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.

The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer.

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

No evidence of association between apolipoprotein E genotype and phenotypic severity in childhood onset proximal spinal muscular atrophy.

The survival motor neuron (SMN) gene is present in two copies on chromosome 5q13 and the evidence is now compelling that mutations in the telomeric copy (SMNt) of the gene underlie childhood onset proximal spinal muscular atrophy (SMA). There is a correlation between the number of centromeric SMN gene copies (SMNc) and the clinical severity of the disease but this relationship is not absolute. Allelic variants of the apolipoprotein E (APOE) gene encoded on chromosome 19q are known to influence the prognosis and risk in a number of neurological disorders. We have therefore genotyped 166 unrelated cases of SMA to determine whether the presence of specific APOE genotypes correlates with severity of disease. The study failed to show the influence of any particular APOE genotype on disease severity, with specifically APOE epsilon4 being no more common in the milder SMA forms and APOE epsilon2 not over represented in type I SMA. A limited study of 23 SMA families also failed to show any influence of APOE genotype on SMA disease severity. Factors other than APOE genotype must therefore be responsible for determining SMA disease severity.

Lack of association of the alpha-1-antitrypsin PIZ allele with rheumatoid arthritis or with its extra articular complications.

PIZ allele frequencies were defined by PCR amplification and hybridization using a PIZ SSO (sequence specific oligonucleotide) probe. The groups studied included 64 normal controls, 104 subjects with rheumatoid arthritis (RA) without any extra-articular features, 29 of whom had severe arthritis and 31 of whom had mild RA. The extra-articular subsets include 41 with RA-bronchiectasis (RA-BR), 21 with bronchiectasis without RA (BR), and 23 with RA and pulmonary fibrosis (RA-PF). Fifteen RA subjects with obstructive airways disease (RA-OAD) were compared to 25 RA patients with normal lung function tests. Using Fishers' exact test and chi-squared statistical analysis with Yates correction, no statistically significant associations were found between PIZ and any of the groups studied. Thus in this population there is no evidence that PIZ either increases susceptibility to rheumatoid arthritis or affects the risk of pulmonary complications or the severity of arthritis in subjects with rheumatoid arthritis.

Polymorphisms in the glutamate transporter gene EAAT2 in European ALS patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder characterised by degeneration of upper and lower motor neurons. Whilst the primary pathogenic trigger is unknown in most cases, evidence is mounting to implicate a role for glutamate-mediated neurotoxicity in the disorder. Recent studies have shown reduced levels of the mainly astroglial glutamate transporter EAAT2 in ALS motor cortex and spinal cord and multiple abnormal EAAT2 mRNA species in ALS brain tissue. One cause of the low EAAT2 levels may be that point mutations in the EAAT2 gene, EAAT2, result in an abnormal unstable protein. To test this hypothesis we analysed EAAT2 in 128 sporadic and 23 familial European ALS cases. No variants within the coding sequence of EAAT2 to affect the protein sequence nor in the consensus splice sites of the flanking intronic sequences were found in any cases, similar to findings in other reports. Frequent polymorphisms within the flanking intronic sequences of both exons 2 and 4 were seen but at similar frequencies in controls. Mechanisms other than mutations within the coding region of EAAT2 must therefore be responsible for the low levels of EAAT2 seen in most cases of ALS.

Initial assessment of a model relating intratumoral genetic heterogeneity to radiological morphology.

Tumour heterogeneity has major implications for tumour development and response to therapy. Tumour heterogeneity results from mutations in the genes responsible for mismatch repair or maintenance of chromosomal stability. Cells with different genetic properties may grow at different rates and exhibit different resistance to therapeutic interventions. To date, there exists no approach to non-invasively assess tumour heterogeneity. Here we present a biologically inspired model of tumour growth, which relates intratumoral genetic heterogeneity to gross morphology visible on radiological images. The model represents the development of a tumour as a set of expanding spheres, each sphere representing a distinct clonal centre, with the sprouting of new spheres corresponding to new clonal centres. Each clonal centre may possess different characteristics relating to genetic composition, growth rate and response to treatment. We present a clinical example for which the model accurately tracks tumour growth and shows the correspondence to genetic variation (as determined by array comparative genomic hybridisation). One clinical implication of our work is that the assessment of heterogeneous tumours using Response Evaluation Criteria In Solid Tumours (RECIST) or volume measurements may not accurately reflect tumour growth, stability or the response to treatment. We believe that this is the first model linking the macro-scale appearance of tumours to their genetic composition. We anticipate that our model will provide a more informative way to assess the response of heterogeneous tumours to treatment, which is of increasing importance with the development of novel targeted anti-cancer treatments.

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.

CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding.

In order to identify potential markers of renal cancer, the plasma membrane protein content of renal cell carcinoma (RCC)-derived cell lines was annotated using a proteomics process. One unusual protein identified at high levels in A498 and 786-O cells was CD70 (TNFSF7), a type II transmembrane receptor normally expressed on a subset of B, T and NK cells, where it plays a costimulatory role in immune cell activation. Immunohistochemical analysis of CD70 expression in multiple carcinoma types demonstrated strong CD70 staining in RCC tissues. Metastatic tissues from eight of 11 patients with clear cell RCC were positive for CD70 expression. Immunocytochemical analysis demonstrated that binding of an anti-CD70 antibody to CD70 endogenously expressed on the surface of A498 and 786-O cell lines resulted in the rapid internalisation of the antibody-receptor complex. Coincubation of the internalising anti-CD70 antibody with a saporin-conjugated secondary antibody before addition to A498 cells resulted in 50% cell killing. These data indicate that CD70 represents a potential target antigen for toxin-conjugated therapeutic antibody treatment of RCC.

A quantitative analysis of lymphatic vessels in human breast cancer, based on LYVE-1 immunoreactivity.

This study was undertaken to determine the highly sensitive method for detecting tumour lymphatic vessels in all the fields of each slide (LV), lymphatic microvessel density (LMVD) and lymphatic vessel invasion (LVI) and to compare them with other prognostic parameters using immunohistochemical staining with polyclonal (PCAB) and monoclonal antibodies (MCAB) to the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and the pan-endothelial marker factor VIII in a series of 67 human breast cancers. In all LYVE-1-stained sections, LV (some of which contained red blood cells) were frequently found localised in extralobular stroma, dermis, connective tissue stroma and adjacent to artery and vein, but were rare within the intralobular stroma or the tumour body (3/67 cases) or areas of widespread invasion. In contrast small blood vessels were observed in intra- and extralobular stroma in the factor VIII-stained sections. Quantitation of vessel numbers revealed that LYVE-1/PCAB detected a significantly larger number of LV than either H&E or LYVE-1/MCAB (P<0.0001). LYVE-1/PCAB detected LVI in 25/67 cases (37.3%) and their presence was significantly associated with both lymph node metastasis (chi(2)=4.698, P=0.0248) and unfavourable overall survival (OS) (P=0.0453), while not relapse- free survival (RFS) (P=0.2948). LMVD had no influence for RFS and OS (P=0.4879, P=0.1463, respectively). Our study demonstrates that immunohistochemistry with LYVE-1/PCAB is a highly sensitive method for detecting tumour LV/LVI in breast cancer and LVI is a useful prognostic indicator for lymphatic tumour dissemination.

Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe.

We have synthesized capsid proteins of human papillomavirus types 6 (HPV 6) and 16 (HPV 16) in fission yeast Schizosaccharomyces pombe and produced virus-like particles (VLP). The capsid proteins were localized in the nucleus by indirect immunofluorescence and cell fractionation analyses. The VLP were produced in both yeast clones synthesizing L1 alone and L/L2 and purified by sulfato-cellulofine chromatography. Electron microscopic examination showed that these VLP were similar in structure to native HPV particles. Two HPV 16 L1 variants (16 B27L1 and 16 T3L1), isolated from benign cervical samples, produced many more (68- and 14-fold) VLP than the prototype L1 (16 PL1) derived from cervical carcinoma. Coexpression of the HPV 6 L2 protein with 6 L1 and 16 B27L1 proteins increased the production level of the VLP four- and twofold, respectively. The L2 was not detected in the VLP purified with sulfato-cellulofine column, although the L2 was purified in the same fraction containing HPV 6 and 16 B27-VLP by size-fractionation using Sepharose column. Interaction between 6 L2 and 6/16 L1 proteins was not detected by the coimmunoprecipitation assays with either L1 or L2 antibodies. These results suggest that the L2 is not incorporated into the VLP synthesized in yeast.

Relative contributions of HLA-DQA and complement C4A loci in determining susceptibility to systemic lupus erythematosus.

The objective of this study was to reassess the role of C4A null alleles in systemic lupus erythematosus (SLE) susceptibility after taking into account the association of DQA*0501 with this disease. The frequency of C4A null alleles in 82 SLE patients and 59 controls was determined using both immunofixation and a TaqI RFLP method. HLA-DQA and DQB alleles were identified by sequence-specific oligonucleotide typing. Empirical logistic analysis was used to assess the interactive effects of C4 and DQA alleles. It was found that the strongest association with SLE was for the combination of DQA*0501 and C4A*Q0 [odds ratio (OR) = 5.4, 95% confidence interval (CI) 2.5-11.7]. Both DQA*0501 (P = 0.02) and C4A*Q0 (P = 0.03) appeared to have significant individual effects on SLE susceptibility, with a significant statistical interaction between the two loci (P = 0.01). However, when anti-La antibody negative patients were examined only C4A*Q0 had a significant individual effect (P = 0.04). A significant statistical interaction between DQA*0501 and C4A*Q0 was again detected (P = 0.02). These results support the hypothesis that susceptibility to SLE is influenced by several genes with differing functions: HLA-DQA*0501 may predispose to autoantibody formation while C4A*Q0 impairs immune complex clearance.

Vascular patterns in reactive lymphoid tissue and in non-Hodgkin's lymphoma.

The few studies published on angiogenesis in lymphoma have raised the question of whether or not microvessel density (MVD) is associated with more aggressive disease and have reported the observation that in follicular lymphomas, vessels are mature rather than immature. We investigated MVD and the vascular phenotype within follicular or diffuse large B-cell lymphomas, reactive nodes and tonsils. Vascular phenotype was defined by the expression or loss of reactivity to the antibody LH39 (detecting the LH39 laminin epitope of the basement membrane in mature vessels) and by detection of alpha V beta 3 (expressed on immature vessels). In reactive nodes and in follicular lymphomas, MVD was higher in the paracortex than in germinal centres or in neoplastic follicles. However, in neoplastic follicles an increase in alpha V beta 3-positive endothelium suggested the activation of an angiogenic pathway different from that present in the reactive follicles. In large B-cell lymphomas, MVD was higher than in reactive and neoplastic follicles but lower than in the reactive paracortex. The number of immature vessels (LH39 negative) and of alpha V beta 3-positive vessels was higher than in reactive lymph nodes and follicular lymphoma suggesting that a switch to a different angiogenic pathway has occurred. Finally, we have demonstrated that within reactive and neoplastic follicles vascular regression is occurring, perhaps constraining the growth of reactive follicles alongside other phenomena such as apoptosis. Vascular regression was previously believed to occur in adults only in ovarian and endometrial tissue. We conclude that different types of angiogenesis are present in follicular lymphomas and large B-cell lymphomas. This has implications for possible future therapies.