Biomarker Discovery and Identification in Laser Microdissected Head and Neck Squamous Cell Carcinoma with ProteinChip® Technology, Two-dimensional Gel Electrophoresis, Tandem Mass Spectrometry, and Immunohistochemistry

Abstract

Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip® technology. For this, cryostat sections from head and neck tumors (n ‫؍‬ 57) and adjacent mucosa (n ‫؍‬ 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p ‫؍‬ 0.000029) was isolated by twodimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexinpositive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal

Enhanced Interferon Signaling Pathway in Oral Cancer Revealed by Quantitative Proteome Analysis of Microdissected Specimens Using 16O/18O Labeling and Integrated Two-dimensional LC-ESI-MALDI Tandem MS

“…However, relatively few studies have used these techniques to identify OSCC/HNSCC-associated proteins in tissue specimens from patients with OSCC/HNSCC (15,17,29). For example, Melle et al (29) used ProteinChip technology and 2D gel electrophoresis to examine the up-regulation of annexin V in microdissected HNSCC tissues.…”

Proteomic profiling of cholangiocarcinoma: Diagnostic potential of SELDI-TOF MS in malignant bile duct stricture

“…[9][10][11][12][13][15][16][17][18][19][20][21]25 The most interesting discovery of the study was the finding that a SELDI-derived peak (m/z 4,462) was as effective at discriminating cancer from benign serum as the tumor markers CEA or CA19.9, while combining these three serum markers marginally improved classification. Classification could be further improved with data generated from a panel of peaks, suggesting analysis of proteomic profiles, rather than individual proteins, may yield improved diagnostic ability.…”

“…[9][10][11][12][13][15][16][17][18][19][20][21]25 A potential source of error in the tissue group not present in the serum study may be sampling error. The high desmoplastic nature of CCs means the sampled tissue may miss the malignant portion of the tumor and may mainly consist of stroma rather than cholangiocytes proper.…”

“…Proteomic profiling of serum has been used with a number of other malignancies to discover potential biomarkers. 7,8 In particular, affinity-based arrays-such as the protein chips analyzed via surface-enhanced laser desorption/ionization time-offlight mass spectrometry (SELDI-TOF MS) used in this study-have been used to successfully identify malignant patterns in serum from ovarian, [9][10][11] pancreatic, [12][13][14] head and neck, 15,16 prostate, 17 transitional cell, 18 renal cell, 19,20 and breast 21 cancers. TOF-MS separates ions derived from proteins or protein fragments that are differentially detected according to their mass-to-charge ratio.…”

Direct-Tissue SELDI-TOF Mass Spectrometry Analysis: A New Application for Clinical Proteomics

“…This effect has been demonstrated for protein lysate and is a main focus of SELDI-TOF MS technology (15)(16)(17). Whole tumor biopsy lysate as starting material is too heterogeneous for marker detection, however, and is not feasible for clinical samples.…”