Overexpression of TWIST2 correlates with poor prognosis in head and neck squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of tumors with variable presentation and clinical behavior. Despite improvements in surgical and radiation therapy techniques, the 5-year survival rate has not improved significantly over the past decades. Thus, there is an urgent need to identify novel markers that may allow for the development of personalized therapeutic approaches. In the present study we evaluated the prognostic role of the expression of genes related to the induction of epithelial mesenchymal transition (EMT). To this aim, a consecutive series of 69 HNSCC were analyzed for the expression of TWIST1, TWIST2, SNAI1, SNAI2, E-Cadherin, N-Cadherin and Vimentin.TWIST1, TWIST2, SNAI1 and SNAI2 were significantly overexpressed in HNSCC, with TWIST2, SNAI1 and SNAI2 being more markedly increased in tumors compared to normal mucosae. The expression of TWIST1 and SNAI2 was associated with upregulation of mesenchymal markers, but failed to correlate with pathological parameters or clinical behaviour. In contrast, we found that upregulation of TWIST2, which was independent of the activation of a mesenchymal differentiation program, correlated with poor differentiation grade (p=0.016) and shorter survival (p=0.025), and identifies a subset of node-positive oral cavity/pharynx cancer patients with very poor prognosis (p less than 0.001). Overall our study suggests that the assessment of TWIST2 expression might help to stratify HNSCC patients for risk of disease progression, pointing to TWIST2 as a potential prognostic marker.     

Elevated expression of the c-myc oncoprotein correlates with poor prognosis in head and neck squamous cell carcinoma

We quantitated c-myc oncoprotein in 44 squamous cell carcinomas of the head and neck using an enzyme-linked immunosorbence assay. The clinicopathological parameters of these patients were followed up for between 3 and 60 months and analysed for any correlations with observed levels of c-myc protein using the Kruskal-Wallis one-way analysis of variance method. Although no statistical correlation was found between different clinicopathological parameters (patient age, sex, TNM staging, number of lymph nodes invaded, extracapsular rupture of the tumour, its histopathological differentiation, or its site), the survival periods of patients with tumours possessing elevated levels of c-myc protein were found to be statistically shorter than those with lower levels of c-myc expression, (P less than 0.02). This indicates that c-myc expression may be an effective prognostic indicator in head and neck cancer.     

Severe anemia is associated with poor tumor oxygenation in head and neck squamous cell carcinomas

PURPOSE: To investigate the relationship between tumor oxygenation and the blood hemoglobin (Hb) concentration in patients with squamous cell carcinoma of the head and neck (SCCHN). METHODS AND MATERIALS: A total of 133 patients with SCCHN underwent pretreatment polarographic pO2 measurements of their tumors. In 66 patients measurements were also made in sternocleidomastoid muscles. The patients were divided into three groups according to their Hb concentration-severe anemia (Hb < 11.0 g/dl), mild anemia (female: Hb 11.0-11.9 g/dl; male: Hb 11.0-12.9 g/dl), and normal Hb concentration (female: Hb > or =12.0 g/dl; male: > or =13.0 g/dl). RESULTS: No significant difference in tumor oxygenation could be detected between mildly anemic patients and patients with a normal Hb level. However, the tumor oxygenation in the severely anemic group was significantly below that of each of the other two groups (p < 0.0001). There was no significant difference between the Hb groups in oxygenation of sternocleidomastoid muscles. In a multivariate analysis including Hb group, tumor volume, smoking habits, gender, T-stage, N-stage, and histologic grade a Hb level < 11 g/dl was found to be the strongest predictor for a poor tumor oxygenation. Smoking also had a marginal influence on median pO2. CONCLUSION: Our data suggest that a low Hb concentration and cigarette smoking contribute to inadequate oxygenation of SCCHN and thus for increased radioresistance. Consequently, Hb correction and abstinence from smoking may significantly improve tumor oxygenation.     

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.     

Hypermethylation of the RECK gene predicts poor prognosis in oral squamous cell carcinomas

The RECK gene is a novel tumor suppressor gene that regulates matrix metalloproteinases (MMPs) to inhibit tumor angiogenesis, invasion and metastasis. We investigated the methylation status of the RECK gene in 40 primary oral squamous cell carcinomas (OSCC) and 20 paired adjacent normal mucosa by methylation-specific PCR. Furthermore, we determined the prognostic importance of RECK hypermethylation in OSCC patients. Our findings showed that the RECK gene was methylated in 52.5% (21 of 40) of the primary OSCC. Among the 20 cases with corresponding normal tissues, RECK hypermethylation was detected in both primary tumor (55%, 11 of 20) and adjacent normal mucosa (30%, 6 of 20). Methylation of the RECK gene was not detected in all normal oral mucosa samples of the 12 healthy controls. In univariate analysis, RECK hypermethylation was inversely correlated with recurrence-free survival (p=0.027) and overall survival (p=0.023) of the OSCC patients. Multivariate analysis showed that the methylation status of the RECK gene was the only independent prognostic factor affecting overall survival (p=0.037). The result indicates that hypermethylation of RECK promoter is a common event in human OSCC, occurs concurrently in tumor-adjacent normal mucosa and is correlated with poor prognosis in OSCC patients. Although additional work is needed, hypermethylation of the RECK gene is a promising biomarker in early detection and prognosis for oral cancer patients.     

Gene amplification and overexpression of EGF receptor in squamous cell carcinomas of the head and neck

Tumours of the head and neck were examined for gene amplification and expression of the epidermal growth factor (EGF) receptor by Southern blot and Western blot analyses. The EGF receptor gene was found to be amplified in four (19%) of 21 squamous cell carcinomas. The EGF receptor was overexpressed in eight (53%) of 15 squamous cell carcinomas examined, including all four tumours showing gene amplification. No amplification or overexpression of the EGF receptor gene was detected in any of nine malignant or eight benign tumours of other types of the head and neck. The tumours showing amplification and/or overexpression of the EGF receptor gene (8/15) were all identified histologically as well differentiated squamous cell carcinomas, whereas none of the histologically less differentiated squamous cell carcinomas (0/9) showed amplification and/or overexpression of the EGF receptor gene. Within our sample set, no correlation was evident between amplification and/or overexpression and the clinical stage or tumour site. Our results support the possible involvement of gene amplification and overexpression of the EGF receptor in a subclass of squamous cell carcinomas of the head and neck.     

Overexpression of cell cycle regulatory proteins correlates with advanced tumor stage in head and neck squamous cell carcinomas

Squamous cell carcinoma of the head and neck region (HNSCC) is the sixth most frequent cancer worldwide. In the USA, 30,000 new cases and 8,000 deaths are reported each year. Differences between normal epithelium and cancer cells from the upper aerodigestive tract arise from alterations in expression of specific genes controlling proliferation and immortalization. The protein products of these genes include growth factor receptors, cell cycle regulators, and tumor suppressors which affect a variety of intracellular signaling pathways. To determine how altered expression of these gene products contribute to HNSCC progression, we examined expression of epidermal growth factor receptor (EGFR), cyclins, p16INK4A, c-myc, proliferating cell nuclear antigen (PCNA), and telomerase in archival pathology specimens by immunohistochemistry. A substantial majority of HNSCC tumors showed loss of p16INK4A expression and dramatic overexpression of EGFR. Overexpression of this receptor correlated with increased cyclin A levels and high mitotic index. EGFR, cyclins A, -B1, -E, and c-myc overexpression was significantly increased in stage III and IV tumors compared to early stage cancers. hTERT was expressed in all tumors and primarily in the basal layer cells of dysplastic epithelial lesions. Suprabasal expression of hTERT was found in a significantly higher number of HNSCC cases than in dysplastic lesions. These results indicate that overexpression of cell cycle regulatory proteins correlates with advanced tumor stage in HNSCC.     

Downregulation of SERPINB13 expression in head and neck squamous cell carcinomas associates with poor clinical outcome

Tumorigenesis of head and neck squamous cell carcinomas (HNSCC) is associated with various genetic changes such as loss of heterozygosity (LOH) on human chromosome 18q21. This chromosomal region maps a gene cluster coding for a family of intracellular serine protease inhibitors (serpins), including SERPINB13. As SERPINB13 expression in HNSCC has recently been shown to be downregulated both at the mRNA and protein levels, here we investigated if such a low SERPINB13 expression is associated with histopathological and clinical parameters of HNSCC tumors and patient survival. By generating specific antibodies followed by immunohistochemistry on a well‐defined cohort of 99 HNSCC of the oral cavity and oropharynx, SERPINB13 expression was found to be partially or totally downregulated in 75% of the HNSCC as compared with endogenous expression in non‐neoplastic epithelial cells. Downregulation of SERPINB13 protein expression in HNSCC was significantly associated with the presence of LOH at the SERPINB13 gene in the tumors (p = 0.006), a poor differentiation grade of the tumors (p = 0.001), the presence of a lymph node metastasis (p = 0.012), and a decreased disease‐free (p = 0.033) as well as overall (p = 0.018) survival of the patients. This is the first report demonstrating that downregulation of SERPINB13 protein expression in HNSCC is positively associated with poor clinical outcome. Therefore, SERPINB13 seems to act as an important protease inhibitor involved in the progression of HNSCC. © 2009 UICC     

Amplification of the int-2 gene in human head and neck squamous cell carcinomas

Head and neck squamous cell carcinomas (SCC) from 21 patients were analyzed for structurally rearranged or amplified proto-oncogenes by Southern blot hybridization. The int-2 proto-oncogene was amplified 3-5 fold in 5 (50%) of 10 laryngeal SCC and 2-3 fold in 5 (45%) of 11 nonlaryngeal SCC of the head and neck. Adjacent histologically normal tissue from the same patients had single int-2 gene copy number. Coamplification of int-2 and the epidermal growth factor receptor (c-erbB-1) gene was found in one laryngeal SCC and one SCC metastatic to the neck. No amplification or structural alterations of proto-oncogenes c-erbB-2/HER2, c-myc, H-ras-1, or K-ras-2 was detected in any of the head and neck tumors. In a survey of head and neck tumor-derived cell lines, int-2 was amplified 9 fold in a hypopharyngeal tumor cell line (FaDu), but not amplified in 3 laryngeal tumor cell lines. int-2 has been localized to the q13 band of chromosome 11. We used chromosome 11 specific probes to demonstrate that int-2 amplification was not due to complete or partial chromosome 11 duplication. int-2 amplification was localized to 11q13, but did not extend to the ets-1 locus 11q23. The results indicate that int-2 is frequently amplified in SCC of the head and neck and suggest that int-2 amplification may correlate with clinical disease progression.     

Current trends in head and neck squamous cell carcinomas

Squamous cell carcinomas of the head and neck remain frequent in France with an evolving epidemiology. Progress was observed both in diagnostic and treatments. Increasing possibilities of curative treatment should be provided within next years through surgery, radiotherapy, chemotherapy and target therapies as well as high performance imaging modalities and molecular biology and tumoral markers. Surgical techniques have improved function preservation while non surgical means have increased efficacy through chemo-radiotherapy. Assessment of optimal sequential therapeutic strategies is required, including both oncologic efficacy and quality of life.     

MAPKs activation in head and neck squamous cell carcinomas

Cancer of head and neck (HN) represents a diffused group of neoplasia; despite advances in clinical treatment its mortality rate has remained high, mainly because it is frequently diagnosed only at an advanced stage. Molecular markers allowing the diagnosis at an early stage would be useful. Mitogen-activated protein kinases (MAPK) are a family of protein kinases, which transduces extracellular stimuli into intracellular responses, controlling proliferation, differentiation and apoptosis. Given their key role in the regulation of cell survival and proliferation, MAPKs have been deeply studied in cancer. Furthermore, they are of particular interest in HN cancer progression, since they are downstream of one of the most frequently found alterations in this cancer: the over-expression of the epidermal growth factor receptor. Although a deregulation of MAPK expression and activation has been reported in several type of cancers, data regarding their role in HN cancer progression are contrasting, thus the aim of this short review is to summarize the data from literature regarding MAPKs activation in this type of cancer.     

Targeting EGF-receptor-signalling in squamous cell carcinomas of the head and neck

Despite significant advances in the use of surgery, chemotherapy and radiotherapy to treat squamous cell carcinoma of the head and neck (SCCHN), prognosis has improved little over the past 30 years. There is a clear need for novel, more effective therapies to prevent relapse, control metastases and improve overall survival. Improved understanding of SCCHN disease biology has led to the introduction of molecularly targeted treatment strategies in these cancers. The epidermal growth factor receptor (EGFR) is expressed at much higher levels in SCCHN tumours than in normal epithelial tissue, and EGFR expression correlates with poor prognosis. Therefore, much effort is currently directed toward targeting aberrant EGFR activity (e.g. cell signalling) in SCCHN. This review discusses the efficacy of novel therapies targeting the EGFR (e.g. anti-EGFR antibodies and EGFR tyrosine kinase inhibitors) that are currently tested in SCCHN patients.     

Cyclin D1 overexpression correlates with poor prognosis in patients with tongue squamous cell carcinoma

Tongue squamous cell carcinoma makes up a large percentage of head and neck cancers, and the incidence among young patients is increasing. The aim of this study was to reveal the correlation between cyclin D1 (CCND1) expression and clinical and histologic features. We performed an immunohistochemical study on the level of CCND1 expression in tumor specimens obtained from 94 patients with tongue squamous cell carcinoma. The relationship between the expression and the following features such as age, sex, smoking and alcohol intake history, T, N, histologic grade, and multiple primary cancer was analyzed. Eighteen patients (19%) showed CCND1 overexpression (tumor cell nuclei positivity >/=50%). The 5-year survival rate of high CCND1 expressors was 39%, which was significantly poor (p=0.04). N classification correlated with CCND1 expression. CCND1 overexpression is associated with poor survival associated with progression of lymph node spread in patients with tongue squamous cell carcinomas. CCND1 expression may be a useful biologic marker for prognosis.     

Oncogenic mutations of the PIK3CA gene in head and neck squamous cell carcinomas

Phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases that regulate cellular activities such as proliferation, survival, motility and morphology. Recent studies reported that the p110alpha (PIK3CA), catalytic subunit of PI3-kinase is somatically mutated in human cancers. Hot- spot mutations (E542K, E545K and H1047R) are reported to have higher oncogenic potential. Although PIK3CA mutations were reported in head and neck squamous cell carcinomas (HNSCC) of limited ethnicity, the functional consequences of HNSCC-associated PIK3CA mutations have not been examined. Status of PI3K signaling related genes (PTEN-RAS-EGFR) in the presence of PIK3CA mutation have not been reported. In this study, we analyzed exons 9 and 20 of PIK3CA in 54 samples, including 17 HNSCC cell lines, 19 Indian and 18 Vietnamese primary tumors. We found mutations in 29.4% (5/17) of HNSCC cell lines, 10.5% (2/19) of Indian tumors and no mutation (0/18) in Vietnamese tumors. Two homozygous PIK3CA mutations were found in cell lines and a novel insertion mutation with oncogenicity in Indian tumor. Analysis of PI3K signaling related genes showed that PIK3CA and PTEN mutations were mutually exclusive, though PTEN mutation is uncommon in HNSCC. However, PIK3CA mutation coexisted with H-RAS mutation. Furthermore, PIK3CA mutations were mutually exclusive to EGFR amplification. All the 5 mutants that we found in HNSCC, showed increased PI3 kinase activities, followed by growth factor independent higher colony forming efficiency, changes in morphology, higher rates of migration and invasion compared with PIK3CA wild-type. Our study is the first to examine the oncogenic potential of PIK3CA mutants associated with HNSCC and report on PIK3CA mutations in Indian and Vietnamese ethnicity. These results suggest that PIK3CA mutations in HNSCC are likely to be oncogenic and may significantly contribute to HNSCC carcinogenesis and pave attractive target for therapeutic prevention.     

人类乳头状瘤病毒感染与头颈部鳞状细胞癌

大量基础和临床研究的证据显示,人类乳头状瘤病毒(HPV)感染和头颈部鳞状细胞癌(HNSCC)的发生发展有关,但两者间确切发病机制尚不明确.HPV表达E6和E7导致被感染细胞发生恶性转化是其主要的致癌机制.HPV阳性HNSCC对放化疗更为敏感,预后更好.HPV疫苗可能成为预防和治疗HNSCC的重要手段.     

Time-dose-response relationships in postoperatively irradiated patients with head and neck squamous cell carcinomas.

BACKGROUND AND PURPOSE: To define the influence of the dose and time on the response to treatment in postoperatively irradiated head and neck cancer patients and to establish a good prediction of failure. METHODS AND MATERIALS: From January 1985 to December 1995, 214 patients with histologically proven head and neck squamous cell carcinomas were irradiated after radical surgery or single tumour resection according to surgical and histopathological findings. The total doses given ranged between 50 and 75 Gy to the primary bed tumour and between 42 and 56 Gy to the neck with fraction sizes of 1.7-2 Gy/day. The median length of the time interval between surgery and radiotherapy, time of irradiation and total treatment time were 81, 59 and 139 days, respectively. The end-point analyzed was the local-regional tumour control rate at the primary tumour bed and neck for 5 years from the beginning of radiotherapy. Univariate and multivariate analyses were used to determine predictors of failure from among the following studied variables: (i), clinical stage (T/N) of the patients; (ii), tumour grade; (iii), neck surgery; (iv), tumour margins; (v), histological tumour nodal extension; (vi), chemotherapy; (vii), normalized total dose; (viii), time interval between surgery and radiotherapy; (ix), time of irradiation; and (x), total treatment time. RESULTS: The actuarial 5-year tumour control rate for the entire group was 72%, and 92% of the patients who achieved local control are currently alive without disease. Tumour control was inversely related to T stage (83% for T2 vs. 57% for T4) and the probability of local control within each stage was dependent on the N status (> or =71% for T3-T4/N0 vs. 31-44% for T3-T4/N1-N3). Histological N status and tumour margins, but not tumour grade, impacted significantly on tumour control. When local control was analyzed as a function of the dose to the primary, a non-significant negative dose-response relationship was found. The total treatment time was a significant prognostic factor, and the time interval between surgery and irradiation proved to be an independent predictor of failure. CONCLUSIONS: Despite the absence of a statistically significant dose-response relationship, the present results suggest that postoperative irradiation treatment given to patients with head and neck squamous cell carcinomas should not be unduly prolonged, in order to minimize the amount of tumour cell proliferation. In these patients, nodal involvement, positive margins of the resected specimens and time interval between surgery and irradiation were the most important prognostic factors.     

Intratumoural level of SDF-1 correlates with survival in head and neck squamous cell carcinoma

The SDF-1/CXCR4 pathway has been suggested to play a role in the metastatic dissemination of various tumours. We assessed the prognostic impact of SDF-1 and CXCR4 expression in head and neck squamous cell carcinoma (HNSCC). Seventy-one HNSCC samples collected at the time of initial diagnosis were retrospectively analysed. SDF-1 and CXCR4 expression levels were measured using real-time RT-PCR and correlated to survival. After a median follow-up of 45 months, 25 patients (35%) died of cancer (group D), and 46 patients (65%) were alive or dead without evidence of HSNCC evolution (group A). The median level of CXCR4 expression was 0.33 and 0.29 in groups A and D, respectively (P=0.93), showing no correlation with recurrence or survival. By contrast, the median level of SDF-1 expression was significantly different in the A and D groups (2.41 vs 1.16, respectively, P=0.018). Using the median level as a cut-off, patients with low SDF-1 had poorer metastasis-free (P=0.026), disease-free (P=0.006) and overall specific survival rates (P=0.002). The prognostic value of SDF-1 was confirmed by a multivariate analysis. In this series of 71 HNSCC patients, the SDF-1 expression level correlated significantly with metastatic evolution and overall survival.