Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours.
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Antigens, CD↗
Biomedical subjects
A L Harris
Publications and source records attributed to A L Harris.
A critical review of vascular endothelial growth factor (VEGF) analysis in peripheral blood: is the current literature meaningful?
Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor with a key role in many physiological and pathological processes. Investigation into the implications of circulating levels of this cytokine is progressing at an exponential rate. However, there are important inconsistencies between reports ranging from method of sample collection, processing, software manipulation and data interpretation and controversy as to whether plasma, serum or whole blood will provide the best prognostic information. Different techniques of centrifugation and temperature on sample handling and the impact of in vitro collection of blood on subsequent VEGF results have not been fully appreciated. We provide a critical review of the literature, report the results of our further investigations, suggest a uniform protocol for handling blood samples and highlight previously unsuspected problems in data interpretation.
Blood Specimen Collection↗
The angiogenic "vascular endothelial growth factor/flk-1(KDR) receptor" pathway in patients with endometrial carcinoma: prognostic and therapeutic implications.
BACKGROUND: Vascular endothelial growth factor (VEGF) is an important endothelial cell mitogen associated with increased angiogenesis and aggressive tumor behavior. Its stimulating effect on endothelial cells basically is dependent on the presence of specific VEGF receptors, such as the flk-1(KDR) receptor. This study investigates the roles of VEGF and of a functionally intact angiogenic pathway, "VEGF/flk-1(KDR)," in patients with endometrial carcinoma and their significance in prognosis and therapy. METHODS: A series of 121 endometrial carcinomas were studied. The expression of VEGF by endometrial tumor cells was assessed using the monoclonal antibody (MoAb) VG1. VEGF/KDR complexes on tumor endothelium or activated microvessel density (aMVD) were identified using the MoAb 11B5. In addition, the standard microvessel density (sMVD) was assessed with anti-CD31. In all tumors, the alkaline phosphatase/antialkaline phosphatase technique was employed. A Fisher exact test or an unpaired, two-tailed t test was used for testing correlations between categoric tumor variables, whereas a log-rank test was used to determine statistical differences between life tables. A Cox proportional hazards model was used to assess the effect of tumor variables on overall survival. RESULTS: Cytoplasmic VEGF expression in > 50% of tumor cells was associated significantly with aMVD (P < 0.0001) and with sMVD (P < 0.003). In univariate survival analysis, VEGF (P = 0.0002), aMVD (P = 0.001), and sMVD (P = 0.0009) were significant prognostic variables. Equally important were the histologic parameters tumor type (P = 0.03), tumor grade (P = 0.003), and disease stage (P < 0.0001). In multivariate analysis, disease stage was the most important independent prognostic factor (P < 0.0001), followed by VEGF/KDR (P < 0.01), and VEGF (P < 0.04). Furthermore, VEGF and VEGF/KDR were the only independent prognostic variables for patients with Stage I endometrioid adenocarcinoma. CONCLUSIONS: sMVD and the angiogenic factor VEGF are important indicators of a poor prognosis in patients with endometrial carcinoma. VEGF/KDR complexes define a subgroup of patients with endometrial carcinoma with an even worse prognosis.
Adult↗
Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer.
Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular active site, is involved in the reversible metabolism of the carbon dioxide to carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible factor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res., 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4%) had strong membrane/cytoplasmic expression of CA9 and were grouped as positive. The staining was confined around areas of necrosis, and a significant association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did not express CA9. CA9 expression was more frequent in the squamous cell histology (P = 0.001) and with advanced T stage (P = 0.009). A significant coexpression of CA9 with platelet-derived endothelial cell growth factor and basic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microvessel density in the areas expressing CA9 than in negative areas (P = 0.0005). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of expression were diffuse (not confined in the necrotic areas). A direct association of CA9 expression with epidermal growth factor receptor, c-erbB-2, and MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expression is a significant prognostic factor independent of angiogenesis. We conclude that CA9 is an important molecule in non-small cell lung cancer, the up-regulation of which occurs in highly hypoxic/necrotic regions of the tumors. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of CA9 with poor outcome.
Adenocarcinoma↗
Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway.
Many oncogenes induce expression of vascular endothelial growth factor (VEGF), a key factor in tumor angiogenesis. Phosphatidylinositol 3'-kinase (PI3K)/Akt is a common signaling pathway for oncogenes and tumor suppressor genes and is involved in VEGF regulation. Because hypoxia is a major stimulus for VEGF production, we examined the effects of LY294002, a selective PI3K inhibitor, on hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression and on endogenous VEGF responses to hypoxia. A panel of breast cancer cell lines reflecting the different genetic changes occurring in human breast cancer was analyzed. LY294002 inhibited HIF-1alpha induction and phosphorylation under hypoxia. However, HIF-2alpha expression was not affected. Basal and hypoxia-inducible VEGF expression was reduced at both mRNA and protein levels by 50%. V12-ras overexpression resulted in an increase in hypoxia-induced HIF-1alpha and HIF-2alpha expression. This effect was blocked by PI3K inhibitor, demonstrating one mechanism for ras synergy with hypoxia-mediated induction of genes. The decreased HIF-1alpha expression was not dependent on VHL interaction because a renal carcinoma cell line with VHL mutation and constitutive high HIF-1alpha expression also showed down-regulation of HIF-1alpha after treatment with LY294002. These results have implications for the use of PI3K inhibitors to inhibit synergistic effects of hypoxia with a wide range of common oncogenes.
Basic Helix-Loop-Helix Proteins↗
HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors.
Solid tumors contain regions of hypoxia, a physiological stress that can activate cell death pathways and, thus, result in the selection of cells resistant to death signals and anticancer therapy. Bcl2/adenovirus EIB 19kD-interacting protein 3 (BNIP3) is a cell death factor that is a member of the Bcl-2 proapoptotic family recently shown to induce necrosis rather than apoptosis. Using cDNA arrays and serial analysis of gene expression, we found that hypoxia induces up-regulation of BNIP3 and its homologue, Nip3-like protein X. Analysis of human carcinoma cell lines showed that they are hypoxically regulated in many tumor types, as well as in endothelial cells and macrophages. Regulation was hypoxia inducible factor-1-dependent, and hypoxia inducible factor-1 expression was suppressed by von Hippel-Lindau protein in normoxic cells. Northern blotting and in situ hybridization analysis has revealed that these factors are highly expressed in human tumors compared with normal tissue and that BNIP3 is up-regulated in perinecrotic regions of the tumor. This study shows that genes regulating cell death can be hypoxically induced and are overexpressed in clinical tumors.
Animals↗
Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival.
Hypoxia inducible factors HIF1alpha and HIF2alpha are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1alpha and 2alpha protein levels, as a consequence of a redox-sensitive stabilization. The HIFalphas enter the nucleus, heterodimerize with the HIF1beta protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1alpha and HIF2alpha expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2alpha expression with HIF1alpha expression showed a significant association (P< 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P< 0.004), PD-ECGF (P< 0.003) and bFGF (P< 0.04) was noted. HIF1alpha correlated with the expression of bek-bFGF receptor expression (P = 0.01), while HIF2alpha was associated with intense VEGF/KDR-activated vascularization (P = 0.002). HIF2alpha protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P = 0.006). Analysis of overall survival showed that HIF2alpha expression was related to poor outcome (P = 0.008), even in the group of patients with low MVD (P = 0.009). HIF1alpha expression was marginally associated with poor prognosis (P = 0.08). In multivariate analysis HIF2alpha expression was an independent prognostic indicator (P = 0.006, t-ratio 2.7). We conclude that HIF1alpha and HIF2alpha overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC.
Adenocarcinoma↗
Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix.
There is increasing evidence that hypoxia-regulated gene expression influences tumor aggressiveness, contributing to the poorer outcome of patients with hypoxic tumors. The role of the transcriptional complex hypoxia-inducible factor-1 as an important mediator of hypoxia-regulated gene expression is one of the best documented pathways. Recently, it has emerged that certain tumor-associated carbonic anhydrases (CAs) can be added to the list of known hypoxia-inducible factor-responsive genes. Here we show that the immunohistochemical expression of the tumor-associated CA IX is correlated with the level of hypoxia in human cervical tumors. We performed a prospective study in 68 patients where needle electrodes were used to make direct measurements of tumor oxygenation levels. CA IX expression was evaluated immunohistochemically in pretreatment tumor biopsies. There was a significant positive correlation between the level of tumor hypoxia (HP5) and the extent of CA IX expression. A retrospective study of 130 squamous cell cervical carcinomas demonstrated that a semiquantitative immunohistochemical analysis of CA IX expression in tumor biopsies is a significant and independent prognostic indicator of overall survival and metastasis-free survival after radiation therapy. These studies provide clinical evidence that CA IX expression is up-regulated in hypoxic human cervical tumors and is associated with a poor prognosis. CA IX may act as an intrinsic marker of tumor hypoxia and poor outcome after radiation therapy. The level of CA IX expression may be used to aid in the selection of patients who would benefit most from hypoxia-modification therapies or bio-reductive drugs.
Antigens, Neoplasm↗
Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma.
PURPOSE: To assess the frequency of expression and the prognostic significance of a hypoxia-regulated marker, carbonic anhydrase IX (CA IX), in a cohort of patients with invasive breast cancer. PATIENTS AND METHODS: CA IX expression was evaluated by immunohistochemistry with a murine monoclonal antibody, M75, in a series of 103 women treated surgically for invasive breast cancer. The majority of patients were treated with adjuvant hormonal or chemotherapy. The frequency of CA IX expression, its association with recognized prognostic factors, and the relationship with outcome was evaluated by univariate and multivariate statistical analyses. RESULTS: CA IX expression was present in 49 (48%) of 103 cases. The level of CA IX expression was found to be significantly associated with tumor necrosis (P <.001), higher grade (P =.02), and negative estrogen receptor status (P <.001). Furthermore, CA IX expression was associated with a higher relapse rate (P =.004) and a worse overall survival (P =.001). By multivariate analysis, CA IX was also shown to be an independent predictive factor for overall survival (hazard ratio, 2.61; 95% confidence interval, 1.01 to 6.75, P =.05). CONCLUSION: CA IX expression was associated with worse relapse-free survival and overall survival in an unselected cohort of patients with invasive breast carcinoma. The potential role of CA IX as a marker of hypoxia within breast carcinomas was also indicated by a significant association with necrosis. Further work assessing its prognostic significance in breast cancer is warranted, particularly interactions with radiotherapy and chemotherapy resistance.
Adult↗
Hypoxia and oxidative stress in breast cancer. Hypoxia and tumourigenesis.
The microenvironmental hypoxia that arises as a consequence of the development of a solid tumour also acts to promote tumour growth. Hypoxia induces the expression of key components of the angiogenic and apoptotic signalling cascades, the glycolytic pathway and various cell-cycle control proteins. At the cellular level it mediates the infiltration and accumulation of tumour-associated macrophages within avascular tumour regions. Complex interactions between tumour cell and macrophage hypoxia-regulated gene products and their associated pathways form the basis for the hypoxic promotion of tumourigenesis and malignant progression.
Apoptosis↗
Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis, and microvessel density.
Carbonic anhydrase IX (CA IX) is a transmembrane glycoprotein with an active extracellular enzyme site. We have shown previously that it was hypoxia inducible and may therefore be an endogenous marker of hypoxia. It is overexpressed in some tumors, particularly renal cell carcinoma. The aim of this study was to examine the expression and localization of CA IX in head and neck squamous cell carcinoma (HNSCC) and relate this to the location of tumor microvessels, angiogenesis, necrosis, and stage. Expression of CA IX was determined by immunoblotting in three HNSCC cell lines grown in normoxia and hypoxia (pO(2) 0.1%) and three paired tumor and normal tissue samples of HNSCC. Archived paraffin sections (79) of HNSCC were immunostained with antibodies to CA IX and CD34 to determine microvessel density (MVD). By double staining sections with CA IX and CD34, the distance between blood vessels and the start of CA IX expression and necrosis was calculated. CA IX was induced by hypoxia in all three HNSCC cell lines and overexpressed in HNSCC tumor tissue. Overexpression was localized to the perinecrotic area of the tumor on immunostaining, and the percentage area of the tumor expressing CA IX was significantly higher with more tumor necrosis (P = 0.001), a high MVD (P = 0.02), and advanced stage (P = 0.033) on univariate analysis and necrosis (P = 0.0003) and MVD (P = 0.0019) on multivariate analysis. The median distance between a blood vessel and the start of CA IX expression was 80 microm (range, 40-140 microm). CA IX is overexpressed in HNSCC because of hypoxia and is a potential biomarker for hypoxia in this tumor. Overexpression may help to maintain the intracellular pH, giving tumor cells a survival advantage and enhancing resistance to radiotherapy and chemotherapy. CA IX is a potential target for future therapy in HNSCC.
Antigens, Neoplasm↗
Mutant epidermal growth factor receptor enhances induction of vascular endothelial growth factor by hypoxia and insulin-like growth factor-1 via a PI3 kinase dependent pathway.
Over-expression of truncated epidermal growth factor receptor (EGFR) occurs in a variety of malignancies including glioblastoma multiforme, breast and lung cancer. The truncation deletes an extracellular domain and results in constitutive activation of the receptor. NIH3T3 cells were transfected with full length or truncated human EGFR and differences in growth rates in vivo and in vitro analysed. A growth advantage was seen for cells expressing mutant receptor compared to full length EGFR in vivo only. Administration of an anti-mutant EGFR antibody to mice transiently reduced the growth rates of mutant tumours, confirming that the mutant receptor itself was important in this enhanced tumorigenicity. This showed that stimuli present in vivo and not in vitro may be contributing to growth. We therefore analysed the regulation of the angiogenic factor vascular endothelial growth factor (VEGF). Although levels of secreted VEGF did not differ significantly between wild-type and mutant EGFR cell lines when grown in vitro under normoxic conditions, following exposure to 0.1% hypoxia levels of VEGF produced by mutant cells increased 3.5-6.6 fold compared to 2 or less for full length EGFR cells. The fold induction was influenced by experimental conditions, including cell confluence and percentage of fetal bovine serum, but was consistently higher for mutant cell lines. The increase in VEGF under hypoxic conditions was blocked by the addition of PI3 kinase inhibitors, indicating that the latter pathway is important in the hypoxic stress response. Basal levels were not affected. Addition of insulin-like growth factor-1 also increased levels of VEGF under normoxic conditions in the mutant cells and no further increase was seen when added to cells exposed to 0.1% oxygen, indicating that levels of VEGF were already maximally stimulated. These results show that the mutant EGFR interacts with other growth factors and hypoxia to regulate VEGF via a PI3 kinase pathway, and suggests a specific role for anti-mutant EGFR antibodies and PI3 kinase inhibitors as therapy of this specific tumour target.
3T3 Cells↗
Nuclear expression of human apurinic/apyrimidinic endonuclease (HAP1/Ref-1) in head-and-neck cancer is associated with resistance to chemoradiotherapy and poor outcome.
PURPOSE: HAP1/Ref-1 endonuclease is involved in the repair of DNA strand breaks and in the activation of DNA binding of several transcription factors. HAP1 is also a potent activator of wild type p53. It therefore has multiple possible roles in the response of human cancer to radiotherapy and chemotherapy. METHODS AND MATERIALS: The nuclear expression of HAP1 and p53 proteins was studied by immunohistochemistry in paraffin-embedded material from 95 patients with locally advanced squamous cell head-and-neck cancer (HNC) treated with radical radiotherapy (38 cases with induction platinum-based chemotherapy and 57 with concurrent platinum chemoradiotherapy). RESULTS: HAP1 was present in the nuclei of normal epithelium and stromal cells. Loss of HAP1 nuclear expression was frequently noted in cancer cells. Tumors with high HAP1 nuclear expression (% of positive cells > mean; mean = 11%) were of good differentiation (p = 0.06) and presented frequently with advanced nodal disease (p = 0.01). High nuclear HAP1 expression was significantly associated with poor complete response rate (p = 0.00001), shorter local relapse-free interval (p < 0.0001), and poorer survival (p < 0.0008). HAP1 nuclear reactivity was inversely associated with p53 nuclear accumulation (p = 0.003). The inverse correlation between HAP1 expression and prognosis was independent of p53 status. CONCLUSION: HAP1 nuclear expression in HNC is inversely associated with p53 nuclear accumulation and directly related to resistance to chemoradiotherapy and poor survival. Further clinical investigation is required to confirm these findings.
Carbon-Oxygen Lyases↗
Vascular endothelial growth factor-B and vascular endothelial growth factor-C expression in renal cell carcinomas: regulation by the von Hippel-Lindau gene and hypoxia.
Angiogenesis is essential for tumor growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B and VEGF-C, two new VEGF family members, have been identified that bind to the tyrosine kinase receptors flt-1 (VEGFR1), KDR (VEGFR2), and flt-4 (VEGFR3). Although the importance of VEGF-A has been shown in renal carcinomas, the contribution of these new ligands in kidney tumors is not clear. We have, therefore, measured the mRNA level of VEGF-B and VEGF-C together with their receptors by RNase protection assay (RPA) in 26 normal kidney samples and 45 renal cell cancers. We observed a significant up-regulation of VEGF-B (P = 0.002) but not VEGF-C (P = 0.3) in neoplastic kidney compared with normal tissues. In addition, although VEGF receptors were higher in tumors than normal kidney, there was a significant up-regulation of only flt-1 (P = 0.003) but not KDR (P = 0.12) or flt-4 (P = 0.09). There was also a significant correlation between VEGF-C and both of its receptors flt-4 (P = 0.006) and KDR (P = 0.03) but no association between VEGF-B and its receptor flt-1 (P = 0.23). A significant increase was observed in flt-1 (P < 0.001), KDR (P = 0.02), and flt-4 (P = 0.01) but not VEGF-B (P = 0.82) or VEGF-C (P = 0.52) expression in clear cell compared with chromophil (papillary) carcinomas. No significant association was demonstrated between VEGF-B, VEGF-C, flt-1, KDR, and flt-4 with patient sex, patient age, or tumor size (P > 0.05). The effect of von Hippel-Lindau (VHL) gene and hypoxia on VEGF-B and VEGF-C expression in the renal carcinoma cell line 786-0 transfected with wild-type and mutant VHL was determined by growing cells under 21% O2- and 0.1% O2. In wild-type VHL cells, whereas VEGF-A was significantly up-regulated under hypoxic compared with normoxic conditions (P < 0.001), expression of VEGF-C was reduced (P < 0.002). Nevertheless, the repression of VEGF-C was lost in mutant VHL cell lines under hypoxia. In contrast VEGF-B was not regulated by VHL despite clear up-regulation in vivo. These findings strongly support an enhanced role for this pathway in clear cell carcinomas by regulating angiogenesis and/or lymphangiogenesis. The study shows that clear cell tumors are able to up-regulate angiogenic growth factor receptors more efficiently than chromophil (papillary), that clear cell tumors can use pathways independent of VHL to regulate angiogenesis, and that this combined regulation may account for their more aggressive phenotype, which suggests that targeting VEGFR1 (flt-l) may be particularly effective in these tumor types.
Adenocarcinoma, Clear Cell↗
Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C.
PURPOSE: N-Benzoyl staurosporine (PKC412) is a protein kinase C inhibitor with antitumor activity in laboratory models. We determined the toxicity of oral PKC412 administered daily for repeat cycles of 28 days. PATIENTS AND METHODS: Thirty-two patients with advanced solid cancers were treated at seven dose levels (12.5 to 300 mg daily) for a total of 68 cycles. RESULTS: The most frequent treatment-related toxicities were nausea, vomiting, fatigue, and diarrhea. At the two top dose levels (225 and 300 mg/d), 15 of 16 patients experienced nausea/vomiting (common toxicity criteria [CTC], version 1), grade 2 in nine of 16 and grade 3 in three of 16 patients; and six of 16 patients developed CTC grade 2 diarrhea. After 1 month of treatment, there were significant reductions in circulating lymphocyte (P <.02) and monocyte (P <.01) counts in patients receiving doses > or = 100 mg/d. Nevertheless, only two patients developed myelosuppression (both grade 2). Of two patients with progressive cholangiocarcinoma, one attained stable disease lasting 4.5 months and one a partial response lasting 4 months. There was a linear relationship between PKC412 dose and area under the curve (0-24 hours) and maximum plasma concentration with marked interpatient variability. The estimated median elimination half-life was 1.6 days (range, 0.9 to 4.0 days), and a metabolite with a median half-life of 36 days was detected. Steady-state PKC412 plasma levels at the top three dose cohorts (150 to 300 mg) were five to 10 times the cellular 50% inhibitory concentration for PKC412 of 0.2 to 0.7 micromol/L. CONCLUSION: PKC412 can be safely administered by chronic oral therapy, and 150 mg/d is suitable for phase II studies. The pharmacokinetics and lack of conventional toxicity indicate that pharmacodynamic measures may be additionally needed to optimize the drug dose and schedule.
Administration, Oral↗
Hypoxia inducible factor (HIF-1a and HIF-2a) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy.
Hypoxia inducible factor 1a and 2a (HIF-1a and HIF-2a) are key proteins regulating cellular response to hypoxia. Because the efficacy of photodynamic therapy (PDT) is dependent on the presence of oxygen, the assessment of HIF-1a and HIF-2a expression may be of value in predicting clinical response to PDT. Using recently produced MoAbs, we examined the expression of HIF1a and HIF2a in a series of 37 early-stage esophageal cancers treated with PDT and with additional radiotherapy in case of incomplete response after PDT. Strong expression of the HIF1a and of HIF2a proteins in all optical fields examined was noted in 51% and in 13% of cases, respectively. High expression was associated with a low complete response (CR) rate and with the absence of bcl-2 protein expression. On the contrary, bcl-2 expression was associated with a high CR rate. Combined analysis of HIF1a and bcl-2 protein expression revealed that of 16 cases with high HIF1a expression and the absence of bcl-2 reactivity, only 1 (7%) responded completely to PDT (P = 0.007). Bivariate analysis showed that HIF1a expression was independently related to response to PDT (P = 0.04; t ratio = 2.8), whereas bcl-2 approached significance (P = 0.07; t-ratio = 1.8). The final response to radiotherapy was high (70%) and independent of the HIF and bcl-2 status, which may be a result of reoxygenation after cellular depletion mediated by PDT. The present study suggests that assessment of HIF and of bcl-2 expression are important predictors of in vivo sensitivity to PDT. Modulation of PDT response with bioreductive drugs and/or drugs targeting bcl-2 (i.e., taxanes) may prove of significant therapeutic importance in a subgroup of patients with high HIF expression.
Basic Helix-Loop-Helix Proteins↗
The HIF pathway: implications for patterns of gene expression in cancer.
Regulation of the growth and metabolism of large organisms is tightly constrained by the need for precise oxygen homeostasis. Work on control of the haematopoietic growth factor erythropoietin has led to the recognition of a widespread transcriptional response to hypoxia which provides insights into how this is achieved. The central mediator of this response is a DNA binding complex termed hypoxia inducible factor 1 (HIF-1), which plays a key role in the regulation by oxygen of a large and rapidly growing panel of genes. In cancer, activity of the HIF system is up-regulated both by microenvironmental hypoxia and by genetic changes. The clearest example of genetic activation is seen in the hereditary cancer syndrome von Hippel-Lindau (VHL) disease. In normal cells the product of the VHL tumour suppressor gene targets the regulatory HIF subunits (HIF-1alpha and HIF-2alpha) for oxygen-dependent proteolysis, acting as the substrate recognition component of an E3 ubiquitin ligase. In pVHL defective cells this process is blocked leading to constitutive up-regulation of HIF-1alpha subunits, activation of the HIF complex and overexpression of HIF target genes. Using gene array screens we have defined a large number of VHL-regulated genes. The majority of these show hypoxia-inducible responses, supporting the central involvement of pVHL in gene regulation by oxygen. In addition to known HIF target genes involved in angiogenesis, glucose metabolism and vasomotor control, these new targets include examples with functions in matrix metabolism, apoptosis, carbon dioxide metabolism and secondary cascades of transcriptional control. Thus activation of HIF provides insights into the classical metabolic alterations in cancer cells, and into the mechanisms by which microenvironmental hypoxia might influence tumour behaviour. In the case of VHL disease, this activation can be linked to mutations in a defined tumour suppressor gene. Equally regulation of the HIF-1alpha/pVHL interaction in normal cells should provide insights into the physiological mechanisms operating in cellular oxygen sensing.
Basic Helix-Loop-Helix Proteins↗
VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis.
Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B, a new VEGF family member that binds to the tyrosine kinase receptor flt-1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF-B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF-B and its receptor flt-1 by ribonuclease protection assay and the pattern of VEGF-B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt-1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF-B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF-B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF-B and flt-1 (p=0.2) or tumour vascularity (p=0.4). VEGF-B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF-B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF-B may contribute to tumour progression by a non-angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF-B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti-VEGF receptor therapy targeted to flt-1 (VEGFR1) as well as kdr (VEGFR2).
Adult↗