Rosario Rivera Buery et al.: Evaluation of Oral Melanotic Lesions Journal of Hard Tissue Biology 19[1] (2010) p57-64 © 2010 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Online ISSIN 1888-828X Original Clinico-pathological Evaluation of Oral Melanotic Macule, Oral Pigmented and Oral Mucosal

Rosario Rivera Buery1) ,Chong Huat Siar2), Naoki Katase3), Masae Fujii 3), Han Liu3), Midori Kubota3), Ryo Tamamura3), Hidetsugu Tsujigiwa3) and Hitoshi Nagatsuka3)

1)College of Dentistry, University of the East, Manila, Philippines. 2)Department of Oral , Oral Medicine and Periodontology, Faculty of Dentistry, University of Malaya, Malaysia. 3)Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama City, Japan. (Accepted for publication, February 8, 2010)

Abstract: Focal pigmented melanocytic lesions rarely occur in the oral cavity but should not be taken for granted for they may represent markers or risks for oral mucosal melanoma (OMM). The study investigated the clinical and pathological features of focal pigmented lesions of melanocytic origin focusing on oral melanotic macule (oral MM), oral pigmented nevus (OPN) and OMM. Immunohistochemistry was employed with S100, HMB-45, Melan A, c-kit and Ki-67. Oral MM mostly occurred on the gingiva and buccal mucosa while OPN mostly occurred on the buccal mucosa. OMM occurred on the palate and gingiva. A female gender predilection was observed in oral MM. Most of the benign lesions were less than 6 mm in diameter while OMM had greater than 10 mm in diameter. Benign lesions occurred in almost the same location as that of OMM. S100 and c-kit were detected in most benign cases while HMB-45 and Melan A were focally detected in some cases. S100, HMB-45 and Melan A expressions were detected in all cases of OMM. Ki-67 was only detected at the epithelial basal layer in oral MM and was completely negative in nevus cells. In OMM, Ki-67 was more than 80%. In conclusion, oral MM and OPN may not be markers of risk for OMM but excisional is highly recommended for clinically undefined lesions greater than 6 mm in diameter. The combination of S100, c-kit, HMB-45 and Melan A can be utilized to support the diagnosis of OMM.

Keywords: Oral melanotic macule, Oral pigmented nevus, Oral mucosal melanoma, Immunohistochemistry, Melanoma markers

Introduction tumor so-called oral malignant melanoma (OMM). This justifies Pigmented lesions in the oral cavity may be classified as that melanocytic lesions should not be ignored and a definite melanotic and non-melanotic lesions 1). One factor causing oral diagnosis must be established. pigmentation is over production of melanin pigments. Melanocytes In contrast to cutaneous melanoma, predisposing factors and may be found in the but may not be noticeable because precursor lesions have not been thoroughly clarified in OMM. of their low level of pigment production. However, when they are Precise distinction of a pigmented melanocytic lesion is essential active in pigment production or proliferation, they may be as it may represent a benign stimulant, a potential precursor or as responsible for several oral pigmentations ranging from focal to a marker of risk for OMM. Consequently, focal pigmentations diffuse and physiologic to malignant neoplasm 1). Diffuse such as oral melanotic macule (oral MM), oral pigmented nevus pigmentations are mostly systemic-related, drug-induced or caused (OPN) and OMM were investigated. Oral MM is a benign by exogenous pigments while most focal pigmentations are pigmented lesion caused by increased melanin production without brought about by over production of melanin. Although most increased melanocyte proliferation. They are most commonly seen pigmented lesions are benign, it is necessary to distinguish the on the gingiva, buccal mucosa and palate. Those occurring on the origin of pigmentation for melanocytic lesion may be a malignant lower lip are simply called melanotic macule 2, 3). OPN is a very rare occasion in the oral cavity characterized by the accumulation Correspondence to: Hitoshi Nagatsuka, DDS, PhD, Department of Oral of nevus cells at the junctional epithelium or in the submucosa or Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University 2-5-1 Shikata-cho, a combination of both 4). The most common of which are found in Okayama City, Japan 700-8525, Tel No: +81-86-235-6650, Fax No: +81- the buccal mucosa with nevus cells accumulating in the submucosa 86-235-6654, Email: [email protected] 57 J.Hard Tissue Biology Vol. 19(1):57-64, 2010 5). OMM is a malignant pigmented melanocytic tumor of the oral Table 1. Oral melanotic macule cavity characterized by tremendous proliferation of tumor cells in the epithelium or in the connective tissues or a combination of Case Age/gender Location Size (mm) both. OMM has been regarded as a separate entity and different 1 50/F BM Not specified from its cutaneous counterpart in many aspects and for reason 2 14/F Lower lip 3* that it is not related to ultra-violet exposure 6 - 9). 3 30/F Gingiva Not specified Melanocytic lesions are renowned as the most difficult 4 67/F BM Not specified diagnostic fields in histopathology 10). For instance, melanoma 5 32/F Gingiva Not specified may have varying morphological features. Particularly, non- 6 70/F Gingiva 5x15 cutaneous primary melanoma as well as metastatic 7 51/F Soft palate Not specified may resemble other neoplasms ranging from carcinoma, sarcoma, 8 64/M Gingiva 5x5 plasmacytoma, lymphoma and germ cell tumors 11). Another 9 62/M BM Not specified challenging feature might be a case of 10 74/M BM Not specified having epithelioid shape cells. Moreover, atypical melanocytic 11 47/F Lower lip 4* proliferation may occur in the oral mucosa, having equivocal 12 46/F Palate 5x7 features but not sufficient to categorize as melanoma in situ 6). 13 63/F Gingiva 3* Nevertheless, atypical melanocytic proliferation may suggest the 14 63/F BM Not specified earliest stage of OMM progression 12, 13). 15 43/M Gingiva 7* Although H and E remains the basis of diagnosis, the advent of *Greatest diameter immunohistochemistry(IHC) has greatly expanded the histological analysis. Melanoma differentiation markers are used to distinguish pigmented nodular type is an amelanotic lesion without radial poorly differentiated melanomas from non-melanocytic tumors. growth phase. The histological classification of OMM had been Among the panel of markers, S100, HMB-45, Melan A are mainly previously described 12). used to establish the diagnosis of melanoma 14 - 19). Nevertheless, variations in the expressions of these molecules in different types Immunohistochemistry of melanomas have been reported 9). This study aimed to determine New sections of 3µ were cut for IHC. For comparative purposes, the clinical and pathological features of focal pigmented sections of neuroma, neurofibroma and schwannoma were also melanocytic lesions occurring in the oral cavity as well as the included in the study. Sections were deparaffinized in series of expressions of different melanocytic markers in these lesions. No xylene and dehydrated in series of alcohol. Sections were blocked sign of malignant feature both clinically or histologically, was for endogenous peroxidase activity using 3% hydrogen peroxide observed in oral MM and OPN, nonetheless, it is recommended in methanol for 30 min and washed with Tris-buffered saline that focal pigmentation with a diameter of greater than 6 mm on solution. IHC protocols were optimized for each antibody. Antigen preferred sites for OMM be subjected to biopsy. The combination retrieval for S100 and HMB-45 was done by immersing the slides of S100, HMB-45 and Melan A is a reliable aid in the diagnosis in pre-warmed (37oC) 0.1% trypsin for 15 min. For Melan, A, c- of OMM. kit and Ki-67, slides were immersed in citrate buffer, pH 6.0 (heat pressure for 5 min at 121°C). The slides were covered with serum- Materials and methods free protein block (DakoCytomation, Carpinteria, USA) for 15 Case selection from paraffin-embedded tissues min, followed by primary antibodies with the following dilutions: Cases were obtained from the archive of the Department of Oral HMB-45, c-kit and Ki-67 with 1:100, Melan A with 1:50 and Pathology and Medicine, Okayama University conforming to the S100 is ready to use. Slides were incubated overnight at 4°C. ethics imposed by the university hospital. H and E sections were Immunoreaction was carried out with Envision™ Detection thoroughly reviewed, representative paraffin blocks were chosen Reagent Peroxidase (DakoCytomation) and detected with 3- and new sections for H and E staining were made. Cases selected amino-ethylcarbazole substrate chromogen (DakoCytomation). for the study were limited to those diagnosed as oral MM, OPN and OMM. Clinical findings of OMM were classified based on Results Tanaka et al’s report 20). Briefly, pigmented macular type is a flat, Oral MM pigmented lesion with radial growth phase, pigmented nodular All fifteen cases were observed in patients over 30 years of age type is a pigmented tumor with vertical growth phase and rarely except for one case, which was observed in a 14-year old patient. radial growth phase, pigmented mixed type is characterized by There seemed to be a female gender predilection observed in 11/ the presence of radial growth phase surrounding the tumor, non- 15 cases (Table 1). Most cases were located on the gingiva (6/15)

58 Rosario Rivera Buery et al.: Evaluation of Oral Melanotic Lesions

Table 2. Oral pigmented nevus

Case Age/gender Location Size (mm) Histological type 1 69/M BM Not specified Subepithelial 2 30/F Palate 2x4 Subepithelial 3 36/M BM 4* Subepithelial 4 38/M BM 4* Subepithelial 5 22/F BM 2-3* Subepithelial 6 32/F BM 5* Subepithelial *Greatest diameter

Table 3. Oral mucosal melanoma

Case A/G Location Size (mm) Clinical findings Histological type Thickness (mm) 1 66/F Palate 25x22 Pigmented macular In situ NA 2 67/F Palate 30x15 Pigmented mixed Invasive with in situ 2 3 75/M Palate 10x25 Pigmented mixed Invasive with in situ 8 4 73/M Gingiva 25x22 Non-pigmented nodular Invasive 1.2 5 68/M Palate/gingiva 25x15 Pigmented nodular Invasive Not specified 6 79/F Palate/gingiva 25x15 Non-pigmented mixed Invasive with in situ 5 7 58/M Palate 20x13 Pigmented mixed Invasive with in situ 10 8 61/F Palate/gingiva 30x20 Pigmented mixed Invasive with in situ 7 9 29/M Gingiva NS Pigmented nodular Invasive >4 10 51/M Gingiva NS Pigmented mixed Invasive with in situ >4 11 24/F Gingiva NS Pigmented nodular Invasive >4 12 50/F Gingiva NS Pigmented nodular Invasive Not specified 13 43/F Gingiva 30x30 Pigmented mixed Invasive with in situ >4 14 59/F Palate NS Pigmented nodular Invasive Not specified 15 82/M BM/palate 20x20 Pigmented nodular Invasive >4 16 71/F Gingiva 30x40 Non-pigmented nodular Invasive Not specified 17 30/M Palate NS Pigmented nodular Invasive Not specified 18 56/M Palate 30* Pigmented mixed Invasive with in situ >4 19 64/F Gingiva NS Pigmented mixed Invasive with in situ Not specified *Greatest diameter NS - not specified Case 9 - region from 34 to 41 Case 10 - mandibular premolar to molar region Case 11 - region from 36 to 46 Case 12 - region from 23 to 25 Case 14 - region from 14 to 18 Case 17 - region from 11 to 24

and buccal mucosa (5/15) followed by palate and lower lip (2/15 Histologically, melanocytes were dendritic in shape and they each). All cases were also asymptomatic. Other histories like past were limited at the basal layer of the epithelium (Fig. 1a). Some medical, social and family histories were not significant. melanocytes contained remnants of melanin pigments they Unfortunately, the actual clinical size in eight cases was not produced. Some melanin pigments were seen scattered in the specified. However, for others, the greatest diameter ranged from underlying connective tissues and some were phagocytosed by 3 to 15 mm with a mean diameter of 6.2 mm. tissue macrophages. No epithelial change or inflammatory reaction

59 J.Hard Tissue Biology Vol. 19(1):57-64, 2010

Table 4. Comparison of IHC results Specimen S100 HMB-45 Melan A C-kit Ki-67 Oral melanotic macule 15/15 9/15* 4/15* 14/15* basal cells only Oral melanotic nevus 6/6 2/6* 3/6* 5/6* 0% Oral mucosal melanoma 19/19 19/19 19/19 17/19 >80% Neuroma 3/3 0/3 0/3 3/3 Not done Neurofibroma 10/10 0/10 0/10 10/10* Not done Schwannoma 9/9 0/9 0/9 9/9* Not done *Focal staining

Fig. 1. Oral melanotic macule (oral MM). 1a shows the H and E section of oral MM. Fig. 1b revealed melanocytes and other dendritic cells at the basal layer. Fig. 1c shows HMB-45 intracellular reactivity most probably indicative of premelanosomes. Ki-67 immunoreactivity was limited at the basal cell layer (Fig. 1d).

was detected. Mitosis and atypia were not observed either. In all from 2 to 5 mm in their greatest diameter. All cases had a flat cases, melanocytes intensely expressed S100 (Fig. 1b). In some appearance except for one, which was nodular (case 3). cases, HMB-45 (Fig. 1c) was focally detected intracellularly. C- Histologically, nevus cells were localized at the subepithelial kit expression was detected in almost all cases. Ki-67 expression surface. Nevus cells were round in shape and were fully pigmented was limited at the basal cell layer (Fig. 1d). Table 4 shows the without any dysplastic change or sign of mitosis (Fig. 2a). The summary of the IHC study. nevus cells also formed nests or clusters. No inflammatory reaction was observed in all areas. Nevus cells were intensely positive to OPN S100. In some cases, focal reactivity to HMB-45 (Fig. 2b), Melan OPN were observed in patients from 22 to 69 years old. No gender A and c-kit (Fig. 2c) was observed. Similar to oral MM, almost predilection was noted. All cases occurred on the buccal mucosa all cases were positive to c-kit. Incidentally, nevus cells were except for one, which was located on the palate. The size ranged completely negative to Ki-67 (Table 4). 60 Rosario Rivera Buery et al.: Evaluation of Oral Melanotic Lesions

Fig. 2. Oral pigmented nevus (OPN). 2a shows the H and E section of OPN. Nevus cells are round in shape, heavily pigmented and arrange in clusters. HMB-45 reactivity was observed intracellularly in some nevus cells (2b arrows) as well as c-kit reactivity (2c, arrows).

Fig. 3. Oral malignant melanoma (OMM). Junctional activity is a prominent finding in the in situ component of OMM (3a). In invasive component, multinucleated, bizarre-looking cells may also be found in some OMM cases (3b). HMB-45 (3c) and Melan A (3d) reactivities in OMM. Both proteins were positively detected in all cases having dusty appearance inside the tumor cells.

61 J.Hard Tissue Biology Vol. 19(1):57-64, 2010 OMM clinical and pathological examination must be accomplished in All cases occurred either on the palate or gingiva except for order to avert the progress of a malignant tumor in its early stage. one, which occurred both on the buccal mucosa and gingiva. The In this study, oral MM mostly occurred on gingiva and buccal age ranged from 24 to 82 years with an average of 58.2 years. No mucosa. OPN mostly occurred on the buccal mucosa similar to gender predilection was observed. The size ranged from 10 to 40 previous reports though recently OPN was found out to be more mm in their greatest diameter. In cases where actual size was not common on the palate 3, 21). Both benign lesions were mostly less specified, affected regions were mentioned. Macroscopic findings than 6 mm in diameter. On the other hand OMM had more than 6 revealed that most cases were of pigmented mixed and pigmented mm in diameter and mostly were located on the most common nodular types. There were three cases of non-pigmented nodular regions reported for OMM 6, 8, 22, 23). The clinical findings concurred type and one case of pigmented macular type. to previous reports that most benign melanocytic lesions are less Histologically, cases were mostly classified as invasive and than 6 mm in diameter in contrast to OMM, which is larger than invasive with in situ component and only one case was purely in the benign lesions. However, most of these lesions whether benign situ. Junctional activity was a prominent finding in the in situ or malignant may be found on the gingiva and palate suggesting component (Fig. 3a). The underlying connective tissues were the need for meticulous clinical examination of focal pigmented abundant in inflammatory cells and some even invaded the lesions affecting these regions. It is therefore suggested that focal epithelium. In cases of purely invasive OMM, no epithelial pigmented lesion having greater than 6 mm in diameter located component was observed or a thin epithelium devoid of tumor on these preferred sites be subjected to histo-pathological analysis. cells lined the connective tissues. Tumor cells in the connective This is the only way to rule out OMM. tissues formed architectures ranging from nodular nests to sheet- Histologically, oral MM and OPN did not show any sign of like nests 12). The tumor cells varied in shape ranging from spindle, malignancy or transformation to malignancy. Routine histological epithelioid, bizarre and multinucleated tumor cells (Fig. 3b). The H and E was rather sufficient in establishing the diagnosis except nucleus had irregular chromatin pattern and some with deep for one case of OPN whose diagnosis was quite challenging (case eosinophilic nucleolus. Tumor cells located in deep areas and those 6) for reason that initial sections did not clearly reveal melanin that were invading the bone tissues were less pigmented or not pigments. OMM is more difficult to diagnose compared to other pigmented at all. Atypical mitoses were very commonly tumors in the oral cavity for it may highly resemble other tumors encountered. Ten cases had greater than 4 mm thickness. For cases and may present in a non-pigmented state. For the studied cases, whose thickness was not specified, loss of follow-up and/or patient the in situ component with its junctional activity clearly depicted refused further treatment after initial biopsy and so precise the diagnosis of OMM. However, as mentioned earlier, diagnosis thickness was not determined. All cases were positive to S100, of non-pigmented nodular type was rather formidable and IHC HMB-45 and Melan A regardless melanotic or not (Table 4). S100 examination supplemented the diagnosis. expressioon was very intense while HMB-45 (Fig. 3c) and Melan S100, HMB-45 and Melan A are the most commonly employed A (Fig. 3d) appeared like dusty intracellular stainings. No markers in the diagnosis of melanoma 14 - 19). S100 is expressed by significant variation between HMB-45 and Melan A expressions normal melanocytes and nevus cells and approximately 98% of was observed although some cases revealed a stronger expression melanomas 24 - 25). This study disclosed an equally impressive of HMB-45 and others to Melan A. Almost all cases expressed c- expression of S100 in benign and malignant melanocytic lesions kit. Specific c-kit reaction of these cases had been previously as well as those of the benign neuronal tumors. This further reported except for case 1 13). Ki-67 index was more than 80% in affirmed that S100 is a highly sensitive but less specific marker most cases but false negative reaction was obtained in 2 cases for OMM. (cases 2 and 12). HMB-45 (homatropine methyl bromide) identifies gp100, a For comparative purposes, sections of neuroma, neurofibroma premelanosomal protein 16). It may be expressed in reactive and schwannoma were also subjected to the same panel of melanocytes and is usually negative in resting melanocytes and antibodies except to Ki-67. Tumor cells in these cases were 100% nevus cells 26). The protein is organelle specific rather than lineage positive to S100 but completely negative to HMB-45 and Melan specific. HMB-45 is a fairly specific and reliable marker for A. Focal c-kit staining was observed in all cases (Table 4). melanoma expressed in 90% to 100% in different studies. It is more often expressed in epithelioid melanomas and very rare or Discussion never in spindled or desmoplastic types 11, 27). Melan A is the Pigmented lesion of melanocytic origin is a rare occurrence in product of MART-1 gene, is expressed in resting melanocytes the oral cavity, which justifies precise distinction to non- and nevus cells. It is also organelle specific and has essentially melanocytic lesions. As the exact etiology, precursor lesion and the same sensitivity and specificity as HMB-45 26). HMB-45 was pathogenesis of OMM have yet to be unraveled, meticulous more sensitive than Melan A in oral MM suggesting that HMB-

62 Rosario Rivera Buery et al.: Evaluation of Oral Melanotic Lesions 45 is expressed in actively producing-melanin cells. However, no and Technology (21592326 [c] and 20791337). The authors would significant variation was observed between HMB-45 and Melan like to thank Kazuko Funakoshi for her technical expertise. A expressions in OMM. The 100% reactivity of both antibodies highly suggests their usefulness in the aid of diagnosing OMM. References C-kit is a protein encoded by the KIT gene expressed by melanocytes as well as other cells of neuronal origin and 1. Regezi J, Sciubba J and Jordan R. Pigmented lesions. In: hematopoietic cells. In contrast to HMB-45 and Melan A, c-kit is Oral Pathology: Clinical Pathological Correlations, Saunders lineage specific. It is expressed by melanocyte, precursor cells Elsevier, Missouri, 2008, pp 127-137. and OMM cells 13, 28, 29). C-kit like S100 was the most sensitive 2. 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