Developing a digital data collection platform to measure the prevalence of sepsis in Wales.

OBJECTIVE: To develop a secure, efficient, and easy-to-use data collection platform to measure the prevalence of sepsis in Wales over 24 hours. MATERIALS AND METHODS: Open Data Kit was used on Android devices with Google App Engine and a digital data collection form. RESULTS: A total of 184 students participated in the study using 59 devices across 16 hospitals, 1198 datasets were submitted, and 97% of participants found the Open Data Kit form easy to use. DISCUSSION: We successfully demonstrated that by combining a reliable Android device, a free open-source data collection framework, a scalable cloud-based server, and a team of 184 medical students, we can deliver a low-cost, highly reliable platform that requires little training or maintenance, providing results immediately on completion of data collection. CONCLUSION: Our platform allowed us to measure, for the first time, the prevalence of sepsis in Wales over 24 hours.

PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.

Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer.

PURPOSE: To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC). METHODS: Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers. RESULTS: Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by (18)FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P<0.00001) and in 9 out of 13 patients at the definitive surgery compared with baseline (P<0.03). There was also a significant suppression of CD31 and VEGF-A expression in response to treatment (P=0.01 and P=0.007, respectively). CONCLUSIONS: The LCS combination is feasible and tolerable. The tumour response and target biomarker modulation indicate that the combination is clinically and biologically active.

A nonlinear approach to surface-enhanced sensing in the short-wave infrared.

The surface-enhanced hyper-Raman scattering (SEHRS) of three analyte molecules has been obtained with excitation wavelengths at 1.55 and 1.8 µm. At 1.55 µm, excellent signal-to-noise ratios under modest experimental parameters demonstrate compelling potential for practical applications in chemical analysis and molecular imaging.

Hypoxia regulates FGFR3 expression via HIF-1α and miR-100 and contributes to cell survival in non-muscle invasive bladder cancer.

BACKGROUND: Non-muscle invasive (NMI) bladder cancer is characterised by increased expression and activating mutations of FGFR3. We have previously investigated the role of microRNAs in bladder cancer and have shown that FGFR3 is a target of miR-100. In this study, we investigated the effects of hypoxia on miR-100 and FGFR3 expression, and the link between miR-100 and FGFR3 in hypoxia. METHODS: Bladder cancer cell lines were exposed to normoxic or hypoxic conditions and examined for the expression of FGFR3 by quantitative PCR (qPCR) and western blotting, and miR-100 by qPCR. The effect of FGFR3 and miR-100 on cell viability in two-dimensional (2-D) and three-dimensional (3-D) was examined by transfecting siRNA or mimic-100, respectively. RESULTS: In NMI bladder cancer cell lines, FGFR3 expression was induced by hypoxia in a transcriptional and HIF-1α-dependent manner. Increased FGFR3 was also in part dependent on miR-100 levels, which decreased in hypoxia. Knockdown of FGFR3 led to a decrease in phosphorylation of the downstream kinases mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), which was more pronounced under hypoxic conditions. Furthermore, transfection of mimic-100 also decreased phosphorylation of MAPK and PKB. Finally, knocking down FGFR3 profoundly decreased 2-D and 3-D cell growth, whereas introduction of mimic-100 decreased 3-D growth of cells. CONCLUSION: Hypoxia, in part via suppression of miR-100, induces FGFR3 expression in bladder cancer, both of which have an important role in maintaining cell viability under conditions of stress.

Diagnostic utility of aP2/FABP4 expression in soft tissue tumours.

Adipocyte P2 (aP2), also known as fatty acid-binding protein 4 (FABP4), is a fatty acid-binding protein found in the cytoplasm of cells of adipocyte differentiation. In this study, we examined a large number of soft tissue tumours with a commercial polyclonal anti-aP2/FABP4 antibody and a newly developed mouse monoclonal antibody raised against this protein to determine the diagnostic utility of aP2/FABP4 as a marker of tumours of adipose differentiation. A mouse monoclonal antibody, clone 175d, was raised against a mixture of synthetic peptides corresponding to the amino acid sequence of residues 10-28 and 121-132 of the human aP2/FABP4 protein. Antigen expression with polyclonal and monoclonal antibodies was immunohistochemically determined in paraffin sections of normal adipose tissue and a wide range of benign and malignant primary soft tissue tumours (n = 200). aP2/FABP4 was expressed around the cytoplasmic lipid vacuole in white and brown fat cells in benign lipomas and hibernomas. Immature fat cells and lipoblasts in spindle cell/pleomorphic lipoma, atypical lipomatous tumour/well-differentiated liposarcoma, myxoid/round cell liposarcoma and pleomorphic liposarcoma also reacted strongly for aP2/FABP4. No specific staining was seen in non-adipose benign and malignant mesenchymal and non-mesenchymal tumours. aP2/FABP4 is expressed by mature and immature fat cells and is a marker of tumours of adipose differentiation. Immunophenotypic aP2/FABP4 expression is useful in identifying lipoblasts, and immunohistochemistry with polyclonal/monoclonal anti-aP2/FABP4 antibodies should be useful in distinguishing liposarcoma from other malignancies, particularly round cell, myxoid and pleomorphic soft tissue sarcomas.

Prognostic impact of thymidine phosphorylase expression in breast cancer--comparison of microarray and immunohistochemical data.

Contrary findings exist according to the prognostic and predictive impact of thymidine phosphorylase (TP) expression in breast cancer. Goal of our study was to investigate TP expression on the mRNA level by microarray analysis in a large cohort of 1781 breast cancers and to analyse its prognostic impact. Furthermore we compared mRNA expression and immunohistochemical data to explain discrepancies between different studies. The prognostic value of TP mRNA expression was analysed among n=622 untreated patients. Strong expression in the subgroup of n=213 ER-negative cancer correlates with improved survival (P=0.012). In contrast, no difference in survival was detected in the ER-positive group. We also failed to observe a prognostic value of TP mRNA among n=435 endocrine-treated patients as well as n=111 CMF-treated patients. In an unsupervised analysis, TP clustered together with genes expressed in immune cells. Moreover, among normal tissues the highest TP mRNA expression was found in tissues of the immune system. The profile of TP expression in breast cancers correlates to a metagene of interferon induction whereas the expression of TP among normal tissues correlates to a metagene for macrophages. When comparing microarray data with immunohistochemistry from the same n=51 samples, there was no correlation with stained carcinoma cells. In contrast, the correlation with stromal staining was highly significant (P<0.001). Thus TP mRNA from microarray mainly reflects expression in stromal and immune cells. This could account for discrepant results from mRNA and IHC studies. In conclusion, the tumour infiltrating immune cells seem to be a major source of TP expression and predict a favourable prognosis in ER-negative breast cancer. Our data point to a role of TP in host immune response.

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer.

BACKGROUND: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4-Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. METHOD: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). RESULTS: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P<0.0001). The expression of VEGF was significantly associated with HIF-2alpha (P<0.0001) and Dll4 (P=0.010). Only HIF-2alpha had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01-2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. CONCLUSION: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies.

Hypoxia-inducible factor-1alpha and -2alpha are expressed in most rectal cancers but only hypoxia-inducible factor-1alpha is associated with prognosis.

The hypoxia-mediated response of tumours is a major determining factor in growth and metastasis. Understanding tumour biology under hypoxic conditions is crucial for the development of antiangiogenic therapy. Using one of the largest cohorts of rectal adenocarcinomas to date, this study investigated hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha protein expression in relation to rectal cancer recurrence and cancer-specific survival. Patients (n=90) who had undergone surgery for rectal adenocarcinoma, with no prior neoadjuvant therapy or metastatic disease, and for whom adequate follow-up data were available were selected. Microvessel density (MVD), HIF-1alpha and HIF-2alpha expressions were assessed immunohistologically with the CD34 antibody for vessel identification and the NB100-131B and NB100-132D3 antibodies for HIF-1alpha and HIF-2alpha, respectively. In a multifactorial analysis, results were correlated with tumour stage, recurrence rate and long-term survival. Microvessel density was higher across T and N stages (P<0.001) and associated with poor survival (hazard ratio (HR)=8.7, P<0.005) and decreased disease-free survival (HR=4.7, P<0.005). hypoxia-inducible factor-1alpha and -2alpha were expressed in >50% of rectal cancers (HIF-1alpha, 54%, 48/90; HIF-2alpha, 64%, 58/90). HIF-1alpha positivity was associated with both TNM stage (P<0.05) and vascular invasion (P<0.005). In contrast, no associations were demonstrated [corrected] between HIF-2alpha [corrected] and any pathological features or [corrected] outcome. The study showed an independent association between HIF-1alpha expression and advanced TNM stage with poor outcome. Our results indicate that HIF-1alpha, but not HIF-2alpha, might be used as a marker of prognosis, in addition to methods currently used, to enhance patient management.

The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy.

Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and factor inhibiting HIF (FIH)-1, and carbonic anhydrase (CA) IX stained in 456 breast tumours. Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1alpha, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy (P<0.001 and P=0.03, respectively). The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients.

Assessment of microvessel density and carbonic anhydrase-9 (CA-9) expression in rectal cancer.

AIM: The mechanism by which neoplasias respond to hypoxia determines their biological behavior and prognosis. Understanding the biology of tumors under hypoxic conditions is crucial for the development of anti-angiogenic therapy. Using the largest cohort of rectal adenocarcinomas to date, this study aimed to assess microvessel density (MVD) and carbonic anhydrase-9 (CA-9) expression and to correlate the results with recurrence and cancer-specific survival. MATERIALS AND METHODS: Patients (n=101) who underwent surgery for rectal adenocarcinoma without previous neoadjuvant therapy or metastatic disease were selected. MVD and CA-9 expression were assessed immunohistologically by using the CD34 antibody and the MN/CA9 M75 antibody, respectively. In a multifactorial analysis, the results were correlated with tumor stage, recurrence rate, and long-term survival. RESULTS: MVD was higher with increased T- and N-stages (p<0.01) and associated positively with poor survival (hazard ratio (HR) 1.3 per 10 vessel increase, p<0.01). CA-9 was expressed in 73% of cancers. Negative lymph node status correlated with CA-9 positivity (p<0.05), reflected in a higher rate of CA-9 positivity in earlier Dukes' stages (p<0.05). CA-9 positivity across tumor node metastasis (TNM) stages approached significance (Stage I/II: 80% CA-9 positive vs. 20% CA-9 negative; Stage III: 63% CA-9 positive vs. 37% negative, p=0.051). A trend was seen towards better cancer-specific survival in patients with CA-9 positive carcinomas (HR 0.51, p=0.07) on univariate analysis. DISCUSSION: MVD was higher in more advanced T- and N-stages and may be used as a determinant of survival in patients with rectal adenocarcinomas. CA-9 expression was seen more often in earlier Dukes' stages, possibly representing an early tumor hypoxic response. CA-9 expression by adenocarcinoma cells may confer long-term survival advantage in surgically treated rectal cancer.

Prognostic significance of microvessel density and other variables in Japanese and British patients with primary invasive breast cancer.

The purpose of this study is to investigate the associations of microvessel density (MVD) and other pathological variables with survival, and whether they accounted for survival differences between Japanese and British patients. One hundred seventy-three Japanese and 184 British patients were included in the study. British patients were significantly older (56.3+/-11.4 years vs 52.5+/-12.9 years; P<0.01) and had smaller tumours (2.2+/-1.3 vs 2.7+/-1.8 cm; P<0.01), which were more frequently oestrogen receptor positive (78.8 vs 57.2%, P<0.01), had more grade III tumours (29.9 vs 21.4%, P=0.04) and more infiltrating lobular carcinomas (13.6 vs 4.0%, P<0.01) and a higher MVD compared with Japanese patients (57.9+/-19.8 vs 53.2+/-18.6; P=0.01). However, no difference in the prevalence of lymph-node metastasis was found between them (39.1 vs 37.5%, P=0.75). Younger British patients (age <50 years) had the highest MVD compared with Japanese and older British patients (P<0.01). Japanese patients were proportionately more likely to receive chemotherapy than endocrine therapy (P<0.01). British patients had a significantly worse relapse-free survival and overall survival compared with Japanese patients, after statistical adjustment for variables (hazard ratio=2.1, 2.4, P<0.01, P<0.01, respectively), especially, in T2 stage, low MVD and older subgroup (HR: 3.6, 5.0; 3.1, 3.3; 3.2, 3.9, respectively), but only in ER negative cases (P=0.04, P=0.01, respectively). The present study shows that MVD contributes to the Japanese-British disparity in breast cancer. However, the MVD variability did not explain the survival differences between Japanese and British patients.

Activated VEGFR2/KDR pathway in tumour cells and tumour associated vessels of colorectal cancer.

BACKGROUND: Vascular endothelial cell growth factor (VEGF) acts by phosphorylating specific tyrosine kinase receptors on endothelial cell membrane promoting angiogenesis. The study of the activation status of VEGF receptors in human malignancies has recently become feasible by means of specific monoclonal antibodies recognising the phosphorylated form of these receptors. MATERIALS AND METHODS: In the current study, we investigate the expression of the phosphorylated VEGFR2/KDR receptor in normal colon and colorectal adenocarcinomas in parallel with histopathological parameters, prognosis and the expression of the 'hypoxia inducible factor' HIF1alpha. RESULTS: pVEGFR2/KDR was weakly expressed in the normal colon, but it was expressed strongly in the cytoplasm and nuclei of cancer cells and in the tumour associated vasculature, mainly at the invading tumour edge. pVEGFR2/KDR expression in cancer cells was significantly associated with a tumour diameter > 6 cm (P = 0.04), poor histological differentiation (P = 0.004) and with high CEF1alpha expression (P = 0.05). High pVEGFR2/KDR expressing vascular density was significantly related with a high VEGF and HIF1alpha expression in cancer cells (P = 0.02 and 0.03, respectively). This was also related significantly to high pVEGFR2/KDR expression in cancer cells. In multivariate analysis, the most significant predictors for death were lympho-vascular invasion (P < 0.001) followed by VEGF (P = 0.014), node status (P = 0.015), standard vascular density (P = 0.022) and necrosis (P = 0.032). CONCLUSIONS: pVEGFR2 receptors are largely expressed in colon cancer cells and intratumoural vasculature. As VEGF targeting agents enter the clinical practice, the role of monoclonal antibodies recognising the phosphorylated form of VEGF receptors as predictors of response to targeted therapies should be sought in clinicopathological trials.

The angiogenic factor thymidine phosphorylase up-regulates the cell adhesion molecule P-selectin in human vascular endothelial cells and is associated with P-selectin expression in breast cancers.

Thymidine phosphorylase (TP) is an angiogenic enzyme, catalysing the reversible phosphorylation of thymidine to thymine and 2-deoxyribose. TP is up-regulated in neoplasia, being associated with advanced tumour stage, microvessel density and prognosis in several tumour types. Although TP is a non-mitogenic migratory factor for endothelium, the mechanism by which TP mediates these effects is still unclear. We compared the gene expression profile of endothelial cells grown in vitro in the presence or absence of TP by cDNA microarray analysis. To determine the time-course of TP angiogenic induction, endothelial cells were stimulated with TP (10 ng/ml) for 5 and 18 h. Gene expression levels of Tie2, angiopoietin (Ang)1 and Ang2, measured by RNase protection assay (RPA), showed maximal alteration at 18 h. cDNA from human umbilical vein endothelial cells (HUVEC) grown for 18 h in the presence or absence of TP (10 ng/ml) was hybridized to a human cDNA cytokine array representing 375 angiogenic genes. Significantly altered expression occurred in 89 human angiogenic genes (72 genes were up-regulated and 17 down-regulated). Changes in five genes relevant to vascular remodelling biology (Tie2, nNos, P-selectin, ephrin-B1 and TP) were validated in triplicate experiments by real-time RT-PCR. But only P-selectin gene expression remained significant. Correlation between P-selectin and TP was assessed by immunohistochemistry on 161 human breast cancers, using human tissue microarray. Tumour cell TP correlated with tumour cell P-selectin but not with endothelial cell P-selectin. These data show that TP stimulates changes in mRNA expression maximally after 18 h culture in vitro. It confirms a role for TP in vascular remodelling involving several classes of genes, including the cell adhesion molecule, P-selectin. Although confirmation of the role of TP-mediated cell adhesion molecule (CAM) induction is required; however, this pathway may provide an attractive therapeutic target, since it is likely to affect several important tumour processes, including angiogenesis and metastasis.

BNIP3 expression in follicular lymphoma.

AIMS: To investigate the role of BNIP3, a 19-kDa interacting protein of the Bcl-2 family, alongside Bcl-2 in follicular lymphoma in comparison with reactive lymphoid hyperplasia. The results were compared with those from p53 and caspase-3 (apoptotic markers) and Ki67 (proliferation marker). METHODS AND RESULTS: Immunohistochemistry using monoclonal antibodies showed BNIP3 to be strongly expressed in most follicular lymphomas but to be weak to negative in all of the reactive cases. There was also an inverse relationship with Bcl-2 expression. There was no correlation of BNIP3 immunoreactivity with proliferation and caspase and p53 were virtually negative in all follicular lymphomas and reactive lymphoid cases. CONCLUSIONS: BNIP3 is strongly expressed in most follicular lymphomas, especially those that are Bcl-2 negative. BNIP3 may serve as a marker of more aggressive behaviour in follicular lymphoma and be useful diagnostically in the distinction from reactive lymphadenitis.

Selective silencing of the hypoxia-inducible factor 1 target gene BNIP3 by histone deacetylation and methylation in colorectal cancer.

Hypoxia, via the hypoxia-inducible factors 1 and 2 (HIF-1 and HIF-2), upregulates many genes involved in cell survival. However, proapoptotic pathways are also induced. BCL-2/adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) represents a paradigm of a cell death protein that is hypoxically upregulated via HIF-1 in most cancers. We found that in contrast to many other cell types, 6/8 colorectal cancer (CRC) cell lines show little hypoxic induction of BNIP3 despite an intact HIF signalling system. Colorectal tumour tissue also loses BNIP3 expression relative to matched normal samples. Downregulation of hypoxic BNIP3 in CRC cells was independent of the expression of other BCL-2 family members, or BNIP3L. That BNIP3 plays a functional role in hypoxic survival in CRC cells was demonstrated by the fact that CRC cell lines that do not upregulate BNIP3 or have been treated with BNIP3 RNA interference were insensitive to hypoxia-induced cell death. Promoter methylation and histone deacetylation were shown to silence BNIP3 in these CRC cell lines. Of significance, hypoxic induction of BNIP3 was restored in 4/6 cell lines by trichostatin-A treatment alone. These data suggest that BNIP3 plays an important role in hypoxic cell death and epigenetic mechanisms selectively silence its expression in CRC.

Use of novel monoclonal antibodies to determine the expression and distribution of the hypoxia regulatory factors PHD-1, PHD-2, PHD-3 and FIH in normal and neoplastic human tissues.

AIMS: The cellular response to hypoxia includes the hypoxia inducible factor (HIF)-induced transcription of genes involved in diverse processes such as glycolysis, angiogenesis and the growth of experimental tumours. Regulation of the level of hypoxia inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) is a primary determinant of HIF activity. Recent biochemical and candidate gene approach studies have led to the discovery of three HIF-regulatory prolyl hydroxylases, PHD-1, -2 and -3 and an asparaginyl hydroxylase, also known as FIH (factor inhibiting HIF). In this study, we raised and characterized monoclonal antibodies against PHD-1, PHD-2, PHD-3 and FIH. METHODS AND RESULTS: Immunohistochemistry of normal tissues with these monoclonal antibodies demonstrated a wide distribution in epithelial cells, stromal cells and leucocytes, with cytoplasmic staining predominating over nuclear staining. A preliminary study of tumours showed variable staining in tumour, stromal and inflammatory cells. While all tumour types showed some positive staining with each antibody, the overall pattern suggested a slight decrease in the amount of staining seen with PHD-1, -2 and -3 and an increase in FIH staining in neoplasia compared with corresponding normal tissues. CONCLUSIONS: These monoclonal antibodies will allow further larger scale studies to determine the significance of PHD and FIH expression in neoplasia.

Angiogenesis and hypoxia in lymph node metastases is predicted by the angiogenesis and hypoxia in the primary tumour in patients with breast cancer.

Hypoxia and angiogenesis are important factors in breast cancer progression. Little is known of hypoxia and angiogenesis in lymph node metastases of breast cancer. The aim of this study was to quantify hypoxia, by hypoxia-induced marker expression levels, and angiogenesis, by endothelial cell proliferation, comparing primary breast tumours and axillary lymph node metastases. Tissue sections of the primary tumour and a lymph node metastasis of 60 patients with breast cancer were immunohistochemically stained for the hypoxia-markers carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1alpha (Hif-1alpha) and DEC-1 and for CD34/Ki-67. Endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. On haematoxylin-eosin stain, the growth pattern and the presence of a fibrotic focus were assessed. Hypoxia-marker expression, ECP% and TCP% in primary tumours and in lymph node metastases were correlated to each other and to clinico-pathological variables. Median ECP% and TCP% in primary tumours and lymph node metastases were comparable (primary tumours: ECP%=4.02, TCP%=19.54; lymph node metastases: ECP%=5.47, TCP%=21.26). ECP% correlated with TCP% (primary tumours: r=0.63, P<0.001; lymph node metastases: r=0.76, P<0.001). CA9 and Hif-1alpha expression were correlated (primary tumours P=0.005; lymph node metastases P<0.001). In primary tumours, CA9 and Hif-1alpha expression were correlated with DEC-1 expression (P=0.05), presence of a fibrotic focus (P<0.007) and mixed/expansive growth pattern (P<0.001). Primary tumours and lymph node metastases with CA9 or Hif-1alpha expression had a higher ECP% and TCP% (P<0.003); in primary tumours, mixed/expansive growth pattern and fibrotic focus were characterised by higher ECP% (P=0.03). Furthermore, between primary tumours and lymph node metastases a correlation was found for ECP%, TCP%, CA9 and Hif-1alpha expression (ECP% r=0.51, P<0.001; TCP r=0.77, P<0.001; CA9 and Hif-1alpha P<0.001). Our data demonstrate that the growth of breast cancer lymph node metastases is angiogenesis dependent and that angiogenesis and hypoxia in the primary tumour predict angiogenesis and hypoxia in the lymph node metastases. Together with previous findings in breast cancer liver metastases, which grow in 96% of cases angiogenesis independently, these data suggest that both the intrinsic growth characteristics and angiogenic potential of breast cancer cells and the site-specific tumour microenvironment determine angiogenesis and hypoxia in breast cancer.

Expression of hypoxia-inducible factors is correlated with the presence of a fibrotic focus and angiogenesis in pancreatic ductal adenocarcinomas.

AIMS: To study the expression of hypoxia-regulated markers in pancreatic ductal adenocarcinomas (PA) in relationship to the presence of a fibrotic focus, angiogenesis quantification and clinical outcome. METHODS AND RESULTS: The expression of hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) was immunohistochemically detected in 50 PA and correlated with tumour characteristics, microvascular density (MVD) and survival. HIF-1alpha was expressed within tumour cells in 68%, HIF-2alpha in 46%, CA9 in 78% and VEGF in 52% of the cases. Stromal expression was also noted for HIF-2alpha and CA9 in, respectively, 42% and 48% of the cases. Tumour CA9 expression was associated with that of VEGF (P=0.004) and that of stromal HIF-2alpha (P=0.013), with the presence of a fibrotic focus (P=0.046) and with an increased MVD (P=0.034). Tumour VEGF expression correlated with the presence of a fibrotic focus (P=0.039) and a greater MVD (P=0.047). Both the presence of a fibrotic focus (P=0.0002) and high tumour CA9 expression (P=0.029) were associated with reduced overall survival. CONCLUSION: The strong association of the presence of a fibrotic focus with CA9 expression and lower survival demonstrates that hypoxia-driven angiogenesis plays an important role in the progression of PA.

The expression and cellular localization of phosphorylated VEGFR2 in lymphoma and non-neoplastic lymphadenopathy: an immunohistochemical study.

AIMS: To study the expression of phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2), a membrane-bound tyrosine kinase receptor to vascular endothelial growth factor, in lymphoma and non-neoplastic lymphadenopathy. METHODS AND RESULTS: Archival cases (89 cases of lymphoma and 17 cases of non-neoplastic lymphadenopathy) were studied immunohistochemically with three monoclonal antibodies to the different autophosphorylation sites in the cytoplasmic tail of the receptor. There was increased expression of this receptor in lymphoma and particularly in all cases of peripheral T-cell lymphoma. In this category, there was nuclear re-location of this receptor. CONCLUSIONS: This very interesting finding raises the possibility that VEGFR2 may be involved in the transcriptional regulation of this disease. Small molecule inhibitors to this receptor may therefore be a useful adjunct in the therapy of this disease.