SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma.

BACKGROUND: Extracapsular spread (ECS) in cervical lymph nodes is the single-most prognostic clinical variable in oral squamous cell carcinoma (OSCC), but diagnosis is possible only after histopathological examination. A promising biomarker in the primary tumour, alpha smooth muscle actin (SMA) has been shown to be highly prognostic, however, validated biomarkers to predict ECS prior to primary treatment are not yet available. METHODS: In 102 OSCC cases, conventional imaging was compared with pTNM staging. SERPINE1, identified from expression microarray of primary tumours as a potential biomarker for ECS, was validated through mRNA expression, and by immunohistochemistry (IHC) on a tissue microarray from the same cohort. Similarly, expression of SMA was also compared with its association with ECS and survival. Expression was analysed separately in the tumour centre and advancing front; and prognostic capability determined using Kaplan-Meier survival analysis. RESULTS: Immunohistochemistry indicated that both SERPINE1 and SMA expression at the tumour-advancing front were significantly associated with ECS (P<0.001). ECS was associated with expression of either or both proteins in all cases. SMA+/SERPINE1+ expression in combination was highly significantly associated with poor survival (P<0.001). MRI showed poor sensitivity for detection of nodal metastasis (56%) and ECS (7%). Both separately, and in combination, SERPINE1 and SMA were superior to MRI for the detection of ECS (sensitivity: SERPINE1: 95%; SMA: 82%; combination: 81%). CONCLUSION: A combination of SMA and SERPINE1 IHC offer potential as prognostic biomarkers in OSCC. Our findings suggest that biomarkers at the invasive front are likely to be necessary in prediction of ECS or in therapeutic stratification.

A microRNA-based prediction algorithm for diagnosis of non-small lung cell carcinoma in minimal biopsy material.

BACKGROUND: Diagnosis is jeopardised when limited biopsy material is available or histological quality compromised. Here we developed and validated a prediction algorithm based on microRNA (miRNA) expression that can assist clinical diagnosis of lung cancer in minimal biopsy material to improve clinical management. METHODS: Discovery utilised Taqman Low Density Arrays (754 miRNAs) in 20 non-small cell lung cancer (NSCLC) tumour/normal pairs. In an independent set of 40 NSCLC patients, 28 miRNA targets were validated using qRT-PCR. A prediction algorithm based on eight miRNA targets was validated blindly in a third independent set of 47 NSCLC patients. The panel was also tested in formalin-fixed paraffin-embedded (FFPE) specimens from 20 NSCLC patients. The genomic methylation status of highly deregulated miRNAs was investigated by pyrosequencing. RESULTS: In the final, frozen validation set the panel had very high sensitivity (97.5%), specificity (96.3%) and ROC-AUC (0.99, P=10(-15)). The panel provided 100% sensitivity and 95% specificity in FFPE tissue (ROC-AUC=0.97 (P=10(-6))). DNA methylation abnormalities contribute little to the deregulation of the miRNAs tested. CONCLUSION: The developed prediction algorithm is a valuable potential biomarker for assisting lung cancer diagnosis in minimal biopsy material. A prospective validation is required to measure the enhancement of diagnostic accuracy of our current clinical practice.

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) testing in oropharyngeal squamous cell carcinoma (OPSCC) is now advocated. Demonstration of transcriptionally active high-risk HPV (HR-HPV) in fresh tumour tissue is considered to be the analytical 'gold standard'. Clinical testing has focused on formalin-fixed paraffin-embedded (FFPE) tissue at the expense of sensitivity and specificity. Recently, a novel RNA in situ hybridisation test (RNAscope) has been developed for the detection of HR-HPV in FFPE tissue; however, validation against the 'gold standard' has not been reported. METHODS: A tissue microarray comprising FFPE cores from 79 OPSCC was tested using HR-HPV RNAscope. Analytical accuracy and prognostic capacity were established by comparison with the reference test; qRT-PCR for HR-HPV on matched fresh-frozen samples. RESULTS: High-risk HPV RNAscope had a sensitivity and specificity of 97 and 93%, respectively, against the reference test. Kaplan-Meier estimates of disease-specific survival (DSS, P=0.001) and overall survival (OS, P<0.001) by RNAscope were similar to the reference test (DSS, P=0.003, OS, P<0.001) and at least, not inferior to p16 immunohistochemistry +/- HR-HPV DNA-based tests. CONCLUSION: HR-HPV RNAscope demonstrates excellent analytical and prognostic performance against the 'gold standard'. These data suggest that the test could be developed to provide the 'clinical standard' for assigning a diagnosis of HPV-related OPSCC.

The epigenetic landscape of oral squamous cell carcinoma.

BACKGROUND: There is relatively little methylation array data available specifically for oral squamous cell carcinoma (OSCC). This study aims to compare the DNA methylome across a large cohort of tumour/normal pairs. METHODS: DNA was extracted from 44 OSCCs and paired normal mucosa. DNA methylation analysis employed the Illumina GoldenGate high-throughput array comprising 1505 CpG loci selected from 807 epigenetically regulated genes. This data was correlated with extracapsular spread (ECS), human papilloma virus (HPV) status, recurrence and 5-year survival. RESULTS: Differential methylation levels of a number of genes distinguished the tumour tissue sample from the matched normal. Putative methylation signatures for ECS and recurrence were identified. The concept of concordant methylation or CpG island methylator phenotype (CIMP) in OSCC is supported by our data, with an association between 'CIMP-high' and worse prognosis. Epigenetic deregulation of NOTCH4 signalling in OSCC was also observed, as part of a possible methylation signature for recurrence, with parallels to recently discovered NOTCH mutations in HNSCC. Differences in methylation in HPV-driven cases were seen, but are less significant than that has been recently proposed in other series. CONCLUSION: Although OSCC seems as much an 'epigenetic' as a genetic disease, the translational potential of cancer epigenetics has yet to be fully exploited. This data points to the application of epigenetic biomarkers and targets available to further the development of therapy in OSCC.

Quantitative promoter methylation differentiates carcinoma ex pleomorphic adenoma from pleomorphic salivary adenoma.

BACKGROUND: potential epigenetic biomarkers for malignant transformation to carcinoma ex pleomorphic adenoma (Ca ex PSA) have been sought previously with and without specific comparison with the benign variant, pleomorphic salivary adenoma (PSA). Previous analysis has been limited by a non-quantitative approach. We sought to demonstrate quantitative promoter methylation across a panel of tumour suppressor genes (TSGs) in both Ca ex PSA and PSA. METHODS: quantitative methylation-specific real-time polymerase chain reaction (qMSP) analysis of p16(INK4A), CYGB, RASSF1, RARß, human telomerase reverse transcriptase (hTERT), Wilms' tumour 1 (WT1) and TMEFF2 gene promoters was undertaken on bisulphite-converted DNA, previously extracted from archival fixed tissue specimens of 31 Ca ex PSA and an unrelated cohort of 28 PSA. All target regions examined had formerly been shown to be hypermethylated in salivary and/or mucosal head and neck malignancies. RESULTS: the qMSP demonstrated abnormal methylation of at least one target in 20 out of 31 (64.5%) Ca ex PSA and 2 out of 28 (7.1%) PSA samples (P<0.001). RASSF1 was the single gene promoter for which methylation is shown to be a statistically significant predictor of malignant disease (P<0.001) with a sensitivity of 51.6% and a specificity of 92.9%. RARß, TMEFF2 and CYGB displayed no apparent methylation, while a combinatory epigenotype based on p16, hTERT, RASSF1 and WT1 was associated with a significantly higher chance of detecting malignancy in any positive sample (odds ratio: 24, 95% CI: 4.7-125, P<0.001). CONCLUSIONS: we demonstrate the successful application of qMSP to a large series of historical Ca ex PSA samples and report on a panel of TSGs with significant differences in their methylation profiles between benign and malignant variants of pleomorphic salivary adenoma. qMSP analysis could be developed as a useful clinical tool to differentiate between Ca ex PSA and its benign precursor.

EUELC project: a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration.

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular-pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARbeta genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes.

Cytoglobin is upregulated by tumour hypoxia and silenced by promoter hypermethylation in head and neck cancer.

BACKGROUND: Cytoglobin (Cygb) was first described in 2002 as an intracellular globin of unknown function. We have previously shown the downregulation of cytoglobin as a key event in a familial cancer syndrome of the upper aerodigestive tract. METHODS: Cytoglobin expression and promoter methylation were investigated in sporadic head and neck squamous cell carcinoma (HNSCC) using a cross-section of clinical samples. Additionally, the putative mechanisms of Cygb expression in cancer were explored by subjecting HNSCC cell lines to hypoxic culture conditions and 5-aza-2-deoxycitidine treatment. RESULTS: In clinically derived HNSCC samples, CYGB mRNA expression showed a striking correlation with tumour hypoxia (measured by HIF1A mRNA expression P=0.013) and consistent associations with histopathological measures of tumour aggression. CYGB expression also showed a marked negative correlation with promoter methylation (P=0.018). In the HNSCC cell lines cultured under hypoxic conditions, a trend of increasing expression of both CYGB and HIF1A with progressive hypoxia was observed. Treatment with 5-aza-2-deoxycitidine dramatically increased CYGB expression in those cell lines with greater baseline promoter methylation. CONCLUSION: We conclude that the CYGB gene is regulated by both promoter methylation and tumour hypoxia in HNSCC and that increased expression of this gene correlates with clincopathological measures of a tumour's biological aggression.

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy.

BACKGROUND: Epigenetic silencing of O(6)-methylguanine-DNA-methyltransferase (MGMT) by promoter methylation is associated with improved survival in glioblastomas treated with alkylating agents. In this study, we investigated MGMT promoter methylation in glioblastomas treated with temozolomide and radiotherapy in a single UK treatment centre. METHODS: Quantitative methylation data at individual CpG sites were obtained by pyrosequencing for 109 glioblastomas. RESULTS: Median overall survival (OS) was 12.4 months with 2-year survival of 17.9%. Pyrosequencing data were reproducible with archival samples yielding data for all glioblastomas. Variation in methylation patterns of discrete CpG sites and intratumoral methylation heterogeneity were observed. A total of 58 out of 109 glioblastomas showed average methylation >non-neoplastic brain in at least one clinical sample; 86% had homogeneous methylation status in multiple samples. Methylation was an independent prognostic factor associated with prolonged progression-free survival (PFS) and OS. Cases with methylation more than 35% had the longest survival (median PFS 19.2; OS 26.2 months, 2-year survival of 59.7%). Significant differences in PFS were seen between those with intermediate or high methylation and unmethylated cases, whereas cases with low, intermediate or high methylation all showed significantly different OS. CONCLUSIONS: These data indicate that MGMT methylation is prognostically significant in glioblastomas given chemoradiotherapy in the routine clinic; furthermore, the extent of methylation may be used to provide additional prognostic stratification.

The LLP risk model: an individual risk prediction model for lung cancer.

Using a model-based approach, we estimated the probability that an individual, with a specified combination of risk factors, would develop lung cancer within a 5-year period. Data from 579 lung cancer cases and 1157 age- and sex-matched population-based controls were available for this analysis. Significant risk factors were fitted into multivariate conditional logistic regression models. The final multivariate model was combined with age-standardised lung cancer incidence data to calculate absolute risk estimates. Combinations of lifestyle risk factors were modelled to create risk profiles. For example, a 77-year-old male non-smoker, with a family history of lung cancer (early onset) and occupational exposure to asbestos has an absolute risk of 3.17% (95% CI, 1.67-5.95). Choosing a 2.5% cutoff to trigger increased surveillance, gave a sensitivity of 0.62 and specificity of 0.70, while a 6.0% cutoff gave a sensitivity of 0.34 and specificity of 0.90. A 10-fold cross validation produced an AUC statistic of 0.70, indicating good discrimination.If independent validation studies confirm these results, the LLP risk models' application as the first stage in an early detection strategy is a logical evolution in patient care.

Family history and risk of lung cancer: age-at-diagnosis in cases and first-degree relatives.

To investigate the little known risk of lung cancer at an early age when a first-degree relative has had such a diagnosis, 579 incident cases and 1157 population controls were studied in Liverpool between 1998 and 2004 using standardised questionnaires covering demography and lifestyle. A history of lung cancer in first-degree relatives was associated with a significantly increased risk in the proband where in both individuals the cancers were diagnosed before the age of 60 years (odds ratio (OR)=4.89; 95% confidence interval (CI): 1.47-16.25). A significantly elevated risk of lung cancer was also observed in association with a relative affected before the age of 60 years, regardless of age-at-onset of the disease (OR=2.08; 95% CI: 1.20-3.59). This finding is strongly consistent with a genetic component in early-onset lung cancer risk.

Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing.

Methylation profiling of cancer tissues has identified this mechanism as an important component of carcinogenesis. Epigenetic silencing of tumour suppressor genes through promoter methylation has been investigated by a variety of means, the most recent of which is pyrosequencing. We have investigated quantitative methylation status in oral squamous cell carcinoma patients. Fresh tumour tissue and normal control tissue from resection margin was obtained from 79 consecutive patients undergoing resection of oral squamous cell carcinoma. DNA was extracted and bisulphite treated. PCR primers were designed to amplify 75-200 bp regions of the CpG rich gene promoters of p16, RARbeta, E-cadherin, cytoglobin and cyclinA1. Methylation status of 4-5 CpG sites per gene was determined by pyrosequencing. Significant CpG methylation of gene promoters within tumour specimens was found in 28% for p16, 73% for RARbeta, 42% for E-cadherin, 65% for cytoglobin and 53% for cyclinA1. Promoter methylation was significantly elevated in tumours compared to normal tissue for p16 (P = 0.048), cytoglobin (P = 0.002) and cyclin A1 (P = 0.001) but not in RARbeta (P = 0.088) or E-cadherin (P = 0.347). Concordant methylation was demonstrated in this tumour series (P = 0.03). Significant differences in degree of methylation of individual CpG sites were noted for all genes except RARbeta and these differences were in a characteristic pattern that was reproduced between tumour samples. Cyclin A1 promoter methylation showed an inverse trend with histological grade. Promoter methylation analysis using pyrosequencing reveals valuable quantitative data from several CpG sites. In contrast to qualitative data generated from methylation specific PCR, our data demonstrated p16 promoter methylation in a highly tumour specific pattern. Significant tumour specific methylation of cyclin A1 promoter was also seen. Cytoglobin is a novel candidate tumour suppressor gene highly methylated in upper aero-digestive tract squamous cancer.

Colorectal adenomas with a low frequency of ki-ras mutations: a sample from a population with a low mortality rate from colorectal cancer.

OBJECTIVE: To detect and analyse ki-ras point mutations in colorectal adenomas and carcinomas in patients from Greece, where mortality rate from colorectal cancer is lower than that in other European countries and the USA. PATIENTS AND METHODS: As a method we used PCR-RFLP technique, to examine ki-ras codons 12 and 13 mutations in 450 paraffin embedded tissues, consisting of 141 normal mucosas, 165 adenomas and 144 sporadic colorectal cancers taken from 141 patients. RESULTS: Frequency of ki-ras mutations was 3.2% (3/93) in small adenomas, 5.2% (3/57) in greater than 1 cm adenomas, 20% (3/15) in tissues where adenoma and cancer coexisted and 39.5% (57/144) in colorectal cancer. CONCLUSION: These data showed that the frequency of ki-ras mutations was low in all sizes of polyps and high in carcinomas in a sample of a population, characterized by a low mortality rate from colorectal cancer. Ki-ras mutations were not an early event of colon carcinogenesis in these patients and the low frequency of ki-ras mutations in adenomas may probably have some relevance to the low mortality rate. Our findings could be explained, not by the dietary habits of this population, but by the presence of other hitherto unknown environmental factors.

Methylation discriminators in NSCLC identified by a microarray based approach.

Aberrant DNA methylation is a frequent phenomenon in non-small cell lung cancers. We have used a microarray approach to assess the methylation status of 245 CpG positions in 59 candidate genes in 26 squamous cell carcinomas, and 22 adenocarcinomas as well as 26 normal adjacent lung tissue samples from smokers to identify genes that show a distinct methylation status difference between the two different tumour type tissues and normal adjacent tissue. Tumour tissue samples were grouped together and compared to the normal tissue sample group. A multivariate test was performed, taking into account all CpG positions that were analyzed for a particular gene, to calculate p-values for each gene based on the observed methylation difference between the two groups, p-values obtained were corrected for multiple testing. The highest degree of differential DNA methylation in squamous cell carcinoma compared to normal was observed in ARHI, MGMT, GP1bbeta, RARbeta and TMEFF2 genes, while TMEFF2, MGMT and CDKNIC genes differentiated between adenocarcinomas and normal tissue. It is of note that some of the genes for which differential methylation status was observed, have not been previously described in lung cancer. Our results provide compelling evidence that different histological types of lung cancer may be distinguished from normal tissue based on methylation profiles of specific genes.

Absence of association with cancer risk and low frequency of alterations at a p53 responsive PIG3 gene polymorphism in breast and lung carcinomas.

The mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. One of them PIG3, is induced by p53 through a microsatellite in its promoter region. This microsatellite was found to acquire its full structure and p53-functional dependence only in Hominoidea (apes and humans) and has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. Microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 TGYCC repeats), at this locus have been hypothesized to provide an increased risk for cancer development. Therefore, in the present analysis we examined this polymorphism in two common human cancers, lung and breast and compared it with corresponding control cases. Furthermore, for lung cancer we employed two different ethnic groups, Greek and British. Analysis of this locus in this types of tumors showed: (1) a very low frequency of microsatellite instability and loss of heterozygosity (1.4% and 4%, respectively) in the examined carcinomas, (2) the homozygous presence of the 10 repeats allele only in the control cases, and (3) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to control ones. The last two observations were found in both Greek and British populations. Taken together, these data do not support the notion that this PIG3 polymorphism is associated with an increased risk for cancer susceptibility. Larger studies including other types of cancer should also be performed.

METH-2 silencing and promoter hypermethylation in NSCLC.

The antiangiogenic factor METH-2 (ADAMTS-8) was identified in a previous dual-channel cDNA microarray analysis to be at least two-fold under-represented in 85% (28 out of 33) of primary non-small-cell lung carcinomas (NSCLCs). This observation has been validated in an independent series of NSCLCs and adjacent normal tissues by comparative multiplex RT-PCR, and METH-2 mRNA expression was dramatically reduced in all 23 tumour samples analysed. Immunohistochemical analysis of the same sample set demonstrated that METH-2 was strongly expressed in 14 out of 19 normal epithelial sites examined but only one out of 20 NSCLCs. DNA methylation analysis of the proximal promoter region of this gene revealed abnormal hypermethylation in 67% of the adenocarcinomas and 50% of squamous cell carcinomas, indicating that epigenetic mechanisms are involved in silencing this gene in NSCLC. No homozygous deletions of METH-2 were found in lung cancer cell lines. Allelic imbalance in METH-2 was assessed by an intronic single nucleotide polymorphism (SNP) assay and observed in 44% of informative primary samples. In conclusion, the downregulation of METH-2 expression in primary NSCLC, often associated with promoter hypermethylation, is a frequent event, which may be related to the development of the disease.

Mutational analysis of CDKN2A genes in patients with squamous cell carcinoma of the skin.

BACKGROUND: Nonmelanoma skin cancers [squamous cell carcinomas (SCC) and basal cell carcinomas (BCC)] are the most common neoplasias of the Caucasian population. OBJECTIVES: The purpose of our study was to determine the involvement of CDKN2A genes in the development of sporadic nonmelanoma skin cancer in Greek patients. PATIENTS AND METHODS: Allelic imbalance analysis was performed in 22 SCC and five Bowen's disease specimens. Mutational analysis was performed on exons 1alpha, 1beta and 2 of the CDKN2A locus in 22 SCC, five Bowen's disease and 39 BCC specimens. Exon 1alpha was additionally screened in 28 BCC specimens to complete the mutational analysis of a previous study. RESULTS: Overall, 52% (14 of 27) of the SCC and Bowen's disease specimens exhibited loss of heterozygosity (LOH) in at least one microsatellite marker, whereas, only two of 27 (7%) exhibited microsatellite instability. LOH in 9p appears to be equally involved in both BCC and SCC tumours. Exons 1alpha, 1beta and 2 of the CDKN2A locus were screened for mutations. A Val28Gly substitution in exon 1alpha and a CCC-->TTT (Ala57Val and Arg58Ter) substitution in exon 2, resulting in a change in the amino acid sequence, are reported for the first time in two SCCs, the latter being indicative of a combination of an ultraviolet (UV) radiation-induced mutation and a point mutation. A previously described polymorphism of CDKN2A, the gene for p16INK4a, Ala148Thr, was also detected in an allelic frequency of 3.72%. No mutation was found in any of the five Bowen's disease specimens, or in exon 1beta of CDKN2A, also the gene for p14ARF. CONCLUSIONS: Mutations and the high incidence of 9p LOH detected in our SCC samples imply that inactivation of CDKN2A genes, via allelic loss and/or mutation (probably UV-induced) may play a significant role in nonmelanoma skin cancer development, particularly in the more aggressive SCC type.

Absence of mutations in the VHL gene but frequent loss of heterozygosity at 3p25-26 in non-small cell lung carcinomas.

In this study we have examined 79 primary non-small cell lung tumours for the presence of mutations of the VHL gene as well as for allelic imbalance at the gene surrounding loci. While allelic imbalance was found in 83% of specimens, frequently affecting the whole 3p25-p26 region, no mutations were detected in the VHL coding region. The fractional regional loss (FRL) was significantly higher in squamous cell carcinomas (0.746) than adenocarcinomas (0.493) (Wilcoxon P=0.002). This is the first investigation of the VHL gene mutational status in primary lung tumours. Our results indicate that mutation is not a common means of VHL inactivation in NSCLC.

Allelic imbalance at the DNA mismatch repair loci, hMSH2, hMLH1, hPMS1, hPMS2 and hMSH3, in squamous cell carcinoma of the head and neck.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is one of the 10 most frequently occurring cancers in the world. Defective mismatch repair, as exhibited by the phenomenon of microsatellite instability, has been observed in SCCHN although no reports of mismatch repair gene mutations or altered protein expression have been published. In a variety of microsatellite instability (MSI) positive cancers where mutations in the mismatch repair (MMR) genes were not observed, allelic imbalance at the loci of the MMR genes was prevalent. OBJECTIVE: To investigate whether allelic imbalance at the MMR genetic loci contributes to the development of SCCHN. MATERIALS AND METHODS: 35 matched normal/tumour SCCHN pairs were studied using 29 microsatellite markers located within and adjacent to six known DNA mismatch repair genes. In addition, mutational analysis and protein expression of hMSH2 and hMLH1 were investigated. RESULTS AND CONCLUSIONS: We demonstrated that 36 and 17% of the analysed SCCHN specimens exhibited allele imbalance at the hMLH1 and hMSH3 genetic loci, respectively. Allelic instability at these two loci was found to be correlated with the MSI status of the SCCHN tumours. Allelic instability was found to be uncommon at the other MMR gene loci analysed. One mutation was found in hMSH2 and none in hMLH1 in this series of tumours. 23 of 24 (96%) of the examined SCCHN tumours showed reduced expression of either hMSH2 or hMCH1 genes. Allelic instability in the MMR genes, hMLH1 and hMSH3, is proposed to be involved in the aetiology of SCCHN tumours.