• 9/23/2006
  • San Francisco, CA
  • Maricris Macabeo-Ong et al.
  • Molecular Diagnostics in Cancer Therapeutic Development, Sep 12-15, 2006

Diagnostic Technologies and Molecular and Cellular Profiling: Biomarkers in Early Detection and Diagnosis

Introduction:
Cervical lymph node metastasis (N+ node) is the critical prognostic factor for oral cancer patients. Detection of N+ node remains problematic because there are no biomarkers available to predict regional spread. Therefore, the goal of this study is to use proteomics technology to identify candidate biomarkers that may be associated with cervical lymph node metastasis.

Method:
Frozen tissues of a buccal mucosa SCC and associated node containing metastatic tumor (N+) were obtained from one patient. Lesional tissue was microdissected, proteins extracted and separated by two-dimensional (2D) gel electrophoresis. The gels were stained with Colloidal Coomassie Blue and imaged using ImageMaster 2D Platinum software. The 2D gels were differentially analyzed to identify proteins both common and unique to the primary tumor and N+ node. Proteins less than 50 kDa were cut from the gels, trypsin digested and identified using MALDI-TOF peptide mass fingerprinting and LC MS/MS analysis.

Immunohistochemistry (IHC) was used on the tissues to confirm, localize and compare the expression of proteins identified by mass spectrometry.

Results:
353 proteins were resolved in the primary SCC while 376 proteins were resolved in tumor within the lymph node. 210 proteins between the primary tumor and N+ node showed identical migration and percent spot volume. 166 and 143 proteins were unique spots to primary tumor and N+ node, respectively.

Based on differential analysis of protein spots between the primary tumor and N+ node, 18 spots were selected for identification by mass spectrometry yielding a total of 41 unique proteins.

Among the unique proteins identified in the N+ node were: albumin, ß-2-microglobulin, calgranulin A, coactosin-like protein, eosinophil lysophospholipase, fatty acid binding protein 5, galectin 1, histone H3/b, cathepsin C isoform b, immunoglobulin heavy chain, pituitary adenylate cyclase activating polypeptide (PACAP), profilin 1, Pterin-4-apha-carbinolamine dehydratase, cytosolic ribosomal protein S12, S100 calcium binding protein, Shujun-1, 11 hemoglobin related proteins, 6 keratin proteins and 2 trypsin precursor protein. PACAP, not previously associated with oral cancer metastasis, is a protein that has been associated to stimulate c-fos gene expression and cell proliferation in lung cancer. Our immunohistochemistry analysis confirmed overexpression of PACAP in the N+ node.

Conclusions:
Using a proteomic discovery approach, we identified a series of proteins both common and unique to a primary oral SCC and the associated neck metastasis. These several candidate proteins may be associated with the metastatic process from oral SCC to cervical lymph nodes. Differential analysis of the candidate proteins might yield a predictive metastatic marker that could be clinically tested.

Authors:
Maricris Macabeo-Ong, Richard C.K. Jordan, Markus Hardt, Steven Hall, Scott Dixon, Stephanie Lim and Brian L. Schmidt

Authors’ affiliation:
University of California, San Francisco, CA