口腔鱗狀上皮細胞癌是一好發在頭頸部具侵犯性的口腔黏膜惡性病變，其癌化的進程被認為是一種漸進性癌化的發生過程，其中涉及多重基因的突變，導致正常的上皮黏膜，可能出現如白斑、紅斑、疣狀增生等口腔癌前病變(precancerous lesion)。其中部分的病變會再繼續癌化最終成為口腔癌。在這些癌前病變中，疣狀增生(verrucous hyperplasia)、病理特徵最接近口腔癌的上皮嚴重異生(severe dysplasia)與原位癌(carcinoma in situ)，是較獨特且危險的癌前病灶。回顧過去的文獻，發現在侵犯前緣之腫瘤細胞質中有Galectin-1蛋白過度表現，尤其是早期的口腔癌與頭頸部轉移腫瘤細胞表現更明顯。此外，在先前另一部分蛋白質體的研究中，也發現在口腔癌組織中會有上皮性脂肪酸鍵結蛋白(epidermal-type fatty acid binding protein ,E-FABP)的過度表現，然而這兩種蛋白質在口腔癌癌前病變中的可能機轉並不清楚。因此，本研究的目標，是利用免疫組織染色方法來研究Galectin-1與E-FABP這兩種蛋白質，在疣狀增生、口腔上皮細胞嚴重異生中的表現情況。並利用免疫螢光染色法，進一步鑑別在組織中是何種細胞過度表現這些蛋白質。從免疫組織染色方法的結果發現：(一).Galectin-1會表現在部分(16%)上述癌前病變的上細胞中，從癌前病變至口腔癌中似有增加的趨勢(p=0.16)。(二).在間質組織(stroma)中，有56%的癌前病變組織會有Galectin-1過度表現的情況，且從正常組織到口腔癌癌前病變，Galectin-1的表現量亦似有增加的趨勢，但是並無統計上的意義 (p=0.1)；在口腔癌組織中的過度表現比率達71%，與正常組織相比有明顯的增加(p=0.06)。(三).從E-FABP的免疫組織染色結果發現，76%癌前組織的上皮細胞中會有過度表現的情況，與正常組織比有明顯增加(p=0.009)，且部份組織有分佈不均的情況；但是在口腔癌中的上皮癌細胞，E-FABP的表現量則會較癌前組織有減少的趨勢(p=0.067)。這些結果顯示出Galectin-1與E-FABP在細胞惡性變性的過程中扮演著某些相關的角色。從免疫螢光染色方法的結果發現：(一).許多在間質表現Galectin-1的細胞也呈現纖維母細胞的標記vimentin。(二).發現Galectin-1的螢光也會表現在血管的內皮細胞，與部分免疫細胞球。(三).E-FABP的表現在組織切片中產生較多不規則濃染的現象，與細胞增生標記Ki-67的螢光染色比對後，並無發現兩者有重疊表現的現象。總而言之，透過以疣狀增生、口腔上皮細胞嚴重異生組織的實驗中，可以發現病變的上皮細胞與緊鄰的間質在癌化的不同時期與不同的位置，皆過度表現異於正常上皮組織的特別蛋白質，進一步了解這些蛋白質的作用機轉後，將有助於腫瘤生成微環境中更深入的研究，及臨床上所代表的意義。

Oral squamous cell carcinoma (OSCC) is an invasive lesion and is the most common cancer in head and neck region. The development and progression of OSCC is thought to be a multi-step process in which accumulation of damages to genes occurred through clonally evolution. In the carcinogenesis process, the normal epithelium will transform into precancerous lesions (PML), and then some of these lesions may get worse to OSCC. Among these precancerous transformations, verrucous hyperplasia and severe dysplasia, and carcinoma in situ are thought to be must intriguing and dangerous. In Prof. Chen’s previous study, altered expression of Galectin-1 and epidermal-type fatty acid binding protein (E-FABP) in oral cancerous tissue were detected but their roles in the progression from precancerous to cancerous stages remained unclear. The aims of this study are to analyze the immunohistochemical profiles of Galectin-1 and E-FABP in human oral verrucous hyperplasia, severe dysplasia, and carcinoma in situ. For further examination of the expression and location, immunofluorescent assay was used to identify which were over-expressed Galectin-1 and E-FABP in the precancerous tissue. The results of the immunohistochemical (IHC) staining showed that Galectin-1 was over-expressed in the epithelial cells in some samples (16%), and the over-expression rate increased in the cancer group. Second, Galectin-1 was also found over-expressed in 56% in the stroma layer of the precancerous cases (p=0.1). The over-expression of galectin-1 also increased from the PML group to the OSCC group (p=0.06). E-FABP was over-expressed in the epithelial cells in 76% of the PML group. There is a statistically significant increase in over-expression rate comparing to that of normal tissue (p=0.009). However, the E-FABP expression rate decreased from PML group to the OSCC group (p=0.067). In the immunofluorescent staining, we found that Galectin-1 immunoreactivities are mostly co-localized with the vimentin (the marker of fibroblast) in the double immunofluorescent staining. We also observed the expression of galectin-1 in the endothelial cells and immune cells. While in the E-FABP IHC staining, the irregular expression in epithelium layer was supposed to correlate with the cellular proliferation, we did not find the co-localized fluorescent staining in E-FABP and Ki-67. These results indicate that Galectin-1 and E-FABP may play some role in cell malignant transformation. In conclusion, to profiling the proteins expression in the different precancerous lesions may help to understand the carcinogenesis progression of OSCC.

1. de Visser KE, Korets LV, Coussens LM. De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell 2005;7(5):411-23.
2. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420(6917):860-7.
3. Kurkivuori J, Salaspuro V, Kaihovaara P, et al. Acetaldehyde production from ethanol by oral streptococci. Oral oncology 2007;43(2):181-6.
4. Lippman SM, Sudbo J, Hong WK. Oral cancer prevention and the evolution of molecular-targeted drug development. J Clin Oncol 2005;23(2):346-56.
5. Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 2005;7(3):211-7.
6. Syrjanen S. Human papillomaviruses in head and neck carcinomas. N Engl J Med 2007;356(19):1993-5.
7. Lippman SM, Hong WK. Molecular markers of the risk of oral cancer. N Engl J Med 2001;344(17):1323-6.
8. Sudbo J, Kildal W, Risberg B, Koppang HS, Danielsen HE, Reith A. DNA content as a prognostic marker in patients with oral leukoplakia. N Engl J Med 2001;344(17):1270-8.
9. Sudbo J, Lippman SM, Lee JJ, et al. The influence of resection and aneuploidy on mortality in oral leukoplakia. N Engl J Med 2004;350(14):1405-13.
10. Batsakis JG, Suarez P, el-Naggar AK. Proliferative verrucous leukoplakia and its related lesions. Oral Oncol 1999;35(4):354-9.
11. Hsue SS, Wang WC, Chen CH, Lin CC, Chen YK, Lin LM. Malignant transformation in 1458 patients with potentially malignant oral mucosal disorders: a follow-up study based in a Taiwanese hospital. J Oral Pathol Med 2007;36(1):25-9.
12. Kujan O, Khattab A, Oliver RJ, Roberts SA, Thakker N, Sloan P. Why oral histopathology suffers inter-observer variability on grading oral epithelial dysplasia: an attempt to understand the sources of variation. Oral Oncol 2007;43(3):224-31.
13. Kujan O, Oliver RJ, Khattab A, Roberts SA, Thakker N, Sloan P. Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol 2006;42(10):987-93.
14. Swinson B, Jerjes W, El-Maaytah M, Norris P, Hopper C. Optical techniques in diagnosis of head and neck malignancy. Oral Oncol 2006;42(3):221-8.
15. Lindeboom JA, Mathura KR, Ince C. Orthogonal polarization spectral (OPS) imaging and topographical characteristics of oral squamous cell carcinoma. Oral Oncol 2006;42(6):581-5.
16. Rumboldt Z, Day TA, Michel M. Imaging of oral cavity cancer. Oral oncology 2006;42(9):854-65.
17. Fyfe EC, Guest RG, St Rose L, Eveson JW. Focal acantholytic dyskeratosis arising in an intraoral skin flap. Int J Oral Maxillofac Surg 2002;31(5):560-1.
18. Yeh CJ. Treatment of verrucous hyperplasia and verrucous carcinoma by shave excision and simple cryosurgery. Int J Oral Maxillofac Surg 2003;32(3):280-3.
19. Chen YK, Hsuen SS, Lin LM. Increased expression of inducible nitric oxide synthase for human oral submucous fibrosis, verrucous hyperplasia, and verrucous carcinoma. Int J Oral Maxillofac Surg 2002;31(4):419-22.
20. Shear M, Pindborg JJ. Verrucous hyperplasia of the oral mucosa. Cancer 1980;46(8):1855-62.
21. Chen J, He QY, Yuen AP, Chiu JF. Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis. Proteomics 2004;4(8):2465-75.
22. Patel V, Leethanakul C, Gutkind JS. New approaches to the understanding of the molecular basis of oral cancer. Crit Rev Oral Biol Med 2001;12(1):55-63.
23. He QY, Chen J, Kung HF, Yuen AP, Chiu JF. Identification of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics. Proteomics 2004;4(1):271-8.
24. Barondes SH, Castronovo V, Cooper DN, et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994;76(4):597-8.
25. Rorive S, Belot N, Decaestecker C, et al. Galectin-1 is highly expressed in human gliomas with relevance for modulation of invasion of tumor astrocytes into the brain parenchyma. Glia 2001;33(3):241-55.
26. Camby I, Belot N, Lefranc F, et al. Galectin-1 modulates human glioblastoma cell migration into the brain through modifications to the actin cytoskeleton and levels of expression of small GTPases. Journal of neuropathology and experimental neurology 2002;61(7):585-96.
27. Paz A, Haklai R, Elad-Sfadia G, Ballan E, Kloog Y. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 2001;20(51):7486-93.
28. Rabinovich GA, Iglesias MM, Modesti NM, et al. Activated rat macrophages produce a galectin-1-like protein that induces apoptosis of T cells: biochemical and functional characterization. J Immunol 1998;160(10):4831-40.
29. Bohn H, Kraus W, Winckler W. Purification and characterization of two new soluble placental tissue proteins (PP13 and PP17). Oncodev Biol Med 1983;4(5):343-50.
30. Saussez S, Nonclercq D, Laurent G, et al. Toward functional glycomics by localization of tissue lectins: immunohistochemical galectin fingerprinting during diethylstilbestrol-induced kidney tumorigenesis in male Syrian hamster. Histochemistry and cell biology 2005;123(1):29-41.
31. Danguy A, Camby I, Kiss R. Galectins and cancer. Biochimica et biophysica acta 2002;1572(2-3):285-93.
32. Rabinovich GA. Galectin-1 as a potential cancer target. Br J Cancer 2005;92(7):1188-92.
33. Chmurzynska A. The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism. Journal of applied genetics 2006;47(1):39-48.
34. Siegenthaler G, Hotz R, Chatellard-Gruaz D, Didierjean L, Hellman U, Saurat JH. Purification and characterization of the human epidermal fatty acid-binding protein: localization during epidermal cell differentiation in vivo and in vitro. The Biochemical journal 1994;302 ( Pt 2):363-71.
35. Zimmerman AW, Veerkamp JH. New insights into the structure and function of fatty acid-binding proteins. Cell Mol Life Sci 2002;59(7):1096-116.
36. Liu Y, Longo LD, De Leon M. In situ and immunocytochemical localization of E-FABP mRNA and protein during neuronal migration and differentiation in the rat brain. Brain research 2000;852(1):16-27.
37. Hagens G, Masouye I, Augsburger E, Hotz R, Saurat JH, Siegenthaler G. Calcium-binding protein S100A7 and epidermal-type fatty acid-binding protein are associated in the cytosol of human keratinocytes. The Biochemical journal 1999;339 ( Pt 2):419-27.
38. Masouye I, Saurat JH, Siegenthaler G. Epidermal fatty-acid-binding protein in psoriasis, basal and squamous cell carcinomas: an immunohistological study. Dermatology (Basel, Switzerland) 1996;192(3):208-13.
39. Rauch J, Ahlemann M, Schaffrik M, et al. Allogenic antibody-mediated identification of head and neck cancer antigens. Biochemical and biophysical research communications 2004;323(1):156-62.
40. Paterson IC, Eveson JW, Prime SS. Molecular changes in oral cancer may reflect aetiology and ethnic origin. European journal of cancer 1996;32B(3):150-3.
41. Folkman J. Tumor angiogenesis: therapeutic implications. The New England journal of medicine 1971;285(21):1182-6.
42. Beacham DA, Cukierman E. Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Seminars in cancer biology 2005;15(5):329-41.
43. Clausse N, van den Brule F, Waltregny D, Garnier F, Castronovo V. Galectin-1 expression in prostate tumor-associated capillary endothelial cells is increased by prostate carcinoma cells and modulates heterotypic cell-cell adhesion. Angiogenesis 1999;3(4):317-25.
44. Perillo NL, Pace KE, Seilhamer JJ, Baum LG. Apoptosis of T cells mediated by galectin-1. Nature 1995;378(6558):736-9.
45. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nature reviews 2002;2(6):442-54.
46. Zhou BP, Deng J, Xia W, et al. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nature cell biology 2004;6(10):931-40.
47. Singh S, Sadacharan S, Su S, Belldegrun A, Persad S, Singh G. Overexpression of vimentin: role in the invasive phenotype in an androgen-independent model of prostate cancer. Cancer research 2003;63(9):2306-11.
48. Gilles C, Polette M, Mestdagt M, et al. Transactivation of vimentin by beta-catenin in human breast cancer cells. Cancer research 2003;63(10):2658-64.
49. Webb M, Emberley ED, Lizardo M, et al. Expression analysis of the mouse S100A7/psoriasin gene in skin inflammation and mammary tumorigenesis. BMC cancer 2005;5:17.
50. Munz M, Zeidler R, Gires O. The tumour-associated antigen EpCAM upregulates the fatty acid binding protein E-FABP. Cancer letters 2005;225(1):151-7.
51. Uma RS, Naresh KN, D'Cruz AK, Mulherkar R, Borges AM. Metastasis of squamous cell carcinoma of the oral tongue is associated with down-regulation of epidermal fatty acid binding protein (E-FABP). Oral Oncol 2007;43(1):27-32.
52. Ostergaard M, Rasmussen HH, Nielsen HV, et al. Proteome profiling of bladder squamous cell carcinomas: identification of markers that define their degree of differentiation. Cancer research 1997;57(18):4111-7.

53.http://pir.georgetown.edu/cgi-bin/ipcEntry_cat?id=Q01469