Analysis of tumor suppressor p53 status in head and neck squamous cell carcinoma

Head and neck cancer belongs to the most common types of cancer in both males and females with a mortality rate of approximately 50%. More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC). Carcinogenesis of this disease involves activation of proto-oncogenes and inactivation of tumor suppressor genes. Among them, aberrations of p53 tumor suppressor gene are common events. The aim of this study was to assess the frequency of the tumor suppressor p53 aberrations in Czech population by using a functional test in yeast (FASAY) and by two immunochemical methods. We compared results of the methods and assessed the relationship between the presence of p53 aberration and some clinico-pathological parameters. The following observations were made: i) the accumulated p53 protein was detected in 33 of 50 tested samples (66%) by immunohistochemical analysis and in 27 of 49 tested samples (55.1%) by immunoblotting; ii) the presence of p53 mutation was detected in 36 of 50 tested samples (72%); iii) 6 of 36 p53 mutations detected by FASAY were temperature sensitive (16.7%); iv) 2 independent p53 mutations were found in at least 2 of the 36 positive cases; v) no statistically significant relationship was found between p53 aberration and overall survival.     

Sequential p53 mutation analysis of pre-invasive and invasive head and neck squamous carcinoma

Single-stranded conformation polymorphism (SSCP) and direct sequencing were performed on uninvolved mucosa, severe dysplasia and invasive carcinoma samples from 20 patients with head and neck squamous carcinoma. Seven (35%) of the non-invasive lesions and 15 (75%) of the invasive carcinomas manifested p53 mutations. Although the majority of mutations were mis-sense, resulting in single amino acid substitution, a silent mutation encoding for the same amino acid and 2 non-sense mutations encoding a stop codon were also observed. Mutations in invasive carcinoma were mostly in exon 8 and involved codons 296, 288 and 298; non-invasive lesions showed more mutations at exons 5 to 7. Five lesions showed simultaneous mutations in 2 different exons; in 3 both non-invasive and invasive carcinomas showed primary mutation at exons 5 to 7, and invasive carcinoma showed a secondary mutation at exon 8. Different codon mutations in the same exon between dysplastic and the corresponding carcinoma samples were found in 2 cases. p53 alterations were not observed in any of the normal mucosa samples. No apparent association between p53 mutations and conventional clinicopathologic parameters, including DNA content, was found in this cohort. Our study indicates that (i) p53 alteration is an early event in the genesis of a subset of head and neck squamous carcinomas, (ii) normal mucosa within the resected specimens lacked p53 mutation, (iii) sequential mutations of different exons of the p53 gene suggests accumulation of genetic alterations during the neoplastic transformation of these lesions and (iv) the difference in codon mutation of the same exon between dysplastic and corresponding carcinoma suggests an independent clonal development. © 1995 Wiley-Liss, Inc.     

p53 mutation, but not p53 overexpression, correlates with survival in head and neck squamous cell carcinoma.

Survival in squamous cell carcinoma of the head and neck (HNSCC) was compared with overexpression and mutation of the p53 gene. Archival tissue from 77 tumours was analysed for protein expression using immunohistochemistry (IHC) with the monoclonal antibody Do-7, and for the presence of mutation in exons 5-8 using single-stranded conformation polymorphism (SSCP), followed by DNA sequencing in SSCP-positive cases. p53 expression was scored as high (>70% nuclei stained) in 25 (32%) tumours, as intermediate (10-70% nuclei stained) in 19 (25%) tumours and as low (<10% nuclei stained) in 33 (43%) tumours. Twelve (18%) tumours exhibited gene mutation (ten missense and two nonsense mutations) and an additional five tumours contained changes that could not result in amino acid substitution or protein truncation. There was no correlation between gene expression and mutation, mutations being equally frequent in tumours with either high (4/25), intermediate (4/19) or low protein expression (4/33). Fifty-eight patients were eligible for survival analysis. There was a strong correlation between p53 mutation and cause-specific survival; median survival among mutated cases was 12.5 months compared with >160 months among non-mutated patients (P < 0.005). There was no correlation between p53 overexpression and survival. The results suggest that p53 mutation status is an important prognostic factor in HNSCC, and that IHC analysis of protein overexpression is an inadequate measure of gene mutation in these tumours.     

Detection of head and neck squamous cell carcinoma among exfoliated oral mucosal cells by microsatellite analysis.

Prompt detection of head and neck squamous cell carcinoma (HNSCC) is vital to successful patient management. In this feasibility study, we used microsatellite analysis to detect tumor-specific genetic alterations in exfoliated oral mucosal cell samples from patients with known cancer. Exfoliated mucosal cells in pretreatment oral rinse and swab samples were collected from 44 HNSCC patients and from 43 healthy control subjects (20 nonsmokers and 23 smokers). We tested a panel of 23 informative microsatellite markers to assay DNA from the matched lymphocyte, tumor (from cancer cases), and oral test samples. Loss of heterozygosity or microsatellite instability of at least one marker was detected in 38 (86%) of 44 primary tumors. Identical alterations were found in the saliva samples in 35 of these 38 cases (92% of those with markers; 79% overall) including 12 of 13 cases with small primaries [stage Tt or Tx (occult primary)] and 4 of 4 cases of patients that had undergone prior radiation. Microsatellite instability was detectable in the saliva in 24 (96%) of 25 cases in which it was present in the tumor, and loss of heterozygosity was identified in the test sample in 19 (61%) of 31 cases. No microsatellite alterations were detected in any of the samples from the healthy control subjects. This approach must now be refined and validated for the detection of clinically occult disease. Microsatellite analysis of oral samples may then become a valuable method for detecting and monitoring HNSCC.     

头颈部鳞状细胞癌中与p53突变相关基因的鉴定

目的:探讨在头颈部鳞状细胞癌（HNSCC）中与p53突变相关的潜在关键基因。方法:从GEO数据库中下载芯片数据GSE107591,用R语言加权重基因共表达网络分析(weighted gene co-expression network analysis,WGCNA)包构建基因共表达网络并划分模块。选择与p53变异相关的基因模块进行GO分析和KEGG通路分析,并结合cytoscape筛选中枢基因。结果:基因共表达网络包括7个模块。其中蓝色（blue）模块与p53变异呈正相关,GO富集分析结果为细胞外基质组织等,KEGG通路为ECM受体相互作用等。蓝色模块的中枢基因为TPX2,CCNB2和DLGAP5。绿松石（turquoise）模块与p53变异呈负相关,GO富集分析结果为转录、DNA模板化等,KEGG通路为细胞黏附分子等。绿松石模块的中枢基因包括VWA3A,ARMC4,CFAP46和C11orf88（并列第三）。结论:通过WGCNA的方法能够从转录组数据中挖掘到头颈部鳞状细胞癌中与p53变异相关的重要基因,为疾病研究提供新的候选基因和分子机制。     

Adenoviral p53 gene therapy in head and neck squamous cell carcinoma cell lines

We reconstructed the recombinant p53-expressing adenovirus and examined its infections and effects in head and neck squamous cell carcinoma cell lines. Eight human head and neck squamous cell carcinoma cell lines were infected by the recombinant adenovirus harboring the lacZ gene (AxCAiLacZ) or the wild-type p53 gene (AxCAip53), and the effects were investigated. The eight cell lines were successfully infected by AxCAiLacZ at a level of more than 50%. The survival of all 8 squamous cell lines were inhibited in the range from 8 to 26.7% by only one treatment of the AxCAip53 infection. This result suggested that p53 gene therapy might become a useful tool in head and neck squamous cell carcinoma treatment.     

Expression of p53 isoforms in squamous cell carcinoma of the head and neck

Recent data indicate that, similar to p63 and p73, several different p53 isoforms can be produced in humans through alternative initiation of translation, usage of an internal promoter and alternative splicing. These isoforms are reported to have varying functions and expressions. In squamous cell carcinoma of the head and neck (SCCHN), disruption of the p53 pathway is one of the most common genetic alterations. However, to our knowledge, no studies regarding the expression of different p53 isoforms in SCCHN have so far been performed. We screened for the expression of different p53 isoforms in SCCHN and clinically normal oral epithelia using nested RT-PCR. p53 mRNA was expressed in all tumours, all matched clinically normal tissue adjacent to the tumour and in buccal mucosa from healthy volunteers. Of the novel isoforms, p53beta was detected in the majority of samples analysed, and all of the recently described isoforms were also detected in at least some tumour and normal epithelium samples, with the exception of Deltap53 isoforms. We conclude that p53 variant mRNAs are expressed in both normal oral stratified epithelium and SCCHN. Improvements in methodologies and reagents to detect and quantify p53 isoform expression in clinical material will be required to correlate p53 status with clinical outcomes.     

Radioresistance in oral squamous cell carcinoma with p53 DNA contact mutation

Reliable variables to predict the radiosensitivity of each tumor have not been identified. Recent studies have demonstrated that specific regions of mutations within the core domain of p53 protein correlate with responses to chemotherapy and radiotherapy in some tumor types. In this study, we evaluated the relationship between specific p53 mutations and radiosensitivity in 49 patients with oral squamous cell carcinomas (SCCs) who underwent preoperative radiotherapy. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. We detected p53 mutations in 27 (55.1%) cases. DNA contact mutations were detected in 11 (40.7%) of these 27 cases in L3 loop, loop-sheet-helix motif, and zinc-binding residues. Tumors containing p53 DNA contact mutations had significantly poorer responses to radiation than the other tumors, although no statistically significant difference between tumors with and without p53 mutations was found. These data indicate that DNA contact mutation of p53 could be a useful marker to predict the radioresistance of oral SCCs.     

外周血循环肿瘤细胞的检测在头颈部鳞癌中的应用

目的:检测头颈部鳞状细胞癌患者的外周血中循环肿瘤细胞（circulating tumor cells,CTCs）的数量,探讨CTCs与患者临床病理特征及预后的相关性。方法:选取我院2016年6月至2017年6月住院的60例头颈部鳞癌患者为实验组,10例健康志愿者作为阴性对照组,通过莱尔生物科技循环肿瘤细胞检测技术检测患者外周血中CTCs的表达数量,分析其相关的临床意义。结果:10例健康对照者均未检测到CTCs,60例头颈部鳞癌患者CTCs阳性率是76.7%（46/60）,二者差异有统计学意义（P<0.001）。单因素分析显示CTCs与淋巴结N分期（P=0.023）、临床TNM分期（P<0.001）相关,多因素分析显示CTCs检出仅与临床TNM分期（P=0.002）相关。生存分析的Kaplan-Meier法显示CTCs与患者无进展生存期（progression free survival,PFS）相关（P=0.039）,与总生存期（overall survival,OS）无关;Cox多因素回归分析则显示CTCs检出与PFS、OS均无明显相关。结论:头颈部鳞癌患者外周血CTCs检出与临床分期相关,可用于指导临床病情的评价,并有一定的预后价值。     

p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma: p53 mutations in primary tumor and matched lymph node metastases

In order to define the diagnostic value of p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma (HNSCC), we investigated p53 mutations in primary tumors (PT) and matched lymph node metastases (LNM); the underlying question being whether differentiation between metastatic disease of a known PT or (a metastasis of) a synchronous or metachronous second tumor is possible by means of p53 sequencing-based mutation analysis. In 15 PT, the p53 status was analyzed, following RNA isolation, cDNA synthesis and polymerase chain reaction amplification, by direct sequencing full-length mRNA. Mutations thus found were confirmed by DNA sequencing analysis of the corresponding exon in the PT. When RNA isolation was defective, DNA sequencing analysis of exons 1 through 11 was performed. In the matched LNM, DNA analysis of the corresponding exon was performed to prove the presence of the same p53 mutation. In the event of small clones not detectable by direct sequencing, an oligo ligation assay was developed to detect a specific mutation. The presence of germline mutations was excluded by DNA sequencing analysis of the corresponding exon of peripheral blood leucocytes. In 14 PT (94%), a mutation was identified. In one PT, no p53 mutation could be identified either after full-length mRNA sequencing or after sequencing exons 1 through 11. In all cases of PT and matched LNM, the mutations proved to be identical. We conclude that p53 mutations develop in carcinogenesis before metastases occur and are maintained during metastasis. Consequently, p53 may serve as a clonal marker not susceptible to change during tumor metastasis. This merits further exploration of the application of p53 mutation analysis in differentiating between metastatic disease from a known PT versus a metastasis of another second PT.     

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV‐positive than non‐oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV‐positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus‐unrelated HNSCC and virus‐related HNSCC consisting of nasopharyngeal and HPV‐positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus‐unrelated HNSCC. A disruptive mutation was never found in virus‐related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus‐unrelated HNSCC. Moreover, in virus‐unrelated HNSCC, G:C to T:A transversions were more frequent in ever‐smokers than in never‐smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever‐smokers than in never‐smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus‐related and virus‐unrelated subgroups. In virus‐related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus‐unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.     

Thermoradiotherapy combined with adenoviral p53 gene therapy in head and neck squamous cell carcinoma cell lines

We examined effects of recombinant p53-expressing adenovirus combined with thermoradiotherapy in 8 head and neck squamous cell carcinoma (SCC) cell lines to improve the outcomes of the treatment of advanced head and neck cancer. The p53 gene therapy did not improve the discrepancy between thermoradiosensitivities among the 8 SCC cell lines. However, p53 gene therapy improved the effects of thermoradiotherapy in all 8 cell lines, and there were significant differences in four situations of the HSC4 44 degrees C (p=0.032), SAS at 44 degrees C (p=0.029), the KB at 43 degrees C (p=0.025), and the Ca9-22 43 degrees C (p=0.020). In comparing the survival rates of thermoradiotherapy with those of thermotherapy and radiotherapy, thermoradiotherapy demonstrated actual survival rates less than theoretical survival rates based on the survival rates of thermotherapy multiplied by the survival rates of radiotherapy in almost all treatments of thermoradio-gene therapy of the 8 SCC cell lines. These results demonstrate that thermoradiotherapy combined with p53 gene therapy may be a useful tool in treating SCC cells.     

Bcl-xL inhibits p53- but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line

Nonfunctional p53 and especially upregulation of Bcl-x(L) result in advanced disease and poor prognosis of patients suffering head and neck squamous cell carcinoma (HNSCC). Aberrancies of Bcl-x(L) and/or p53 in HNSCC lead to inability of anticancer drugs to induce apoptosis. Bcl-x(L) and/or mutated p53 inhibit the apoptotic process by preventing the mitochondrial release of cytochrome c and/or activation of execution caspases. Here, we report that expression of the avian virus-derived apoptin protein resulted in induction of apoptosis in the HNSCC-derived cell line UMSSC-14B despite the presence of nonfunctional p53. Apoptin activated the execution caspase 3 and induced the release of mitochondrial cytochrome c. Upregulation of Bcl-x(L) in UMSCC-14B cells did not interfere with the apoptin-induced apoptosis, whereas it clearly negatively affected the p53-induced one. Bcl-x(L) significantly decreased the p53-induced cytochrome c release, but not the apoptin-triggered one. Our data demonstrate that apoptin induces apoptosis independent of Bcl-x(L) and p53 and may constitute a potential therapeutic agent for treatment of HNSCC.     

p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck squamous cell carcinoma: a prospective series.

PURPOSE: The tumor suppressor gene p53 plays a crucial role in cell cycle control and apoptosis in response to DNA damages. p53 gene mutations and allelic losses at 17p are one of the most common genetic alterations in primary head and neck squamous cell carcinoma (HNSCC). Alterations of the p53 gene have been shown to contribute to carcinogenesis and drug resistance. PATIENTS AND METHODS: In this prospective series, patients with HNSCC were treated with cisplatin-fluorouracil neoadjuvant chemotherapy. p53 status was characterized in 106 patients with HNSCC (p53 mutations, allelic losses at p53 locus, and plasma anti-p53 antibodies) to determine the existence of a relationship between p53 gene status and response to neoadjuvant chemotherapy. RESULTS: Exons 4 to 9 of the p53 gene were analyzed, and mutations were found in 72 of 106 patients with HNSCC. p53 mutations were associated with loss of heterozygosity at chromosome 17p (P <.001). The prevalence of p53-mutated tumors was higher in the group of patients with nonresponse to neoadjuvant chemotherapy than in the group of responders (81% v 61%, respectively; P <.04). When compiling p53 mutations and anti-p53 antibodies in plasma, the correlation between p53 status and response to chemotherapy was significant (87% v 57%, respectively; P =.003). A multivariate analysis showed that p53 status is an independent predictive factor of response to chemotherapy. CONCLUSION: This prospective study suggests that p53 status may be a useful indicator of response to neoadjuvant chemotherapy in HNSCC.     

DNA copy number gains in head and neck squamous cell carcinoma

Gene amplification, a common mechanism for oncogene activation in cancer, has been used as a tag for the identification of novel oncogenes. DNA amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC) and potential oncogenes have already been reported. We applied restriction landmark genome scanning (RLGS) to study gene amplifications and low-level copy number changes in HNSCC in order to locate previously uncharacterized regions with copy number gains in primary tumor samples. A total of 63 enhanced RLGS fragments, indicative of DNA copy number changes, including gains of single alleles, were scored. Enhanced sequences were identified from 33 different chromosomal regions including those previously reported (e.g. 3q26.3 and 11q13.3) as well as novel regions (e.g. 3q29, 8q13.1, 8q22.3, 9q32, 10q24.32, 14q32.32, 17q25.1 and 20q13.33). Furthermore, our data suggest that amplicons 11q13.3 and 3q26.3-q29 may be divided into possibly two and three independent amplicons, respectively, an observation supported by published microarray expression data.