, the Epithelial rests of genic cyst. Br Dent J. 1976;141:9-14. Malassez theory cannot explain why paradental cysts 3. Main DMG. Epithelial jaw cysts: a clinicopathological reap- praisal. Br J Oral Surg. 1970;8:114-125. arise mainly in the third molar region. 4. Shear M, Speight P. Nasopalatine duct (incisive canal) cyst. Cysts Based on the present results, the most probable of the Oral and Maxillofacial Regions. Oxford, U.K: Wiley- pathogenesis suggests periodontal pocket-related pro- Blackwell; 2007:143-149. cesses. Such pathogenic pathways have already been 5. Philipsen HP, Reichart PA, Ogawa I, Suei Y, Takata T. The in- fl proposed by several groups. We agree with the sug- ammatory paradental cyst: a critical review of 342 cases from a literature survey, including 17 new cases from the author’s files. gestion of Vedtofte and Praetorius that paradental cyst 35 J Oral Pathol Med. 2004;33:147-155. is initiated by pericoronitis. However, it is difficult to 6. Saku T, Shibata Y, Koyama Z, Cheng J, Okabe H, Yeh Y. Lectin accept their conceptual speculation on the lining histochemistry of cystic jaw lesions: an aid for differential diag- epithelial origin from epithelial rests of Malassez or nosis between cystic ameloblastoma and odontogenic cysts. reduced enamel epithelium35 as mentioned previously. J Oral Pathol Med. 1991;20:108-113. 7. Tsuneki M, Yamazaki M, Cheng J, Maruyama S, Kobayashi T, Although reduced enamel epithelium has been Saku T. Combined immunohistochemistry for the differential considered the origin of the paradental cyst by other diagnosis of cystic jaw lesions: its practical use in surgical pa- 2,36 groups, this seems unlikely because, in contrast thology. Histopathology. 2010;57:806-813. with dentigerous cysts, paradental cysts never arise in 8. Tsuneki M, Cheng J, Maruyama S, Ida-Yonemochi H, the tooth crown.1,4 The role of remnants of the dental Nakajima M, Saku T. Perlecan-rich epithelial linings as a back- ground of proliferative potentials of keratocystic odontogenic lamina in conditions occurring in adulthood seems to be tumor. J Oral Pathol Med. 2008;37:287-293. doubtful, and in terms of immunohistochemical pro- 9. Tsuneki M, Cheng J, Yamazaki M, et al. Lateral periodontal cyst: files, the paradental cyst differs markedly from the a clinicopathological study of 23 cases and an immunohisto- dentigerous cyst. chemical analysis of its characteristic epithelial plaques in the Terms such as “globulomaxillary cyst,”1 “branchial lining. Oral Med Pathol. 2008;12:89-96. ”37 “ ”11 10. Yamazaki M, Cheng J, Hao N, et al. Basement membrane-type cyst, and nasopalatine duct cyst have also been heparan sulfate proteoglycan (perlecan) and low-density lipo- widely applied for many years for cysts arising in protein (LDL) are co-localized in granulation tissues: a possible their specific locations, based on the assumption that pathogenesis of cholesterol granulomas in jaw cysts. J Oral such cysts result from fetal tissue remnants. However, Pathol Med. 2004;33:177-184. fl given the lack of solid scientific support for the 11. Tsuneki M, Maruyama S, Yamazaki M, et al. In ammatory his- topathogenesis of nasopalatine duct cyst: a clinicopathological histopathogenesis of such entities, we believe it is Oral Dis 1,11,37 study of 41 cases. . 2013;19:350-359. time to abandon these 3 terms. 12. Ida-Yonemochi H, Ikarashi T, Nagata M, Hoshina H, Takagi R, Colgan et al. concluded that food impaction at the site Saku T. The basement membrane-type heparan sulfate proteo- of erupting teeth was the major cause of paradental cysts glycan (perlecan) in ameloblastomas: its intercellular localization by showing foreign body reactions in their series.38 We in stellate reticulum-like foci and biosynthesis by tumor cells in culture. Virchows Arch. 2002;441:165-173. also found foreign bodies in the present study. The union 13. Ida-Yonemochi H, Ahsan MS, Saku T. Differential expression between the oral epithelium and the cyst lining profi les between a-dystroglycan and integrin b1 in amelo- 39,40 epithelium has also been reported, and we have blastoma: two possible perlecan signaling pathways for cellular demonstrated the continuity between the two in growth and differentiation. Histopathology. 2011;58:234-245. Figure 2D. The presence of foreign bodies also suggests 14. Tsuneki M, Maruyama S, Yamazaki M, Cheng J, Saku T. Podo- planin expression profiles characteristic of odontogenic tumor- the continuity between cystic lumina and periodontal specific tissue architectures. Pathol Res Pract. 2012;208:140-146. pockets during the process of paradental cyst 15. Abé T, Maruyama S, Yamazaki M, et al. Intramuscular keratocyst 41 formation, including tooth eruption steps. In other as a soft tissue counterpart of keratocystic odontogenic tumor: words, the cystic space may be completed by closure differential diagnosis by immunohistochemistry. Hum Pathol. of periodontal pockets, hence the paradental cyst is 2014;45:110-118. 16. Maruyama S, Itagaki M, Ida-Yonemochi H, et al. Perlecan- a kind of inclusion cyst of the junctional/sulcular enriched intercellular space of junctional epithelium provides epithelium, which is generated by a mechanism similar primary infrastructure for leucocyte migration through squamous to that for epidermal inclusion cyst of the skin. epithelial cells. Histochem Cell Biol. 2014;26:390-396. OOOO ORIGINAL ARTICLE Volume 120, Number 2 Maruyama et al. 237

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Alvarado CG, Maruyama S, Cheng J, et al. Nuclear translocation Chuo-ku of beta-catenin synchronized with loss of E-cadherin in oral Niigata 951-8514 epithelial dysplasia with a characteristic two-phase appearance. Japan Histopathology. 2011;59:283-291. [email protected]