Membrane type -1 matrix metalloproteinase and CD-44 expression in oral cavity carcinomas with micrometastasis

Ross Germani, MD, Elizabeth Franzmann, MD, Francisco Civantos, MDFrom the Department of Otolaryngology Head and Neck Surgery,

University of Miami/ Miller School of Medicine* Acknowledgements: Geroge Elgart, MD and Brenda Roberts PhD, Department of Dermatopathology

Introduction

The standard of care today for most oral cavity squamous cell carcinomas (SCCA) includes elective neck dissection (END) due to the risk of occult metastasis to regional lymph node basins. Identifying molecular markers in the primary tumor that predict the presence of micrometatstatic disease, would avoid unnecessary neck dissections in a number of patients with oral cavity SCCA.

The matrix metalloproteinases (MMP’s) are a family of endopeptidases that play a major role in extracellular matrix (ECM) degradation related to cancer cell invasion and metastasis. Membrane type -1 matrix metalloproteinase (MT1-MMP), a 582 amino acid protein with a transmembrane domain, is an important member of this family. It is known to play a central role in ECM degradation through its inherent collagenase activity and by activation of other MMP’s. It also cleaves CD44 from the surface of cells, resulting in increased cell migration. For this reason, expression and tissue localization of MT1-MMP and CD44 in early metastatic SCCA is an active area of investigation

Lymphoscintigraphy and sentinel lymph node biopsy (SLNB) is a minimally invasive technique that allows examination of a tumor’s most proximal lymph node basin for micrometastatic disease. Current applications include use as a predictive tool to guide treatment for many solid tumors and as a research tool to study early disease spread. Correlation between SLNB status and activity of molecular markers at the primary tumor site may help to define the molecular events associated with early tumor metastasis.

By studying MT1-MMP and CD-44 expression in oral cavity carcinomas with evidence of micrometastasis, we hope to be able to find a molecular marker that correlates with early lymph node metastasis.

There were two groups identified based on results of sentinel lymph node biopsy: patients with negative sentinel lymph node biopsies who represented true clinically and pathologically negative necks (N0; group I), and those with confirmed lymph node metastasis (N+; group II). Group I represented 19/30 (63%) cases, and group II the remaining 11/30 (37%) cases. The American Joint Committee on Cancer (AJCC) staging of primaries and locations by subsite are depicted below (Fig 3,4).

For CD-44, there were 29 cases that underwent successful immunohistochemical staining. In group I (negative SLNB), there were 3/19 (16%) cases with type 1 or peripheral staining, 8/19 (42%) cases with type 2 or mixed staining and 8/19 cases with type 3 or universal staining. In group II (positive SNLB), staining patterns were as follows: 1 = 2/10 (20%), 2 = 5/10 (50%), 3 = 3/10 (30%). Differences between groups for each staining pattern were calculated by the fisher exact method and were not statistically significant; P = 0.65, 0.38 and 0.51 for types 1, 2, and 3 respectively.

For MT1-MMP, there were 21 cases (group I, N = 15; group II, N = 6) thatunderwent successful immunohistochemical staining as demonstrated by a strong internal positive control (salivary gland tissue). In group I, there were 3/15 (20%) specimens with positive staining. In group II, there were 4/6 (67%) specimens with positive staining. This difference did not reach statistical significance ( P = 0.13).

Results

1. Umamaheswar, D, Simental AA, D’Angelo, G, et al. Elective neck dissection and survival in patients with squamous cell carcinoma of the oral cavity and oropharynx. Laryngoscope 2004; 114(12):2228-2234.

2. Myers, EN and Gastaman, BR. Neck dissection: an operation in evolution: Hayes Martin lecture. Arch. Otolaryngol. Head Neck Surg. 2003; 129(1):14-25.

3. Pillsbury, HC and Clark, M. A rationale for therapy of the N0 neck. Laryngoscope 1997; 107(10):1294-1300.4. Myers, JN, Greenberg, JS, Mo, V, et al. Extracapsular spread. A significant predictor of treatment failure in patients with squamous cell carcinoma

of the tongue. Cancer 2001; 92(12):3030–3036.5. Beenken SW, Krontiras H, Maddox WA, et al. T1 and T2 squamous cell carcinoma of the oral tongue: prognostic factors and the role of elective

lymph node dissection. Head Neck. 1999 Mar; 21(2):124-30.6. Liotta LA. Tumor invasion and metastases–role of the extracellular matrix: Rhoads Memorial Award lecture.Cancer Res 1986; 46(1):1–7. 7. Egeblad, M and Werb, Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2002 Mar; 2(3):161-74. 8. Rosenthal, EL and Matrisian, LM. Matrix metalloproteases in head and neck cancer. Head Neck. 2006 Jul; 28(7):639-48.9. Sato H, Takino T, Okada Y, et al. A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature. 1994 Jul 7; 370(6484):61-

5. 10. Ohuchi, E, Imai, K, Fujii, Y, et al. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix

macromolecules. J Biol Chem. 1997; 272: 2446-2451. 11. Katayama, A, Bandoh, N, Kishibe, K, et al. Expression of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive

indicators for tumor metastases and prognosis. Clin Cancer Res. 2004 Jan 15; 10(2): 634-40.12. Kurahara, S, Shinohara, M, Ikebe, T, et al. Expression of MMP’s, MT-MMP, and TIMP’s in squamous cell carcinoma of the oral cavity:

correlations with tumor invasion and metastasis. Head Neck. 1999 October; 21(7):627-38.13. Imanishi, Y, Fujii, M, Tokumaru, Y, et al. Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix

metalloproteinase-2 in human head and neck squamous cell carcinoma. Hum Pathol. 2000 Aug; 31(8): 895-904.14. Civantos, FJ, Gomez, C, Duque, C, et al. Sentinel node biopsy in oral cavity cancer: correlation with PET scan and immunohistochemistry. Head

Neck. 2003 Jan; 25(1): 1-9. 15. El-Sayed, IH, Singer, MI and Civantos, F. Sentinel lymph node biopsy in head and neck cancer. Otolaryngol Clin North Am. 2005 Feb; 38(1):

145-60. 16. Ross, GL, Shoaib, T, Soutar DS, et al. The first international conference on sentinel node biopsy in mucosal head and neck cancer and adoption

of a multicenter trial protocol. Ann. Surg. Oncol. 2002; 9(4):406-410.17. Kajita, M, Itoh, Y, Chiba, T, et al. Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J Cell Biol. 2001 May ;

153(5) : 893-904.18. Mori, H, Tomari, T, Koshikawa, N, et al. CD44 directs membrane-type 1 matirx metalloproteinase to lamellipoda by associating with its

hemopexin-like domain. EMBO J. 2002; 21(15): 3949-3959.19. Franzmann, EJ, Reategui, EP, Carraway, KL, et al. Salivary souble CD44: a potential molecular marker for head and neck cancer.Cancer

Epidemiol Biomarkers Prev 2005;14(3):735–9.

Bibliography

Samples were collected in a prospective fashion from patients who underwent sentinel lymph node biopsy for oral cavity squamous cell carcinoma, with or without subsequent cervical lymphadenectomy, at the University of Miami between 1998 and 2006 (N = 30). Patient demographics and tumor location and stage were recorded.

Specimens (N=30) were fixed in 10% formalin and then embedded in paraffin. Blocks were sectioned at 5μ and attached to positively charged slides. The slides were placed in a section drying oven for 30 minutes at 580C, de-parrafinized in xylene and rehydrated through graded alcohols. Next, the slides were briefly washed in de-ionized water (DDI) and placed in phosphate buffered saline (PBS). The slides were then incubated in a 3% solution of H2O2 in PBS to block the staining of endogenous peroxidase. For CD-44, high temperature epitope retrieval was accomplished by exposing the slides to an Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) in a pressure cooker. Pronasedigestion was used to expose the Antigen site for MT1-MMP. Two sections from each primary tumor were then sequentially incubated with horse normal serum (Vector Laboratories, Burlingame, CA) and either monoclonal antibody MT1-MMP (R&D Systems, Minneapolis, MN; clone 5h2) or monoclonal antibody anti-sol CD-44 (Bender MedSystems Inc., Burlingame, CA ) at a dilution of 1:1,000 for one hour at room temperature and then washed in PBS for 2 x 5 minutes. The sections were then incubated for 30 minutes with anti-mouse IgG biotinylatedantibody (Vector Laboratories, Burlingame, CA), washed for 5 minutes in PBS and further incubated for 30 minutes with Avidin Biotin Complex (Vector Laboratories, Burlingame, CA). Following 2 x 5 minute washes in PBS, the sections were exposed to a freshly prepared solution of H2O2and 3, 3’ diaminobenzidine (DAB) to visualize the antigen-antibody site. The sections were then counterstained in Mayers Hematoxylin for 1 minute, blued for 5 minutes in running water, then dehydrated through graded alcohols, cleared in xylene and coverslipped. Omission of the primary antibody served as the negative control. Known positive and reactive cells in the skin served as positive controls.

Immunohistochemical reactivity of the specimens for MT1-MMP and sol CD-44 was characterized by an experienced pathologist blinded to SLNB status. Staining patterns were classified as follows for MT1-MMP: 0 = no staining, 1 = positive staining (Fig 1) and for CD-44: 0 = no staining, 1 = peripheral staining, 2 = mixed staining, and 3 = universal staining (Fig 2).

The statistical significance of differences between groups I and II for each type of staining pattern was calculated using the Fisher exact method. P-values <0.05 were considered to indicate statistical significance.

Methods and Materials

CD44: Peripheral staining pattern

CD44: Universal staining pattern

CD44: Mixed staining pattern

There was no statistically significant difference in staining patterns for CD44 between subjects with (group II) and without (group I ) micrometastases . This may suggest that CD44 expression does not directly correlate with early tumor metastasis. Alternatively, the low sample size in group II (N=10) may not have provided sufficient power to detect a statistical difference.

There was a striking difference in MT1-MMP staining between groups I (20%) and II (67%), that did not reach statistical significance (p=0.13). We only included specimens with a strong internal control to differentiate true staining from possible background staining with our anti-MT1-MMP antibody. This reduced the power of our study (group II, N =6). It is possible that by increasing the number of specimens, we may detect a significant difference. Quantitative RT-PCR measurements of MT1-MMP levels in these tissues may further differentiate early node positive and node negative specimens.

Lymphoscintigraphy followed by sentinel lymph node biopsy is a sensitive means to detect early tumor spread. By studying this unique subset of patients we hope to identify a reliable molecular marker for metastasis, which may ultimately change the way we manage the neck for early oral cavity cancer.

Conclusions

Fig 2: CD-44 Staining Patterns

Fig 1: MT1-MMP Staining Patterns

MT1-MMP: 0 = no staining of tumor (10x)

MT1-MMP: 1= positive staining of tumor (10x and 100x)

Fig 3: AJCC Staging Fig 4: Location by Subsite

T1N0: 13/30 (43%)

T2N0: 6/30 (20%)

T1N1: 6/30 (20%)

T2N1: 3/30 (10)%

T3N1: 2/30 (7%)

Lip 7%Floor of Mouth 13%

Retromolar Trigone 10%

Buccal Mucosa 7%

Tongue: 63%