β-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms

Oral Oncology (2005) 41, 170–174

http://intl.elsevierhealth.com/journals/oron/

b-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms Trent H. Westernoff a,b, Richard C.K. Jordanb,c,d, Joseph A. Regezib,c,d, Daniel M. Ramosb,d, Brian L. Schmidt a,d,* a

Department of Oral and Maxillofacial Surgery, 521 Parnassus Avenue, C-522, University of California, San Francisco, CA 94143-0440, United States b Oral Pathology, Department of Stomatology, University of California, San Francisco, United States c Department of Pathology, School of Medicine, University of California, San Francisco, United States d Comprehensive Cancer Center, University of California, San Francisco, United States Received 28 June 2004; accepted 6 August 2004

KEYWORDS

Summary b-6 Integrin, tenascin-C, and MMP-1 (matrix metalloproteinase-1) are invasion-related proteins that are frequently overexpressed in many human malignancies. The objective of this study was to determine whether there is overexpression of these molecules in three types of salivary neoplasms showing markedly different behavior. A total of 55 formalin-fixed, paraffin-embedded archived specimens comprising 19 adenoid cystic carcinomas (ACC), 18 polymorphous low-grade adenocarcinomas (PLGA) and 18 pleomorphic adenomas (PA) were utilized in this study. A standard immunohistochemical technique was used to determine the expression levels of b-6 integrin, tenascin-C, and matrix metalloproteinase-1 (MMP-1) proteins. Sections were assessed semiquantitatively, and tumors were divided into two groups, low-expressors (0–1+) and high-expressors (2–3+) for statistical analysis. Staining was graded as 0 (<1% positive tumor cells), 1+ (<25% positive tumor cells), 2+ (25–50% positive tumor cells), and 3+ (>50% positive cells). The results showed that the malignant tumors were higher expressors of b-6 than the benign tumors. ACCs showed significantly higher expression of b-6 than PAs (p = 0.04). No significant difference was observed between ACCs and PLGAs. b-6 expression was rarely seen in normal salivary gland epithelium and was occasionally present in mucosa overlying the tumors. PAs were high-expressors of tenascin-C with a significant difference relative to ACCs (p = 0.03). A majority of tumors in all three tumor types showed high expression of MMP1 with expression significantly greater in the PAs compared to ACCs (p = 0.008). We conclude that ACCs and PLGAs

Adenoid cystic carcinoma; Polymorphous low grade adenocarcinoma; Pleomorphic adenoma; b-6 Integrin; Tenascin-C; MMP-1; Salivary gland neoplasm

*

Corresponding author. Tel.: +1 415 502 3297. E-mail address: [email protected] (B.L. Schmidt).



1368-8375/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2004.08.002

b-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms

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express b-6, tenascin-C, and MMP-1, but that their expression patterns are not significantly different. b-6 appears to be more closely associated with the malignant tumors, and MMP-1 more closely associated with the benign tumors. We believe that b-6, tenascin-C, and MMP-1 proteins are part of the molecular repertoire used by salivary tumors for malignant invasion and benign tumor expansion. c 2004 Elsevier Ltd. All rights reserved.



Introduction Adenoid cystic carcinoma (ACC) is a relatively slow growing malignancy, but is highly invasive. Recurrences due to extensive local-regional infiltration and late distant metastases account for the very poor prognosis associated with ACC.1–3 Polymorphous low-grade adenocarcinoma (PLGA) is also a locally invasive neoplasm but does not have the infiltrative capacity of ACC as reflected in its relatively good prognosis. PLGAs infrequently recur at the primary site but are unlikely to metastasize.4,5 Pleomorphic adenoma (PA) is a benign neoplasm that does not exhibit the inherent infiltrative behavior that is characteristic of ACC or PLGA. Instead, PAs grow and expand circumferentially causing compression of surrounding tissues and thus, has an excellent prognosis. The markedly different behaviors associated with the above three neoplasms suggest that they utilize different biological mechanisms for invasion and local tumor expansion. Several proteins have been identified that are associated with stromal invasion by neoplastic epithelial cells. b-6 integrin is a subunit of an integrin heterodimer, amb-6, and functionally binds to tenascin-C. The interaction between these two molecules is important for keratinocyte adhesion and motility. b-6 integrin is exclusively expressed by epithelium, and only during wound healing and carcinogenesis (neoexpression).6 Neoexpression in oral squamous cell carcinoma has been noted in both invasive and in situ lesions.7 Tenascin-C, a glycoprotein ligand of b-6 integrin is found in the extracellular matrix of normal developing embryonic tissue as well as epithelial tumors.8–10 It is found around normal salivary ducts and in the stroma of ACC.11–14 Tenascin-C is involved in loss of cell adhesion, assisting cells in migration during wound healing and carcinoma invasion.15–19 Tenascin-C expression has not been related to clinical behavior in malignant salivary

tumors, but may correlate with recurrence of benign tumors.20 The matrix metalloproteinases (MMP) are a family of zinc dependent endopeptidases which are responsible for a wide range of enzymatic activities including cell migration, neural growth, wound healing, and salivary gland morphogenesis. These proteins, especially MMP-1, -2, -3, and -9 have also been associated with invasion and metastasis of many human tumors.21–23 A recent study of MMP2 and MMP-9 expression in salivary gland tumors showed that both proteins, particularly MMP-2, may be related to the invasive properties of these neoplasms.24 However, MMP-1 protein has not been evaluated in salivary gland tumors. We hypothesize that b-6 integrin, tenascin-C, and MMP-1 are overexpressed in malignant salivary tumors, and that expression levels are related to their relative invasiveness. We compared expression of these molecules in highly invasive ACCs, mildly invasive PLGAs and non-invasive PAs.

Materials and methods Case selection Sequentially accessioned cases of ACC, PLGA, and PA were selected from UCSF Oral Pathology Laboratory specimen file between the period 1980 and 2003. Slides were reviewed and diagnoses confirmed by two oral pathologists (J.A.R. and R.C.K.J). There were 19 ACCs, 18 PLGAs and 18 PAs that were of adequate size and quality for study inclusion.

Immunohistochemistry Tissue sections (5 lm) were deparaffinized and stained by means of a standard immunohistochemical technique using a high-temperature water bath

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for antigen enhancement. Dako EnVision + (Dako Corporation, Carpenteria, CA) reagents was used in a peroxidase-based system to identify antigen– antibody conjugates. Aminoethylcarbazole was used as a chromagen. The sections were incubated with the following primary antibodies: anti-b-6 (undiluted rabbit monoclonal antibody produced by immunization with purified recombinant human amb-6 that by Western blot analysis recognized the b-6 subunit),25 anti-tenascin-C (1:50, clone TN2; Dako), and anti-MMP1 (1:100; Oncogene, clone 41-1E5, Cambridge, Mass). Normal mouse and rabbit sera containing mixed immunoglobulins at a concentration approximating primary antibodies were used as negative controls. Three oral squamous cell carcinomas (SCC), known to overexpress b-6 integrin, tenascin and MMP-1, were used as positive controls. Immunohis-

Table 1

tochemically stained sections from the three groups were semiquantitatively assessed. Staining was graded as 0 (<1% positive tumor cells), 1+ (<25% positive tumor cells), 2+ (25–50% positive tumor cells), and 3+ (>50% positive cells). Because tenascin-C staining is extracellular, grading for this protein was percent area of positive tumor.

Statistical analysis We used a contingency table approach using FisherÕs exact test to test the statistical associations between tumor types and expression levels for each of the three markers. For analysis we dichotomized results by comparing low-expressors (0–1+) to high-expressors (2–3+). All analyses were two tailed with a significance level set at 0.05.

Summary of results of immunohistochemical staining Score

ACC

PLGA

PA

b-6

Negative 1+ 2+ 3+

12 2 5 0

4 11 1 2

8 10 0 0

Tenascin

Negative 1+ 2+ 3+

17 0 2 0

5 6 2 5

3 7 8 0

MMP-1

Negative 1+ 2+ 3+

0 7 5 7

1 1 2 14

0 0 5 13

Figure 1 Immunohistochemical stain showing b-6 expression in polymorphous low-grade adenocarcinoma (left) and adenoid cystic carcinoma (right). The positive-staining cells in PLGA are part of the peripheral invasive component from the main tumor mass (upper left). In the ACC, cells at the periphery of the tumor nests stain positive (aminoethylcarbazole chromagen and hematoxylin counterstain x400).

b-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms

Results b-6 Integrin staining was usually cytoplasmic, although several cases showed both cytoplasmic and membrane staining. b-6 was expressed by some tumors in all three types of neoplasms (Table 1; Fig. 1). There were significantly more high-expressors of this protein in the ACC group compared to the PA group (p = 0.04), although 12 of the 19 ACCs were negative for b-6. The PAs were low-expressors of b-6 with positive staining located predominantly in cells at the periphery of the tumors. Most PLGAs were low-expressors of b-6. Normal salivary gland was generally negative for b-6 staining, although rare positivity was evident in ductal epithelium. Infrequently, mucosa directly overlying neoplasms was positive. Tenascin-C staining was predominantly stromal, but occasionally there was cytoplasmic staining in tumor cells (Fig. 2). Tenascin-C was expressed in tumors from all three salivary groups, although 17 of 19 ACCs were negative. The two ACCs that were positive were high-expressors with greater than

Figure 2 Immunohistochemical stain for tenascin-C in PLGA showing intercellular staining pattern (aminoethylcarbazole chromagen and hematoxylin counterstain x400).

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50% of tumor cells positive. Statistically, there was a significantly higher expression of tenascin-C in the PAs relative to ACCs (p = 0.03). Cytoplasmic MMP1 staining was found in a vast majority of specimens at a 2–3+ level (Fig. 3). In PAs, MMP-1 staining intensity was most prominent at the epithelial-connective tissue interface. MMP-1 positivity was found in normal salivary ducts and myoepithelial cells.

Discussion In this study we demonstrated that there is a subset of ACCs that are high-expressors of b-6 integrin. This suggests that in some cases, b-6 may be of importance in the stromal invasion that characterizes ACCs. However, because a few PLGAs also highly expressed b-6, it appears that this protein may be functional in the invasion process of other salivary malignancies. In any event, because there was no significant difference in the expression of b-6 in ACCs and PLGAs, b-6 cannot account for the biological differences associated with these two neoplasms. High levels of b-6 integrin were not seen in the PAs suggesting that overexpression is not associated with growth of this benign neoplasm. While tenascin-C expression was evident in some tumors of all groups, it was most often associated with PLGAs and PAs. Because it is a ligand for b6, we expected more tumors in the ACC group to be positive. This suggests that the tenascin-C epitope may have been compromised in some ACC cases. The expression of tenascin-C in PAs is likely related to peripheral expansion of this tumor. This is further supported by the fact that the staining for this protein was most intense at the tumor– stromal interface. All but one case of PLGA expressed MMP-1 and a majority of tumors, both benign and malignant, showed high expression. This MMP along with MMP-2 and MMP-924 appears to be functional in the invasion/expansion of salivary gland tumors.

Figure 3 Positive cytoplasmic immunohistochemical stain for MMP1 in PLGA (left) and ACC (right) (aminoethylcarbazole chromagen and hematoxylin counterstain x400).

174 We conclude that b-6, tenascin-C, and MMP-1 are variably expressed in ACCs, PLGAs, and PAs. The greater expression of b-6 integrin in the salivary gland malignancies studied suggests that this molecule may be a significant factor in stromal invasion. The high expression of tenascin-C and MMP-1 in PAs suggests that these molecules may be important in benign tumor expansion.

Acknowledgements The authors are indebted to Nusi P. Dekker for performing the immunohistochemistry. This work was supported the following grants: NIH/NIDCR K23DE00443, NIH/NCI CA095231, NIH/NIDCR PO1DE13904; NIH/NIDCR DE14609, NIH/NIDCR ROI DEO11930, NIH/NIDCR ROI DE12856, Tobaccorelated disease research program grants 11RT0141 and 12KT-0166.

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