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Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.Experimental Design: Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers.Results: Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing–induced apoptosis. K-Ras–mutated, c-Met–amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways.Conclusion: Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. Clin Cancer Res; 18(2); 380–91. ©2011 AACR.

PDF file - 3.6K, Cancer cells with K-Ras activation mutation (HCT116/Hkh-2 isogenic pair) are more susceptible to Ran silencing-induced apoptosis.

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PDF file - 1.6MB, Percentage of sub-G1 phase apoptotic MCF10a and MDA MB231 cells grown in serum-containing medium after Ran silencing. (A-B) Cells were infected with shRan1 and shRan2. Medium was changed to serum-free medium (DMEM) 24hr post-infection. Cells were harvested at 72hr and 96hr post-infection for (A) cell cycle and (B) Western blot analyses. (A) Significantly more sub-G1 phase cells were observed in MDA MB231 breast cancer cells than in immortalized breast epithelial cells 72 and 96 hr post-infection of the shRNAs. (B) More cleaved PARP, caspase 9 and caspase 3 were observed in MDA MB231 than in MCF10A cells upon Ran silencing, suggesting a higher apoptotic response in MDA MB231 than in MCF10a cells. Levels of Ran knockdown were similar in both cell lines. Actin was used as a loading control. (C-D) Cells were infected with shRan1 and shRan2, and harvested 72hr post-infection for (C) cell cycle and (D) Western blot analyses. (C) More apoptotic cells were observed in MDA MB231 than MCF10a cells upon Ran-silencing 72hr post-infection of shRNAs, when these two cell lines were grown in their normal medium. (D) Higher levels of cleaved PARP, caspase 9 and caspase 3 were observed at 72hr post-infection of shRNAs. Results are plotted as histogram showing the mean � SD from three independent experiments. Key; * represents p < 0.05 and ** represents p

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PDF file - 13.3MB, High level of Ran is correlated with shorter survival time of lung cancer patients.(A) Representative images for IHC staining in lung cancer specimens. Lung cancer specimens stained with different patterns for Ran. Upper panel, 40x magnification; Lower panel, 200x magnification of the same cases. (B) Kaplan-Meier plot of the survival of lung cancer patients using combined nuclear and cytoplasmic expression of Ran; High level, high level expression of both nuclear and cytoplasmc Ran; Intermediate level, high level expression of either nuclear or cytoplasmic Ran; Low level, low level expression of both nuclear and cytoplasmic Ran. Cumulative proportion of lung cancer patients surviving with time is plotted for high, intermediate and low Ran groups. (C) Kaplan-Meier plot of survival of lung cancer patients from seven microarray datasets available in GEO database. Cumulative proportion of lung cancer patients surviving with time is plotted for high (top 25% of patients expressing a high level of Ran) and low (bottom 25% of patients expressing a low level of Ran) Ran groups.

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PDF file - 3.5, MTT assay showing that cancer cells with K-Ras activation mutation (HCT116/Hkh-2 and DLD1/DKO3 isogenic pairs) and c-Met (HCC827 parental/GR5 pair) are more susceptible to Ran silencing-induced apoptosis. (A) In HCT116/Hkh-2 colon cancer isogenic pair, HCT116 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 5 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, Hkh-2. (B) In DLD1/DKO3 colon cancer isogenic pair, DLD1 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, DKO3. (C) In HCC827 parental/GR5 lung cancer pair, the GR5 cells, which contain c-Met amplification, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the wild-type HCC827 cells. Results are plotted as histogram showing the mean � SD from three independent experiments. Key; *, ** and *** represent p < 0.05

PDF file - 1.8K, Percentage of sub-G1 phase apoptotic cells and apoptotic pathway induced by Ran silencing in different cell lines. (A) Cells were harvested 72 hours post-infection, fixed and stained with Propidium iodide. Significantly fewer Sub-G1 phase apoptotic cells were observed in the immortalized human embryonic kidney 293 (HEK 293) cell line compared to cancer cell lines from breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (HCT116 and DLD-1) upon Ran silencing (p < 0.01). (B) Western blots of apoptosis mediators in immortalized HEK293, breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (DLD-1 and HCT116) cancer cell lines at 72 hours post-infection with Scr or Ran shRNAs.

PDF file - 1.9MB, Ran silencing induced apoptosis is concentration dependent. Effect of Ran silencing on apoptotic induction was tested using five different shRNAs (shRan1-5) targeting different regions of Ran compared to scramble shRNA (shScr) control in MDA MB231 breast cancer and A549 lung cancer cells. (A) mRNA and (B) protein levels of Ran, and (C) Flow cytometric analysis for DNA content, in MDA MB231 cells infected with different shRNAs targeting Ran. (D) Protein level and (E) Flow analysis for DNA content in A549 cells infected with different shRNAs targeting Ran. Note that the decrease in Ran mRNA level was associated with the decrease in Ran protein levels (r = 0.950, p < 0.01) in MDA MB231 cells infected with shRan1-5. In addition, the percentage of sub-G1 phase apoptotic cells induced by Ran silencing was significantly inversely associated with the levels of Ran in protein levels in MDA MB231 breast cancer cells (r = -0.880, p

PDF file - 4.8, Effect of chemical inhibitors on the activity of signaling pathways in MDA MB231 and A549 cells expressing various levels of Ran. Western blots of phospho- and total Akt, ERK and S6K proteins in MDA MB231 and A549 cells expressing various levels of Ran grown in different growth conditions. Representative blots from two independent experiments are shown.

PDF file - 725K, Ran silencing-induced apoptosis depends on the growth stimulation in MDA MB231 and A549 cells using MTT assay.Percentage of cell survival in (A) MDA MB231 and (B) A549 cells expressing various levels of Ran grown in different conditions as determined by MTT assay. Results are plotted as histograms showing the mean � SD from three independent experiments. Key; *, ** and *** represent p < 0.05

Isobornyl acrylate (IBOA) has recently caused a number of cases of allergic contact dermatitis (ACD) from its use in medical devices. We would like to enhance the awareness of this issue with the reporting of three Australian cases, involving two adults and a child. We also report a successful solution by using hydrocolloid wafer (Stomahesive®) as a barrier. As IBOA is not usually found on baseline patch test series and does not cross-react with other acrylic monomers, ACD to IBOA may be missed by clinicians.

Multiple dermatofibromas is a rare entity consisting of more than fifteen lesions. Multiple clustered dermatofibroma is a distinct variant of multiple dermatofibromas and is defined as a well-demarcated plaque composed of individual dermatofibromas. We report a 16-year-old boy with multiple clustered dermatofibroma in a segmental distribution, which has previously not been reported in the literature.

Purpose To assess the outcomes of small optical zone (OZ) ablations used in conjunction with large transition zones (TZ) and a highly aspheric treatment profile. Methods Interventional case series of 39 consecutive patients with myopia or myopic astigmatism. Patient data included pre and postoperative refraction and visual acuities, laser treatment settings and pre and postoperative corneal topography as well as questionnaire responses about the use of glasses and the quality of vision postoperatively. Results The mean preoperative spherical equivalent was −4.50 ± 2.11 dioptres (D) and the mean OZ and TZ diameters were 4.5 ± 0.5 mm and 8.1 ± 0.4 mm, respectively. The mean patient age was 40.7 ± 10.4 years. Manifest spherical refraction was within ±0.5 D in 87% of patients (±1.0 D in 99%) and cylindrical refraction within 0.5 D in 79% (≤1.0 D in 95%). The need to wear distance glasses postoperatively was associated with dissatisfaction with the quality of daytime vision ( p = 0.05) and unhappiness with night vision was associated with symptoms of halos ( p = 0.03) and starbursts ( p = 0.02). The proportion of patients reporting symptoms of dysphotopsias included: ghosting 0%; glare 2%; halos 10%; and starbursts 15%. There was a significant difference in the measured mean effective OZ diameter (4.8 ± 0.3 mm) compared to the mean programmed OZ (4.5 ± 0.5 mm, p = 0.00). Conclusions Small ablation zones, when used in conjunction with a large diameter TZ, do not lead to a greater incidence of unwanted visual phenomena over that reported by many studies with larger OZs.

Purpose. To assess near visual acuity in a presbyopic age group following hyperopic photorefractive keratectomy (PRK).Setting. Private practice in Siena, Italy.Methods. In this retrospective single-surgeon comparative study, PRK with mitomycin C was performed to correct hyperopia using Bausch & Lomb 217z laser for 120 eyes of 60 patients in the presbyopic age group (mean spherical equivalent SE +2.38 D ± 0.71 D and mean age ). 120 eyes of 60 age-matched controls (mean age ) had their unaided near vision measured.Results. At 12 months mean SE was −0.10 D ± 0.27 D in the PRK group. Mean best corrected visual acuity (BSCVA) was log MAR; 2 eyes lost ≥0.1 log MAR. Mean uncorrected visual acuity was log MAR. Mean distance corrected near visual acuity (DCNVA) in the PRK group was . This was statistically better () than the mean unaided near visual acuity in the control group .Conclusion. PRK was found to be safe, predictable, and an effective way of correcting hyperopia in this age group. It was also found to give better than expected near vision.

Background Dermatological conditions account for a substantial proportion of the global burden of disease in low and middle income countries (Bickers D, Lim H, Margolis D, et al. The burden of skin diseases: 2004. A joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. J Am Acad Dermatol 2006; 55: 490–500) and place major pressures on primary healthcare centers (Satimia F, McBride S, Leppard B. Prevalence of skin disease in rural Tanzania and factors influencing the choice of health care, modern or traditional. Arch Dermatol 1998; 134: 1363–1366). In mountainous North India, where limited resources are available for skin care, no dermatological data exists on prevalence, treatment patterns, or associations. The study aimed to measure prevalence and treatment of dermatological conditions and associated factors in Uttarakhand so to inform delivery of dermatological care and prevention programs in India. Methods Single stage cluster randomized sampling generated seven cluster units or villages. Household members (n = 1275) from each cluster were interviewed, and where possible, examined and offered treatment. Results Dermatological conditions were prevalent (45.3%), with 33% being of infectious etiology. Atopic dermatitis (9.2%), scabies (4.4%), tinea corporis (4.1%), and pityriasis alba (3.6%) were most prevalent. Multivariate analysis showed that cohabitation with animals (OR = 1.62, 95% CI-1.35, 1.95) was a predictor of any skin diseases. A health practitioner was not consulted in 64.7% of dermatological conditions, and where consulted, approximately 69% received inappropriate or ineffective treatments. Excessive spending on dermatological care was commonplace. Limitations associated with cross-sectional cluster methodology included the underrepresentation of seasonal conditions and conditions of short duration. Caste proved difficult to randomize across clusters given villages were often composed according to caste. Conclusion These results demonstrate a high prevalence of dermatological conditions and a pattern of conditions somewhat distinctive to this mountainous area of North India. These findings will assist development of appropriate and cost-effective dermatological services in these mountainous regions.

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non‐adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression. (Cancer Sci 2012; 103: 1145–1154)

To evaluate the risks of flap displacement after LASIK.Retrospective case series.We included 41 845 consecutive adults who underwent LASIK surgery at Optical Express in the United Kingdom, including 81 238 eyes, of which 14 555 were hyperopic and 66 681 myopic or mixed astigmatic. We treated 57 241 eyes with the IntraLase FS-60 femtosecond laser and 23 997 with the Moria S.A. ONE Use-Plus automated microkeratome.We calculated the incidence of all flap displacements in the study population during an observational time period of ≥12 months after surgery. Independent variables were entered into logistic regression models to identify risk factors. Postoperative outcomes were assessed.The incidence and odds ratios (OR) of flap displacement in the study population and in categories of refractive error and flap surgery technique.The incidence of flap displacements was 10 in 81 238 LASIK procedures (0.012%), including 8 hyperopic eyes (0.055%) and 2 myopic eyes (0.003%). All flap displacements occurred within 48 hours of surgery and none were preceded by ocular trauma. They were classified as "early flap displacements" (EFD). The incidence of EFD after microkeratome surgery was 0.033% (n = 8), and after femtosecond laser it was 0.003% (n = 2). In hyperopic eyes having microkeratome surgery, the incidence was 0.179% (n = 7). In a logistic regression model, the strongest predictor of EFD after LASIK was hyperopia, recording an OR of 19.29 (P0.001). The OR of developing an EFD after microkeratomy was 10.53 times higher than after femtosecond laser (P0.005). In hyperopes, the OR of an EFD was 18.87 times higher after microkeratomy than after femtosecond treatment. Four of 10 displaced flaps needed secondary surgery, and 1 eye lost 2 lines of best-corrected visual acuity.The incidence of flap displacements during a 12-month period after LASIK was extremely low (0.012%). Although the small number of displacements with the femtosecond laser limits conclusions, the risk of EFD was higher after microkeratome surgery than femtosecond laser.

Background: We have previously demonstrated that Tcf-4 regulates osteopontin (OPN) in rat breast epithelial cells, Rama37. In this report, we have examined the importance of this regulation in human breast cancer. Methods: The regulatory roles of Tcf-4 on cell invasion and OPN expression were investigated. The mRNA expression of Tcf-4 and OPN, and survival of breast cancer patients were correlated. Results: Tcf-4 enhanced cell invasion in both MCF10AT and MDA MB 231 breast cancer cells by transcriptionally activating OPN expression. Osteopontin was activated by Wnt signalling in MDA MB 231 cells. Paradoxical results on Tcf-4-regulated OPN expression in MCF10AT (activation) and Rama37 (repression) cells were shown to be a result of differential Wnt signalling competency in MCF10AT and Rama37 cells. High levels of OPN and Tcf-4 mRNA expression were significantly associated with survival in breast cancer patients. Most importantly, Tcf-4-positive patients had a poorer prognosis when OPN was overexpressed, while OPN-negative patients had a better prognosis when Tcf-4 was overexpressed. Conclusion: Our results suggest that Tcf-4 can act as a repressor or activator of breast cancer progression by regulating OPN expression in a Wnt-dependent manner and that Tcf-4 and OPN together may be a novel prognostic indicator for breast cancer progression.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.

Dermatological conditions account for a substantial proportion of the global burden of disease in low and middle income countries (Bickers D, Lim H, Margolis D, et al. The burden of skin diseases: 2004. A joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. J Am Acad Dermatol 2006; 55: 490-500) and place major pressures on primary healthcare centers (Satimia F, McBride S, Leppard B. Prevalence of skin disease in rural Tanzania and factors influencing the choice of health care, modern or traditional. Arch Dermatol 1998; 134: 1363-1366). In mountainous North India, where limited resources are available for skin care, no dermatological data exists on prevalence, treatment patterns, or associations. The study aimed to measure prevalence and treatment of dermatological conditions and associated factors in Uttarakhand so to inform delivery of dermatological care and prevention programs in India.Single stage cluster randomized sampling generated seven cluster units or villages. Household members (n = 1275) from each cluster were interviewed, and where possible, examined and offered treatment.Dermatological conditions were prevalent (45.3%), with 33% being of infectious etiology. Atopic dermatitis (9.2%), scabies (4.4%), tinea corporis (4.1%), and pityriasis alba (3.6%) were most prevalent. Multivariate analysis showed that cohabitation with animals (OR = 1.62, 95% CI-1.35, 1.95) was a predictor of any skin diseases. A health practitioner was not consulted in 64.7% of dermatological conditions, and where consulted, approximately 69% received inappropriate or ineffective treatments. Excessive spending on dermatological care was commonplace. Limitations associated with cross-sectional cluster methodology included the underrepresentation of seasonal conditions and conditions of short duration. Caste proved difficult to randomize across clusters given villages were often composed according to caste.These results demonstrate a high prevalence of dermatological conditions and a pattern of conditions somewhat distinctive to this mountainous area of North India. These findings will assist development of appropriate and cost-effective dermatological services in these mountainous regions.

Background: Co-localisation is a widely used measurement in immunohistochemical analysis to determine if fluorescently labelled biological entities, such as cells, proteins or molecules share a same location. However the measurement of co-localisation is challenging due to the complex nature of such fluorescent images, especially when multiple focal planes are captured. The current state-of-art co-localisation measurements of 3-dimensional (3D) image stacks are biased by noise and cross-overs from non-consecutive planes.Method: In this study, we have developed Co-localisation Intensity Coefficients (CICs) and Co-localisation Binary Coefficients (CBCs), which uses rich z-stack data from neighbouring focal planes to identify similarities between image intensities of two and potentially more fluorescently-labelled biological entities. This was developed using z-stack images from murine organotypic slice cultures from central nervous system tissue, and two sets of pseudo-data. A large amount of non-specific cross-over situations are excluded using this method. This proposed method is also proven to be robust in recognising co-localisations even when images are polluted with a range of noises.Results: The proposed CBCs and CICs produce robust co-localisation measurements which are easy to interpret, resilient to noise and capable of removing a large amount of false positivity, such as non-specific cross-overs. Performance of this method of measurement is significantly more accurate than existing measurements, as determined statistically using pseudo datasets of known values. This method provides an important and reliable tool for fluorescent 3D neurobiological studies, and will benefit other biological studies which measure fluorescence co-localisation in 3D.

Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design: Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results: Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing–induced apoptosis. K-Ras–mutated, c-Met–amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion: Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. Clin Cancer Res; 18(2); 380–91. ©2011 AACR.

Background The bioenergetic status of non-small cell lung cancer correlates with tumour aggressiveness. The voltage dependent anion channel type 1 (VDAC1) is a component of the mitochondrial permeability transition pore, regulates mitochondrial ATP/ADP exchange suggesting that its over-expression could be associated with energy dependent processes including increased proliferation and invasiveness. To test this hypothesis, we conducted an in vivo gene-expression meta-analysis of surgically resected non-small cell lung cancer (NSCLC) using 602 individual expression profiles, to examine the impact of VDAC1 on survival. Methodology/Principal Findings High VDAC1 expression was associated with shorter overall survival with hazard ratio (HR) = 0.6639 (95% confidence interval (CI) 0.4528 to 0.9721), p = 0.035352 corresponding to 52 versus 101 months. VDAC1 predicted shorter time to recurrence and was shown to be an independent prognostic factor compared with histology, gender, age, nodal stage and tumour stage in a Cox multivariate analysis. Supervised analysis of all the datasets identified a 6-gene signature comprising HNRNPC, HSPA4, HSPA9, UBE2D2, CSNK1A1 and G3BP1 with overlapping functions involving regulation of protein turnover, RAS-RAF-MEK pathway and transcription. VDAC1 predicted survival in breast cancer and myeloma and an unsupervised analysis revealed enrichment of the VDAC1 signature in specific subsets. Conclusions In summary, gene expression analysis identifies VDAC1 gene expression as a predictor of poor outcome in NSCLC and other cancers and is associated with dysregulation of a conserved set of biological pathways, which may be causally associated with aggressive tumour behaviour.

Background: Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide.Methodology: This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap.Conclusion: This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.

Introduction The molecular mechanism underlying mitochondrial BAK activation during apoptosis remains highly controversial. Two seemingly conflicting models have been proposed. In one, BAK requires so-called activating BH3 only proteins (aBH3) to initiate its conformation change. In the other, displacement from inhibitory pro-survival BCL-2 proteins (PBPs) and monomerization of BAK by PBP selective dissociator BH3-only proteins (dBH3) is sufficient. Methodology/Principal Findings To better understand the kinetic implications of these conflicting but highly evidence-based models, we have conducted a deterministic, dynamical systems analysis to explore the kinetics underlying the first step of BAK activation, as a non-linear reaction system. We show that dBH3 induced BAK activation is efficient, even in the absence of aBH3s, provided constitutive interaction of PBPs with open conformation BAK occurs in an adenoviral E1B 19K-like manner. The pattern of PBP expression robustly predicts the efficacy of dBH3s. Conclusion Our findings accommodate the prevailing BAK activation models as potentially coexisting mechanisms capable of initiating BAK activation, and supports a model based approach for predicting resistance to therapeutically relevant small molecule BH3 mimetics.

PURPOSE To investigate the effect of tear hyperosmolarity and signs of clinical ocular surface pathology on conjunctival goblet cell population. METHODS 111 participants were assessed using tear osmolarity (TO) measurements and a comprehensive selection of clinical ophthalmic tests. The resultant clinical database was assessed for evidence of patterns of composite increasing pathology. The total, filled, and empty goblet cell numbers were measured: total number of goblet cells as per cytokeratin 7 (CK7) immunofluorescence and number of filled goblet cells as per periodic acid Schiff's reagent (PAS) or lectin helix pomatia agglutinin (HPA). Goblet cell profile was correlated with composite clinical pathologic grades. RESULTS No significant correlation was found between TO and goblet cell number or function (as indicated by number of filled or unfilled goblet cells). Distinct composite clinical pathologic groups 0-IV with increasing pathology were created based on the frequency of positive pathologic signs, which adhered to the Dry Eye Workshop purported mechanism. Only in group IV was there significantly increased mean tear osmolarity of 344 mOsm/L (P < 0.000) along with significantly decreased empty goblet cell number (CK7+ and HPA-) compared to filled (CK7+ and HPA+, P = 0.000). When the number of filled goblet cells (PAS+) was analyzed there was significant increase in tear osmolarity for the two most severe grades; 3 and 4. CONCLUSIONS The goblet cell population does not appear to be affected by isolated tear hyperosmolarity. Hyperosmolarity when combined with other ocular surface pathology or inflammation alters the goblet cell population.

1599 Background: To capture the influence of multiple clinicopathological factors on NSCLC recurrence following surgical resection, machine learning methods (MLMs) can provide clear stratification between subgroups of patients. In this study a clinical signature associated with recurrence-free survival is considered, comparing the performance of MLMs against Cox regression analysis (CRA). Methods: Recurrences 60 months from surgery were filtered, yielding 846 patients with full data comprising 14 clinicopathological factors. Neural Network classification was applied with correlation feature selection and discretisation, using the target values of recurrence and survival status. Ten-fold cross-validation was used, ranked by AUC values. Likewise, Decision Trees were applied, with Kaplan-Meier analysis (KMA) based on time to recurrence (TTR) or last follow-up. Initial results led to the removal of non-recurring patients with < 60 months follow-up time, leaving 477 patients. Results: CRA identified p...

7029 Background: The likelihood of relapse following surgery for non-small cell lung cancer (NSCLC) is determined by clinical and genetic factors. Metabolic status of tumors may influence outcome. ...

e13537 Background: Personalising therapy for non-small cell lung cancer has been validated as an effective treatment paradigm. Somatic gene alterations confer sensitivity to inhibition of growth su...