Max Partridge

Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines.

One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed in about half (44%) of OSCCs and its expression inversely correlated with p16/MTS1 expression. In conclusion, we show that abnormalities of the RB pathway are a common mechanism of oral carcinogenesis.

Expression of epidermal growth factor receptor on oral squamous cell carcinoma.

The expression of the receptor for epidermal growth factor (EGF) in normal oral mucosa, papillomas and squamous cell carcinoma (SCC) has been determined by immunohistology and autophophorylation studies. Immunoreactive receptor was localised using two antibodies which recognise the receptor; EGFR1 which reacts with sequences in the external domain of the receptor and F4 which recognises sequences in the internal domain. EGFR was present on basal, suprabasal and some spinous cells of normal oral mucosa. Regional variation in the distribution of receptor was apparent. A similar pattern of receptor expression was seen on oral papillomas. The distribution and intensity of epidermal growth factor receptor (EGFR) expression varied between 20 patients with oral SCC. The staining patterns seen with the two antibodies were similar on all tissue types. The protein tyrosine-kinase activity of the receptor present on eight oral SCC was also examined by immunoprecipitation and autophosphorylation studies. This procedure also demonstrated variations in the amount of functional EGFR in these tumours. There was no significant correlation between the level of EGFR expression and tumour behaviour.

LOH at 3p correlates with a poor survival in oral squamous cell carcinoma.

We analysed chromosome 3p for loss of heterozygosity (LOH) in 48 primary oral squamous cell carcinomas (SCCs) using 15 markers and constructed a deletion map for this chromosome arm. LOH at one or more loci was found in 34/48 (71%) of tumours. The data support the existence of at least three distinct regions of deletion at 3p24-26, 3p21.3-22.1 and 3p12.1-14.2. A significant correlation was observed between the number of regions showing allele loss at 3p and tumour stage, consistent with the progressive accumulation of genetic errors during the development of oral SCC. There were also significant associations between LOH at 3p and disease-free and overall survival of patients with early stage disease. This study is the first to demonstrate the prognostic significance of LOH at 3p for oral cancer and may help to identify patients who should receive more aggressive treatment.

Expression of the tumour suppressor gene p53 in oral cancer

In this preliminary series, the product of the tumour suppressor gene p53 was detected in 12/15 cases of oral squamous cell carcinoma (SCC) and in two cases of leukoplakia. The tumours either expressed the mutant form of p53 throughout the specimen or contained focal areas of positive cells. p53 expression was commonly observed in tumours obtained from patients who were heavy smokers and drinkers suggesting that alterations in the p53 gene may be one of the sites of genetic damage in this group of patients.

Field cancerisation of the oral cavity: comparison of the spectrum of molecular alterations in cases presenting with both dysplastic and malignant lesions.

Dysplastic lesions and invasive oral squamous cell carcinoma (SCC) from patients with field change were screened by restriction fragment length polymorphism (RFLP) and microsatellite assay. All tumours contained more genetic changes than the matched dysplasia which are likely to represent progression. Four of the 15 dysplastic lesions harboured the same abnormalities detected in the tumour and some paired lesions showed identical novel microsatellite alleles. The finding of identical genetic fingerprints in dysplastic lesions and invasive carcinoma from the same patient provides strong evidence that these dysplasias are precursor lesions and that multiple lesions have probably arisen due to transfer of the progeny of an altered cell. Eight of the 15 dysplastic lesions showed alterations which were not present in the matched cancer, showing that evolution of subclones, or fusion of multiple clones also occurs. A further case showed loss of different alleles in the paired samples. These findings highlight the complexity of the genetic abnormalities present in the mucosa of patients with field change and suggests that the origin of these altered foci may be diverse.

Genetics/epigenetics of oral premalignancy: current status and future research*

Squamous cell carcinoma (SCC) of the oral and oropharyngeal region is the sixth most common malignancy in the world today. Despite numerous advances in treatment, long-term survival from this disease remains poor. Early detection can decrease both morbidity and mortality associated with this neoplasm. However, screening for potentially malignant disease is typically confounded by difficulty in discriminating between reactive/inflammatory lesions vs those lesions that are premalignant in nature. Furthermore, the histologic diagnosis of dysplasia can be subjective and is thus prone to a considerable range of interpretation. Similarly, no definitive, validated criteria exist for predicting which dysplastic lesions are most likely to progress to cancer over time. Given this state of science, the presence of dysplasia can only be used to indicate that an oral lesion may have an increased risk of malignant transformation. Molecular biomarkers capable of identifying the subset of lesions likely to progress to cancer are required to eliminate this clinical diagnostic dilemma. The purpose of this review is to assess the current state of knowledge regarding genetic/epigenetic alterations observed in oral mucosal premalignancy. In addition, recommendations for future research studies directed at defining the predictive capacity of specific biomarkers in this modeling are presented.

The role of angiogenesis in the spread of oral squamous cell carcinoma

Microvessels were counted in 41 primary oral squamous cell carcinomas using JC70 antibody to PECAM (CD31). The counts were compared with clinical and pathological indicators of tumour behaviour including lymph node status, tumour stage, type of histological differentiation, size and velocity of tumour growth. Tumour microvessel counts correlated with lymph node metastasis (p < 0.001). This association was independent of tumour size, velocity and type of histological differentiation and when all the variables were analysed by multivariate analysis only vascular count showed a significant association with lymph node metastasis.

Location of candidate tumour suppressor gene loci at chromosomes 3p, 8p and 9p for oral squamous cell carcinomas.

To help define the location of tumour suppressor genes implicated in the pathogenesis of oral squamous cell carcinoma (SCC), we have used microsatellite assay and restriction fragment length polymorphism (RFLP) analysis to screen 48 primary SCC for allelic imbalance (AI) with 32 polymorphic markers at chromosome 3p, and prepared a detailed deletion map. The finding of a high frequency of AI at specific regions, together with the presence of multiple small interstitial deletions involving these loci, identifies 5 areas at this chromosome arm that may harbour tumour suppressor genes. No sequence aberrations affecting the von Hippel Lindau (VHL) and fragile histidine triad (FHIT) genes, which reside within the candidate tumour suppressor gene areas at this chromosome arm, were identified. A more limited analysis of polymorphic sequences at 8p and 9p supports the existence of at least 2 areas that harbour tumour suppressor genes at 8p and evidence that additional targets for deletion reside centromeric and telomeric to the p16 gene at 9p21. Int. J. Cancer 83:318–325, 1999.

Spectral Clustering of Microarray Data Elucidates the Roles of Microenvironment Remodeling and Immune Responses in Survival of Head and Neck Squamous Cell Carcinoma

PURPOSEnTo identify functionally related prognostic gene sets for head and neck squamous cell carcinoma (HNSCC) by unsupervised statistical analysis of microarray data.nnnPATIENTS AND METHODSnMicroarray analysis was performed on 14 normal oral epithelium and 71 HNSCCs from patients with outcome data. Spectral clustering (SC) analysis of the data set identified multiple vectors representing distinct aspects of gene expression heterogeneity between samples. Gene ontology (GO) analysis of vector gene lists identified gene sets significantly enriched within defined biologic pathways. The prognostic significance of these was established by Cox survival analysis.nnnRESULTSnThe most influential SC vectors were V2 and V3. V2 separated normal from tumor samples. GO analysis of V2 gene lists identified pathways with heterogeneous expression between HNSCCs, notably focal adhesion (FA)/extracellular matrix remodeling and cytokine-cytokine receptor (CR) interactions. Similar analysis of V3 gene lists identified further heterogeneity in CR pathways. V2CR genes represent an innate immune response, whereas high expression of V3CR genes represented an adaptive immune response that was not dependent on human papillomavirus status. Survival analysis demonstrated that the FA gene set was prognostic of poor outcome, whereas classification for adaptive immune response by the CR gene set was prognostic of good outcome. A combined FA&CR model dramatically exceeded the performance of current clinical classifiers (P < .001 in our cohort and, importantly, P = .007 in an independent cohort of 60 HNSCCs).nnnCONCLUSIONnThe application of SC and GO algorithms to HNSCC microarray data identified gene sets highly significant for predicting patient outcome. Further large-scale studies will establish the usefulness of these gene sets in the clinical management of HNSCC.

New insights into p53 protein stabilisation in oral squamous cell carcinoma

p53 is a transcription factor which regulates cell proliferation and apoptosis to prevent division of potentially malignant cells. In many tumours mutation of the p53 gene leads to stabilisation of this protein which can be detected by immunohistochemistry (IHC). However, there are many reports describing detection of p53 by IHC in the absence of gene mutation, and in these cases other factors stabilise p53. To shed light on the mechanisms which permit detection of this protein in these mutation-negative cases we have examined 45 primary oral squamous cell carcinomas (SCCs) by IHC and gene sequencing for p53 (exons 4-8) and related the results to a FAL score (determined using microsatellite assay and expressing the number of loci showing allelic imbalance as a fraction of the total number of informative markers for each case). We also investigated the pattern of MDM2 expression in these tumours. High levels of p53 protein were detected in 24/45 cases and point mutations involving exons 4-9 were seen in 11 cases. A further four cases harboured deletions or a stop codon. For 6/48 cases there was concordance of AI within the p53 gene and mutation. However nine cases showed p53 mutation only and 5 AI without mutation, suggesting that oral tumours frequently retain one normal p53 allele. Detection of p53 by IHC correlated strongly with the FAL score. Thus whilst it is possible that some tumours harbour p53 mutations outside the open reading frames examined, or are missed due to sequencing a mixture of normal and tumour tissue, a subgroup of tumours may express high levels of wild-type p53 as a reflection of the high FAL score and ongoing genomic stress. Levels of MDM2 transcripts and protein were similar in all SCCs examined. However, MDM2 may be non-functional, or there may be defects affecting other important regulatory proteins in tumours which which express wild type p53 protein.