p73 Expression for Human Buccal p73 Expression for Human Buccal Epithelial Dysplasia & Squamous Cell Epithelial Dysplasia & Squamous Cell

Carcinoma: Does It Correlate with Nodal Carcinoma: Does It Correlate with Nodal Status of Carcinoma & Is There a Status of Carcinoma & Is There a

Relationship with Malignant Change of Relationship with Malignant Change of Epithelial Dysplasia?Epithelial Dysplasia?

高雄醫學大學 口腔醫學院 高雄醫學大學 口腔醫學院 牙醫學系 口腔病理科 牙醫學系 口腔病理科

2727thth, August, 2009, August, 2009

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

A Series of Tumor Markers Studies

Studies on Human Oral & DMBA-Induced Studies on Human Oral & DMBA-Induced Hamster Buccal-PouchHamster Buccal-Pouch

Squamous cell CarcinogenesisSquamous cell Carcinogenesis

Studies on Human Oral & DMBA-Induced Studies on Human Oral & DMBA-Induced Hamster Buccal-PouchHamster Buccal-Pouch

Squamous cell CarcinogenesisSquamous cell Carcinogenesis

*Year of First Publication

NSC Projects Derived From thep53 Family Studies

NSC Projects Derived From thep53 Family 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)

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)

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)

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

Human Studies

Publications Derived From thep53 Family Studies

Publications Derived From thep53 Family Studies

Animal Studies

1. 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)

Animal Studies

1. 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

1. Clin Otolaryngol 2003;28:451-5 (12)*

2. Int J Oral Maxillo- fac Surg 2004;33: 493-7 (9)

3. Head & Neck 2004;26:945-52 (5)

4. J Oral Pathol Med 2005;34:232-9 (8)

Human Studies

1. Clin Otolaryngol 2003;28:451-5 (12)*

2. Int J Oral Maxillo- fac Surg 2004;33: 493-7 (9)

3. Head & Neck 2004;26:945-52 (5)

4. J Oral Pathol Med 2005;34:232-9 (8)

* Frequency to be cited

I.F.2008= 2.603 (1/31)

Introduction

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

p73 also shares some common functions with the p53 protein, including inhibiting cellgrowth & 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

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

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.

Materials & Methods

75 samples of human buccal ED, including mild, moderate, & severe ED (25 samples in each category), aged 36-65 yrs (mean, 44 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.

10 samples of human normal buccal mucosa, aged 36 to 62 yrs (mean, 47 yrs).

Materials & Methods Paraffin-embedded tissues

Human buccal SCCs

Immunohistochemistry: p73 protein

Normal oral mucosaHuman buccal EDs

Fresh tissues

RT-PCR: p73 mRNA

Normal oral mucosa

Human buccal SCCs

Materials & Methods

(a) Performed by avidin-biotin peroxidase complex (ABC) method.

(b) Primary rabbit polyclonal anti-p73 Ab (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100 dilution).

(c) Samples with 10% or more positive nuclear-stained keratinocytes were classified as positive staining.

Immunohistochemical staining

Materials & MethodsRT-PCR

RNA extraction Guanidium isothiocyanate- caesium chloride method

Reverse transcription reaction cDNA synthesis

18S 28S

O.D.:1.8-2.0 (260/280nm)

Materials & MethodsThermocycling conditions

PCR cycle for p73

PCR cycle for -actinTaKaRa MP, Tokyo, Japan

Oligoprimers from GenbankPrimers Sequences Product

p73 Forward: 5‘-CTCCCCGCTCTTGAAGAAAC-3'Reverse: 5‘-GTTGAAGTCCCTCCCGAGC-3’

180-bp

-actin Forward: 5‘-AACCGCGAGAAGATGACCCAGATCATGTTT-3'Reverse: 5‘-AGCAGCCGTGGCCATCTCTTGCTCGAA GTC'-3’

350-bp

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).

A B C D

Results

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

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).

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).

p73180-bp

p73180-bp

-actin 350-bp

-actin 350-bp

M 1 2 3 4 5 6 7 8 9 10 11 12 N NC

M 13 14 15 16 17 18 19 20 21 22 23 24 25 N NC

Results

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.

p73 protein p73 mRNA

(+) (-) (+) (-)

Mild ED 17* 8 Not studied

Moderate ED 18* 7 Not studied

Severe ED 18* 7 Not studied

Squamous cell carcinoma

17* 8 17* 8

Normal mucosa 2 8 2 8

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).

Results

p73 expression (protein & mRNA levels) correlated significantly with cervical lymph node metastasis for cases of buccal SCC.

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 protein (+)/mRNA (+)

p73 protein (-)/mRNA (-)

Buccal SCC with cervical node

metastasis15/14 2/3

Results

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.

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).

Moderate ED* Severe ED

p73protein (+)

p73protein (-)

p73protein (+)

p73protein (-)

With malignant transformation

3 0 5 0

Without malignant transformation

15 7 13 7

Discussion 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 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 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 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 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 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

A corroborative conclusion could be achieved by comparing p73 activity between patients with oral cancer who are not habitualbetel-quid chewers & those who do chew betel quid but exhibit no disease in their buccal mucosa.

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.

Summaries 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 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.

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