P53, p16 and Ki67 immunoexpression in oral squamous carcinomas

In this study, we have analyzed clinically, histopathologically and immunohistochemically a total of 34 cases of oral squamous carcinoma in 11 of the cases being identified adjacent epithelial dysplastic lesions. Carcinomas were diagnosed in patients aged 40-60 years, males, with chronic exposure to tobacco and/or alcohol, being located especially on the lips. Well-differentiated carcinomas have been predominant (52.9%) in stage I/II tumoral (88.3%). Immunoexpression analysis of p53, p16 and Ki67 did not reveal statistically significant differences between the expression of markers and clinical or histopathological parameters, except Ki67 whose increased expression was associated to the decrease of the degree of tumoral differentiation and with high degree dysplasia. The positivity index and the intensity of reaction were increased at the level of dysplasic epithelium for p16 and at the level of tumoral invasion front for the p53 and Ki67. The study highlights the value of the immunostain for p16 in identifying dysplasic lesions and predictive importance of p53 and Ki67 markers in identifying the aggressive forms of oral carcinomas.     

Immunohistochemical expression of p16 protein in oral squamous cell carcinoma and lichen planus

Epithelial carcinogenesis is a multistep process. Specific genetic events lead to malignant transformation of oral epithelium. Oral squamous cell carcinoma (OSCC) may be preceded by potentially malignant lesions such as oral lichen planus (OLP). The p16 protein functions as a negative regulator of the cell cycle progression. Altered pattern of p16 serves as a biomarker for oral mucosal dysplasia and malignant growth. The purpose of this study was to evaluate p16 expression in OSCC and OLP to determine whether it can be a useful marker for early detection of carcinogenesis. We examined p16 expression in 45 OSCCs (15 grade I, 15 grade II, and 15 grade III), 15 OLPs without dysplasia, and 8 normal mucosal specimens with immunohistochemistry. p16 was interpreted as positive if more than 70% of tumor cells showed brown nuclear and cytoplasmic staining. All of the OSCC and control group samples showed negative immunoreactivity, whereas 26.7% of OLP samples were positive for p16. Our findings suggest that p16 expression could not be used as a helpful marker for detection of development toward malignancy in OLP samples.     

Deregulated Bag-1 protein expression in human oral squamous cell carcinomas and lymph node metastases

Bag-1 is an anti-apoptotic protein that promotes metastasis in some tumour cell types. To determine whether Bag-1 expression is altered in 64 oral squamous cell carcinomas, tumour samples were compared with 17 samples of normal oral epithelium. Normal oral epithelia had pronounced nuclear staining in the basal and maturation layers and weak cytoplasmic staining that was most pronounced in the basal and suprabasal layers. Oral squamous cell carcinomas demonstrated a tendency for reduced nuclear staining intensity (p=0.036). Cytoplasmic staining intensity was not significantly different between tumour and normal tissue. However, many tumours were observed to have less of a difference between nuclear staining intensity and cytoplasmic staining intensity than normal oral epithelium. Furthermore, in lymph node metastases, cytoplasmic Bag-1 staining was stronger in 8/13 cases than in corresponding primary tumours (p=0.021). Western blotting using nine oral primary carcinoma cell lines and four normal keratinocyte cultures showed that the isoforms Bag-1s, Bag-1M, and Bag-1L were expressed in normal and malignant oral epithelial cells. Bag-1L unique sequences were shown to adopt an exclusively nuclear, and predominantly nucleolar, localization by use of transiently transfected N-terminal Bag-1L-EGFP. However, levels of Bag-1L in carcinoma cells did not differ significantly from those of normal keratinocytes. Therefore the reduced nuclear staining observed in oral squamous cell carcinomas compared with normal epithelium may reflect changes in the localization of Bag-1 isoforms, rather than decreased expression of Bag-1L. Alterations in the relative proportions of Bag-1S, Bag-1M, and Bag-1L were detected in 6/9 oral carcinoma cell lines; 5/9 oral carcinoma cell lines had a significantly greater proportion of Bag-1M than normal keratinocytes and in another cell line, Bag-1L was significantly underrepresented. Overall, the results suggest that Bag-1 deregulation plays a role in oral carcinogenesis at two different stages: during primary carcinoma development and during lymph node metastasis.     

Abnormalities of tumor suppressor genes P16 and P15 in primary maxillofacial squamous cell carcinomas.

As members of the same gene family, tumor suppressor genes P16/CDKN2/INK4A and P15/INK4B have a high degree of structural and functional homology with both P16 and P15 proteins involved directly in the regulation of cell cycles. However, the status of P16 and P15 genes in primary maxillofacial squamous cell carcinomas (MSCC) has not been reported. Studies on abnormalities of these genes including homozygous deletion, methylation of the 5'CpG islands, and mutations were carried out in 65 primary MSCC with polymerase chain reaction (PCR), methylation-specific PCR (MSP), PCR-SSCP (single-strand conformation polymorphism), and DNA sequencing techniques. Of the 65 tumors, 22 (34%) were methylated; 7 (11%) displayed point mutations. The total frequency of alteration of the P16 gene was 43% (28/65). The methylation rate of P15 was 12% (8/65). No homozygous deletion was found in either the P16 gene or P15 gene. In all MSCC samples, almost half (49%) harbored an alteration of the P16 or P15 gene. The P16 gene was altered more frequently than P15, and therefore is inactivated by methylation or mutation in a significant proportion of MSCC. The P15 gene appeared to play a lesser role in tumorigenesis of these tumors.     

PDCD5和Smac在口腔鳞癌中的表达及临床意义

目的研究凋亡相关新基因PDCD5与Smac蛋白在口腔正常黏膜、口腔鳞癌中表达的相关性及其意义。方法采用免疫组化方法检测68例口腔鳞癌组织和43例癌旁正常黏膜组织中PDCD5和Smac的表达,并分析两者的表达与临床病理的关系以及两者之间相互关系。结果正常口腔黏膜组PDCD5染色阳性率为80.2%(P0.05),口腔鳞癌组PDCD5阳性率为29%(P0.05),明显低于癌旁组织,并且表达与TNM分期、淋巴结转移相关性有统计学意义(P0.05)。Smac在正常口腔黏膜组染色阳性率为41.2%(P0.05),明显高于口腔鳞癌组织11.7%(P0.05),且与肿瘤的分化程度、TNM分期、淋巴结转移的相关性均有统计学意义(P0.05)。PDCD5和Smac蛋白呈明显正相关(r=0.892,P0.05)。结论PDCD5和Smac蛋白在口腔鳞癌中表达下调,提示PDCD5与Smac蛋白的改变可能与口腔鳞癌的发生、发展相关,这两项指标可作为辅助口腔黏膜癌变的基因标志物。     

Cytogenetic abnormalities in 106 oral squamous cell carcinomas

We report karyotypic features of 106 short-term cultured oral squamous cell carcinomas (SCC), 51 new and 55 previously reported cases, with clonal chromosome aberrations. The major cytogenetic findings were as follows: simple karyotypic changes were present in 38 cases (36%) and 68 tumors (64%) displayed complex karyotypes. The most common numerical changes were +7, +8, +9, +16, +18, +20, and -4, -10, -13, -14, -18, -19, -21, -22, and -Y. Structural rearrangements frequently (43% of the breaks) affected the centromeric regions, resulting in the formation of isochromosomes and whole-arm translocations. Among the recurrent structural aberrations identified, the most common were i(1q), i(3q), i(5p), i(8q), del(16)(q22), and hsr. With the exception of chromosomal band 11q13, which was involved in 25 tumors, only centromeric or near-centromeric bands were commonly involved: 3p11 approximately q11 (59 cases), 8p11 approximately q11 (57), 1p11 approximately q11 (48), 13p11 approximately q11 (46), 5p11 approximately q11 (41), 14p11 approximately q11 (41), and 15p11 approximately q11 (37). Losses of genetic material dominated over gains. The most frequent imbalances included loss of 2q33 approximately qter, 3p, 4p, 6q, 8p, 10p, 11q, 13p, 14p, and 15p, and chromosomes 18, 21, 22, and Y, and gain of chromosomes 7 and 20, 8q, and 11q13. No major karyotypic differences could be discerned between the present series of oral SCC and a previously reported series of laryngeal SCC, indicating that common genetic pathways are involved in the initiation and progression of SCC irrespective of site of origin.     

Parathyroid hormone related protein in oral squamous cell carcinomas invading the mandible.

AIM--To assess parathyroid hormone related protein (PTHrP) as a candidate biochemical marker of invasion of the mandible by oral squamous cell carcinoma. METHODS--Tumour PTHrP concentrations were quantitated by immunoassay, and PTHrP was detected by immunohistochemistry, in a cohort of 24 primary squamous cell carcinomas of the mandible. RESULTS--PTHrP was identified in all tumours examined, but no correlation was found between scores of the intensity and/or consistency of staining or tumour PTHrP concentrations and the histological classification of tumour invasion. CONCLUSION--Although PTHrP was present in all squamous tumours studied, there was no correlation between PTHrP expression and pattern of tumour invasion. However, tumour derived PTHrP may act locally to influence tumour growth and differentiation and resorption of bone.     

EGFR protein expression and gene amplification in squamous intraepithelial lesions and squamous cell carcinomas of the cervix

The purpose of this study was to evaluate the protein expression and gene amplification of epithelial growth factor receptor (EGFR) in intraepithelial neoplasias and squamous cell carcinoma of the cervix and to determine the value of EGFR in carcinogenesis, progression, and prognosis of cervical cancer. EGFR protein expression and gene amplification involved gene copy number in 75 cases of cervical various lesions were evaluated using immunohistochemistry and by fluorescence in situ hybridization (FISH) techniques. Expression of EGFR was observed in 76.00% of the high-grade CIN and 79.17% of the invasive carcinomas. In contrast, there were low levels of EGFR expression in chronic cervicitis (1/10) and low-grade CIN (7/16). There were statistically significant differences among them (P<0.05). Gene amplification was detected in 20.51% high-grade CIN and invasive carcinoma, but there only 4.35% EGFR gene amplification was observed in chronic cervicitis and low grade CIN. Among the 42 patients with negative or low levels of EGFR expression, 26 patients (61.90%) were found to have diploidy and 11 patients (26.20%) to have balanced triploidy. However, among the 20 patients with an intermediate and high levels of EGFR protein expression, 13 (65.00%) were found to have balanced polyploidy or gene amplification. All cases of EGFR gene amplification involved intermediate and high levels of protein expression. EGFR may be involved in the carcinogenesis of the cervix and may be an early event during the carcinogenesis. Overexpression of EGFR protein may result from gene amplification and increases in gene copy number.     

乳腺癌细胞中PELP1基因表达与启动子区CpG岛甲基化相关性

目的建立针对PELP1基因启动子区CpG岛的甲基化特异性PCR(Methylation specific PCR,MSP)检测方法,分析乳腺癌细胞中PELP1基因表达与启动区CpG岛甲基化的相关性。方法设计针对PELP1基因启动子区的MSP引物组,以甲基化和非甲基化DNA为模板验证MSP引物的特异性,建立针对PELP1基因启动子区的MSP检测方法。以DNA甲基转移酶抑制剂5'-氮杂-脱氧胞苷磷酸(5'-Aza-d C)分别处理MCF-7乳腺癌细胞、MCF-10正常乳腺导管上皮细胞,采用MSP检测PELP1基因启动子区甲基化状态变化,采用Western blot检测蛋白表达。结果针对PELP1基因启动子区设计的MSP引物组特异性良好,甲基化特异性引物仅在甲基化DNA模板获得阳性扩增条带,非甲基化引物仅在非甲基化DNA模板获得阳性条带。MCF-7乳腺癌细胞中PELP1基因启动子区呈非甲基化状态,MCF-10正常乳腺导管上皮细胞中PELP1基因启动子区呈甲基化状态,MCF-10细胞中PELP1蛋白表达水平为MCF-7细胞的1/16(P0.05)。采用5'-Aza-dC去除MCF-10细胞PELP1基因启动子区甲基化后,PELP1蛋白表达水平上升了9.7倍(P0.05)。结论所建立的PELP1基因启动子区MSP检测方法特异性良好,去甲基化可能是导致乳腺癌细胞中PELP1基因过表达的重要机制。     

Frequent methylation of DAB2, a Wnt pathway antagonist,in oral and oropharyngeal squamous cell carcinomas

Background

Aberrations in Wnt signaling pathway are related to the pathogenesis of head and neck carcinomas and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of the methylation of DAB2, which acts as a negative regulator of Wnt signaling, and correlate it with clinicopathological features in a group of oral cancer patients.

癌基因c-erbB2、c-myc和抑癌基因p16、p53在口腔鳞癌中的蛋白表达及其协同作用

目的：探讨癌基因c—erbB2、c-myc和抑癌基因p16、p53在口腔鳞癌(OSOC)中的蛋白表达及其协同作用。方法：用免疫组化结合图像分析对口腔鳞癌中4种基因的蛋白表达进行定性、定位、定量研究。结果：口腔鳞癌中c—erbB2、c—myc、p16和p53的蛋白表达阳性率依次为46．67％、60％、86．67％和63。33％。肿瘤部位不同,erbB2蛋白表达有显著性差异;腭癌和口底癌中的erbB2蛋白表达都明显高于唇癌和牙龈癌中的erbB2表达。c-myc蛋白表达与p16蛋白表达之间具有显著性相关。结论：以上4种基因的蛋白表达增高在口腔鳞癌发生发展中具有重要作用,c—erbB2蛋白过表达在腭癌和牙龈癌中具有更重要的意义;c—myc和p16蛋白表达间具有一定的内在联系。     

Human papillomavirus DNA in oral squamous cell carcinomas and normal oral mucosa

To elucidate the putative etiologic role of human papillomaviruses (HPV) in oral carcinogenesis, a comparative study was carried out on 62 tissue specimens of oral squamous cell carcinoma (OSCC) and on 62 specimens of histologically normal oral mucosa obtained from the individuals who matched the subjects with OSCC in age, gender, localization of obtained tissue specimens, drinking and smoking habits. Internal control amplification showed that amplifiable DNA was recovered from 59/62 and 61/62 tissue samples of OSCC and normal oral mucosa, respectively. The amplification with two different HPV L1 and one HPV E6 consensus primer sets showed the presence of the HPV DNA genotypes 16, 33, 58 in 5/59 (8.4%) OSCC specimens and HPV genotypes 11, 16, 31, 68 in 4/61 (6.6%) tissue samples of normal oral mucosa tested. In the study in which a comparative examination of the presence of HPV DNA was for the first time performed on the tissue samples of the patients with OSCC and the age- and gender-matched control subjects there was no significant difference in the prevalence of HPV DNA among both study groups. Our results suggest that occasional findings of HPV DNA in OSCC tissue specimens may be the result of an incidental HPV colonization of oral mucosa, rather than of viral infection, and that HPVs play a limited role in the etiopathogenesis of the majority of OSCC.     

Immunohistochemical expression of angiogenesis-related markers in oral squamous cell carcinomas with multiple metastatic lymph nodes

The aim of this study was to evaluate the histopathologic features and the expression of angiogenesis-related markers in primary tumors and metastatic lymph nodes of oral squamous cell carcinomas (SCCs) with multiple lymph node involvement in comparison with oral SCCs without nodal metastasis. The protein levels of the angiogenesis inhibitor endostatin, as well as those of the related molecules collagen XVIII, collagen-binding protein (CBP) 2/heat shock protein (HSP) 47, and cathepsin L, were evaluated by immunohistochemical analysis. Compared with nonmetastatic cases, primary tumors of the metastatic group exhibited significantly decreased protein levels of endostatin and its precursor collagen XVIII. Comparison between primary tumors and positive nodes of the metastatic cases revealed decreased expression of collagen XVIII and CBP2/HSP47 in metastases. Angiogenesis is essential for tumor growth and metastasis; accordingly, the observed differences in the immunohistochemical expression of angiogenesis-related proteins in oral SCC with multiple lymph node involvement may provide an explanation for the increased metastatic potential of these tumors.     

p53, p16 and RB expression in adenosquamous and squamous cell carcinomas of the stomach

The expression of p53, p16 and RB proteins and their clinicopathologic correlation were investigated in 15 cases of primary gastric adenosquamous carcinoma and 2 cases of squamous cell carcinoma of the stomach. The male to female ratio of the patients was 13:4 and the average age was 55.7 years. None of the cases was early gastric carcinoma, and none of the adenocarcinoma components were of the diffuse or signet ring cell types. Fourteen cases showed metastasis to regional lymph nodes and/or other organs at the time of surgery. The adenocarcinoma component was metastasized to lymph nodes in 12 cases, and both adenocarcinoma and squamous cell carcinoma components were metastasized in three cases. The altered expression of p53 correlated with the advanced stage, but did not correlate with the depth of invasion, lymph node metastasis or recurrence. The altered expression of p16 and RB proteins did not correlate with any of the above clinico-pathologic factors. Both the adenocarcinoma and squamous cell carcinoma components revealed an inverse correlation between the expression pattern of p16 and RB proteins (p < 0.05). This suggests that the two proteins share a role in the carcinogenesis of these tumors. The expression pattern of p53 proteins in the adenocarcinoma and squamous cell carcinoma components was exactly the same in all of the cases. The expression patterns of p16 and RB protein were also identical in most of the cases. The expression patterns of all three proteins in the metastatic lesions were also identical to those in the primary lesions. The fact that the alteration of the three tumor suppressor gene products shares the same pattern suggests that squamous and adenocarcinoma components in the stomach originate from the same or a genetically related clone.     

Immunohistochemical comparison of p16 expression in actinic keratoses and squamous cell carcinomas of the skin.

There are approximately 200,000 new cases of cutaneous squamous cell carcinoma diagnosed each year in the United States, with between 1300 and 2300 deaths per year from metastatic disease. The tumor suppressor p16, encoded by the CDKN2/INK4a locus, has been reported mutated in >or=24% of squamous cell carcinomas. Mutations of the p16 gene have also been found in actinic keratoses, the first identifiable lesion in the continuum from normal skin to squamous cell carcinoma. We hypothesized that there may be an appreciable difference in expression of p16 between normal skin, actinic keratoses, squamous cell carcinoma in situ, and invasive squamous cell carcinoma. Ten actinic keratoses, 10 in situ squamous cell carcinomas, and 10 invasive squamous cell carcinomas were examined using the immunoperoxidase method with antigen retrieval for anti-p16(INK4a) antibody. All 10 actinic keratoses were positive for weak to moderate p16 staining in the lower third to lower half of the epidermis (especially the basal keratinocytes). This staining was significant when compared with the lack of staining seen in normal skin controls. Twenty percent of in situ squamous cell carcinomas had moderate to strong staining in only the lower half to lower two thirds of the epidermis, whereas 70% of the in situ squamous cell carcinomas exhibited full-thickness p16 staining, with no staining in the dermis. Thirty percent of invasive squamous cell carcinomas had full-thickness staining of the in situ component of the lesion, and 100% of invasive squamous cell carcinomas exhibited moderate to strong staining of the invasive component of the lesion. These findings indicate correlation between the increased expression of p16 during the progression of skin from actinic keratosis to in situ squamous cell carcinoma to invasive squamous cell carcinoma. These data may lend further support to the view of the actinic keratosis as a precursor lesion to squamous cell carcinoma.     

Comparison of integrin,cadherin, and catenin expression in squamous cell carcinomas of the oral cavity

In addition to their role in maintenance of tissue integrity, cell adhesion molecules regulate the growth and differentiation of stratified squamous epithelia. Reduced expression of E-cadherin and the α₂β₁, α₃β₁ and α₆β₄ integrins is already reported to correlate with poor histological differentiation in oral squamous cell carcinomas. However, it is not clear how closely cadherin and integrin loss are related in any given tumour, nor whether cadherin loss is correlated with changes in expression of the cytoplasmic regulatory proteins known as catenins. Double-label immunofluorescence has been used to stain a panel of 22 oral squamous cell carcinomas with antibodies to ten proteins, including E- and P-cadherin, the major keratinocyte integrin subunits, and α-, β- and γ-catenin. Overall, E-cadherin expression and integrin expression correlated well with tumour grade, while P-cadherin staining was more variable. All tumours, regardless of differentiation status, showed reduced staining for at least two of the catenins, implying that the adhesive function of E- and P-cadherin could be impaired even when cadherin expression is normal. It is concluded that in all squamous cell carcinomas, regardless of degree of histological differentiation, there is some perturbed expression of cell adhesion molecules and that integrin and E-cadherin loss are closely related. © 1998 John Wiley & Sons, Ltd.     

Relationship between the extent of chromosomal losses and the pattern of CpG methylation in gastric carcinomas

The extent of unilateral chromosomal losses and the presence of microsatellite instability (MSI) have been classified into high-risk (high- and baseline-level loss) and low-risk (low-level loss and MSI) stem-line genotypes in gastric carcinomas. A unilateral genome-dosage reduction might stimulate compensation mechanism, which maintains the genomic dosage via CpG hypomethylation. A total of 120 tumor sites from 40 gastric carcinomas were examined by chromosomal loss analysis using 40 microsatellite markers on 8 chromosomes and methylation analysis in the 13 CpG (island/non-island) regions near the 10 genes using the bisulfite-modified DNAs. The high-level-loss tumor (four or more losses) showed a tendency toward unmethylation in the Maspin, CAGE, MAGE-A2 and RABGEF1 genes, and the other microsatellite-genotype (three or fewer losses and MSI) toward methylation in the p16, hMLH1, RASSF1A, and Cyclin D2 genes (p<0.05). The non-island CpGs of the p16 and hMLH1 genes were hypomethylated in the high-level-loss and hypermethylated in the non-high-level-loss sites (p<0.05). Consequently, hypomethylation changes were related to a high-level loss, whereas the hypermethylation changes were accompanied by a baseline-level loss, a low-level loss, or a MSI. This indicates that hypomethylation compensates the chromosomal losses in the process of tumor progression.