Epidermal growth factor receptor expression in ameloblastoma

Oral Oncology 39 (2003) 138–143 www.elsevier.com/locate/oraloncology

Epidermal growth factor receptor expression in ameloblastoma Marilena Vereda, Izhar Shohatb, Amos Buchnera,* a

Department of Oral Pathology and Medicine, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel b Department of Oral and Maxillofacial Surgery, Chaim Sheba Medical Center, Tel Hashomer, Israel Received 30 May 2002; accepted 5 June 2002

Abstract Ameloblastoma is a locally aggressive tumor with possible lethal potential. Currently extensive surgery is the most acceptable treatment modality. Aim: To investigate the presence of EGFR in ameloblastoma in order to consider using newly developed antiEGFR therapeutic agents in cases of unresectable tumors. The study consisted of 58 formalin-fixed, paraffin-embedded specimens of ameloblastoma that were immunohistochemically stained with a monoclonal anti-EGFR antibody (clone 31G7). Positive and negative controls determined specificity of the antibody. A staining score based on the staining intensity and the proportion of stained cells was established, ranging between 0 and 2. All specimens were EGFR positive; 8 (14%) exhibited the maximum score of 2 and 19 (33%) scored between 1 and 2. Since ameloblastomas are EGFR-positive tumors, anti-EGFR agents could be considered to reduce the size of large tumors and to treat unresectable tumors that are in close proximity to vital structures. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ameloblastoma; EGFR; Anti-EGFR immunotherapy

1. Introduction Ameloblastoma, the most common epithelial odontogenic tumor of the jawbones [1], is divided into two biologic-microscopic subtypes: solid (multicystic) and unicystic [2,3]. The solid ameloblastoma, which is the most common subtype, is a locally aggressive tumor that may produce marked facial deformity and serious debilitation. Since it tends to infiltrate between cancellous bone trabeculae, the actual margin of the tumor extends beyond the radiographic margins and recurrence rates of 55–90% have been reported after curettage. Marginal resection past the radiographic borders of the tumor is the most widely accepted surgical treatment, yet it yields recurrence rates of up to 15% [2,3]. Solid ameloblastomas of the posterior maxilla are particularly dangerous because of their close proximity to vital structures and the difficulty of obtaining clean surgical margins. Ameloblastoma of the maxilla has been attributed as the cause of death in several cases [4]. The * Corresponding author. Tel.: +972-3-6407904; fax: +972-36409250. E-mail address: [email protected] (A. Buchner).

unicystic subtype of ameloblastoma is less aggressive and is usually treated as a cystic lesion, by enucleation or curettage. Recurrence rate of about 10% have been reported when this method was the primary form of treatment. Growth factors and their receptors play a key role in the growth of normal tissues and the development and progression of human neoplasms [5]. Epidermal growth factor (EGF), a peptide growth hormone widely spread in human tissues, regulates proliferation of both normal and neoplastic cells [5,6]. The actions of this factor are mediated by binding to the external domain of its specific transmembrane receptor. The binding of EGF to its receptor results in stimulation of tyrosine kinase, an integral part of the internal domain of the receptor [7]. Expression of epidermal growth factor receptor (EGFR) has been observed in normal epithelia, including the oral mucosa, and has also been detected in tissues of developing teeth. This suggests that EGF and its receptor participate in the differentiation of ameloblasts and in the control of their cellular functions [8,9]. Ameloblastoma, as a tumor originating from EGFRexpressing odontogenic epithelium, has not been fully investigated for the presence of EGFR. Only four

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studies on the immunohistochemical detection of EGFR are found in the English language literature [6,10–12]. These studies are inconsistent and contradicting as to the percentage of EGFR-positively stained cases, ranging from 0% [6] and up to about 100% [10–12]. Enhanced activity or overexpression of EGFR has been associated with tumor progression in many epithelial neoplasms from different locations, e.g. head and neck, gastrointestinal tract, lung, breast and brain [13,14]. Recently, therapeutic modalities against EGFR have been developed to down regulate EGFR in overexpressing neoplasms and therefore to lead to tumor regression [5]. The purpose of the present study was to investigate the presence of EGFR in a large series of ameloblastomas with improved immunohistochemical methods. If ameloblastomas indeed demonstrate the presence of EGFR, then immunotherapeutic agents against EGFR could be considered as an adjunctive therapy to reduce the size of large tumors and avoid mutilating surgical resection, as well as to treat tumors that are in close proximity to vital structures. Furthermore, immunotherapy could be used in cases of recurrence to avoid additional extensive surgical procedures.

2. Materials and methods 2.1. Study cases The study comprised of 58 specimens of ameloblastomas (41 solid, 15 unicystic, one desmoplastic and one peripheral), which were retrieved from the archives of the Department of Oral Pathology, School of Dental Medicine, Tel Aviv University. Of the specimens, 52 were in the mandible and 6 in the maxilla (Table 1). Selection was based on strict histological criteria according to the WHO classification [15] and the presence of sufficient tissue in the paraffin blocks. 2.2. Staining procedure For the immunohistochemical detection of EGFR on the formalin-fixed, paraffin-embedded material, a primary mouse antihuman monoclonal antibody (Zymed, San Francisco, CA, USA; clone 31G7), dilution 1:20, was used. A tissue section, 3 mm width, was mounted on positive-charged microscope slides (OptiplusTM, Biogenex, San Ramon, CA, USA), incubated for 60 min in a heat bath at 60  C, deparaffinized and rehydrated. Slides were treated with ready to use pepsin (Digest-all kitTM, Zymed, San Francisco, CA, USA) at 37  C for 10 min and washed in PBS for 5 min. Immunodetection kit HistostatinTM plus (Zymed, San Francisco, CA, USA) was used strictly according to the manufacturer’s

Table 1 Expression of EGFR in patients with ameloblastoma Case No.

Age (years)

Gender

Site

Staining score

Tumor type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

54 21 22 45 60 36 25 52 42 22 44 40 30 48 51 51 68 69 53 55 64 78 50 50 60 59 43 21 65 19 52 56 70 31 34 34 44 50 61 25 83 22 19 23 21 19 27 20 31 36 20 20 23 25 34 49 52 79

F F M M M M M F M F M F M M F M M M M F M F M M M M M M M M M F M F M M M F M F F M M M F M M M M F M F M F M M M M

Maxilla Mandible Mandible Mandible Maxilla Mandible Mandible Mandible Maxilla Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Maxilla Mandible Maxilla Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Maxilla Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible Mandible

0.05 0.1 0.2 0.2 0.2 0.25 0.4 0.5 0.5 0.5 0.6 0.7 0.8 0.8 0.8 0.8 0.8 0.85 0.9 0.9 0.9 0.9 1.0 1.0 1.0 1.4 1.6 1.6 1.6 1.8 1.8 1.8 1.8 1.8 1.8 1.8 2.0 2.0 2.0 2.0 2.0 0.3 0.3 0.5 0.7 0.8 0.8 0.8 1.0 1.0 1.0 1.2 1.8 2.0 2.0 2.0 2.0 0.6

Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Solid Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Unicystic Desmoplastic Peripheral

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instructions. Slides were counterstained in Mayer’s hematoxylin (Pioneer Research Chemicals, Colchester, Essex, UK) and covered by a non-aqueous GVA mounting medium (Zymed, San Francisco, CA, USA). Surgical specimens of squamous cell carcinoma and normal skin, known to be EGFR positive, served as positive control. Overlying mucosa, if present, served as internal positive control and absence of staining of the blood vessels served as internal negative control. The specificity of the reaction was further determined using non-immune serum instead of primary antibody as negative control. All slides were stained on the same day under identical conditions to minimize variability of staining.

between 0.05 and 2.0. Within solid tumor islands, both peripheral and central cells were stained (Figs. 1 and 2). In the unicystic ameloblastoma, staining of the basal cells was more intense than that of the suprabasal layers. Solid ameloblastomas showed an EGFR-score ranging between 0.05 and 2.0, and five tumors (12%) exhibited the maximum score of 2 (Fig. 3). The unicystic ameloblastoma showed an EGFR-score ranging between 0.3 and 2.0 and three tumors (20%) exhibited the maximum score of 2. The staining score of the desmoplastic ameloblastoma was 2 and that of the peripheral ameloblastoma was 0.6.

4. Discussion 2.3. Scoring criteria Membrane staining or both membrane and cytoplasm staining were evaluated. The proportion of stained cells and staining intensity were combined to assess the immunohistochemical staining. The proportion of stained cells was scored as the percentage of positively stained cells from the entire tumor cell population per high-power field in the tissue sections. Staining intensity was evaluated on a semi-quantitative three-point scale: 0—no staining, 1—weak and 2—strong staining. The final EGFR staining score was calculated by multiplying the percentage of positively stained tumor cells by the staining intensity. Accordingly, the highest score for a given tumor would be equal to 2. A similar scoring method has been used to assess EGFR staining in SCC [16].

Treatment of solid ameloblastoma requires wide resection of the jawbones. The local aggressiveness of the tumor and the infiltration between bone trabeculae

3. Results All specimens demonstrated EGFR-positively stained tumor cells. Staining was either membranous, or both membranous and cytoplasmic, with scores ranging

Fig. 2. Plexiform solid ameloblastoma exhibiting membranous (wide arrow) and cytoplasmic (small arrow) EGFR staining (immunohistochemical staining with anti-EGFR antibody, original magnification 100).

Fig. 1. Follicular solid ameloblastoma exhibiting extensive membranous staining of EGFR in both peripheral and stellate reticulum-like tumor cells (immunohistochemical staining with anti-EGFR antibody, original magnification 100).

Fig. 3. Ameloblastoma exhibiting a maximum EGFR staining score of 2. Staining is conspicuous in both membrane and cytoplasm of tumor cells (immunohistochemical staining with anti-EGFR antibody, original magnification 200).

M. Vered et al. / Oral Oncology 39 (2003) 138–143

often extend the actual margins beyond the apparent radiographic margins [1,3]. The most widely accepted surgical approach is marginal resection with at least 1.0 cm past the tumor’s radiological margins. Such wide resections in the facial area could lead to considerable functional difficulties and esthetic problems. Furthermore, this surgical procedure could be hampered by the proximity to vital structures, so that surgeons are faced with a compromised treatment that results in a recurrence rate of about 15%. Less aggressive surgical approaches for solid ameloblastoma lead to a much higher recurrence rate, between 55 and 90% [3]. Recently, new and promising strategies for treatment of various types of neoplasms have been developed and clinically investigated, i.e. monoclonal antibodies against growth factors and small molecule inhibitors of the tyrosine kinase enzymatic activity that inhibit downstream intracellular signaling of growth factors [5]. With regard to epithelial derived neoplasms, EGFR is an attractive target for these new therapeutic strategies, since its activation and downstream signaling trasduction cascades regulate multiple cellular processes, such as proliferation, differentiation, survival and transformation [17]. Ameloblastoma is the most common epithelial odontogenic neoplasm derived from EGFR-expressing odontogenic epithelial sources [2]. All specimens of ameloblastoma in the present study, irrespective of their type (solid, unicystic, desmoplastic or peripheral), were immunohistochemically positive for EGFR and some were remarkably positive in both the extent and intensity of the staining. Results of previous similar studies are inconsistent. Shrestha et al. [6] claim that of the 23 cases of solid ameloblastomas that they examined, none demonstrated EGFR expression. However, Li et al. [10] reported that EGFR was detected in all six of their cases of ameloblastoma (five solid and one unicystic). Ueno et al. [11] examined 39 cases of solid ameloblastoma and EGFR expression was found in 30 (88%). Nouri et al. [12] found that 24 out of 25 cases of ameloblastoma (96%) were EGFR-positive. The discrepancy among these studies could be in the technical procedures associated with using immunohistochemistry to detect EGFR. A close inspection of their methods reveals that different types of fixatives and different times for tissue fixation were used, a variety of primary anti-EGFR antibodies were utilized, and different incubation conditions with these antibodies were applied (Table 2). Antigen retrieval procedure was performed in only one study [10]. In another [11], the specificity of the antiEGFR antibody was not examined. Furthermore, these studies also differ in the interpretation of the results, some considered only the membranous staining [6,10], while others considered both membranous and cytoplasmic staining [11]. Staining assessment was based on the presence of positive tumor cells [6,12] or on the

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intensity and distribution of the staining [10,11]. In the present study special consideration was given to fixation time, antigen-retrieval procedure and use of commercially available primary antibody and accessory kits. Positive and negative controls were used to verify specificity of the primary antibody and interpretation of the results was performed in accordance with scoring criteria of other neoplasms that combine both the extent and intensity of the staining [16]. Extensive EGFR presence was unequivocally demonstrated in ameloblastomas in the present study. Therefore, it is apparent that this locally aggressive tumor can be a candidate for anti-EGFR treatment, particularly in large infiltrative tumors or recurrences. As to whether EGFR-expression is needed for a certain tumor to benefit from the new treatment modalities, there is still no evidence for a direct relationship between EGFRexpression levels and responsiveness to anti-tumor activity of the therapeutic agents [18]. Thus, ameloblastoma can be included in the group of EGFR-positive tumors that can benefit from these agents. Phase III clinical trials with either anti-EGFR monoclonal antibody (C225) or EGFR-tyrosine kinase inhibitor (ZD1839) are underway in EGFR-positive neoplasms, especially head and neck squamous cell carcinoma [19]. There is accumulating evidence that these anti-EGFR agents, combined with conventional radiotherapy, have a synergistic effect with significant clinical regression in otherwise unresponsive tumors [20]. Ameloblastoma is considered to be a radioresistant tumor [2]. However, in light of this evidence, combining antiEGFR agents with radiotherapy could result in a favorable response, particularly in cases of extensive maxillary tumors, which spread toward the base of the skull. This will ultimately limit the extent of the required curable surgery, either in primary or recurrent ameloblastoma. C225 and ZD-1839 appear to be well tolerated alone or in combination with conventional radiotherapy. Adverse events have been observed in less than 15% of patients [18,21]. With ameloblastoma, these adverse events could be avoided by intralesional administration of the anti-EGFR agents. This has been suggested for treatment of superficial bladder cancer with intravesical administration of EGF conjugated with cytostatic drugs or suitable radionucleotides [22]. Since ameloblastoma is histologically a multicystic tumor, intratumoral injection of the therapeutic agents is quite feasible. Recently, a fully human anti-EGFR monoclonal antibody, ABX-EGF was developed. Its potential to act as a monotherapeutic agent, leading to regression and eradication of multiple human tumors that demonstrate different levels of EGFR-expression has been shown, in vivo [23]. This could also be an optional treatment for surgically compromised cases of ameloblastoma whenever it enters clinical use.

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Table 2 Prevalence of EGFR in ameloblastoma Laboratory procedure No. of cases

Fixation

Type and concentration of EGFR antibody

Temperature/time of reaction

Antigen retrieval

Control tissues

Scoring criteria

EGFR-positive cases (%)

Shrestha et al., 1992 [6]

21

10% formalin, 12 h

MoAB, Biomarker, Israel, 1:80

30 mina

None

Positive—normal oral mucosa Negative—omission of primary antibody

Presence of membranous staining

0%

Li et al., 1993 [10]

6

10% formalin, 18–24 h

MoAb: E30—Bio Diagnostic, 1:60; C11—Serotec, 1:600; EGFR1—Amersham, 1:10; F4—Sigma, 1:200

4  C /over night

Trypsin

Positive—normal oral mucosa Negative—omission of primary antibody

Presence of membranous or membranous and cytoplasmic staining +++ strong ++medium +weak negative

E30—100% strong membranous staining; C11 and F4—patchy variable membranous/ cytoplasmic staining.

Ueno et al., 1994 [11]

39

4% para-formaldehyde in phosphate buffer or 100% ethanol at 4  C, 24 h

MoAb F4, Sigma, USA, 1:200

Moist chamber, room temperature/48 h

None

None

Presence of membranous and/or cytoplasmic staining

88%

Nouri et al., 1997 [12]

25

Frozen sections

MoAb, (ICRF, London); not commercially available

45 mina

None

Positive: pan-keratin MoAb positivity Negative: omission of primary antibody and negative stromal cells

Location of staining not mentioned. Negative Intermediate: weak staining or staining detected only on a proportion of tumor cells Strong: equal intensity of expression of tumor cells

16%—strong 80%—intermediate 4%—negative

Present study

58

10% formalin, 24 h

MoAB 31G7, Zymed, CA, USA, 1:20

60  C/60 min

Pepsin

Positive: normal skin, squamous cell carcinoma, oral mucosa. Negative: absence of staining of blood vessel cells; omission of primary antibody

Presence of membranous or membranous and cytoplasmic staining. 0— No staining 1—weak staining 2—strong staining. Staining intensity multiplied by percentage of positive cells; maximum score—2; minimum score—0.

100% 14% score=2 33% score51 53% score <1 (minimum score 0.05)

a

Complete information not available.

M. Vered et al. / Oral Oncology 39 (2003) 138–143

Reference

M. Vered et al. / Oral Oncology 39 (2003) 138–143

The present study, which was conducted with improved immunohistochemical methods, has unequivocally demonstrated that ameloblastomas are EGFRexpressing tumors and, as such, are a candidate for the new anti-EGFR treatment modalities, especially in cases where the surgical approach is limited (i.e. maxillary tumors) or has failed to control the disease. These modalities, which are clinically successful, could be either administered intralesionally as monotherapy or used in combination with conventional radiotherapy.

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Acknowledgements The study was supported by the Ed and Herb Stein Chair in Oral Pathology, Tel Aviv University. The authors would like to thank Ms. Rita Lazar for editorial assistance and Mrs. Hana Vered for technical assistance.

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