RPS6KA2, a putative tumour suppressor gene at 6q27 in sporadic epithelial ovarian cancer.

We had previously defined by allele loss studies a minimal region at 6q27 (between D6S264 and D6S297) to contain a putative tumour suppressor gene. The p90 ribosomal S6 kinase-3 gene (p90 Rsk-3, RPS6KA2) maps in this interval. It is a serine-threonine kinase that signals downstream of the mitogen-activated protein kinase pathway. It is expressed in normal ovarian epithelium, whereas reduced or absent in tumours or cell lines. We show that RPS6KA2 is monoallelically expressed in the ovary suggesting that loss of a single expressed allele is sufficient to cause complete loss of expression in cancer cells. Further, we have identified two new isoforms of RPS6KA2 with an alternative start codon. Homozygous deletions were identified within the RPS6KA2 gene in two cell lines. Re-expression of RPS6KA2 in ovarian cancer cell lines suppressed colony formation. In UCI101 cells, the expression of RPS6KA2 reduced proliferation, caused G1 arrest, increased apoptosis, reduced levels of phosphorylated extracellular signal-regulated kinase and altered other cell cycle proteins. In contrast, small interfering RNA against RPS6KA2 showed the opposite effect in 41M cells. The above results suggest that RPS6KA2 is a putative tumour suppressor gene to explain allele loss at 6q27.

A case-control study confirms that microsatellite assay can identify patients at risk of developing oral squamous cell carcinoma within a field of cancerization.

Distinguishing true precursor lesions on the basis of clinical or histological features alone is unreliable but is important so that appropriate intervention can be instigated. Preliminary studies have shown that a microsatellite assay may provide important new prognostic information. To build on these observations, we have performed a case-control study to establish whether we can be confident about incorporating this new information into clinical practice. We have determined the frequency of allelic imbalance (AI) within key chromosomal regions, by matching 39 cases with dysplastic oral lesions that developed a tumor on the same side of the mouth, for as many variables as possible, with controls presenting with similar lesions that did not progress to malignancy when followed for the same period. Our findings confirm that the group that developed tumor had precursor lesions that harbor AI at more loci (P = 0.002). However, no consistent patterns of AI were associated with the three grades of dysplasia: mild, moderate, and severe. One-third of the tumors developed at the same site as the dysplastic lesion and two-thirds at a different site, which revealed that the presence of these aberrations in a dysplastic lesion provided information about the risk of malignant change within a larger field. This suggests that the process of field cancerization is more widespread than previously recognized. On the basis of these findings, we advocate complete excision of all suspicious areas that show AI at two or more key loci, regardless of the degree of dysplasia. However, because the remaining mucosa is also "at risk," these cases should also be targeted to receive dietary advice and chemoprevention, to minimize their risk of tumor formation at a distant site.

Profiling clonality and progression in multiple premalignant and malignant oral lesions identifies a subgroup of cases with a distinct presentation of squamous cell carcinoma.

A cohort of head and neck cancer patients, without exposure to tobacco and alcohol, presented with multiple preneoplastic and neoplastic lesions, the natural history of which may span several decades. Examination of these cases provides an opportunity to study the relationship between genetic, morphological, and clonal progression in these fields and establish whether they represent a unique presentation of squamous cell carcinoma. The presence of a common novel microsatellite allele, a common breakpoint or concordant allelic imbalance at multiple loci, reveals that a high proportion of these serial lesions arise due to spread of a precursor. The tumors arising in these patients were typically nonaggressive, although metastases developed at a late stage, supporting the notion that the genotype results in a phenotype with a propensity for lateral spread, rather than invasion. Different genetic aberrations were detected in morphologically similar phenotypes such that no consistent early or late events were associated with development of premalignant lesions. Combining information about the clinicopathological features and histological examination of the margins with that derived from clonality analysis reveals that a subgroup of patients, without exposure to the traditional risk factors associated with this disease, developed multiple clonally related oral lesions and represents a unique presentation of head and neck squamous cell carcinoma. We suggest the term clonal cancerization to describe multiple premalignant and malignant lesions when there is conclusive evidence that they arise due to lateral spread from a common precursor.

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.

Allelic imbalance at chromosomal loci implicated in the pathogenesis of oral precancer, cumulative loss and its relationship with progression to cancer.

A microsatellite assay was used to screen 31 potentially malignant oral lesions presenting as leukoplakia and erythroplakia, with histological evidence of dysplasia, for genetic abnormalities at loci which frequently show allelic imbalance when oral squamous cell carcinomas (SCC) are examined. The microsatellite and restriction fragment length polymorphism (RFLP) markers selected were at 3p21, 8p21-23, 9p21 and included sequences within the Rb (13q14.2), p53 (17p13.1) and DCC (18q21.1) tumour suppressor genes. 8 patients subsequently developed an invasive tumour at the same site, or within 2 cm of the premalignant lesion. A further 8 patients developed SCC at a distant site. Seventy-seven per cent (24/31) of these potentially malignant lesions showed allelic imbalance (AI) and 55% (17/31) of cases showed microsatellite instability (msi). The probability of developing SCC was much greater for patients with lesions showing AI at two or more relevant loci (P = 0.008 by the logrank test) than the group with AI at fewer loci. The estimated probability of development of SCC in this group by 5 years was 73% (95% Cl: 50-92%). This suggests that determining the number of genetic abnormalities in a potentially malignant lesion can help identify patients with true precancers who should be followed closely to ensure that they receive chemoprevention and appropriate advice to limit risk factors, and to allow the early detection of invasive lesions.

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.

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.

Frequent gene deletions in potentially malignant oral lesions.

Some oral cancers are preceded by premalignant lesions which include leucoplakia and erythroplakia. At present there are no reliable markers to identify lesions that may progress to malignancy. We have analysed 30 potentially malignant oral lesions for deletions at chromosomal regions that harbour tumour-suppressor genes for oral cancer. A total of 16 of 30 cases (53%) showed loss of heterozygosity (LOH) or allele imbalance at TP53, DCC, 3p21.3-22.1 or 3p12.1-13. These genetic alterations were detected in dysplastic lesions but not in histologically normal mucosa and may be early events in the carcinogenic process. A total of 64% of dysplastic lesions that recurred during the study showed LOH or allele imbalance in the initial biopsy and the number of genetic abnormalities increased in the tumours that developed. This type of molecular profiling may help to identify patients with lesions that may recur or acquire additional genetic events and progress to malignancy.

A clinical and genetic study of X-linked recessive ichthyosis and contiguous gene defects.

X-linked recessive ichthyosis (XLI) is caused by a deletion, or mutation, of the steroid sulphatase gene on the distal short arm of the X chromosome (Xp22.3). This region of the X chromosome is particularly susceptible to deletions. Such deletions can occasionally extend to involve neighbouring genes, causing a contiguous gene defect. Therefore, XLI may be associated with Kallmann's syndrome (KS), mental retardation, X-linked recessive chondrodysplasia punctata and short stature. We have reviewed 33 patients with XLI. Nine showed evidence of contiguous gene defects. A further four had neurological deficit sustained at the time of birth. This study highlights the importance of screening patients with X-linked recessive ichthyosis for neighbouring genetic disorders and, in particular, the early identification of KS, as delay in diagnosis may lead to infertility and osteoporosis. Parents should be warned about possible obstetric complications due to prolonged labour in future pregnancies.

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.

The prognostic significance of allelic imbalance at key chromosomal loci in oral cancer.

Forty-eight primary oral squamous cell carcinomas (SCC) were screened for allelic imbalance (AI) at 3p24-26, 3p21, 3p13, 8p21-23, 9p21, 9q22 and within the Rb, p53 and DCC tumour suppressor genes. AI was detected at all TNM stages with stage 4 tumours showing significantly more aberrations than stage 1-3. A factional allelic loss (FAL) score was calculated for all tumours and a high score was associated with development of local recurrence (P = 0.033) and reduced survival (P = 0.0006). AI at one or more loci within the 3p24-26, 3p21, 3p13 and 9p21 regions or within the THRB and DCC genes was associated with reduced survival. The hazard ratios for survival analysis revealed that patients with AI at 3p24-26, 3p13 and 9p21 have an approximately 25 times increase in their mortality rate relative to a patient retaining heterozygosity at these loci. AI at specific pairs of loci, D3S686 and D9S171 and involving at least two of D3S1296, DCC and D9S43, was a better predictor of prognosis than the FAL score or TNM stage. These data suggest that it will be possible to develop a molecular staging system which will be a better predict of outcome than conventional clinicopathological features as the molecular events represent fundamental biological characteristics of each tumour.

Patient-specific mutation databases for oral cancer.

Development of databases, summarising the genetic events associated with oral squamous cell carcinoma (SCC), should increase our understanding of the molecular basis of these lesions. Additionally, databases will help establish whether different cancer subtypes show different growth characteristics, because the multistage carcinogenic process is different in the various tumour subtypes. This new knowledge may also provide new prognostic information, as these aberrations represent fundamental biological characteristics of each tumour. To assess the value of incorporating the results from loss of heterozygosity (LOH) analysis into patient-specific mutation databases, we have carried out microsatellite analysis with 52 polymorphic markers at 13 key chromosomal regions implicated in the pathogenesis of head and neck cancers. Altered expression of the Rb, p53 and DCC tumour suppressor genes has also been studied by immunohistology. Our results shed light on the different pathways that lead to cancer and reveal that a variety of different patterns of allelic imbalance (AI) were detected at all TNM stages, reflecting the different clinical behaviour that tumours classified as being of the same TNM stage may exhibit. Summarising the level of genetic damage as a fractional allelic loss (FAL) score and the presence of AI at 3p22-26, 3p14.3-12.1 and 9p21 was found to be a better predictor of outcome than the TNM system. This finding suggests that molecular data can be incorporated into conventional staging systems to provide more accurate prognostic information for this group of patients.