EGFR mutations in exons 18-21 in sporadic breast cancer.
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Breast Neoplasms↗
Biomedical subjects
A L Harris
Publications and source records attributed to A L Harris.
The relationship of the neo-angiogenic marker, endoglin, with response to neoadjuvant chemotherapy in breast cancer.
Endoglin (CD105) is upregulated in endothelial cells of tissues undergoing neovascularisation. A greater number of CD105-positive vessels predicts poor survival in breast cancer. We examine whether CD105 expression predicts response to neoadjuvant chemotherapy. Fifty-seven women (median age 50 years, range 29-70) received neoadjuvant chemotherapy for operable breast cancer. Immunohistochemical staining using monoclonal antibodies to CD105 and CD34 was performed on pretreatment biopsies and post-treatment surgical specimens. Individual microvessels were counted in 10 random fields at x 200 magnification. Median counts were correlated with clinical and pathological response using the Mann-Whitney U-test. Forty-five out of fifty-seven patients (79%) responded clinically, 22 (39%) responded pathologically. On pretreatment biopsies, clinical responders had significantly lower median CD105-positive vessel counts than nonresponders (median counts 5 and 9.3/high-power field (hpf), median difference=4.0/hpf, 95% CI 0.5-8.0/hpf, P=0.02). For pathological responders and nonresponders, median counts were 4.8 and 5.5/hpf (median difference -0.5/hpf, 95% CI=-2.5-2.0/hpf, P=0.77). CD34 expression (total microvessel density) did not correlate with response. Pretreatment CD105 expression predicts for clinical response to chemotherapy, with a lower initial count being favourable. Patients with high baseline new vessel counts or increased counts after conventional therapy may benefit from additional antiangiogenic therapy.
Adult↗
First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours.
The lymphatic system is the primary pathway of metastasis for most human cancers. Recent research efforts in studying lymphangiogenesis have suggested the existence of a relationship between lymphatic vessel density and patient survival. However, current methodology of lymphangiogenesis quantification is still characterised by high intra- and interobserver variability. For the amount of lymphatic vessels in a tumour to be a clinically useful parameter, a reliable quantification technique needs to be developed. With this consensus report, we therefore would like to initiate discussion on the standardisation of the immunohistochemical method for lymphangiogenesis assessment.
Biomarkers, Tumor↗
Gene therapy targeting to tumor endothelium.
Tumor-associated vasculature is a relatively accessible component of solid cancers that is essential for tumor survival and growth, providing a vulnerable target for cancer gene therapy administered by intravenous injection. Several features of tumor-associated vasculature are different from normal vasculature, including overexpression of receptors for angiogenic growth factors, markers of vasculogenesis, upregulation of coagulation cascades, aberrant expression of adhesion molecules and molecular consequences of hypoxia. Many of these differences provide candidate targets for tumor-selective 'transductional targeting' of genetically- or chemically modified vectors and upregulated gene expression can also enable 'transcriptional targeting', regulating tumor endothelia-selective expression of transgenes following nonspecific gene delivery. Tumor vasculature also represents an important site of therapeutic action by the secreted products of antiangiogenic gene therapies that are expressed in non-endothelial cells. In this review we assess the challenges faced and the vectors that may be suitable for gene delivery to exploit these targets. We also overview some of the strategies that have been developed to date and highlight the most promising areas of research.
Animals↗
Hypoxia inducible factors 1alpha and 2alpha are associated with VEGF expression and angiogenesis in gallbladder carcinomas.
AIMS: To investigate the significance of the hypoxia inducible factors HIF-1alpha and HIF-2alpha in gallbladder adenocarcinomas and their relation to angiogenesis and to the expression of VEGF, an angiogenic factor transcriptionally regulated by HIFalphas. METHODS: HIF-1alpha and 2alpha expression was assessed immunohistochemically in 60 patients with early gallbladder adenocarcinomas, treated with surgery alone. In addition, the vascular density (VD) and the expression of the angiogenic factors VEGF and thymidine phosphorylase (TP) were examined. The results were correlated with clinico-pathological features and prognosis. RESULTS: Overexpression of HIF-1alpha and 2alpha was significantly associated with increased tumor angiogenesis and VEGF expression, while HIF-2alpha was linked with upregulation of TP. None of these factors were associated with T-stage and tumor grade. Although HIFs did not relate significantly with prognosis, patients with HIF-1/2 expression who failed to switch-on VEGF or intratumoral angiogenesis had a favorable outcome. CONCLUSION: Hypoxia inducible factors are upregulated in a large proportion of gallbladder adenocarcinomas, a feature strongly related to increased expression of VEGF and intensified angiogenesis.
Adenocarcinoma↗
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.
Antibodies↗
Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway.
Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.
Acetylcysteine↗
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.
Acetylation↗
Circulating endothelial progenitor cells are inversely correlated with the median oxygen tension in the tumor tissue of patients with cervical cancer.
Tumor hypoxia leads to adaptive responses in cancer cells, including an induction of vasculogenesis initiated by circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs). The aim of the present study was to correlate the number of EPCs and CECs with the oxygenation of cervical cancer. Blood concentrations of EPCs were detected by FACS analysis with antibodies for CD34 and vascular endothelial growth factor receptor 2 (VEGFR2). CECs were evaluated by double staining for 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Di-LDL) and lectin in a cell culture assay. Ten patients with cervical cancer were compared with ten healthy volunteers. Intratumoral oxygen tension was assessed polarographically with the computerized Eppendorf histography system. Analysis of CEC numbers revealed no difference between patients and controls. However, patients had lower concentrations of CD34-positive hematopoietic stem cells (HSCs) but a significantly higher fraction of EPCs related to the number of HSCs (1.09% versus 0.53%). This fraction was significantly inversely correlated to the median oxygen tension (r = -0.74, p = 0.015). Our study shows for the first time a significant inverse correlation between the fraction of EPCs and intratumoral oxygen tension. We conclude that the fraction of EPCs should be further evaluated as a useful and convenient marker in the prediction of tumor tissue oxygenation.
Endothelium, Vascular↗
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.
Adult↗
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.
Adult↗
REporting recommendations for tumour MARKer prognostic studies (REMARK).
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Biomarkers, Tumor↗
Comparison of hypoxia transcriptome in vitro with in vivo gene expression in human bladder cancer.
Hypoxia-inducible genes have been linked to the aggressive phenotype of cancer. However, nearly all work on hypoxia-regulated genes has been conducted in vitro on cell lines. We investigated the hypoxia transcriptome in primary human bladder cancer using cDNA microarrays to compare genes induced by hypoxia in vitro in bladder cancer cell line EJ28 with genes upregulated in 39 bladder tumour specimens (27 superficial and 12 invasive). We correlated array mRNA fold changes with carbonic anhydrase 9 (CA IX) staining of tumours as a surrogate marker of hypoxia. Of 6000 genes, 32 were hypoxia inducible in vitro more than two-fold, five of which were novel, including lactate transporter SLC16A3 and RNAse 4. Eight of 32 hypoxia-inducible genes in vitro were also upregulated on the vivo array. Vascular endothelial growth factor mRNA was upregulated two-fold by hypoxia and 2-18-fold in 31 out of 39 tumours. Glucose transporter 1 was also upregulated on both arrays mRNA, and fold changes on the in vivo array significantly correlated with CA IX staining of tumours (P=0.008). However, insulin-like growth factor binding protein 3 mRNA was the most strongly differentially expressed gene in both arrays and this confirmed its upregulation in urine of bladder cancer patients (n=157, P<0.01). This study defines genes suitable for an in vivo hypoxia 'profile', shows the heterogeneity of the hypoxia response and describes new hypoxia-regulated genes.
Carbonic Anhydrases↗
Phase I trial of intravesical Suramin in recurrent superficial transitional cell bladder carcinoma.
Suramin is an antitrypanosomal agent with antineoplastic activity, but with serious systemic side effects. We administered Suramin intravesically to determine a concentration with low toxicity but with evidence of a pharmacodynamic effect, to recommend a dose level for phase II trials. This was an open-labelled, non-randomized dose-escalation phase I study. In all, 12 patients with a history of recurrent superficial bladder cancer were grouped into four dose levels (10-150 mg ml(-1) in 60 ml saline). Six catheter instillations at weekly intervals were used. Cystoscopy and biopsy were performed before and 3 months after the start of treatment. Suramin was assayed using high-performance liquid chromatography, vascular endothelial growth factor (VEGF) using ELISA (enzyme-linked immunosorbent assay), and urinary protein profile using surface-enhanced laser desorption ionisation mass spectroscopy (SELDI). Minimal systemic absorption of Suramin was found at the highest dose of 150 mg ml(-1). Urinary VEGF was affected by Suramin at doses above 50 mg ml(-1), corresponding to the estimated threshold of saturation of Suramin binding to urine albumin. SELDI showed a specific disappearance of urinary protein peaks during treatment. Intravesical Suramin shows lack of toxicity and low systemic absorption. The results of this phase I trial support expanded clinical trials of efficacy at a dose of 100 mg ml(-1) intravesically.
Administration, Intravesical↗
Cooperative stimulation of vascular endothelial growth factor expression by hypoxia and reactive oxygen species: the effect of targeting vascular endothelial growth factor and oxidative stress in an orthotopic xenograft model of bladder carcinoma.
Elevated thymidine phosphorylase has been shown to correlate with increased angiogenesis and poor prognosis in many cancers including transitional cell carcinoma of the bladder. In vitro studies have demonstrated that thymidine phosphorylase activity causes cellular oxidative stress and increases secretion of vascular endothelial growth factor. In this study, we show that thymidine phosphorylase activity also augments levels of the hypoxia-inducible factor-1alpha during in vitro hypoxia, and that thymidine phosphorylase activity and hypoxia act in concert to increase vascular endothelial growth factor (VEGF) secretion. We also demonstrate that thymidine phosphorylase overexpression confers tumorigenicity on an orthotopically implanted transitional cell carcinoma cell line. Administration of the antioxidant N-acetylcysteine together with a blocking anti-VEGF antibody abrogates the increase in tumorigenicity. Our results support the increased efficacy of combination approaches to antiangiogenic therapy.
Animals↗
Analysis of combination anti-angiogenesis therapy on markers of coagulation, platelet activation and angiogenesis in patients with advanced cancer.
The effects of combination anti-angiogenesis therapy (marimastat, captopril and fragmin) on plasma levels of coagulation initiator tissue factor (TF), platelet marker soluble P-selectin and angiogenic vascular endothelial growth factor (VEGF) were tested in 25 patients with advanced cancer. They had higher soluble P-selectin (P<0.001) and TF (P<0.001), but not VEGF (P=0.066) than 25 age and sex-matched controls. VEGF and TF correlated significantly (r=0.8, P<0.001) in cancer patients. Soluble P-selectin, TF and VEGF did not change at 4- and 8-weeks whilst on treatment. We provide further evidence linking coagulation and angiogenesis but combination anti-angiogenesis therapy does not influence plasma soluble P-selectin, TF or VEGF.
Adult↗
Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression.
Tumour-associated angiogenesis is partly regulated by the hypoxia-inducible factor (HIF) pathway. Endocrine tumours are highly vascularised and the molecular mechanisms of their angiogenesis are not fully delineated. The aim of this study is to evaluate angiogenesis and expression of HIF-related molecules in a series of patients with pancreatic endocrine tumours (PETs). The expression of vascular endothelial growth factor (VEGF), HIF-1alpha, HIF-2alpha and carbonic anhydrase 9 (CA9) was examined by immunohistochemistry in 45 patients with PETs and compared to microvascular density (MVD), endothelial proliferation, tumour stage and survival. Microvascular density was very high in PETs and associated with a low endothelial index of proliferation. Microvascular density was significantly higher in benign PETs than in PETs of uncertain prognosis, well-differentiated and poorly differentiated carcinomas (mean values: 535, 436, 252 and 45 vessels mm(-2), respectively, P < 0.0001). Well-differentiated tumours had high cytoplasmic VEGF and HIF-1alpha expression. Poorly differentiated carcinomas were associated with nuclear HIF-1alpha and membranous CA9 expression. Low MVD (P = 0.0001) and membranous CA9 expression (P = 0.0004) were associated with a poorer survival. Contrary to other types of cancer, PETs are highly vascularised, but poorly angiogenic tumours. As they progress, VEGF expression is lost and MVD significantly decreases. The regulation of HIF signalling appears to be specific in pancreatic endocrine tumours.
Adenoma, Islet Cell↗
Hypoxia and anaemia in head and neck squamous cell carcinoma - mechanisms of therapy failure and provision of new therapeutic targets.
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Anemia↗