Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

Tumour necrosis is an independent prognostic marker in non-small cell lung cancer : correlation with biological variables

Background Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. Methods In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. Results Reproducibility was attained in 36/40 cases, Kappa score=0.8 P<0.001. TN correlated with T-stage (P=0.001), platelet count (P=0.004) and p53 expression (P=0.031). Near significant associations of TN with N-stage (P=0.063) and MMP-9 expression (P=0.058) were seen. No association was found with angiogenesis (P=0.98). On univariate (P=0.0016) and multivariate analysis (P=0.023) TN was prognostic. Conclusion These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Age-related trends in urinary excretion of bisphenol A in Australian children and adults : evidence from a pooled sample study using samples of convenience

Bisphenol A (BPA or 4,4’-(propane-2,2-diyl)diphenol) is a chemical intermediate in the production of polycarbonate and epoxy resins, and used in a wide range of applications. BPA has attracted significant attention in the past decade due to its frequency of detection in human populations worldwide, demonstrated animal toxicity and potential impact on human health, particularly during critical periods of development. The aim of this study was to perform a preliminary assessment of age-related trends in urinary concentration and to estimate daily excretion of BPA in Australian children (aged (>0 – <5 years) and adults (≥15 – <75 years). This was achieved using 79 samples pooled by age and gender, created from 868 individual samples of convenience collected as part of routine, community-based pathology testing. Total BPA was analyzed using online-SPE-LC-MS/MS and detected in all samples with a range of 0.65 – 265 ng/ml. No significant differences were observed between males and females. A urine flow model was constructed from published values and used to provide an estimate of daily excretion per unit bodyweight for each pooled sample. The daily excretion estimates ranged from 26.2 – 18200 ng/kg-d for children; and 20.1 – 165 ng/kg-d for adults. Urinary concentrations and estimated excretion rates were inversely associated with age, and estimated daily excretion rates in infants and young children were significantly higher than in adults (geometric mean: 107 and 47.0 ng/kg-d, respectively). Higher excretion of BPA in children may be explained by their higher food consumption relative to body weight compared to adults and adolescents, and may also reflect alternative exposure pathways and sources. Keywords: bisphenol A, biomonitoring, children, urine flow, Australia

Existence of travelling wave solutions for a model of tumour invasion

The existence of travelling wave solutions to a haptotaxis dominated model is analysed. A version of this model has been derived in Perumpanani et al. (1999) to describe tumour invasion, where diffusion is neglected as it is assumed to play only a small role in the cell migration. By instead allowing diffusion to be small, we reformulate the model as a singular perturbation problem, which can then be analysed using geometric singular perturbation theory. We prove the existence of three types of physically realistic travelling wave solutions in the case of small diffusion. These solutions reduce to the no diffusion solutions in the singular limit as diffusion as is taken to zero. A fourth travelling wave solution is also shown to exist, but that is physically unrealistic as it has a component with negative cell population. The numerical stability, in particular the wavespeed of the travelling wave solutions is also discussed.

Decision fusion from parts and samples for robust iris recognition

Fusion techniques can be used in biometrics to achieve higher accuracy. When biometric systems are in operation and the threat level changes, controlling the trade-off between detection error rates can reduce the impact of an attack. In a fused system, varying a single threshold does not allow this to be achieved, but systematic adjustment of a set of parameters does. In this paper, fused decisions from a multi-part, multi-sample sequential architecture are investigated for that purpose in an iris recognition system. A specific implementation of the multi-part architecture is proposed and the effect of the number of parts and samples in the resultant detection error rate is analysed. The effectiveness of the proposed architecture is then evaluated under two specific cases of obfuscation attack: miosis and mydriasis. Results show that robustness to such obfuscation attacks is achieved, since lower error rates than in the case of the non-fused base system are obtained.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

The role of the Eph and ephrin proteins in prostate cancer

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer related deaths in Australian men. Treatment in the early stages of the disease involves surgery, radiation and/or hormone therapy. However, in late stages of the disease these treatments are no longer effective and only palliative care is available. Therefore, there is a focus on exploration of novel therapies to increase survival and treatment efficacy. Advanced prostate cancer is characterised by bone or other distant metastasis. Spreading of the primary tumour to a secondary location is a complex process requiring an initial loss in cell-cell adhesion followed by increased cell migration and invasion. One gene family that has been known to affect cell-to-cell contact in other model systems are the Eph receptor tyrosine kinases. They are the largest family of receptor tyrosine kinases made up of 14 vertebrate Eph receptors that bind to nine cell membrane bound ephrin ligands. Eph-ephrin interaction is crucial in regulating cell behaviour in developmental processes and it is now thought that the underlying mechanisms involved in development may also be involved in cancer. Aberrant expression has been reported in many human malignancies including prostate cancer. Furthermore, expression has been linked with metastasis and poor prognosis in other tumour models. This study explores the potential role of the Eph receptor family in prostate cancer, in particular the roles of EphA2, EphA3 and ephrin-A5. Gene expression profiles were established for the Eph family in a series of prostate cancer cell lines using quantitative real time RT-PCR. A smaller subset of the most prominently expressed genes was chosen to screen a cohort of clinical samples. Elevated levels of EphA2, EphA3 and their ligands, ephrin-A1 and ephrin-A5 were observed in individual cell lines. Interestingly high EphA3 expression was observed in the androgen responsive cell lines while EphA2 was more prominent in the androgen independent cell lines. However, studies using 5-dihydrotestosterone suggest that EphA3 expression in not regulated by androgen. Cells expressing EphA2 showed a greater ability for migration and invasion while cells expressing EphA3 showed poor migration and invasion. Forced expression of EphA2 in the LNCaP cell line resulted in a more invasive phenotype while forced expression of EphA3 in the PC-3 cell line suggests a possible negative effect for EphA3 on cell migration and invasion. Cell signalling studies show activation of EphA2 decreases activity of proteins thought to be involved in pathways regulating cell movement including Akt, Src and FAK. Changes to the activation status of Rho family members, including RhoA and Rac1, associated with reorganisation of the actin cytoskeleton, an important part of cell migration was also observed. As a result, activation of EphA2 in PC-3 cells resulted in a less invasive phenotype. A novel finding in this study was the discovery of a combination of two EphA2 Mabs able to activate EphA2. Preliminary results show a potential for this antibody combination to reduce prostate cancer invasion in vitro. A unique aspect of Eph-ephrin interaction is the resulting bi-directional signalling that occurs through both the receptor and ligand. In this study a potential role for ephrin-A5 mediated signalling in prostate cancer was observed. LNCaP cells express high levels of EphA3 and its high affinity ligand ephrin-A5. In stripe assays, used to study guidance cues, LNCaP cells show strong attraction/migration to EphA3-Fc stripes but not ephrin-A5-Fc stripes suggesting ephrin-A5 mediated reverse cell signalling is involved. Knockdown of ephrin-A5 using shRNA resulted in a decrease in attraction/migration to EphA3-Fc stripes. Furthermore a reduction in proliferation was also observed in vitro. A subcutaneous xenograft model using ephrin-A5 shRNA cells versus controls showed a decrease in tumour formation. This study demonstrates a difference in EphA2 and EphA3 function in prostate cancer migration/invasion and a potential role for ephrin-A5 in prostate cancer cell adhesion and growth.

Maternal report of young children's eating styles. Validation of the Children's Eating Behaviour Questionnaire in three ethnically diverse Australian samples

The aim of this study was to validate the Children’s Eating Behaviour Questionnaire (CEBQ) in three ethnically and culturally diverse samples of mothers in Australia. Confirmatory factor analysis utilising structural equation modelling examined whether the established 8-factor model of the CEBQ was supported in our three populations: (i) a community sample of first-time mothers allocated to the control group of the NOURISH trial (mean child age = 24 months [SD = 1]; N = 244); (ii) a sample of immigrant Indian mothers of children aged 1–5 years (mean age = 34 months [SD = 14]; N = 203), and (iii) a sample of immigrant Chinese mothers of children aged 1–4 years (mean age = 36 months [SD = 14]; N = 216). The original 8-factor model provided an acceptable fit to the data in the NOURISH sample with minor post hoc re-specifications (two error covariances on Satiety Responsiveness and an item-factor covariance to account for a cross-loading of an item (Fussiness) on Satiety Responsiveness). The re-specified model showed reasonable fit in both the Indian and Chinese samples. Cronbach’s α estimates ranged from .73 to .91 in the Australian sample and .61–.88 in the immigrant samples. This study supports the appropriateness of the CEBQ in the multicultural Australian context.

Assessment of polybrominated diphenyl ethers (PBDEs) in samples collected from indoor environments in South East Queensland, Australia

Polybrominated diphenyl ethers (PBDEs) are compounds that are used as flame retardants. Human exposure is suggested to be via food, dust and air. An assessment of PBDE exposure via indoor environments using samples of air, dust and surface wipes from eight sites in South East Queensland, Australia was conducted. For indoor air, ΣPBDEs ranged from 0.5 -179 pg/m3 for homes and 15 - 487 pg/m3 for offices. In dust, ΣPBDEs ranged from 87 - 733 ng/g dust and 583 - 3070 ng/g dust in homes and offices, respectively. PBDEs were detected on 9 out of 10 surfaces sampled and ranged from non-detectable to 5985 pg/cm2. Overall, the congener profiles for air and dust were dominated by BDE-209. This study demonstrated that PBDEs are ubiquitous in the indoor environments of selected buildings in South East Queensland and suggest the need for detailed assessment of PBDE concentrations using more sites to further investigate the factors influencing PBDE exposure in Australia.

Cancer : the to and fro of tumour spread

The spread of cells from the primary site of a solid tumour to distant sites remains the major cause of disease and death associated with these cancers. For tumour cells to spread, or metastasize, they must modify their 'anchored' state and detach from their neighbouring cells; migrate through tissues into the blood and lymph systems; survive in these circulation systems; and then leave the vessels at an appropriate site to form another tumour1. Many of these events are favoured by conversions between two cellular states — the epithelial and mesenchymal phenotypes. But the role of these transitions in cancer metastasis is controversial. Writing in Cancer Cell, Tsai et al.2 and Ocaña et al.3 help to clarify this issue...

Determining epithelial contribution to in vivo mesenchymal tumour expression signature using species-specific microarray profiling analysis of xenografts

Gene expression profiling using microarrays and xenograft transplants of human cancer cell lines are both popular tools to investigate human cancer. However, the undefined degree of cross hybridization between the mouse and human genomes hinders the use of microarrays to characterize gene expression of both the host and the cancer cell within the xenograft. Since an increasingly recognized aspect of cancer is the host response (or cancer-stroma interaction), we describe here a bioinformatic manipulation of the Affymetrix profiling that allows interrogation of the gene expression of both the mouse host and the human tumour. Evidence of microenvironmental regulation of epithelial mesenchymal transition of the tumour component in vivo is resolved against a background of mesenchymal gene expression. This tool could allow deeper insight to the mechanism of action of anti-cancer drugs, as typically novel drug efficacy is being tested in xenograft systems.

Repeated sequences as genetic markers in pooled tissue samples

We show, using the PDR1 element of pea, that dispersed repeated sequences of moderate copy number can be used simply and efficiently to generate markers linked to a trait of interest. Inspection of hybridization patterns of repeated sequences to DNA mixtures of pooled genotypes is a sensitive way of detecting such markers. The large number of bands in tracks of digests of these mixtures allows the simultaneous sampling of loci at many places in the genome, and the many unlinked loci serve as internal controls. It is also shown that intensity ratios calculated from these band differences can be used to give a rough estimate of linkage distance.

Dormant but migratory tumour cells in desmoplastic stroma of invasive ductal carcinomas

Mortality in breast cancer is linked to metastasis and recurrence yet there is no acceptable biological model for cancer relapse. We hypothesise that there might exist primary tumour cells capable of escaping surgery by migration and resisting radiotherapy and chemotherapy to cause cancer recurrence. We investigated this possibility in invasive ductal carcinoma (IDC) tissue and observed the presence of solitary primary tumour cells (SPCs) in the dense collagen stroma that encapsulates intratumoural cells (ICs). In IDC tissue sections, collagen was detected with either Masson's Trichrome or by second harmonics imaging. Cytokeratin-19 (CK-19) and vimentin (VIM) antibodies were, respectively, used to identify epithelial-derived tumour cells and to indicate epithelial to mesenchymal transition (EMT). Confocal/multiphoton microscopy showed that ICs from acini were mainly CK-19 +ve and were encapsulated by dense stromal collagen. Within the stroma, SPCs were detected by their staining for both CK-19 and VIM (confirming EMT). ICs and SPCs were subsequently isolated by laser capture microdissection followed by multiplex tandem-PCR studies. SPCs were found to be enriched for pro-migratory and anti-proliferative genes relative to ICs. In vitro experiments using collagen matrices at 20 mg/cm 3, similar in density to tumour matrices, demonstrated that SPC-like cells were highly migratory but dormant, phenotypes that recapitulated the genotypes of SPCs in clinical tissue. These data suggest that SPCs located at the breast cancer perimeter are invasive and dormant such that they may exceed surgical margins and resist local and adjuvant therapies. This study has important connotations for a role of SPCs in local recurrence.

Mechanisms of tumour invasion and metastasis : emerging targets for therapy

The progression of a tumour from one of benign and delimited growth to one that is invasive and metastatic is the major cause of poor clinical outcome in cancer patients. The invasion and metastasis of tumours is a highly complex and multistep process that requires a tumour cell to modulate its ability to adhere, degrade the surrounding extracellular matrix, migrate, proliferate at a secondary site and stimulate angiogenesis. Knowledge of the process has greatly increased and this has resulted in the identification of a number of molecules that are fundamental to the process. The involvement of these molecules has been shown to relate not only to the survival and proliferation of the tumour cell but, also to the processes of tumour cell adhesion, migration, and the tumour cells ability to degrade and escape the primary site as well as play a role in angiogenesis. These molecules may provide important therapeutic targets that represent the ability to target specific steps in the process of invasion and metastasis and provide additional therapies. The review focuses on representative key targets in each of these processes and summarises the state of play in each case.

Taxonomic resolution and quantification of freshwater macroinvertebrate samples from an Australian dryland river : The benefits and costs of using species abundance data

In studies using macroinvertebrates as indicators for monitoring rivers and streams, species level identifications in comparison with lower resolution identifications can have greater information content and result in more reliable site classifications and better capacity to discriminate between sites, yet many such programmes identify specimens to the resolution of family rather than species. This is often because it is cheaper to obtain family level data than species level data. Choice of appropriate taxonomic resolution is a compromise between the cost of obtaining data at high taxonomic resolutions and the loss of information at lower resolutions. Optimum taxonomic resolution should be determined by the information required to address programme objectives. Costs saved in identifying macroinvertebrates to family level may not be justified if family level data can not give the answers required and expending the extra cost to obtain species level data may not be warranted if cheaper family level data retains sufficient information to meet objectives. We investigated the influence of taxonomic resolution and sample quantification (abundance vs. presence/absence) on the representation of aquatic macroinvertebrate species assemblage patterns and species richness estimates. The study was conducted in a physically harsh dryland river system (Condamine-Balonne River system, located in south-western Queensland, Australia), characterised by low macroinvertebrate diversity. Our 29 study sites covered a wide geographic range and a diversity of lotic conditions and this was reflected by differences between sites in macroinvertebrate assemblage composition and richness. The usefulness of expending the extra cost necessary to identify macroinvertebrates to species was quantified via the benefits this higher resolution data offered in its capacity to discriminate between sites and give accurate estimates of site species richness. We found that very little information (<6%) was lost by identifying taxa to family (or genus), as opposed to species, and that quantifying the abundance of taxa provided greater resolution for pattern interpretation than simply noting their presence/absence. Species richness was very well represented by genus, family and order richness, so that each of these could be used as surrogates of species richness if, for example, surveying to identify diversity hot-spots. It is suggested that sharing of common ecological responses among species within higher taxonomic units is the most plausible mechanism for the results. Based on a cost/benefit analysis, family level abundance data is recommended as the best resolution for resolving patterns in macroinvertebrate assemblages in this system. The relevance of these findings are discussed in the context of other low diversity, harsh, dryland river systems.