SPRR3 constitutes the largest member of the small proline-rich gene family and is associated with differentiation and the benign phenotype of the human esophageal epithelial cell. MELK is a cell cycle-regulated protein kinase involved in cell cycle progression, proliferation, tumor growth and messenger RNA (mRNA) splicing. MAGE-4 is a cancer-germline gene that is expressed in many tumors. Epidermal growth factor (EGFR) is overexpressed in esophageal tumors and might represent a gene target therapy. However, the clinical significance of these molecular markers has not been well established. Aim: To verify SPRR3, MELK, MAGE-4, and EGFR gene expression in esophageal squamous cell carcinoma (SCC) in selected patients who had undergone potentially curative surgical treatment. Methods: Tissue specimens from 30 esophageal SCC and normal esophageal epithelium within the surgically resected specimens were frozen in liquid nitrogen immediately after their removal (up to 15 minutes). The samples were immediately immersed in liquid nitrogen and send to the laboratory at -80oC freezer. After histopathological verification, the frozen specimens of each case were microdissected. Frozen specimens were cut in cryostat and were histopatologically analyzed, selecting the samples with maximum degree of viable tissue representation, from primary neoplasia and normal esophagus. The levels of mRNA were quantified by Real Time-RT-PCR using SybrGreen method (μL of diluted cDNA), SybrGreen Master Mix (concentrate 1X, 200ηM primer sense e 200ηM primer antisense in 20μL of reaction volume), compared to the constitutive gene β-actin. Results: SPRR3 expression was remarkable decreased in all esophageal SCC compared to the normal mucosa. EGFR was overexpressed in 61.5% of the tumors, and it was associated to the patients outcome (patients alive in the overexpressed group = 36.4% vs. 75% in the low expressed group, p=0.002). MELK was overexpressed in 84.7% of the patients, and MAGE-4 was overexpressed in all tumors. There was no association between downregulation of SPRR3 expression, overexepression of MELK, MAGE-4, and EGFR and clinicopathologic characteristics, including gender, age, T.N.M. stage, or grade of differentiation. Conclusions: 1. SPRR3 is frequently downregulated in esophageal squamous cell carcinoma, and may play a role in the maintenance of normal esophageal epithelium; 2. MELK and MAGE-4 are upregulated and these genes may contribute to the tumorigenesis of esophageal squamous cell carcinoma; 3. These data support the role of targeted therapies against MELK, MAGE-4, and EGFR in the treatment of squamous cell carcinoma of esophagus.