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高雄醫學大學 口腔醫學院 牙醫學系 口腔病理科 27 th , August, 2009

p73 Expression for Human Buccal Epithelial Dysplasia & Squamous Cell Carcinoma: Does It Correlate with Nodal Status of Carcinoma & Is There a Relationship with Malignant Change of Epithelial Dysplasia?. 高雄醫學大學 口腔醫學院 牙醫學系 口腔病理科 27 th , August, 2009.

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高雄醫學大學 口腔醫學院 牙醫學系 口腔病理科 27 th , August, 2009

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  1. p73 Expression for Human Buccal Epithelial Dysplasia & Squamous Cell Carcinoma: Does It Correlate with Nodal Status of Carcinoma & Is There a Relationship with Malignant Change of Epithelial Dysplasia? 高雄醫學大學 口腔醫學院 牙醫學系 口腔病理科 27th, August, 2009

  2. Studies on Human Oral & DMBA-Induced Hamster Buccal-Pouch Squamous cell Carcinogenesis A Series of Tumor Markers Studies • Cytokeratin (1987)* • Lectin (1989) • Creatine kinase isoenzyme (1991) • Placental glutathione S-transferase (P-GST)(1995) • -glutamyltranspeptidase (-GGT) (1997) • Inducible nitric oxide synthase (iNOS) (2000) • p53 family (p53, p63 & p73) (2002) • Inhibitor of apoptosis family (IAP) (2005) • smad family signaling pathway *Year of First Publication

  3. NSC Projects Derived From thep53 Family Studies Human Studies p63於人類口腔癌前期病灶及鱗狀上皮細胞癌中之表現 (92-2314-B-037-073) (2003/8/1-2004/7/31) p73於人類口腔癌前期病灶及鱗狀上皮細胞癌中之表現 (90-2314-B-037-085) (2001/8/1-2002/7/31) Animal Studies DMBA誘發倉鼠頰囊袋鱗狀上皮細胞癌化過程中p63之 免疫組織化學與mRNA表現研究 (91-2314-B-037-260) (2002/8/1-2003/7/31) DMBA誘發倉鼠頰囊袋鱗狀上皮細胞癌化過程中p73之免疫組織化學與mRNA表現研究 (89-2314-B-037-123) (2000/8/1-2001/7/31)

  4. Publications Derived From thep53 Family Studies Animal Studies • Arch Oral Biol 2002;47:695-9(6) 2. Oral Dis 2003; 9:227-34 (11) 3. Oral Dis 2003; 9:235-40 (7) 4. Int J Exp Pathol 2004;85:97-104 (8) • Human Studies • Clin Otolaryngol 2003;28:451-5(12)* • Int J Oral Maxillo- • fac Surg 2004;33: • 493-7 (9) • Head & Neck • 2004;26:945-52(5) • 4. J Oral Pathol Med 2005;34:232-9(8) I.F.2008= 2.603 (1/31) * Frequency to be cited

  5. p73 has been cloned & mapped at chromosome 1p36.2-3, a region that reveals highly frequent loss of heterozygosity for various human cancers. (Kaghad et al, 1997) This gene encodes a protein with structure highly homogeneous to p53 in the three domains: transcriptional activation domain, DNA-binding domain, & oligomerization domain. (Kaghad et al, 1997) Introduction

  6. p73 also shares some common functions with the p53 protein, including inhibiting cell growth & inducing apoptosis. (Jost et al, 1997) Mutations at the TP53 are detected for >50% of many different types of cancers, including human H&N SCCs. (Harris et al, 1993; Ahomadegbe et al, 1995) Introduction

  7. By contrast to p53, a mutant p73 has rarely been found among human tumors. (Nomoto et al, 1998; Yokozaki et al, 1999) Unlike p53, p73 has multiple splice variants (, , , & ), which may have different biologic characteristics & cellular specificities. (Irwin et al, 2001) Introduction

  8. In this study, we examined the p73 expression for human buccal epithelial dysplasia (ED) & SCC to obtain more data for elaboration of its role in human oral squamous cell carcinogenesis. Introduction

  9. 75 samples of human buccal ED, including mild, moderate, & severe ED (25 samples in each category), aged 36-65 yrs (mean, 44 yrs). 10 samples of human normal buccal mucosa, aged 36 to 62 yrs (mean, 47 yrs). 25 samples of human buccal SCCs aged 32 and 70 yrs (mean, 55 yrs); 17 of them with cervical lymph node involvement; 15 samples were well differentiated, & 10 were moderately differentiated. Materials & Methods

  10. Fresh tissues Materials & Methods Paraffin-embedded tissues Normal oral mucosa Human buccal EDs Immunohistochemistry: p73 protein Human buccal SCCs Normal oral mucosa RT-PCR:p73 mRNA Human buccal SCCs

  11. Materials & Methods Immunohistochemical staining • Performed by avidin-biotin peroxidase complex (ABC) method. • Primary rabbit polyclonal anti-p73 Ab (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100 dilution). • Samples with 10% or more positive nuclear-stained keratinocytes were classified as positive staining.

  12. Materials & Methods RT-PCR O.D.:1.8-2.0 (260/280nm) RNA extraction Guanidium isothiocyanate- caesium chloride method Reverse transcriptionreaction cDNA synthesis 28S 18S

  13. Materials & Methods Thermocycling conditions Oligoprimers from Genbank PCR cycle for p73 PCR cycle for -actin TaKaRa MP, Tokyo, Japan

  14. A B C D Results p73 nuclear staining was located in basal cells of normal mucosa (A); in basal & parabasal layers & in more superficial cell layers corresponding to the spinous layer in mild (B), moderate (C), & severe (D) buccal epithelial dysplasia (ABC stain; 100).

  15. p73 protein is chiefly observed in the less-differentiated cells in the periphery of carcinomatous clusters in well-differentiated carcinoma. Lack of staining is noted in areas of keratin (ABC stain 40). Results

  16. Results A sample of original moderate epithelial dysplasia that has undergone malignant transformation to moderately differentiated carcinoma, revealing homogeneous staining of p73 protein in all of the tumor cells. The dysplastic tissue adjacent to the carcinomatous tissue revealing positive p73 protein in basal and parabasal layers and in more superficial cell layers corresponding to the spinous layer (ABC stain 40).

  17. M 1 2 3 4 5 6 7 8 9 10 11 12 N NC p73 180-bp -actin 350-bp M 13 14 15 16 17 18 19 20 21 22 23 24 25 N NC p73 180-bp -actin 350-bp Results Expression of p73 mRNA in buccal SCCs RT-PCR. A band of a 180-bp PCR product corresponding to p73 mRNA is observed for 17 specimens of buccal SCC (lanes 1, 2, 4-7, 9, 10, & 12 in A; lanes 13, 15, 17-20, 22, & 24 in B). Lanes N (A & B) are the two normal buccal mucosa specimens showing a 180-bp PCR product corresponding to p73 mRNA. No band was observed in the negative control sample (lanes NC in A & B). All samples (lanes 1-15 & N in A; lanes 13-25 & N in B) apart from the negative control sample (lanes NC in A & B) reveal bands of -actin (350-bp).

  18. Results Table 1. Expression of p73 (protein & mRNA) for diseased & normal buccal mucosa Abbreviation: ED, epithelial dysplasia. *Statistical significance compared with normal mucosa (Fisher’s exact test, p < .001). The expression of p73 seemed to be significantly elevated for specimens of buccal ED (protein level) & SCC (protein and mRNA levels) compared with the analogous expression for normal control tissue.

  19. Results Table 2. Correlation of p73 protein & mRNA expression for the primary buccal squamous cell carcinoma & the status of cervical lymph node metastasis (Fisher’s exact test, p < .001) Abbreviation: SCC, squamous cell carcinoma. p73 expression (protein & mRNA levels) correlated significantly with cervical lymph node metastasis for cases of buccal SCC.

  20. Results Table 3. Correlation of p73 protein expression for epithelial dysplasia and malignant transformation Abbreviation: ED, epithelial dysplasia. *Fisher’s exact test, p > .05).Fisher’s exact test, p = .05). p73 expression for moderate ED (protein level) was statistically insignificant to SCC transformation, whereas p73 expression for severe ED was marginally insignificant to malignant change.

  21. There is only one published IHC-based report (Choi et al, 2002) of p73 expression in samples of ED taken from the H&N. Our results are compatible with those of this small study of 16 ED lesions (5 mild, 6 moderate, 5 severe). Choi et al demonstrated that 14 (87.5%) of the 16 samples expressed p73; however, no attempt was made to grade degree of staining with respect to the severity of the dysplastic lesions. Discussion

  22. In this study, there seemed to be no significant difference in p73 expression between the lesions of mild, moderate, & severe ED, although we found that p73 was overexpressed in buccal lesions of ED compared with normal tissue. Our result is compatible with our earlier finding that p73 protein was overexpressed in the early stages of DMBA-induced squamous cell carcinogenesis in hamster buccal pouch. Discussion

  23. A small subset of moderate ED & severe variant showing p73 positivity have undergone malignant transformation to develop SCCs; however, no statistical significance was found. This may be the first report to provide follow-up data of p73 expression for lesions of oral ED. Discussion

  24. 3 studies have examined p73 in H&N SCC, including oral SCCs. Our finding supports those previous observations in H&N SCCs. However, the rate of p73 expression in this study was different from that in earlier studies on H&N SCC. Because such differences may be due to the heterogeneity of the H&N SCCs enrolled in the previous studies, a homogeneous & well-characterized series of buccal SCCs was used for our investigation. Discussion

  25. Mutation of p73 has rarely been found in human cancers. It remains possible that wild-type p73, but not mutant p73, might have been overexpressed for oral dysplastic & cancerous keratinocytes in this study, suggesting that p73 may play an important role in oral carcinogenesis through the overexpression of wild type p73 rather than of mutant form as a tumor suppressor. This idea is further supported by the discovery of a new isoform of human p73, Np73, with possible oncogenic potential. Discussion

  26. Results of IHC analyses in this study indicate that p73 proteins are chiefly restricted to less-differentiated cells situated in the basal layers of normal stratified squamous epithelium. p73 proteins are found in less-differentiated cells in the periphery of carcinomatous clusters of well-differentiated carcinomas, whereas moderately differentiated carcinomas revealed more homogeneous (cell) staining, involving almost all of the tumor cells. These observations suggest that p73 protein may be related to the differentiation of oral stratified squamous epithelium. Discussion

  27. No studies have examined the effects, if any, of betel-quid chewing on p73 expression for various types of cancer. All patients in this study were betel-quid chewers. It may be of interest, to test the association between betel-quid chewing & p73 expression. A corroborative conclusion could be achieved by comparing p73 activity between patients with oral cancer who are not habitual betel-quid chewers & those who do chew betel quid but exhibit no disease in their buccal mucosa. Discussion

  28. Our data suggest that p73 expression may be: (1) Associated with the differentiation of oral stratified squamous epithelium caused by the lack of p73 immunoreactivity for areas of keratin pearl & positive p73 expression for the less-differentiated cells of carcinomatous tissue change of oral epithelial dysplastic lesions. Summaries

  29. Our data suggest that p73 expression may be: (2) An early event in human oral carcinogenesis & upregulated during development from normal mucosa to dysplastic or carcinomatous lesions. Our data suggest that p73 expression may be: (3) Associated with the nodal status of patients with oral carcinoma, as well as a possible indicator for malignant change of oral epithelial dysplastic lesions. Summaries

  30. Your constructive comments are appreciated 謝 謝 감 사 합 니 다

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