Bcl‐Xl protein expression in laryngeal squamous cell carcinoma

Bcl‐X1 protein expression in laryngeal squamous cell carcinoma The Bcl‐2 family of proteins regulate one of the steps in an evolutionary conserved apoptotic pathway. The long splice variant of Bcl‐X (Bcl‐Xl) is a potent antagonist of apoptosis. The aim of the study was to evaluate the relation between the presence of immunohistochemically detectable Bcl‐Xl protein in laryngeal squamous cell carcinomas (LSCCs) and clinicopathological data, as well as DNA ploidy status and proliferative activity. In 50 specimens of LSCC, Bcl‐Xl protein expression was evaluated immunohistochemically. Proliferative activity (SG₂M‐phase index) and DNA ploidy were measured by flow cytometry. In our study, Bcl‐Xl protein expression decreased with decreasing tumour differentiation (P = 0.04). The majority of patients with Bcl‐Xl protein immunoreactivity had no metastatic lymph node involvement (P = 0.01). Other factors such as age, gender, primary tumour size (pT) and type of cancer (keratinizing/non‐keratinizing) were not associated with Bcl‐Xl protein level. There was no correlation between Bcl‐Xl protein and SG₂M‐phase index or DNA ploidy status. Our findings show that expression of Bcl‐Xl protein is increased in a great fraction of laryngeal cancers. Further studies, however, are needed to clarify association between Bcl‐Xl protein expression and clinical course of patients.     

Expression of c-myc oncoprotein in laryngeal squamous cell carcinoma

OBJECTIVE: c-myc seems to play a pivotal role in normal growth and development as well in cellular transformation and carcinogenesis. Overexpression of the c-myc oncogene has been observed in many hematopoetic and solid tumors. The role of c-myc protein in squamous cell carcinoma of the head and neck in general and laryngeal squamous cell carcinomas (LSCCs) in particular is far from clear. The aim of this study was to evaluate the relations between the level of c-myc protein in LSCCs and the clinicopathological data of patients, DNA ploidy and the SG2M phase index (PI). MATERIAL AND METHODS: The c-myc protein level was evaluated immunohistochemically in tumor specimens from 50 patients with LSCC. The DNA index and SG2M PI were determined by means of flow cytometry. RESULTS: We found c-myc protein in 34 (68%) tumors. Expression of c-myc protein was demonstrated to be frequent in nonmetastatic cases (p = 0.016). There was no association between c-myc protein level and age, primary tumor size, histological grading or type of cancer. In 13 (26%) cases we observed DNA aneuploid tumors. The mean value of the SG2M PI was 22.5%. Expression of c-myc protein was not related to SG2M PI or DNA ploidy. CONCLUSIONS: We have shown that c-myc oncoprotein may be involved in the genesis of LSCC. Our findings suggest that the detectability of c-myc protein is associated with a lower metastatic potential. The c-myc oncogene is probably not as important in laryngeal cancers compared to other cancers. Further investigations must be performed to establish the value of predicting nodal metastases in LSCC.     

DNA ploidy status as a prognostic marker and predictor of lymph node metastasis in laryngeal carcinoma

In patients with laryngeal carcinoma, nodal metastasis, recurrence after radiotherapy, and prognosis are important factors in clinical decision-making. Parameters such as tumor stage are considered insufficient for predicting these important items. The DNA ploidy status of the tumor may be a useful additional marker. The DNA ploidy status of 38 laryngeal cancers was determined by flow cytometry. Correlations were studied with TNM stage, differentiation, survival rate, relapse risk, recurrence after radiotherapy, and nodal metastasis. A positive correlation of DNA ploidy status with the development of lymph node metastases was found for diploid and peridiploid versus aneuploid tumors (DNA index, <1.4 versus > or = 1.4; p = .007). No correlation was found between ploidy status and recurrence after radiotherapy. The overall survival rate (p = .01), but not the disease-specific survival rate or the relapse risk, showed a correlation with the ploidy status. The DNA ploidy status may be a useful marker for metastatic behavior in head and neck squamous cell carcinoma and may therefore be helpful in decision-making concerning elective treatment of the neck.     

Survival of patients with laryngeal cancer and some prognostic factors

Tumour growth and its progression to a metastatic phenotype involves a serious of genetic events with abnormal activation of oncogenes or inactivation of tumour suppressor genes and others genes connected with proliferation, apoptosis and neovascularisation. The aims of the study were to determine the possible prognostic value of angiogenesis, proliferation index Ki67, p53 and bcl-2 proteins expression in patients with laryngeal cancer. The group of 151 patients with laryngeal cancer, surgically treated with minimum 5 years observation, was multi-variously analysed. Paraffin--embedded tissue sections from each case were stained with a monoclonal antibody raised against FVIII antigen, p53 and bcl-2 proteins and Ki67 proliferation antigen using a peroxidase labelled streptavidin--biotin kit in standard immunohistochemistry techniques. In univariate analysis: staging IV, tumour size T4, nodal metastasis N2 and N3, local and nodal recurrences, high expression of Ki67 and P53, high (over median) IA measured as number of microvessels with FVIII expression were significantly associated with shortened overall survival. Disease-free survival was related to: proliferation index Ki67, expression of P53 protein and angiogenesis measured as microvessels density with expression of FVIII antigen. In multivariate analysis the most important death risk factors for overall survival were: tumour size, nodal metastasis, local and nodal recurrences, P53 protein expression and IA measured as number of microvessels with FVIII expression. In multivariate analysis of disease-free survival only P53 protein expression, proliferative index Ki67 and expression of FVIII had independent prognostic value. Intensity of angiogenesis, proliferation index of Ki67 antigen and expression of P53 protein were independent predictors of patients with laryngeal cancer outcome. In contrary Bcl2 protein seems to be useless in these patients.     

Expression of c-myc oncoprotein in laryngeal squamous cell carcinoma

Objective c-myc seems to play a pivotal role in normal growth and development as well in cellular transformation and carcinogenesis. Overexpression of the c-myc oncogene has been observed in many hematopoetic and solid tumors. The role of c-myc protein in squamous cell carcinoma of the head and neck in general and laryngeal squamous cell carcinomas (LSCCs) in particular is far from clear. The aim of this study was to evaluate the relations between the level of c-myc protein in LSCCs and the clinicopathological data of patients, DNA ploidy and the SG₂M phase index (PI).

Material and Methods The c-myc protein level was evaluated immunohistochemically in tumor specimens from 50 patients with LSCC. The DNA index and SG₂M PI were determined by means of flow cytometry.

Results We found c-myc protein in 34 (68%) tumors. Expression of c-myc protein was demonstrated to be frequent in non-metastatic cases (p=0.016). There was no association between c-myc protein level and age, primary tumor size, histological grading or type of cancer. In 13 (26%) cases we observed DNA aneuploid tumors. The mean value of the SG₂M PI was 22.5%. Expression of c-myc protein was not related to SG₂M PI or DNA ploidy.

Conclusions We have shown that c-myc oncoprotein may be involved in the genesis of LSCC. Our findings suggest that the detectability of c-myc protein is associated with a lower metastatic potential. The c-myc oncogene is probably not as important in laryngeal cancers compared to other cancers. Further investigations must be performed to establish the value of predicting nodal metastases in LSCC.     

Expression of type IV collagen and matrix metalloproteinase-2 (type IV collagenase) in relation to nodal status in laryngeal cancer

Laryngeal carcinoma has a lower incidence of neck metastases than other malignant carcinomas of the head and neck region. However, some cases are very aggressive, showing neck metastases even in the early stages. In this study the expression of collagen IV and type IV collagenase (MMP-2) were examined immunohistologically in 50 patients with laryngeal carcinomas, and the results were compared with the incidence of neck metastases and other clinicopathological factors. The correlation between collagen IV expression and the existence of nodal metastases was statistically significant (P < 0.001). There was also significant correlation between collagen IV expression and the histological grading of the tumour. There was a tendency for samples with continuous collagen IV staining to have no matrix metalloproteinase-2 (MMP-2) immunoreactivity. No significant correlation was seen between MMP-2 protein expression and clinicopathological parameters although the correlation between MMP-2 and existence of nodal metastases was statistically borderline (P = 0.07). Multivariate analysis of the clinicopathological factors that may have an influence on the nodal status in laryngeal cancer revealed that, apart from T stage, collagen IV pattern in the basement membrane surrounding nests of carcinoma is an important prognostic factor.     

Prognostic factors in carcinoma of the larynx: relevance of DNA ploidy, S-fractions and localization of the tumour

The influence of the cell cycle profile and the site of the primary tumour on the overall survival were examined in 36 patients with squamous cell carcinoma of the larynx. DNA ploidy (p = 0.0091), the overall proliferative activity (p = 0.0001), the overall proliferative activity of diploid tumour cells (p = 0.0017) and primary tumour site (p = 0.0008) were found to be significant single prognostic factors of the overall survival. Multivariate analysis showed that only the overall proliferative activity was prognostically significant (p = 0.013). The results of the study show that the supraglottic site of the tumours correlates significantly with DNA ploidy (p = 0.0334) and the overall proliferative activity of tumour cells (p = 0.0159), whereas the correlation with proliferative activity of diploid tumour cells (p = 0.1416) has not been confirmed by this study. Glottic tumours showed a prognostically significant correlation with the overall proliferative activity (p = 0.0037) and proliferative activity of diploid tumour (p = 0.0054). Such a prognostic correlation was not found for DNA ploidy (p = 0.6542).     

C-erbB-2 immunostaining in laryngeal cancer.

Tumour progression is strongly associated with a series of specific genetic changes in protooncogenes and tumour suppressor genes. One of the potential factors involved in tumorogenesis of squamous cell carcinomas is protooncogene c-erbB-2 (also known as neu or HER2). The authors analysed the expression of c-erbB-2 oncoprotein in 154 cases of laryngeal squamous cell carcinomas and its relationship to the clinical outcome of the patients. The difference in c-erbB-2 oncoprotein expression between the control group and cancer patients was on the statistical borderline (p = 0.0470). There was no significant correlation between c-erbB-2 expression and sex and age of the patients. T stage, lymph node status, site and histopathological grading of the tumour and clinical outcome of the patients. Univariate analysis revealed no correlation between c-erbB-2 expression and survival rates. We conclude that immunohistological examination of c-erbB-2 on paraffin section is not a valuable prognostic factor in laryngeal carcinoma.     

DNA index,cellular proliferative activity and nucleolar organizer regions in cancers of the larynx

The DNA index, expression of cell-cycle-related proteins — proliferating cell nuclear antigen (PCNA, cyclin) and Ki-67 — and the content of silver-binding nucleolar organizer regions (AgNORs) were evaluated in 30 unselected consecutive primary squamous cell carcinomas of the larynx. Results were compared and subsequently related to histological grading, lymph node status, pT category, and pathological stage. DNA content was non-diploid in 9 cases (30%). Mean AgNOR counts per tumor ranged from 2.52 to 8.76. PCNA and Ki-67 expressions were similar in 10 cases (33%). In the remaining cases, PCNA-positive cells usually outnumbered Ki-67-positive cells. No significant correlation was found among DNA index, PCNA and Ki-67 expressions, and AgNOR counts. Although there was a positive trend when Ki-67 was compared with histological grading, findings were not statistically significant. In contrast, a significant correlation was found between DNA index and lymph node status (P = 0.035), with a higher incidence of neck node metastases in non-diploid tumors. These data suggest that tumor ploidy can be correlated with lymph node spread in laryngeal squamous cell carcinoma and might be used as an additional prognostic factor when planning treatment.     

喉癌EGFR和PCNA表达及DNA含量的研究

研究喉鳞癌中表皮生长因子受体、增殖细胞核抗原的表达和ＤＮＡ含量,分析各指标间的相关性及与喉癌临床生物学行为和预后的关系。方法：用免疫组化ＳＰ法和图像分析仪检测正常喉粘膜、喉鳞癌的ＥＧＦＲ、ＰＣＮＡ和ＤＮＡ指数,结合临床资料进行分析。结果：喉鳞癌的ＥＧＦＲ的阳性表达率为５４．８％,与正常喉组织的差异有统计学意义,但ＥＧＦＲ的阳性表达与喉癌的病理分级和５年生丰率无显著相关,而ＰＣＮＡ阳性表达和ＤＩ则随     

Expression of type IV collagen and matrix metalloproteinase‐2 (type IV collagenase) in relation to nodal status in laryngeal cancer

Expression of type IV collagen and matrix metalloproteinase‐2 (type IV collagenase) in relation to nodal status in laryngeal cancer Laryngeal carcinoma has a lower incidence of neck metastases than other malignant carcinomas of the head and neck region. However, some cases are very aggressive, showing neck metastases even in the early stages. In this study the expression of collagen IV and type IV collagenase (MMP‐2) were examined immunohistologically in 50 patients with laryngeal carcinomas, and the results were compared with the incidence of neck metastases and other clinicopathological factors. The correlation between collagen IV expression and the existence of nodal metastases was statistically significant (P < 0.001). There was also significant correlation between collagen IV expression and the histological grading of the tumour. There was a tendency for samples with continuous collagen IV staining to have no matrix metalloproteinase‐2 (MMP‐2) immunoreactivity. No significant correlation was seen between MMP‐2 protein expression and clinicopathological parameters although the correlation between MMP‐2 and existence of nodal metastases was statistically borderline (P = 0.07). Multivariate analysis of the clinicopathological factors that may have an influence on the nodal status in laryngeal cancer revealed that, apart from T stage, collagen IV pattern in the basement membrane surrounding nests of carcinoma is an important prognostic factor.     

Expression of selected markers for apoptosis, proliferation and metastasis in evaluation of laryngeal cancer invasiveness dynamics

Evaluation of the biology of laryngeal cancer cell is connected either with many process inside the cell or reactions between cancer cell itself and extracellular matrix. The main purpose in this paper was the evaluation of p53 protein, bcl-2 protein, Ki-67 antigen and CD44 adhesive molecule expressions in comparison to clinical and histopathological features in patients with laryngeal cancer. Paraffin-embedded tissue sections from 89 patients with laryngeal cancer were stained with a monoclonal antibody raised against p53 and bcl-2 proteins, Ki-67 and CD44 antigens using a peroxidase-labelled streptavidin-biotin kit. There were statistically significant relationships between p-53 protein over-expression and pT, histological grading, survival and Ki-67 and CD44 antigens expressions. There were no correlation between bcl-2 protein expression and clinical and histopathological features. We observed statistically significant correlation between Ki-67 expression and pT, histological grading, recurrences and survival. Expression of CD44 statistically significant correlated only with tumour size. We conclude that comparison of data covering mentioned tumour markers expression gives valuable evaluation of biological activity of cancer cells and may allow to create the immunological panel of tumour markers which simplify the prognosis about nodal metastases, recurrences and survival in patients with laryngeal cancer.     

The expression of vascular endothelial growth factor (VEGF) and VEGF-C in early laryngeal cancer: relationship with radioresistance

Angiogenesis is essential for tumour growth and invasion. Vascular endothelial growth factor (VEGF) is a prime mediator of tumour angiogenesis. VEGF-C is a closely related protein that effects lymphatic endothelial cells and may be important in the process of lymphatic metastasis. The purpose of this study was to evaluate the expression of these cytokines in patients with T1 and T2a glottic, squamous cell carcinoma, in comparison with normal epithelial control tissue, to ascertain any association with radioresistance. Twenty-two tumours treated by radiotherapy (13 radiosensitive, nine radioresistant) and seven normal control tissues were studied. The minimum follow-up was 2 years after radiotherapy. Expression of VEGF and VEGF-C was evaluated by immunohistochemistry of formalin-fixed, paraffin-embedded biopsy specimens. Analysis was carried out using a quantitative computer image analyser. Both VEGF and VEGF-C were detectable in tumour and normal control specimens. There was increased expression in tumour specimens of both VEGF (P = 0.03) and VEGF-C (P < 0.001). In addition, the expression of VEGF-C was associated with tumours of higher histological grade (P = 0.021). There was, however, no difference in VEGF and VEGF-C expression between radioresistant and radiosensitive tumours. The expression of VEGF and VEGF-C is increased in early laryngeal squamous cell carcinoma (SCC). However, measuring the expression of these proteins cannot predict radioresistance in this tumour group.     

基质金属蛋白酶14基因在人喉鳞状细胞癌中表达差异的分析

目的 探讨基质金属蛋白酶14(matrix mettallo-proteinase 14,MMP14)即膜性基质金属蛋白酶(membrane type-1 matrix metalloproteinase,MT1-MMP)基因在喉癌中表达与喉癌临床病理生物学行为的关系。方法 从分子和蛋白质水平,研究MMP14在喉癌组织和其相邻的正常黏膜组织的表达差异,分析MMP14基因及其蛋白表达与喉癌临床病理生物学行为的关系。结果 33例配对喉癌标本中MMP14基因在26例喉癌组织中的表达明显高于其相应的正常黏膜组织,2例喉癌低表达,5例表达差异不明显。MMP14基因在不同分期的声门型喉癌中的表达差异无显著性(P>0.05),但在不同分化程度、有无淋巴结转移的声门型喉癌中,表达差异有显著性(P<0.05);在不同分期、不同分化程度及有无淋巴结转移的声门上型喉癌中,表达差异有显著性(P<0.05)。MMP14蛋白绝大多数位于癌巢细胞质内及癌巢间质内的成纤维细胞质内,在部分病例中有的正常黏膜细胞内不表达,有的表达;MMP14蛋白在绝大多数喉癌组织中的表达明显高于其相邻的正常黏膜组织。MMP14蛋白表达与MMP14基因表达基本是一致的。MMP14蛋白表达差异有显著性组患者的术后生存率与表达差异无显著性组的术后生存率无统计学差异(P>0.05)。结论 MMP14蛋白在喉癌的侵袭中可能只起一定的作用,但在喉癌的淋巴结转移     

Tiam1蛋白在喉癌中的表达及其临床意义

目的检测T淋巴细胞侵袭转移诱导因子1（Tiam1）蛋白在喉癌组织中的表达,并探讨其表达水平与喉癌临床病理特征和预后之间的关系。方法采用免疫组织化学染色技术检测Tiam1蛋白在98例喉癌石蜡组织切片中的表达,并统计分析Tiam1蛋白表达水平与喉癌临床病理特征和预后之间的关系。结果Tiam1蛋白的表达与喉癌的淋巴结转移（P<0.001）、临床分期（Ⅰ期、Ⅱ期/Ⅲ期、Ⅳ期）（P=0.027）、组织病理学分级（P=0.020）及复发（P=0.003）密切相关;Kaplan Meier生存曲线分析结果显示Tiam1高表达组的5年无复发生存率（P=0.001）和5年总生存率（P<0.001）均明显低于Tiam1低表达组;而且通过Kaplan Meier生存曲线分析Tiam1蛋白表达和淋巴结转移情况与头颈鳞癌患者预后之间的关系,发现Tiam1高表达并有淋巴结转移组的5年无复发生存率和5年总生存率明显低于其它（Tiam1低表达或无淋巴结转移）组（P均<0.001）;多因素Cox比例风险回归模型进一步显示,淋巴结转移（P=0.001）及Tiam1表达水平（P=0.020）为喉癌的独立预后因素。结论Tiam1在喉癌组织中表达显著升高,且其高表达与喉癌患者的淋巴结转移、复发及预后密切相关。Tiam1可能在喉癌的发生、发展中起重要作用,其表达水平对喉癌的复发及预后具有评估价值。     

Alterations of cell cycle regulating proteins: Rb, p21 and p16 in laryngeal cancer

In cell cycle, most of the regulatory actions occur at the so-called restriction point (R) in the late G1 phase. Tumor suppressor genes; Rb, p53 and p21 are among the most important of the agents suppressing transition through R point. Changes in the expression of Rb (retinoblastoma) gene correlate with the presence of Rb protein and they are believed to be an early event in carcinogenesis. This issue seems to be not plainly defined in laryngeal cancer. P21 with p16, cyclin D1 and Rb genes that play a critical role in the regulation of the G1-S transition of the cell cycle, are frequently altered in several neoplastic entities. Our purpose was to investigate the possible prognostic value of p21, p16 and Rb proteins in patients with laryngeal cancer. 67 patients with laryngeal cancer was multi-variously analysed. Paraffin-embedded tissue sections were immunohistochemically stained with a monoclonal antibody raised against p21, p16 and Rb proteins using standard immunohistochemistry techniques. Low intensity (< or = 10%, 7/67) of p21 protein expression was significantly correlated with histological grading (p < 0,01) and overall and disease free survival (p < 0,05). We did not observed any correlation between p21 expression and T, N and M status and local or nodal recurrences. Absence of p16 protein expression was observed in 35/67 (52,2%) cases and was significantly correlated with N status (p = 0,03) and nodal recurrences (p = < 0,01). By univariate analysis expression of p16 protein was related with quicker relapse. Rb protein was absent in 7/67 cases (10,4%) and was related to T3 and T4 primary tumour size (p < 0,05). We did not observed any correlation between Rb and other clinocopathological features (p > 0,05). Our study has identified p21 protein expression as important biological marker which may indicate the progression of laryngeal squamous cell carcinoma. P16 protein has a prognostic value in assessment of disease free survival. Based on this findings it can be deduced that investigation of Rb, p16 and p21 proteins makes it easier to understand the process of cancerogenesis in laryngeal cancer and to establish its prognostic value further research and observations need to be attempted.     

基质金属蛋白酶14基因在人喉鳞状细胞癌中表达差异的分析

OBJECTIVE: To investigate the role of expression of matrix mettallo-proteinase 14 (MMP14) in laryngeal carcinomas and the relationship between MMP14 expression and the laryngeal biological behavior. METHODS: The gene expression differences of MMP14 between fresh laryngeal cancer tissues and their surrounding normal mucosa was analyzed by RT-PCR. The expression of MMP14 protein in paraffin-embedded tissues was determined immunohistochemically. All statistical analyses were performed by SPSS version 10.0. RESULTS: In the 33 cases of matched specimens, MMP14 gene expression was much higher in tumor tissues than that in surrounding ing normal tissues in 26 cases and lower in 2 cases, whereas in other 5 cases, no significant difference was observed between the cancer tissue and the surrounding normal tissue. MMP14 gene expression was not different in different stages in laryngeal glottic cancers, but correlated to the differentiation and lymph node metastasis (P < 0.05). There was correlation between MMP14 gene expression and the stage, differentiation and lymph node metastasis in the laryngeal supraglottic cancers (P < .05). MMP14 protein was localized predominantly in the carcinoma cell cytoplasm and in the stromal fibroblasct cytoplasm, and weakly or not expressed in surrounding normal tissue. MMP14 protein expression was much higher in tumor tissue than that in surrounding normal tissue in most of the cases. In general, MMP14 protein expression was the same as MMP14 gene expression and related to the stage, differentiation and lymph node metastasis in the laryngeal cancers. There was no survival difference at 3, 5 and 7-year between the group with higher MMP14 protein expression in tumor tissues than surrounding normal tissues and the group with no difference of MMP14 protein expression (Log Rank, P=0.5535). CONCLUSIONS: The protein MMP14 may play role in laryngeal cancer invasion in a certain extent, but important role in lymph node metastasis of laryngeal cancer. The over-expression of MMP14 protein may be a marker for lymph node metastasis of laryngeal cancer.