Transcriptomic convergence despite genomic divergence drive field cancerization in synchronous squamous tumors.

Introduction: Field cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that eventually replace the epithelium, although without evidence. Methods: We performed deep sequencing analyses to characterize the genomic and transcriptomic landscapes of field change in two patients with synchronous aerodigestive tract tumors. Results: Our data support the emergence of numerous genetic alterations in cancer-associated genes but refutes the hypothesis that founder mutation(s) underpin this phenomenon. Mutational signature analysis identified defective homologous recombination as a common underlying mutational process unique to synchronous tumors. Discussion: Our analyses suggest a common etiologic factor defined by mutational signatures and/or transcriptomic convergence, which could provide a therapeutic opportunity.

Pairing a prognostic target with potential therapeutic strategy for head and neck cancer.

OBJECTIVES: We have previously identified and validated a panel of molecular prognostic markers (ATP13A3, SSR3, and ANO1) for Head and Neck Squamous Cell Carcinoma (HNSCC). The aim of this study was to investigate the consequence of ATP13A3 dysregulation on signaling pathways, to aid in formulating a therapeutic strategy targeting ATP13A3-overexpressing HNSCC. MATERIALS AND METHODS: Gene Set Enrichment Analysis (GSEA) was performed on HNSCC microarray expression data (Internal local dataset [n = 92], TCGA [n = 232], EMBL [n = 81]) to identify pathways associated with high expression of ATP13A3. Validation was performed using immunohistochemistry (IHC) on tissue microarrays (TMAs) of head and neck cancers (n = 333), staining for ATP13A3 and phosphorylated Aurora kinase A (phospho-T288). Short interfering RNA was used to knockdown ATP13A3 expression in patient derived HNSCC cell lines. Protein expression of ATP13A3 and Aurora kinase A was then assessed by immunoblotting. RESULTS: GSEA identified Aurora kinase pathway to be associated with high expression of ATP13A3 (p = 0.026). The Aurora kinase pathway was also associated with a trend towards poor prognosis and tumor aggressiveness (p = 0.086, 0.094, respectively). Furthermore, the immunohistochemical staining results revealed a significant association between Aurora kinase activity and high ATP13A3 expression (p < 0.001). Knockdown of ATP13A3 in human head and neck cell lines showed decrease in Aurora kinase A levels. CONCLUSION: Tumors with high ATP13A3 are associated with high Aurora kinase activity. This suggests a potential therapeutic role of Aurora kinase inhibitors in a subset of poor prognosis HNSCC patients with overexpression of ATP13A3.

An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours.

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

Avian influenza and South Jakarta primary healthcare workers: a controlled mixed-method study.

OBJECTIVE: To study the attitudes, concerns, perceived impact, coping strategies, knowledge on avian influenza (AI) and personal protection measures, and institutional and personal preparedness for AI among all Indonesian primary healthcare workers (PHW). METHODS: Questionnaire survey of PHW from four public primary healthcare clinics in South Jakarta (n = 333), with Singaporean PHW from 18 such clinics as controls (n = 1321). Twelve focus group discussions with 51 South Jakarta PHW were also conducted. Quantitative and qualitative data were analysed separately with statistical and thematic analysis, respectively, then combined. RESULTS: South Jakarta PHW had positive attitudes but major concerns about contracting AI, difficulties in diagnosing human AI and inadequacy of personal protection provided. South Jakarta PHW are less knowledgeable about AI and use of personal protection equipment, and reported poorer awareness, availability and participation in AI preparation activities. Only 3% of South Jakarta PHW received influenza vaccination in the preceding 6 months and few felt prepared for AI. CONCLUSIONS: South Jakarta primary healthcare workers are not well prepared for avian influenza. There is an urgent need to build their primary healthcare capacity to protect them and contain this global health threat.

A cross-sectional study of primary-care physicians in Singapore on their concerns and preparedness for an avian influenza outbreak.

INTRODUCTION: During an avian influenza (AI) pandemic, primary-care physicians (PCPs) are expected to play key roles in the prevention and control of the disease. Different groups of PCPs could have different concerns and preparedness level. We assessed the concerns, perceived impact and preparedness for an outbreak among PCPs in Singapore. MATERIALS AND METHODS: A cross-sectional survey of PCPs working in private practice (n=200) and public clinics (n=205) from March to June 2006 with an anonymous self-administered questionnaire on concerns (12- items), perceived impact (10 items) and preparedness (10 items) for an outbreak. RESULTS: Two hundred and eighty-five PCPs responded - 149 (response rate: 72.7%) public and 136 (response rate: 67.3%) private. The majority were concerned about risk to their health from their occupation (95.0%) and falling ill with AI (89.7%). Most (82.5%) accepted the risk and only 33 (11.8%) would consider stopping work. For perceived impact, most felt that people would avoid them (69.6%) and their families (54.1%). The majority (81.3%) expected an increased workload and feeling more stressed at work (86.9%). For preparedness, 78.7% felt personally prepared for an outbreak. Public PCPs were more likely to be involved in infection-control activities and felt that their workplaces were prepared. CONCLUSIONS: Most PCPs felt personally prepared for an outbreak but were concerned about their exposure to AI and falling ill. Other concerns included social ostracism for themselves and their families. Public PCPs appeared to have a higher level of preparation. Addressing concerns and improving level of preparedness are crucial to strengthen the primary-care response for any AI outbreak.

Concerns, perceived impact and preparedness in an avian influenza pandemic--a comparative study between healthcare workers in primary and tertiary care.

INTRODUCTION: With the potential threat of an avian influenza (AI) pandemic, healthcare workers (HCWs) are expected to play important roles, and they encounter significant stress levels from an expected increase in workload. We compared the concerns, perceived impact and preparedness for an AI pandemic between HCWs working in public primary care clinics and a tertiary healthcare setting. MATERIALS AND METHODS: An anonymous, self-administered questionnaire was given to 2459 HCWs working at 18 public polyclinics (PCs) and a tertiary hospital (TH) in Singapore from March to June 2006. The questionnaire assessed work-related and non-work-related concerns, perceived impact on personal life and work as well as workplace preparedness. RESULTS: We obtained responses from 986 PC and 873 TH HCWs (response rate: 74.6% and 76.7%). The majority in both groups were concerned about the high AI risk from their occupation (82.7%) and falling ill with AI (75.9%). 71.9% accepted the risk but 25.5% felt that they should not be looking after AI patients with 15.0% consider resigning. HCWs also felt that people would avoid them (63.5%) and their families (54.1%) during a pandemic. The majority expected an increased workload and to feel more stressed at work. For preparedness, 74.2% felt personally prepared and 83.7% felt that their workplaces were prepared for an outbreak. TH HCWs were more likely to be involved in infection-control activities but the perception of infection-control preparedness in both groups was high (>80.0%). CONCLUSIONS: HCWs in both public primary and tertiary healthcare settings felt prepared, personally and in their workplaces, for a pandemic. Their main concerns were risks of falling ill from exposure and the possibility of social ostracism of themselves and their families. Preparedness levels appeared high in the majority of HCWs. However, concerns of HCWs could affect their overall effectiveness in a pandemic and should be addressed by incorporating strategies to manage them in pandemic planning.

A three gene immunohistochemical panel serves as an adjunct to clinical staging of patients with head and neck cancer.

BACKGROUND: Current management of head and neck squamous cell carcinoma (HNSCC) depends on tumor staging. Despite refinements in clinical staging algorithms, outcomes remain unchanged for the last two decades. In this study, we set out to identify a small, clinically applicable molecular panel to aid prognostication of patients with HNSCC. MATERIALS AND METHODS: Data from The Cancer Genome Atlas (TCGA) was used to derive copy number aberrations and expression changes to identify putative prognostic genes. To account for cross entity relevance of the biomarkers, HNSCC (n = 276), breast (n = 808) and lung cancer (n = 282) datasets were used to identify robust and reproducible markers with prognostic potential. Validation was performed using immunohistochemistry (IHC) on tissue microarrays of an independent cohort of HNSCC (n = 333). FINDINGS: Using GISTIC algorithm together with gene expression analysis, we identified six putative prognostic genes in at least two out of three cancers analyzed, of which four were successfully optimized for automated IHC. Of these, three were successfully validated; each molecular target being significantly prognostic on univariate analysis. Patients were differentially segregated into four prognostic groups based on the number of genes dysregulated (p < 0.001). The IHC panel remained an independent predictor of survival after adjusting for known survival covariates including clinical staging criteria in a multivariate Cox regression model (p < 0.001). . INTERPRETATION: We have identified and validated a clinically applicable IHC biomarker panel that is independently associated with overall survival. This panel is readily applicable, serving as a useful adjunct to current staging systems and provides novel targets for future therapeutic strategies.

Rhenium(I) tricarbonyl complexes of salicylaldehyde semicarbazones: synthesis, crystal structures and cytotoxicity.

A series of N,N-disubstituted salicylaldehyde semicarbazones (SSCs), HOC(6)H(4)CHN-NHCONR(2), and their rhenium(I) tricarbonyl complexes, [ReBr(CO)(3)(SSC)], have been synthesised and characterised by IR and (1)H NMR spectroscopy. Crystallographic analysis of the complex [ReBr(CO)(3)(H(2)Bu(2))] (H(2)Bu(2)=SSC where R=Bu(n)) showed that the SSC acts as a bidentate ligand via its imino nitrogen and carbonyl oxygen atoms. The [ReBr(CO)(3)(SSC)] complexes exhibit moderate to high cytotoxicities towards MOLT-4 cells (IC(50)=1-24µM, cf. 18µM for cisplatin), and the majority of them are virtually non-toxic against non-cancerous human fibroblasts. Apoptotic assays of [ReBr(CO)(3)(H(2)Bnz(2))] (Bnz=benzyl) revealed that it mediates cytotoxicity in MOLT-4 cells via apoptosis. The complex [ReBr(CO)(3)(H(2)Bnz(2))] reacts with guanosine by proton transfer from the phenolic OH group to N(7) of guanosine. In (CD(3))(2)SO, [ReBr(CO)(3)(H(2)Bnz(2))] undergoes facile conversion to the dimeric complex, [Re(CO)(3)(HBnz(2))](2), via bromide dissociation.