P53 expression in squamous cell carcinomas of the supraglottic larynx and its lymph node metastases: new results for an old question

BACKGROUND: Although p53 overexpression is frequent in head and neck squamous cell carcinomas (HNSCCs), controversy remains regarding the prognostic significance of that overexpression. The objective of this study was to investigate the expression pattern and prognostic significance of p53 expression in HNSCC of the same location, treated in the same way, and with long-term follow-up. METHODS: P53 expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 107 consecutive patients (107 primary squamous cell carcinomas of the supraglottic larynx and 46 matched lymph node metastases). All patients underwent surgical resection and bilateral neck dissection. RESULTS: A strong correlation was observed between p53 expression in the primary tumor and in the matched lymph node metastases (P=.0001). P53 overexpression in the lymph nodes was an independent predictor of regional recurrence (P=.027). Likewise, expression of p53 in the lymph nodes correlated significantly with disease-specific survival (P=.018). Five years after treatment, 70% of patients with p53-negative, metastatic lymph nodes remained alive, whereas only 30% of patients with p53-positive lymph nodes remained alive. In multivariate analysis, lymph node status and p53 expression in the lymph nodes remained associated with survival. CONCLUSIONS: The current data suggested that, although p53 overexpression is common in supraglottic carcinomas, its expression in the primary tumor is of limited clinical significance. However, the results supported the role of p53 in the lymph node metastases as an independent predictor of regional failure and a poor prognosis in patients with HNSCC. A prospective trial is indicated to validate these findings.     

Overexpression of Pim-1 in head and neck squamous cell carcinomas

Despite ongoing developments of treatment protocols head and neck squamous cell carcinomas (HNSCC) show only marginal improvement in outcome, which has been attributed to a lack of therapy individualized to tumor biological properties. We compared mRNA expression profiles of HNSCC and normal epithelial cells using differential display to identify gene fragments showing differential expression in HNSCC cells. We identified a 127-bp long fragment to be overexpressed in HNSCC cells that revealed a 98.4% homology with the Pim-1 mRNA. The differential expression was confirmed by Northern hybridization. Immunohistochemistry showed overexpression of the Pim-1 protein in 98% (41/42) of invasive HNSCC. Analysis of Pim-1 protein expression in relation to TNM stage and histological grade of the tumors exhibited no significant correlation. However, when samples of primary tumor and metastasis retrieved from the same patients (n=26) were analyzed, nearly significant correlation of Pim-1 expression with histological grade was found (p=0.06). The high frequency of the Pim-1 expression of HNSCC of different grades and stages in conjunction with its absence in non-neoplastic head and neck squamous cell epithelium underlines the functional role of Pim-1 in molecular processes of HNSCC.     

Expression of c-erbB receptors, MMPs and VEGF in squamous cell carcinoma of the head and neck

Head and neck squamous cell carcinomas (HNSCC) are characterized by a marked propensity for local invasion and cervical lymph node metastasis. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR), c-erbB-2, vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in tumor samples of 91 HNSCC patients, and to study a possible correlation to various clinico-pathologic parameters. The expression of EGFR, c-erbB-2, VEGF, MMP-2, -3 and -9 was analyzed in the same paraffin embedded tissue by semi-quantitative immunohistochemical staining. High expression of EGFR, c-erbB-2, MMP-2 or -9 was associated with advanced clinical stages, nodal metastases and tumor-stages. However, high expression of VEGF or MMP-3 was not associated with any clinico-pathologic parameters except significant correlation between VEGF and the tumor site. There were significant correlations between EGFR, c-erbB-2, MMP-2 and -9 in HNSCC patients. Conversely, no correlation was found between VEGF or MMP-3 and the other markers. However, significant correlation was found between MMP-3 or -9 and VEGF. The results indicate that the expression of EGFR, c-erbB-2, VEGF or MMPs play an important role in tumor growth, invasion and metastasis in HNSCC. The authors conclude that EGFR, c-erbB-2, MMP-2 and -9 could be good independent prognostic markers, but not VEGF and MMP-3.     

Clinical significance of c-myc and p53 expression in head and neck squamous cell carcinomas

c-myc and p53 genes were frequently deregulated in head and neck squamous cell carcinoma (HNSCC). To determine if the concomitant expression of the two oncogenes might have prognostic value, the survival and free disease time of 140 consecutive HNSCC patients followed up for a median time of 29.9 months was analyzed in the light of p53 and c-myc expression assessed by immunohistochemistry. Positive c-myc and p53 staining was detected respectively in 35.7 and 50.7% of the tumors. Double positivity emerged in 16.4% of the cases. Overall-survival of patients was not associated with the immunoreactivity of p53 or c-myc considered separately or grouped in subsets. Considering only the advanced stages, the concomitant expression of both oncogenes in tumors was associated with worse disease-free survival (P = 0.004) suggesting a role for p53 and c-myc genes in progression of this HNSCC subset. Clinical parameters (presence of lymph nodes, histologic grade and tumor width) remained important indicators of overall survival (OS).     

Loss of Fhit expression is a predictor of poor outcome in tongue cancer

Abnormalities of FHIT, a candidate tumor suppressor gene located at 3pl4.2, have been found frequently in multiple tumor types, including head and neck squamous cell carcinoma. To determine the role of FHIT in tongue cancers, Fhit expression was determined by immunohistochemistry studies in tissue samples from 41 patients with stages II-IV squamous cell carcinomas of the tongue. All of the patients underwent curative surgical treatment with a median of 83 months of follow-up care. We found that 28 (68%) of the 41 tumor specimens demonstrated a lack of or significantly decreased staining for Fhit. Fhit expression tended to be stronger in well-differentiated tumor areas than it was in poorly differentiated areas, although this trend was not statistically significant. There was no significant correlation between Fhit expression and a patient's age or sex or the histological grade or clinical stage of disease. As expected, clinical stage and nodal involvement correlated with prognosis. Interestingly, patients whose tumors demonstrated low levels of or no Fhit expression had a significantly shorter time of disease-free survival than those whose tumors had high Fhit expression (P = 0.035, by log-rank test). This prognostic value of Fhit was independent of other clinical parameters, including stage of disease and nodal status. We conclude that Fhit plays an important role in the development of squamous cell carcinomas of the tongue and that loss of Fhit expression may be an independent prognostic indicator for clinical outcome in patients with this tumor type.     

Combined P16 and human papillomavirus testing predicts head and neck cancer survival

While its prognostic significance remains unclear, p16^INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type‐16 DNA and RNA was detected by MY09/11‐PCR and E6/E7 RT‐PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p < 0.0001). With respect to prognosis, p16‐positive oropharyngeal tumors exhibited significantly better overall survival than p16‐negative tumors (log‐rank test p = 0.04), whereas no survival benefit was observed for nonoropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive nonoropharyngeal tumors had significantly better disease‐specific survival than concordantly negative nonoropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking and drinking [adjusted hazard ratio (HR) = 0.04, 0.01–0.54]. Compared with concordantly negative nonoropharyngeal HNSCC, p16(+)/HPV16(?) nonoropharyngeal HNSCC (n = 13, 7%) demonstrated no significant improvement in disease‐specific survival when HPV16 was detected by RNA (adjusted HR = 0.83, 0.22–3.17). Our findings show that p16 IHC alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV‐associated nonoropharyngeal HNSCC with better prognosis.     

C-erbB receptors in squamous cell carcinomas of the head and neck: clinical significance and correlation with matrix metalloproteinases and vascular endothelial growth factors.

We studied the profile of four c-erbB receptors in head and neck squamous cell carcinomas (HNSCC) and to determine whether their expression was associated with clinicopathological features and key molecules involved in angiogenesis and metastasis. We also assessed the impact of expression on survival. This study included 54 cases of primary HNSCC, of which 27 cases showed lymph node metastasis. The expression of c-erbB receptors, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) family members was analysed in the same tissue homogenates by semi-quantitative RT-PCR. HNSCC frequently co-expressed multiple c-erbB receptors and showed significant correlations amongst their levels. High expression of epidermal growth factor receptor (EGFR), c-erbB-2 or c-erbB-3 was associated with an infiltrating mode of invasion, nodal metastases and advanced pathological stages. EGFR and c-erbB-2 levels were strongly correlated (P=0.0004-0.029) with the expression of MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, VEGF-A and VEGF-C whereas the levels of c-erbB-3 and B-4 showed a weaker correlation (P=0.049-0.01) with some MMPs and VEGF-C. Only nodal metastasis and EGFR levels were significantly associated with poor outcome in uni- and multi-variate analysis. We conclude that co-operative signalling of all four c-erbB receptors may play a significant role in the pathogenesis of HNSCC. Amongst these, EGFR appears to be the dominant component controlling the invasive and angio-/lymphangiogenic phenotype in HNSCC via upregulation of multiple MMPs and VEGFs.     

p53 protein accumulation and genomic instability in head and neck multistep tumorigenesis.

Head and neck cancer develops in a multistep process and is associated with increasing frequencies of p53 alterations and with increasing genomic instability. To study the relationship of p53 alterations and genomic instability during head and neck tumorigenesis, we analyzed p53 protein expression and chromosome 9 and 17 polysomy in 48 squamous cell carcinomas of the head and neck and their adjacent normal epithelium (31 sites), hyperplastic (24 sites), and dysplastic lesions (26 sites). Normal oral epithelium obtained from seven nonsmoking, cancer-free individuals served as negative controls. Six (19%) of 31 lesions in adjacent normal epithelium, 7 (29%) of 24 hyperplastic lesions, 12 (46%) of 26 dysplastic lesions, and 28 (58%) of 48 squamous cell carcinomas expressed p53. In contrast, no normal control epithelium had detectable p53 expression. To determine the relationship between dysregulated p53 expression and genomic instability during tumorigenesis, we compared p53 immunohistochemistry distributions and chromosome polysomy levels (by chromosome in situ hybridization) in different histological groups associated with tissue progression. Although the degree of chromosome polysomy increased for all of the groups during histological progression, lesions with dysregulated p53 expression showed nearly 2-4-fold increased levels of chromosome polysomy. This trend was significant for dysplastic lesions (P = 0.005 and P = 0.002 for chromosomes 9 and 17, respectively) and for squamous cell carcinoma (P = 0.005 and P = 0.002 for chromosomes 9 and 17, respectively). Image analysis studies for 28 p53-expressing tumors and their adjacent premalignant lesions demonstrated a strong spatial correlation between stepwise transitions from low to high p53 expression and increased chromosome polysomy frequencies in 13 (46%) of 28 cases. These findings suggest that altered p53 expression is associated with increased genetic instability in preneoplastic epithelium and may play a driving force for increasing the rate of accumulation of genetic events during head and neck tumorigenesis.     

Expression of p53 and its homologues in primary and recurrent squamous cell carcinomas of the head and neck

The tumour-suppressor protein p53 belongs to a family that includes 2 structurally related proteins, p63 and p73. Because of their structural homology, it has been hypothesized that both homologues serve as "spare mechanisms" in p53 mutations to regulate the cell cycle by inducing apoptosis. We investigated the mutational and protein expression status of p53 in correlation to its homologues, p73 and p63, in primary and recurrent squamous cell carcinomas of the head and neck (HNSCC) and corresponding nonneoplastic mucosa. Expression and mutation of p53 and its homologues p63 (including the 2 major isotypes TAp63 and DeltaNp63) and p73 was examined by direct DNA sequencing and immunohistochemistry in 29 primary and 39 recurrent (secondary) HNSCCs after microdissection. Our results were correlated with pathohistologic stage and grade. p53 mutations were detected in 32/68 (47%) carcinomas of 17 patients, with a discordant mutation pattern of primary and consecutive tumours in all cases. Positive immunostaining for p63 was found in 55/68 (81%) carcinomas of 29 patients. Immunohistochemistry revealed p73 protein expression in 32/68 (47%) tumours. In normal mucosa, p63 and p73 were expressed in 40/68 (59%) and 12/68 (18%) cases, respectively. We failed to detect specific mutations of p73 or p63 in primary and recurrent carcinoma of the head and neck. p73 and p63 were rarely mutated in HNSCC, but both were expressed in a subset of tumours. The lack of correlation between p73/p63 and p53 protein expression suggests that neither p73 nor p63 can replace p53 when it is mutated.     

Genotypic and phenotypic alterations of p53 in head and neck squamous-cell carcinoma

p53 is the most frequently altered suppressor gene in human cancers. The genotypic and corresponding phenotypic abnormalities of this gene in head and neck squamous cell carcinoma (HNSCC) remain undefined. We analyzed the loss of heterozygosity (LOH) by restriction fragment length polymorphism (RFLP) at three polymorphic loci in the p53 gene and performed immunohistochemistry (MC) for its protein on paraffin-embedded tumor tissue from 20 previously sequenced tumor specimens. LOH was noted at one or more of the three polymorphic sites within the p53 gene in 12 (67%) of the 18 informative samples. Concordance between LOH and mutations was observed in 14 (78%) cases. Twelve (60%) tumors with point mutations were immunohistochemically reactive to p53 antibody and two (10%) lacked both genetic and immunohistochemical alterations. In six tumors (30%) contradictory results between immunohistochemistry and molecular analysis were observed. Our data indicate that: (i) simultaneous deletion and mutation of both p53 alleles was observed in the majority of head and neck squamous carcinomas and implicate this gene in the oncogenesis of these neoplasms, (ii) p53 immunohistochemical analysis may not fully account for the different molecular alterations of this gene, and (iii) no correlation between p53 abnormalities and clinicopathologic or DNA content characteristics of HNSCC was found.     

The prognostic value of p53 and c-erbB-2 expression, proliferative activity and angiogenesis in node-negative breast carcinoma

AIMS AND BACKGROUND: p53, c-erbB-2 and Ki-67 protein expression and microvessel density (MVD) determined by CD34 antibody were evaluated by immunohistochemistry and their correlation with clinicopathological parameters including estrogen (ER) and progesterone (PR) receptor status and survival were investigated in patients with axillary lymph node-negative infiltrating ductal breast carcinoma. METHODS: The study population consisted of 47 patients with axillary lymph node-negative infiltrating ductal breast carcinoma. RESULTS: p53 and c-erbB-2 expression was detected in 36.2% and 31.9% of patients, respectively. Median Ki-67 expression was 10%. There were no statistically significant differences in the distribution of p53, Ki-67 and c-erbB-2 protein expression in relation to the age of the patients or to the size, histological grade or ER and PR status of the tumors. p53 protein expression correlated positively with c-erbB-2 and Ki-67 protein expression (P < 0.05). The mean MVD was 63.65 +/- 29.1 and it correlated positively with histological grade and Ki-67 expression (P < 0.05). Survival analysis revealed that age, tumor size, p53 and c-erbB-2 expression and PR status had no significant prognostic impact, whereas histological grade, proliferative activity and angiogenic activity were significant prognostic factors. Although ER-positive patients had a statistically significant overall survival advantage, the difference in disease-free survival was not significant. CONCLUSION: In axillary lymph node-negative breast carcinoma the histological grade and the proliferative and angiogenic activity of the tumor could be useful prognostic indicators.     

Expression of c-erbB-2 oncoprotein in gastric carcinoma. Immunoreactivity for c-erbB-2 protein is an independent indicator of poor short-term prognosis in patients with gastric carcinoma.

Correlations of c-erbB-2 protein expression with clinical outcomes of gastric carcinomas were studied in 189 gastric carcinomas. There were 23 (12.2%) carcinomas with evidence of c-erbB-2 protein in which the reaction was localized to the cell membrane. There was no significant association between c-erbB-2 staining and the macroscopic or histologic type of the carcinomas. c-erbB-2-stained tumors were more likely to be associated with serosal invasion, nodal involvement, and peritoneal metastasis, than c-erbB-2-unstained ones. In addition, c-erbB-2 was stained in none of early gastric carcinomas. The 5-year survival rates of the c-erbB-2 protein-positive and the protein-negative group were 11% and 50%, respectively. When the c-erbB-2 tissue status and seven clinicopathologic variables as conventional prognostic factors were entered simultaneously into the Cox regression model, serosal invasion, hepatic metastasis, peritoneal metastasis, nodal status, and c-erbB-2 tissue status emerged as independent prognostic variables. The results suggested that c-erbB-2 protein expression might be enhanced in advanced stages during the progression of gastric carcinoma. In this particular group of patients, immunoreactivity for c-erbB-2 protein is an indicator of poor short-term prognosis.     

A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8

Besides well-known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV-induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single-strand conformation polymorphism (SSCP) analysis of exons 5-8. Paraffin-embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16(INK4A) overexpression in all 10 HPV-positive tumors. Although FISH is considered to be less sensitive than PCR-based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV-positive carcinomas. However, in none of the latter cases could mutations in exons 5-8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors.     

The expression of c-erbB-1 and c-erbB-2 in Iranian patients with gastric carcinoma

To assess the significance of epidermal growth factor receptor family members, the overexpression of c-erbB-1 and c-erbB-2 was retrospectively investigated in 146 southern Iranian gastric cancer patients. Indirect immunostaining was used to evaluate the expression of these two receptors in formalin-fixed paraffin-embedded tissue samples. c-ErbB-1 expression was observed in 47 (32.2%) and c-erbB-2 expression was observed in 24 (16.4%) of tumors. Significant positive correlations were observed between c-erbB-1 expression and tumor size, local invasion, lymph node involvement and tumor stage. There was also a negative correlation between c-erbB-2 expression and tumor stage. These results may suggest the contribution of c-erbB-1 molecule in progression of gastric carcinomas in southern Iranian patients. Moreover, the relatively high percentage of c-erbB-2 positive tumors may provide a useful target for the immunotherapy of these cancers.     

Prognostic value of the epidermal growth factor receptor (EGRF) and p53 in advanced head and neck squamous cell carcinoma patients treated with induction chemotherapy

We measured the expression of the p53 nuclear protein and epidermal growth factor receptor (EGFR) in 46 biopsy samples from patients with advanced head and neck cancer treated with induction combination chemotherapy of 5-fluorouracil, cisplatin, and paclitaxel. Tumour expression of p53 protein was analysed with the monoclonal D07 antibody and EGFR with monoclonal H11 antibody. The overall response, defined as complete (CR) and partial response (PR) rates to treatment, was 88%. p53 positive staining was significantly more frequent in patients who did not respond to the induction treatment. EGFR expression failed to show any correlation with the response rate. Multivariate analysis indicated that a tumour location in the oral cavity together with p53 expression combined with moderate-to-high EGFR staining were independent prognostic factors of a shorter disease-free survival (DFS). Location of the tumour in the oral cavity and EGFR expression had independent prognostic value for overall survival (OS). We conclude that the EGFR status and an oral cavity location of the tumour have independent prognostic value in patients with advanced head and neck carcinoma treated with induction chemotherapy. The p53 status appears to be a determinant of the tumour chemo-sensitivity in advanced head and neck squamous cell carcinoma (HNSCC). The presence in the tumour of a p53-positive stain and moderate-to-high staining of EGFR is associated with a shorter DFS and time to treatment failure (TTF) probably reflecting a more aggressive tumour phenotype.     

EB-D fibronectin expression in squamous cell carcinoma of the head and neck

ED-B fibronectin (ED-B FN), a glycoprotein involved in cell adhesion and migration, is expressed in fetal and neoplastic tissues and absent in their normal counterparts. The aim of this study is to evaluate the expression of this glycoprotein in relation to the histological and clinical data and to determine whether it has a prognostic value in patients with head and neck squamous cell carcinoma (HNSCC). Ninety-five cases were assessed for ED-B FN expression using immunohistochemistry. Positive ED-B FN expression was significantly associated with tumor grade (p=0.06) and primary tumor site (p=0.02). The larynx was the tumor site associated with the least ED-B FN expression. In univariate analysis, there was no association with disease-free survival (p=0.48), but the mean time to progression was clearly shorter in tumors with positive ED-B FN expression than in those with negative expression (6 vs. 11 months). Patients having tumors expressing the ED-B FN had a trend to a significant lower overall survival in the multivariate analysis (p=0.06). Our study showed that ED-B FN expression might have a prognostic value in patients with HNSCC.