Nationwide randomised trial evaluating elective neck dissection for early stage oral cancer (SEND study) with meta-analysis and concurrent real-world cohort.

BACKGROUND: Guidelines remain unclear over whether patients with early stage oral cancer without overt neck disease benefit from upfront elective neck dissection (END), particularly those with the smallest tumours. METHODS: We conducted a randomised trial of patients with stage T1/T2 N0 disease, who had their mouth tumour resected either with or without END. Data were also collected from a concurrent cohort of patients who had their preferred surgery. Endpoints included overall survival (OS) and disease-free survival (DFS). We conducted a meta-analysis of all six randomised trials. RESULTS: Two hundred fifty randomised and 346 observational cohort patients were studied (27 hospitals). Occult neck disease was found in 19.1% (T1) and 34.7% (T2) patients respectively. Five-year intention-to-treat hazard ratios (HR) were: OS HR = 0.71 (p = 0.18), and DFS HR = 0.66 (p = 0.04). Corresponding per-protocol results were: OS HR = 0.59 (p = 0.054), and DFS HR = 0.56 (p = 0.007). END was effective for small tumours. END patients experienced more facial/neck nerve damage; QoL was largely unaffected. The observational cohort supported the randomised findings. The meta-analysis produced HR OS 0.64 and DFS 0.54 (p < 0.001). CONCLUSION: SEND and the cumulative evidence show that within a generalisable setting oral cancer patients who have an upfront END have a lower risk of death/recurrence, even with small tumours. CLINICAL TRIAL REGISTRATION: NIHR UK Clinical Research Network database ID number: UKCRN 2069 (registered on 17/02/2006), ISCRTN number: 65018995, ClinicalTrials.gov Identifier: NCT00571883.

An integrated genomics approach to identify genetic regions associated with neonatal growth trait in mice.

Neonatal growth during the early post-partum period is closely associated with lactation performance. Neonatal growth reflects milk output and is a complex variable trait among inbred mouse strains, but few studies have compared this trait systematically across more than a few strains. In the present study, 11 inbred strains of mice were measured for a neonatal growth phenotype during the first eight days of lactation. Significant differences in neonatal growth trait were observed with QSi5 (3.71±0.05 g) and DBA/1J (2.67±0.06 g) strains defining the two extremes of the phenotype. In silico association analysis was performed for trait variability using the high density SNP information on inbred strains of mice. We found strong evidence to refine a previously identified large neonatal growth QTL on mouse chromosome 9, Neogq1. When an integrated strategy that combined fine mapping and analysis of mammary transcriptome expression profiles of lactating mice with divergent phenotypes was applied, we identified neogenin (Neo1), a gene important for mammary gland morphogenesis, as a likely quantitative trait gene (QTG) underlying the Neogq1 QTL in mice.

Identification of gene sets and pathways associated with lactation performance in mice.

Mammary transcriptome analyses across the lactation cycle and transgenic animal studies have identified candidate genes for mammogenesis, lactogenesis and involution; however, there is a lack of information on pathways that contribute to lactation performance. Previously we have shown significant differences in lactation performance, mammary gland histology, and gene expression profiles during lactation [lactation day 9 (L9)] between CBA/CaH (CBA) and the superior performing QSi5 strains of mice. In the present study, we compared these strains at midpregnancy [pregnancy day 12 (P12)] and utilized these data along with data from a 14th generation of intercross (AIL) to develop an integrative analysis of lactation performance. Additional analysis by quantitative reverse transcription PCR examined the correlation between expression profiles of lactation candidate genes and lactation performance across six inbred strains of mice. The analysis demonstrated that the mammary epithelial content per unit area was similar between CBA and QSi5 mice at P12, while differential expression was detected in 354 mammary genes (false discovery rate < 0.1). Gene ontology and functional annotation analyses showed that functional annotation terms associated with cell division and proliferation were the most enriched in the differentially expressed genes between these two strains at P12. Further analysis revealed that genes associated with neuroactive ligand-receptor interaction and calcium signaling pathways were significantly upregulated and positively correlated with lactation performance, while genes associated with cell cycle and DNA replication pathways were downregulated and positively correlated with lactation performance. There was also a significant negative correlation between Grb10 expression and lactation performance. In summary, using an integrative genomic approach we have identified key genes and pathways associated with lactation performance.

Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Transcriptome analysis identifies pathways associated with enhanced maternal performance in QSi5 mice.

BACKGROUND: Highly fecund mouse strains provide an ideal model to understand the factors affecting maternal performance. The QSi5 inbred strain of mice was selected for high fecundity and low inter-litter interval, and is very successful at weaning large numbers of offspring when compared to other inbred strains. RESULTS: Post-natal pup weight gain was used to estimate mammary gland output and to compare the performance of QSi5 mice to CBA mice. Cumulative litter weights and individual pup weight gain was significantly higher throughout the first eight days of lactation in QSi5 mice compared to CBA mice. Morphometric analysis of mammary glands during pregnancy in QSi5 mice revealed a 150 percent greater ductal side branching compared to CBA mice (P < 0.001). Ontology and pathway classification of transcript profiles from the two strains identified an enrichment of genes involved in a number of pathways, including the MAPK, tight junction, insulin signalling and Wnt signalling. Eleven of these genes, including six genes from the MAPK signalling pathway, were identified as associated with postnatal growth. Further, positive mediators of Wnt signalling, including Wnt4, Csnk2a1 and Smad4, were over-represented in the QSi5 strain profile, while negative regulators, including Dkkl1, Ppp2r1a and Nlk, were under-represented. These findings are consistent with the role of Wnt and MAPK signalling pathway in ductal morphogenesis and lobuloalveolar development suggesting enhanced activity in QSi5 mice. A similar pattern of phenotype concordance was seen amongst 12 genes from the tight junction pathway, but a pattern did not emerge from the insulin signalling genes. Amongst a group of differentially expressed imprinted genes, two maternal imprinted genes that suppress growth induced via the IGF signalling pathway, Grb10 and Igf2r, were under-represented in QSi5 mice. Whereas Peg3 and Plagl1, both paternally imprinted genes that enhance neonatal growth, were over-represented in QSi5 mice. CONCLUSION: We propose that the combined action of at least three major signalling pathways involved in mammary gland development and milk secretion, namely Wnt, MAPK and tight junction pathways, contribute to the superior maternal performance phenotype in QSi5 mice. Additionally, favourable expression patterns of the imprinted genes Peg3, Plagl1, Grb10 and Igf2r may also contribute.

Quantitative trait loci modifying cardiac atrial septal morphology and risk of patent foramen ovale in the mouse.

Atrial septal defect (ASD) is a common congenital heart disease (CHD) occurring in 5 to 7 per 10,000 live births. Mutations in 5 human genes (NKX2.5, TBX5, GATA4, MYHC, ACTC) are known to cause dominant ASD, but these account for a minority of cases. Human and mouse data suggest that ASD exists in an anatomical continuum with milder septal variants patent foramen ovale (PFO) and atrial septal aneurysm, strongly associated with ischemic stroke and migraine. We have previously shown in inbred mice that the incidence of PFO strongly correlates with length of the interatrial septum primum, defining a quantitative trait underlying PFO risk. To better understand genetic causation of atrial septal abnormalities, we mapped quantitative trait loci (QTL) influencing septal morphology using mouse strains (QSi5 and 129T2/SvEms) maximally informative for PFO incidence and 3 quantitative septal anatomical traits including septum primum length. [QSi5x129T2/SvEms]F2 intercross animals (n=1437) were phenotyped and a whole genome scan performed at an average 17-cM interval. Statistical methodology scoring PFO as a binary phenotype was developed as a confirmatory mapping technique. We mapped 7 significant and 6 suggestive QTL modifying quantitative phenotypes, with 4 supported by binary analysis. Quantitative traits, although strongly associated with PFO (P<0.001), correlated poorly with each other and in all but 1 case QTL for different traits were nonoverlapping. Thus, multiple anatomical processes under separate genetic control contribute to risk of PFO. Our findings demonstrate the feasibility of modeling the genetic basis of common CHD using animal genetic and genomic technologies.

The SECONDI MAPZ(©) system: new approach for the classification of oncological defects of the midface.

The appearance and function of the midface are crucial physiologically, psychologically, and aesthetically, and defects in the region can be devastating. Most of these defects are caused by operations for cancer, for which surgical access and rehabilitation can be challenging. Clinical evidence in midfacial ablative surgery is limited because differences between existing classifications do not allow a uniform approach to data recording, which makes comparison difficult. We explore the history of the classification of midfacial and maxillary defects, we analyse the shortcomings of those currently in use, and propose a new system that enables defects to be mapped simply, logically, and accurately.

An integrative genomic analysis of the superior fecundity phenotype in QSi5 mice.

Laboratory inbred mouse models are a valuable resource to identify quantitative trait loci (QTL) for complex reproductive performance traits. Advances in mouse genomics and high density single nucleotide polymorphism mapping has enabled genome-wide association studies to identify genes linked with specific phenotypes. Gene expression profiles of reproductive tissues also provide potentially useful information for identifying genes that play an important role. We have developed a highly fecund inbred strain, QSi5, with accompanying genotyping for comparative analysis of reproductive performance. Here we analyzed the QSi5 phenotype using a comparative analysis with fecundity data derived from 22 inbred strains of mice from the Mouse Phenome Project, and integration with published expression data from mouse ovary development. Using a haplotype association approach, 400 fecundity-associated regions (FDR < 0.05) with 499 underlying genes were identified. The most significant associations were located on Chromosomes 14, 8, and 6, and the genes underlying these regions were extracted. When these genes were analyzed for expression in an ovarian development profile (GSE6916) several distinctive co-expression patterns across each developmental stage were identified. The genetic analysis also refined 21 fecundity associated intervals on Chromosomes 1, 6, 9, 13, and 17 that overlapped with previously reported reproductive performance QTL. The combined use of phenotypic and in silico data with an integrative genomic analysis provides a powerful tool for elucidating the molecular mechanisms underlying fecundity.

Development of a highly fecund inbred strain of mice.

A highly fecund inbred mouse line has been established from the Quackenbush Swiss (QS) outbred strain by full-sib inbreeding combined with selection for high number of pups born alive (NBA) and low interlitter interval (ILI). After more than 50 generations of inbreeding and selection, this line, named QSi5, has an NBA of 13.4 and an ILI of 29 days, averaged over the first four parities, and a total productivity of 50.7 NBA. With its exceptional reproductive performance, this line will be very useful in the creation of resources (including advanced intercross lines) for analysis of quantitative trait loci for a wide range of traits, and for the cost-effective creation of congenic lines.

Quantitative trait loci for steady-state platelet count in mice.

Platelet count in humans is a strongly genetically regulated trait, with approximately 85% of the interindividual variance in platelet numbers attributable to genetic factors. Inbred mouse strains also have strain-specific platelet count ranges. As part of a project to identify novel factors that regulate platelet count, we identified two inbred mouse strains, CBA/CaH and QSi5, with substantial differences in platelet count (mean values of 581 vs. 1062 x 10(9)/L). An F(2) intercross resource of 1126 animals was bred from these two parental strains for a genomewide scan for quantitative trait loci (QTL) for platelet count. QTL were identified on MMU1 (LOD 6.8, p < 0.0005) and MMU11 (LOD 11.2, p < 0.0005) by selectively genotyping animals from the extremes of the F(2) platelet count distribution. Three other QTL of suggestive statistical significance were also detected on MMU7, 13, and 17. It is noteworthy that no QTL were detected in the vicinity of the genes encoding thrombopoietin ( Thpo), and its receptor ( c-Mpl), both known to influence platelet production. Comparison of gene expression levels between the parental mouse strains by microarrays also showed little difference in the mRNA levels of these known candidate genes. These results represent the first published use of a genetic linkage-based approach in a mouse model toward the identification of genetic factors that regulate platelet count.