Tongue carcinoma infrequently harbor common actionable genetic alterations.

BACKGROUND: Oral tongue squamous cell carcinomas (TSCC) are a unique subset of head and neck cancers with a distinct demographic profile, where up to half of the cases are never smokers. A small proportion of patients with OSCC are known to respond to EGFR TKI. We used a high-sensitivity mass spectrometry-based mutation profiling platform to determine the EGFR mutation status, as well as other actionable alterations in a series of Asian TSCC. METHODS: 66 TSCC patients treated between 1998-2009 with complete clinico-pathologic data were included in this study. Somatic mutation profiling was performed using Sequenom LungCarta v1.0, and correlated with clinical parameters. RESULTS: Mutations were identified in 20/66(30.3%) of samples and involved TP53, STK11, MET, PIK3CA, BRAF and NRF2. No activating EGFR mutations or KRAS mutations were discovered in our series, where just over a third were never smokers. The most common mutations were in p53 (10.6%; n = 7) and MET (10.6%, n = 11) followed by STK11 (9.1%, n = 6) and PIK3CA (4.5%, n = 3). BRAF and NRF2 mutations, which are novel in TSCC, were demonstrated in one sample each. There was no significant correlation between overall mutation status and smoking history (p = 0.967) or age (p = 0.360). Positive MET alteration was associated with poorer loco-regional recurrence free survival (LRFS) of 11 months [vs 90 months in MET-negative group (p = 0.008)]. None of the other mutations were significantly correlated with LRFS or overall survival. Four of these tumors were propagated as immortalized cell lines and demonstrated the same mutations as the original tumor. CONCLUSIONS: Using the Sequenom multiplexed LungCarta panel, we identified mutations in 6 genes, TP53, STK11, MET, PIK3CA, BRAF and NRF2, with the notable absence of EGFR and HER2 mutations in our series of Asian OSCC. Primary cell line models recapitulated the mutation profiles of the original primary tumours and provide an invaluable resource for experimental cancer therapeutics.

Targeting cancer stem cell plasticity through modulation of epidermal growth factor and insulin-like growth factor receptor signaling in head and neck squamous cell cancer.

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.

Realtime exonuclease-mediated allelic discrimination (READ): a simple homogeneous genotyping assay for SNPs at the ABC gene loci.

AIMS: Members of the ATP-binding-cassette transporter family are implicated in the traffic of drugs/xenobiotics. Several SNPs in these ATP-binding-cassette genes were previously identified to show evidence of recent positive selection. These recent positive selection SNPs may confer functional effects and account for variation in drug response. To facilitate association studies between these SNPs and drug response, we report the development of a homogeneous (realtime exonuclease-mediated allelic discrimination) assay to genotype these SNPs. MATERIALS & METHODS: Realtime exonuclease-mediated allelic discrimination involves real-time PCR using a proof-reading enzyme and simultaneous genotype determination by product presence/absence as detected using SYBR Green I stain. RESULTS: A total of 29 recent positive selection SNPs from 17 ATP-binding-cassette transporter genes were evaluated. Of the 777 eealtime exonuclease-mediated allelic discrimination assays, 773 genotypes (approximately 99.5%) were concordant with the Perlegen data and other genotyping methods. CONCLUSION: Therefore, this simple, robust, rapid, cost-effective single-step, closed-tube assay with a scalable and automatable platform has potential applications in population genetic screening and association studies.

Nucleotide sequence analyses of the MRP1 gene in four populations suggest negative selection on its coding region.

BACKGROUND: The MRP1 gene encodes the 190 kDa multidrug resistance-associated protein 1 (MRP1/ABCC1) and effluxes diverse drugs and xenobiotics. Sequence variations within this gene might account for differences in drug response in different individuals. To facilitate association studies of this gene with diseases and/or drug response, exons and flanking introns of MRP1 were screened for polymorphisms in 142 DNA samples from four different populations. RESULTS: Seventy-one polymorphisms, including 60 biallelic single nucleotide polymorphisms (SNPs), ten insertions/deletions (indel) and one short tandem repeat (STR) were identified. Thirty-four of these polymorphisms have not been previously reported. Interestingly, the STR polymorphism at the 5' untranslated region (5'UTR) occurs at high but different frequencies in the different populations. Frequencies of common polymorphisms in our populations were comparable to those of similar populations in HAPMAP or Perlegen. Nucleotide diversity indices indicated that the coding region of MRP1 may have undergone negative selection or recent population expansion. SNPs E10/1299 G>T (R433S) and E16/2012 G>T (G671V) which occur at low frequency in only one or two of four populations examined were predicted to be functionally deleterious and hence are likely to be under negative selection. CONCLUSION: Through in silico approaches, we identified two rare SNPs that are potentially negatively selected. These SNPs may be useful for studies associating this gene with rare events including adverse drug reactions.

Strong linkage disequilibrium at the nucleotide analogue transporter ABCC5 gene locus.

The ABCC5 transporter is a ubiquitously expressed ATP-dependent efflux pump that exports nucleotide analogues, including thiopurine anticancer drugs and antiviral drugs. Polymorphisms within this gene may be associated with differences in response to these drugs between different individuals. Haplotype mapping may facilitate the identification of causal genetic variations in association studies. Here, we report the characterization of the haplotype and linkage disequilibrium (LD) profiles across the entire 100 kb of the ABCC5 gene in five ethnically unique populations. Of 24 single nucleotide polymorphisms (SNPs) examined, 16 were observed to occur at high frequency in all five populations and were used for further haplotype and LD analyses. The ABCC5 gene was found to be in strong LD in all populations with half-length LD (LD0.5) estimated to be between 106 and 293 kb long and useful LD extending beyond 100 kb. Low haplotype diversity was observed in the four non-African populations, where the total number of observed haplotypes constituted less than 22% of the predicted number of haplotypes in a simulated population that has undergone maximum recombination. Four and six tagging SNPs, which could account for approximately 90% of observed haplotypes, were identified in the non-African and African-American populations, respectively.