Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

肺癌组织中PCNA、p63和p53蛋白的表达及临床意义

目的:检测蛋白增殖细胞核抗原(PCNA)、p63和p53在肺癌组织中的表达情况,以探讨三者在肺癌的发生、发展中的生物学作用和临床意义。方法:选取195例肺癌组织(其中57例有癌旁组织),应用组织芯片技术和免疫组织化学方法观察三种蛋白的表达情况,并研究三者之间及其与临床病理参数的关系。结果:PCNA、p63和p53蛋白在肺癌组织中的阳性表达率分别为96.41%、38.46%及58.46%,但三者在癌旁组织中均无表达,差异有统计学意义(均P0.05);在肺癌组织中,PCNA、p63和p53蛋白的表达情况均与组织分型有关(P0.05),且PCNA、p53蛋白表达与分化程度有关(P0.05),分化越差,表达越高;p53表达与PCNA表达呈正相关(r=0.352,P=0.043),p63与p53、PCNA的表达不相关(P0.05)。结论:肺癌组织中PCNA、p63和p53蛋白的表达升高,三者均在肺癌的发生、发展中发挥着重要作用,并且临床可通过检测三者的蛋白水平,作为鉴别肺鳞状细胞癌与其他类型癌的重要参考指标,为病理诊断提供依据。     

The Expression of SIRT1 and DBC1 in Laryngeal and Hypopharyngeal Carcinomas

Rationale and Objective

Sirtuin 1 (SIRT1) plays an important role in tumorigenesis and is increased in many human tumors. DBC1 is a negative regulator of SIRT1 via promotion of p53-mediated apoptosis. It is necessary to investigate the expression of SIRT1 and DBC1 in laryngeal and hypopharyngeal squamous cell carcinomas (LSCC and HSCC) and its correlation with available clinical parameters.

Methods

The mRNA levels of SIRT1 and DBC1 were measured in 54 paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1 and DBC1 were also evaluated in 120 cases of patients with LSCC or HSCC using immunohistochemical staining. The correlation between SIRT1 and DBC1 expression and clinical parameters was analyzed with Pearson chi-square test.

Results

qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1 mRNA was significantly decreased in tumors. The immunohistochemical results indicated that the SIRT1 protein was also downregulated in tumors compared with noncancerous mucosae. Moreover, decreased SIRT1 was significantly correlated with the tumor clinical stage and lymph node metastasis. Additionally, DBC1 mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the DBC1 protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Finally, decreased DBC1 protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53 expression.

Conclusions

SIRT1 and DBC1 might be involved in the pathophysiology of laryngeal and hypopharyngeal squamous cell carcinomas and are associated with lymph node metastasis and p53 positive staining in LSCCs and HSCCs.     

Immunohistochemical detection of early-stage carcinogenesis of oral leukoplakia by increased DNA-instability and various malignancy markers

The degree of DNA instability as determined by immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (the DNA instability test) was used as a marker of malignancy. The test was applied to tissues of oral leukoplakia assessed histopathologically as hyperplasia (38 cases), mild (12 cases), moderate (11 cases) and severe (8 cases) dysplasia, and invasive squamous cell carcinoma (SCC, 20 cases). Tissues were subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA), p53, DNA-fragmentation factor 45 (DFF45), analysis of various AgNORs parameters, and triple immunostaining for vascular endothelial growth factor (VEGF), CD34, and PCNA. The DNA instability test was positive in 20 (100%) SCC cases, 8 (100%) severe dysplasia cases, 8 (72.7%) moderate dysplasia cases, 6 (50.0%) mild dysplasia cases, and 9 (23.7%) hyperplasia cases, indicating malignancy. The proportion of lesions positive for PCNA, p53, DFF45, and values of AgNORs parameters steadily increased from hyperplasia to mild, moderate and severe dysplasia, and SCC, especially in those showing positive DNA instability test, indicative of malignancy. Based on these results, 44.9% of leukoplakia were malignant tissues, namely carcinoma in situ. The proportion of PCNA-positive vascular endothelial cells in the vicinity of VEGF-positive epithelial lesion was significantly higher than that of negative DNA instability lesions, as revealed by immunohistochemical triple staining for VEGF, CD34, and PCNA. Our results suggest that increased DNA instability, enhanced proliferative activity, p53 mutation, and induction of DFF45 and VEGF may allow cancer cell proliferation, enhance their survival by escaping apoptosis, and provide abundant nutrients during early-stage carcinogenesis of oral leukoplakia.     

Expression of vascular endothelial growth factor in the progression of cervical neoplasia and its relation to angiogenesis and p53 status

OBJECTIVE: To evaluate vascular endothelial growth factor (VEGF) expression in the successive steps of cervical neoplasia and to determine its correlation with angiogenesis and p53 status. STUDY DESIGN: Immunohistochemical staining with a VEGF monoclonal antibody was performed on a total of 161 cervical specimens representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 30 CIN 3 and 86 squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody to CD34. Computerized image analysis was used to evaluate microvessel density (MVD). p53 Status was determined by immunohistochemistry and direct sequencing of exons 5-8 of the p53 gene. RESULTS: VEGF expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < .05). MVD increased significantly with cervical neoplasia progression, from normal epithelium, through CIN, to squamous cell carcinoma (P < .001). A strong correlation was observed between VEGF expression and MVD (P < .001). p53 Protein expression was not detected in the normal epithelium or in CIN 1, while 3 (10%) of 30 CIN 3 and 28 (33%) of 86 squamous cell carcinomas were positive for p53. VEGF expression correlated statistically with p53 protein expression (P < .001). In double VEGF- and p53-stained sections, the 2 markers were generally expressed in the same tumor cells. Of the 4 p53 gene mutations, 3 exhibited strong VEGF expression, and 1 exhibited moderate VEGF expression. VEGF expression did not correlate significantly with outcome variables in patients with squamous cell carcinoma. CONCLUSION: Our results suggest that VEGF expression is involved in the promotion of angiogenesis in cervical neoplasia and that p53 is likely to be involved in the regulation of VEGF expression.     

Serum anti-p53 autoantibodies in primary resected non-small-cell lung carcinoma

 Mutated p53 proteins accumulate in the nuclei of tumor cells, and anti-p53 autoantibodies are found in the sera of patients with non-small-cell lung carcinoma (NSCLC). We analyzed the correlation among serum anti-p53 autoantibodies, immunohistochemical staining for p53, and clinical features (age, gender, smoking history, histological type, differentiation, stage, T factor, tumor size, and N factor) in resected non-small-cell lung carcinomas. A total of 62 cases of resected NSCLC were studied (43 men and 19 women; 33 adenocarcinomas, 21 squamous cell carcinomas, 8 large-cell carcinomas). Preoperative serum titers of anti-p53 autoantibodies were detected in 13/62 cases (21.0%). A correlation between histological type and positive titers of serum p53 autoantibodies was seen (large-cell carcinoma versus squamous cell carcinoma and adenocarcinoma, P = 0.031, χ²-test). Out of 25 cases, 10 (40%) with positive immunohistochemical staining for p53 had positive titers, whereas 3 positive titers were found in 37 patients with negative immunohistochemical staining for p53 (P = 0.0025, χ²-test). Serum titers of anti-p53 autoantibodies were present in approximately 20% of the cases of NSCLC, and overexpression of p53 protein in tumor cells was detectable in approximately 40%. Serum anti-p53 autoantibodies may be a clinical parameter for the presence of p53 mutations and p53 overexpression in NSCLC patients. Received: 22 October 1997 / Accepted: 22 April 1998     

Immunohistochemical localization and correlation of p53 and PCNA expression in breast carcinoma

The object of the present study is to detect the p53 tumour suppressor gene and proliferation cell nuclear antigen (PCNA) expression in breast carcinoma by immunohistochemistry and correlate them with the prognostic parameters. Total 35 cases of primary breast carcinoma were studied and classified histologically. Paraffin sections were stained by using monoclonal antibody D07 for p53 protein and PC-10 for PCNA. Out of 35 cases, 16 (45.7%) were p53 positive and 25 (71.4%) were PCNA positive. The mean PCNA labelling index (PCNA LI +/- SD) was 58.97 +/- 22.72 in tumors positive for both p53+ and PCNA+ while cases negative for p53- and positive for PCNA+ has higher PCNA LI +/- SD (59.24 +/- 18.97). The difference in the two groups was not significant. Most cases were positive for both p53+ and PCNA+ in the age group < 30 with higher mean PCNA LI +/- SD (62.20 +/- 27.13) than in the group > 30 (57.88 +/- 18.47). In the pre-menopausal group 57.1% cases were positive for p53+ with higher PCNA LI +/- SD (59.94 +/- 24.22). Maximum p53 and PCNA positivity was observed in grade III tumors (63.2% and 84.2%). The mean PCNA LI +/- SD was also highest in grade III carcinomas (66.83 +/- 13.97). No significant correlation was found between p53 and PCNA status with morphological type and tumour size except that logistic regression showed a positive correlation with tumour grade. Therefore the present study suggests that both p53 expression and PCNA are markers of poor differentiation in breast cancer.     

Glycoprotein CD44 expression in benign, premalignant and malignant epithelial lesions of the larynx: an immunohistochemical study including correlation with Rb, p53, Ki-67 and PCNA.

CD44 is an integral membrane glycoprotein that has diverse functions in cell-cell and cell-substrate interactions. It has been suggested that it may be a determinant of metastatic and invasive behavior in carcinomas. The immunohistochemical expression of CD44 was examined in a series of 34 squamous cell carcinomas, 13 in situ carcinomas, 35 cases with various degrees of epithelial dysplasia, 10 papillomas and 17 cases of keratosis. We used the monoclonal mouse anti-human phagocytic glycoprotein-1 CD44 (clone DF 1485), on formalin-fixed, paraffin-embedded tissue. CD44 expression was correlated with the expression of Rb and p53 proteins, with the proliferative indices Ki-67 and PCNA as well as with conventional clinicopathological data. The mean value of CD44 expression was 78.84 in squamous cell carcinomas, 78.04 in situ carcinomas, 54.93 in dysplasia, 26.8 in papillomas and 24.97 in keratosis. There was no significant difference of CD44 expression between in situ and invasive carcinomas. However, a strong difference of reaction between carcinomas and the other cases was observed. CD44 expression was statistically higher in dysplastic lesions than the cases of keratosis (p < 0.0001) and papillomas (p = 0.01). In the group of invasive carcinomas, CD44 expression was statistically correlated with pRb (p = 0.011), while in preinvasive lesions it was correlated with PCNA (p = 0.016). The relationship with the degree of dysplasia or grade of carcinoma and p53 protein expression was insignificant. These observations suggest that CD44 expression may be involved in the multiple mechanism of the development and progression of laryngeal lesions and may help to predict the risk of transformation of the benign or precancerous lesions to cancer.     

Immunohistochemical expression of retinoblastoma gene product (Rb), p53 protein, MDM2, c-erbB-2, HLA-DR and proliferation indices in human urinary bladder carcinoma

Archival biopsy specimens from transitional cell bladder cancers (n=88) were analysed immunohistochemically for the expression of the retinoblastoma (Rb) gene protein, p53, mdm2, c-erbB-2, HLA-DR antigen and proliferation indices. An altered nuclear expression of Rb, p53 and mdm2 was observed in 55.2%, 33.3% and 18.2% of tumors respectively. Cytoplasmic membrane immunoreactivity (>25% tumor cells) for c-erbB-2 was detected in 14.1% of tumors and aberrant HLA-DR antigen cytoplasmic staining (>5% of tumor cells) in 22.2% of the cases. P53 overexpression was associated with higher tumor grade and stage. Aberrant HLA-DR antigen expression and PCNA were also correlated with the grade of differentiation and tumor stage. MIB1 was statistically correlated with stage. pRb scores and HLA-DR antigen expression were correlated with proliferation activity as determined by PCNA and MIB1 immunostaining. p53 protein was also strongly correlated with the proliferation index PCNA. A strong correlation between PCNA and MIB1 (p<0.0001) was also found. In addition a statistically positive correlation between p53 and HLA-DR antigen expression was observed. Our data show that, although pRb and p53 protein expressions are not associated between them, they may contribute to the growth fraction of the bladder cancer. In addition, p53 and HLA-DR antigen expression could be indicators of aggressive behavior of bladder cancer.     

Nuclear image analysis of immunohistochemically stained cells in breast carcinomas

Hitherto, the relationship between malignancy-associated morphological features in single tumour cells and the expression of markers indicating functional properties of these cells remained widely unknown. This study was aimed at describing differences in the size, shape and chromatin structure between tumour cells with different marker expression for progesterone receptors (PgR) and p53. Two series of breast cancers, consisting of 50 PgR-positive, and 39 p53-negative and 49 p53-positive mammary carcinomas, were investigated. The immunohistochemical staining was performed on paraffin sections using 3-amino-9-ethylcarbazole as the chromogenic substrate. By means of a cytometry workstation equipped with a computer-controlled motorised scanning stage, about 500 positive and negative tumour cells in each case were localised in the microscope and categorised by a scoring system for their staining intensity. After destaining, the tissue sections were Feulgen-stained. Then, all the tumour cells were relocated automatically and analysed by high resolution image cytometry. Among the numerous size, shape, and texture features used in the system, several variables of the nuclear contour and chromatin structure were found to be significantly different between the positive and negative tumour cell populations. Nuclei without PgR had more malignancy-associated morphological features than PgR-positive cells. Whereas p53-negative nuclei had a higher degree of regularity, their positive counterparts exhibited higher DNA ploidy values.     

Early progression stage of malignancy of uterine cervical dysplasia as revealed by immunohistochemical demonstration of increased DNA-instability

The degree of DNA-instability as revealed by the immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. This was applied to mild dysplasia (42 cases), moderate dysplasia (43 cases), severe dysplasia (27 cases), squamous cell carcinoma in situ (CIS) (21 cases), invasive squamous cell carcinoma (SCC) (31 cases) and normal (7 cases) human uterine cervix. The expression of tumour suppressor gene p53 and oncogene bcl-2 was detected immunohistochemically. Proliferative activity was evaluated by PCNA immumohistochemistry and the quantitative analysis of the number, mean area, the largest area and maximum shape irregularities of AgNOR in a nucleus were performed for all these cases. The distribution of numeric chromosomal aberrations of chromosome 17 was also investigated in some of these cases. The results showed that 31 SCC (100%), 21 CIS (100%), 21 severe dysplasia (77.77%), 28 moderate dysplasia (65.11%), and 14 mild dysplasia (33.33%) were positively stained by the DNA-instability test diffusely or sporadically, indicating their malignancy. Reflecting the malignant character, these cases showed a remarkable increase in the PCNA-index with the loss of polarity of PCNA positive cell distribution and also an increase in number, mean and largest sizes and maximum shape irregularity of AgNOR dots. The mean chromosome index for chromosome 17, p53 and bcl-2 immunostaining positivity were also found to be significantly increased in moderate and severe dysplasia and in cancerous cases in comparison to normal and mild dysplasia cases. Moreover, the DNA-instability-test positive dysplasia cases showed statistically significant increased values of PCNA-index, AgNOR parameters, mean chromosome index, p53 and bcl-2 expression in comparison to those of DNA-instability-test negative dysplasia cases. In conclusion, some mild dysplasia (33.33%) and most of the moderate (65.11%) and severe dysplasia (77.77%) were regarded as malignant in nature, existing at an early stage of progression of malignancy.     

PTEN、p57／Kip2和CK19在非小细胞肺癌中的表达及意义研究

探讨非小细胞肺癌中PTEN、p57/Kip2和CK19的表达情况和临床意义。选取58例非小细胞肺癌手术切除标本,采用SP法进行免疫组织化学染色。PTEN、p57/Kip2和CK19的阳性表达率分别为44.8%、56.9%和98.3%。PTEN和p57/Kip2在低分化肿瘤中表达显著降低或缺失,在腺癌中的表达强度高于鳞癌;CK19几乎在所有的肺癌组织中均表达,且在腺癌中的表达强度高于鳞癌。PTEN和p57/Kip2的低表达参与肿瘤的生长分化和进展,并提示预后不良。     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Genetic instability promotes the acquisition of chromosomal imbalances in T1b and T1c breast adenocarcinomas.

In order to evaluate biological and genetic properties of early breast carcinomas we analyzed microdissected tissue from 33 primary breast carcinomas stage T1b and T1c with respect to the nuclear DNA content, the expression pattern of Ki-67, cyclin A, p27KIP1, p53 and p21WAF1, and chromosomal gains and losses. The results show that T1b carcinomas (6-10 mm, n=17) were frequently near-diploid (53%) with low proliferative activity and staining patterns of p53 and p21WAF1 that suggest the presence of wild type protein. The majority (12/16) of the T1c tumors (11-20 mm), however, was aneuploid, and proliferative activity and p53 expression were increased. Larger tumor size correlated with an increasing number of chromosomal copy number changes and in particular with regional amplifications. High level copy number increases (amplifications), however, were found exclusively in the aneuploid tumors. Amplification events correlated with elevated cyclin A and reduced p27 expression, respectively. Our results suggest that the sequential acquisition of genomic imbalances during tumor progression is accelerated in aneuploid tumors, and may contribute to the increased malignancy potential.     

Expression of Mina53 and its significance in gastric carcinoma

AIM: To study the expression of Mina53 and its relationships with clinicopathological characteristics, antioncogene inactivation and tumor proliferation in human gastric carcinoma, and to explore the role of Mina53 in carcinogenesis and tumor progression. METHODS: Expression of Mina53 and proliferating cell nuclear antigen (PCNA) was determined in gastric carcinoma (n=79), gastric dysplasia (n=21) and normal gastric tissues (n=20), while p53 was measured in gastric carcinoma tissues by immunohistochemistry. RESULTS: Mina53 was negatively expressed in all normal mucosa tissues. Dysplasia specimens showed weakly positive staining for Mina53 in 3 of 21 cases. Elevated expression of Mina53 was observed in 72 (91.1%) of the gastric carcinomas. No significant associations were found between Mina53 and clinicopathological characteristics such as sex, age, histological differentiation, distant metastasis and lymph node metastasis (p>0.05). There was a significant association with depth of invasion (X2=5.385, p<0.05) and TMN stage (X2=6.255, p<0.05). In gastric carcinoma, positive staining for p53 was detected in 53 of 79 cases (67.1%), showing a significant association with Mina53 (X2=5.161, p<0.05). The mean (+/- SD) PCNA labeling index for gastric carcinoma was 39.47+/-16.92%. Mina53 expression was positively associated with PCNA level (r=0.756, p<0.01). CONCLUSION: Mina53 was overexpressed in gastric carcinoma and associated with tumor proliferation and antioncogene inactivation. Mina53 could therefore play an important role in the carcinogenesis and progression of gastric carcinoma.     

海藻糖毒性的研究

海藻糖（ｔｒｅｈａｌｏｓｅ）作为一种保护剂,其安全性是一项重要的质量指标。本文通过对酵母中提取的海藻糖进行最大耐受量、急性毒性及过敏性的研究,证明海藻糖对动物无毒无害,无副作用,从而推测出海灌糖在人体全地产生不良反应,即是安全的。     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

p53 and PCNA Expression in Malignant Melanomas of the Head and Neck

Mutation in the p53 tumor suppressor gene is the most common genetic alteration in human cancer. As in mutant p53 the protein is stabilised and the half-life is extended, it becomes detectable by immunohistological staining. p53 immunoreactivity thus seems to be a potential biomarker for the assessment of the oncogenic potential of malignant melanomas. In 103 tissue sections of primary and metastatic malignant melanomas of the head and neck detectable levels of p53 were only found in 3 of the primary tumors and in none of the metastases. At the same time the proliferation status of the malignant melanoma lesions was determined using the cell cycle specific antibody PCNA. 55 primary and metastatic tumors were stained with a PCNA-MAb to determine the proliferation activity of the tumors. The results of our immunohistochemical investigation suggest that immunoreactivity of p53 cannot be used to determine the malignant potential of melanomas in the head and neck. PCNA staining showed that the majority of the tumors and metastases were proliferating rapidly.     

Survivin在肺鳞癌、腺癌中的表达及其与Ki67和p53表达的关系

目的探讨survivin在肺鳞癌、腺癌中的表达和意义及其与Ki67、p53表达的相关性。方法采用免疫组织化学SP法检测survivin、Ki67和p53在112例肺鳞癌、腺癌中的表达情况。结果Survivin、Ki67和p53的阳性率分别为59．8％（67／112）、46．4％（52／112）和51．8％（58／112）,survivin的表达与肺癌的低分化（P=0．009）和淋巴结转移正相关（P=0．014）;Ki67和p53的表达分别与肺鳞癌、腺癌的低分化程度负相关（P〈0．05）。Survivin与Ki67和p53在肺鳞癌、腺癌中的表达分别正相关（P〈0．01）。结论Survivin的表达与肺鳞癌、腺癌的低分化和淋巴结转移正相关;Ki67和p53的表达分别与肺鳞癌和腺癌的低分化程度相关,survivin、Ki67和p53三者的协同表达可能是促进肺癌的恶性进展的重要因素。     

Flow cytometric measurement of p53 protein expression and DNA content in paraffin-embedded tissue from bronchial carcinomas

The nuclear protein p53 has been measured in archival lung cancer biopsies. The monoclonal antibody PAb 1801, which recognizes human p53, was used. After immunostaining, the nuclei prepared from paraffin-embedded tissue were stained with propidium iodide for simultaneous measurement of DNA content; 17 of 24 lung cancers were p53 positive. The S-phase fraction in positive tumors was 22.9 +/- 6.4%, as compared to 13.6 +/- 6.1% in negative tumors (P less than 0.02). In ten of the positive tumors (two small cell carcinomas and eight non-small cell carcinomas), the p53 expression varied through cell cycle, whereas in seven tumors (five small cell carcinomas and two non-small cell carcinomas), no such variation of p53 expression was observed. Freezing the nuclear suspensions did not substantially reduce the p53 signals. Control experiments with the SV40-transformed human foreskin fibroblast cell line HSF4-T12 showed that the enzymatic digestion utilized to dissociate paraffin-embedded tissue did not significantly reduce p53 fluorescence. Immunohistochemical staining of biopsy specimens indicated that only cancer cells were overexpressing p53. In conclusion, using the monoclonal antibody PAb 1801, p53 is detectable in cell nuclei prepared from paraffin-embedded bronchial carcinoma biopsies. P53 positive tumors have increased proliferative activity compared to p53 negative tumors. Furthermore, the lack of cell cycle variation of p53 in small cell carcinomas indicates that this pattern may be related to high-grade malignancy.