High HIF-1alpha Expression Research Articles

10247-ET-8 HHYPOXIA-TARGETING RADIOPHARMACEUTICAL 64CU-ATSM FOR PET MONITORING WITH LOCAL THERAPY IN HIGH-GRADE GLIOMA PATIENT-DERIVED XENOGRAFT MODEL

Abstract High-grade gliomas (HGGs) are highly aggressive brain cancers characterized by the presence of hypoxia within a rapidly-growing tumor mass. Due to invasion to the surrounding brain parenchyma, these tumors commonly recur locally,, and novel local approaches are required. Here, we show a positron emission tomography (PET) integrated local therapy (PETx), to target HGGs. This technique consists of a one-step local theranostic application, followed by PET monitoring, with a hypoxia-targeting radiopharmaceutical64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM). We examined the safety and therapeutic potential of 64Cu-ATSM PETx for HGG patient-derived xenograft (PDX) tumors. PDX models were recapitulated the parent tumor phenotype of high expression of HIF-1 alpha and BNIP3, biomarkers of tissue hypoxia. We confirmed that HIF-1 alpha and BNIP3 were highly upregulated under hypoxia and 64Cu-ATSM was retained with potent cytotoxic effect under hypoxic condition in vitro. We determined that determined the maximum tolerated dose (MTD) to be 3.7 MBq in mouse. PETx using the MTD dose of 64Cu-ATSM indicated high tumor penetration, distribution, and retention of 64Cu-ATSM in PDX tumors, as compared to sham-treated mice. The 64Cu-ATSM PETx promoted DNA double-strand breaks, followed by apoptosis in tumors, and extensively prolonged overall survival with tolerable systemic toxicity. These findings indicate the potential of 64Cu-ATSM PETx to induce high uptake in the hypoxic tumor microenvironment, and strong therapeutic effects in PDX models. These findings establish 64Cu-ATSM PETx as a potential novel theranostic approach to facilitate local control of HGGs.

Whole-tumor histogram analysis of diffusion-weighted imaging and dynamic contrast-enhanced MRI for soft tissue sarcoma: correlation with HIF-1alpha expression.

To investigate the correlation of histogram metrics from diffusion-weighted imaging (DWI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters with HIF-1alpha expression in soft tissue sarcoma (STS). We enrolled 71 patients with STS who underwent 3.0-T MRI, including conventional MRI, DWI, and DCE-MRI sequences. Location, maximum tumor diameter, envelope, T2-weighted tumor heterogeneity, peritumoral edema, peritumoral enhancement, necrosis, tail-like pattern, bone invasion, and vessel/nerve invasion and/or encasement were determined using conventional MRI images. The whole-tumor histogram metrics were calculated on the apparent diffusion coefficient (ADC), Ktrans, Kep, and Ve maps. Independent-samples t test and one-way ANOVA were used for testing the differences between normally distributed categorical data with HIF-1alpha expression. Pearson and Spearman correlations and multiple linear regression analyses were performed to determine the correlations between histogram metrics and HIF-1alpha expression. Survival curves were plotted using the Kaplan-Meier method. Regarding conventional MRI features, only highly heterogeneous on T2-weighted images (55.6 ± 19.9% vs. 45.4 ± 20.5%, p = 0.041) and more than 50% necrotic area (57.3 ± 20.4% vs. 43.9 ± 19.7%, p = 0.002) were prone to indicate STS with higher HIF-1alpha expression. Histogram metrics obtained from ADC (mean, median, 10th, and 25th percentile values), Ktrans (mean, median, 75th, and 90th percentile values), and Kep (90th percentile values) were significantly correlated with HIF-1alpha expression. Multiple linear regression analysis demonstrated that more than 50% necrosis, ADCskewness, Ktrans90th, and grade III were independently associated with HIF-1alpha expression. DWI and DCE-MRI histogram parameters were significantly correlated with HIF-1alpha expression in STS. • DWI and DCE-MRI histogram parameters are correlated with HIF-1alpha expression in STS. • More than 50% necrosis, ADCskewness, Ktrans90th, and grade III were independently associated with HIF-1alpha expression in STS.

Abstract 2056: MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a devastating disease. Recent findings suggest that the EGFR antagonist including erlotinib may induce tumor regression alone or in combination with chemo/radiation. Our preliminary clinical study conducted at KaviKrishna laboratory indicates that many poor patients are taking the highly expensive drug Erlotinib, and or equivalent EGFR antagonists. However, oral cancer lesions are often hypoxic, and the hypoxia-induced cellular mechanisms might contribute to drug resistance. Here, we investigated, whether resistance to EGFR antagonists may involve MYC and HIF-2alpha, two transcription factors upregulated during hypoxia by using established oral cancer cell lines. We have also obtained primary oral cancer cells from patients living in the Kamrup district of Assam, where KaviKrishna laboratory is located. We intend to evaluate the drug sensitivity of these oral cancer cells towards EGFR antagonists. Method: We used SSC-25 and SCC-9 cell lines, as well patient derived primary oral cancer cells (n=5) for the study. Immunomagnetic sorting was performed to obtain ABCG2+ population. The self-renewal was studied using in vitro clonogenic and in vivo serial transplantation assay in NOD/SCID mice. Results: First, we identified a rare ABCG2+ expressing, highly tumorigenic cell population in SSC-25, and SCC-9 having cancer stem cell (CSC) like characteristics. Second, we found that the ABCG2+ cells exhibited sensitivity to PD158780 (10 uM; 62% inhibition within 48 hours), and AG1478 (10 uM; 56% inhibition within 48 hours), two small molecular inhibitors of EGFR tyrosine kinase. These small molecular inhibitors significantly inhibited the clonogenic capacity of the ABCG2+ cells. Next, we found that ABCG2+ cells also showed sensitivity to Erlotinib in the in vitro clonogenic assay. Third, the ABCG2+ cells, when exposed to hypoxia (<0.1% O2, 24 hours), exhibited enhanced expression and transcriptional activity of MYC, and HIF-2alpha. The post-hypoxia ABCG2+ cells exhibited complete resistance to PD158780, AG1478 and Erlotinib treatment, which could be reversed by siRNA silencing of MYC and or HIF-2alpha. ChIP assay revealed that HIF-2alpha directly binds to MYC in ABCG2+ cells. We found similar results in ABCG2+ cells obtained from primary oral cancer samples (n=4). Importantly, ABCG2+ cells directly isolated from patients exhibited hypoxic phenotype, including the high expression of HIF-1alpha, and HIF-2alpha, as well as resistance to erlotinib. Furthermore, erlotinib enhanced the stemness of ABCG2+ cells by activation of the MYC/HIF-2alpha self-renewal pathway (1). Conclusion: These data indicate that MYC and HIF-2alpha co-operate to mediate intrinsic resistance of oral squamous cancer cells to EGFR antagonist in the hypoxia microenvironment. (1). Bhuyan et al. Cancer Research, volume 76 (14), abstract 935; 2016 Note: This abstract was not presented at the meeting. Citation Format: Sukanya Gayan, Reza Bayat-Mokhtari, Bidisha Pal, Anupam Sarma, Joyeeta Talkudar, Sorra Sandhya, Rashmi Bhuyan, Seema Bhuyan, Jaishree Garhyan, Debabrat Baishya, Amal Kataki, Rakesh Bhatnagar, Herman Yeger, Bikul Das. MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2017-2056

HIF-1alpha Expression Profile in Intratumoral and Peritumoral Inflammatory Cells as a Prognostic Marker for Squamous Cell Carcinoma of the Oral Cavity

The HIF-1 transcriptional complex is responsible for controlling transcription of over 100 genes involved in cell hypoxia response. HIF-1alpha subunit is stabilized in hypoxia conditions, creating the HIF-1 nuclear transcription factor. In inflammatory cells, high HIF-1alpha expression induces lymphocytic immunosuppression, decreasing tumoral antigen recognition, which promotes tumor growth. The present work investigated the relationship between HIF-1alpha expression in lymphocytes populating the intratumoral and peritumoral region of 56 patients with oral cancer. Our data indicates a prognostic value for this expression. High HIF-1alpha expression in peritumoral inflammatory cells is significantly related to worse patient outcome, whereas high expression in the intratumoral lymphoid cells correlates with a better prognosis. A risk profile indicating the chance of disease relapse and death was designed based on HIF-1alpha expression in tumoral inflammatory cells, defining low, intermediate and high risks. This risk profile was able to determine that high HIF-1alpha expression in peritumoral cells correlates with worse prognosis, independently of intratumoral expression. Low HIF-1alpha in tumor margins and high expression in the tumor was considered a low risk profile, showing no cases of disease relapse and disease related death. Intermediate risk was associated with low expression in tumor and tumor margins. Our results suggest that HIF-1alpha expression in tumor and peritumoral inflammatory cells may play an important role as prognostic tumor marker.

Overexpression of cyclooxygenase‐2 in urothelial carcinoma in conjunction with tumor‐associated‐macrophage infiltration, hypoxia‐inducible factor‐1α expression, and tumor angiogenesis

This study examines whether the expression of cyclooxgenase-2 (COX-2) in urothelial carcinoma (UC) is associated with macrophage infiltration, hypoxia-inducible factor-1alpha (HIF-1alpha) expression and angiogenesis. We investigated the expression of COX-2 associated with HIF-1alpha and performed double immunohistochemical analysis of 216 UCs for COX-2 expression and the correlation with tumor-associated-macrophage (TAM) density and microvessel density (MVD) in situ. A high expression of COX-2 was positively correlated with tumor invasiveness, histologic grade and HIF-1alpha expression in UC (p<0.0001, p=0.003, p<0.0001, respectively). Quantification of double staining of COX-2/CD34 and COX-2/CD68 showed that a higher MVD and TAM density was found in COX-2 high-expression than in COX-2 low-expression tumor fields (p<0.0001). Adjacent to the principal of COX-2 expression areas, MVD value and TAM density were significantly increased in HIF-1alpha high-expression specimens compared with HIF-1alpha low-expression ones (p<0.0001). Interestingly, our data revealed that high COX-2 expression (p=0.002), high HIF-1alpha expression (p<0.0001) and TAM density (p<0.0001) were all associated with high MVD value. Our results suggest that COX-2 may produce a cooperative effect in promoting tumor progression and may be involved in the process of angiogenesis through increasing TAM infiltration or HIF-1alpha regulation by hypoxia.

Prognostic Significance of Tumor Hypoxia Inducible Factor–1α Expression for Outcome After Radiotherapy in Oropharyngeal Cancer

Head-and-neck squamous cell carcinoma (HNSCC) represents a heterogeneous group of patients in terms of subsite, treatment, and biology. Currently most management decisions are based on clinical parameters with little appreciation of patient differences in underlying tumor biology. We investigated the prognostic significance of clinicopathologic features and tumor hypoxia-inducible factor-1alpha (HIF-1alpha) expression in a homogeneous series of patients who underwent radiotherapy. An audit identified 133 consecutive patients with histologically proven squamous cell carcinoma of the tonsil or tongue base. All patients received primary radiotherapy between 1996 and 2001. Tumor HIF-1alpha expression was examined in 79 patients. Features associated with poor locoregional control were low Hb level (p = 0.05) and advancing T (p = 0.008), N (p = 0.03), and disease (p = 0.008) stage. HIF-1alpha expression was a more significant adverse prognostic factor in the tonsil (hazard ratio [HR], 23.1; 95% confidence interval [CI]. 3.04-176.7) than the tongue-base tumor (HR, 2.86; 95% CI, 1.14-7.19) group (p = 0.03, test for interaction). High tumor HIF-1alpha expression was associated with low blood Hb levels (p = 0.03). In a multivariate analysis HIF-1alpha expression retained prognostic significance for locoregional control (HR, 7.10; 95% CI, 3.07-16.43) and cancer-specific survival (HR, 9.19; 95% CI, 3.90-21.6). There are significant differences in radiation therapy outcome within a homogeneous subsite of the oropharynx related to molecular marker expression. The work highlights the importance of studying homogeneous groups of patients in HNSCC, and the complex interrelationships between tumor biology and clinicopathologic factors. The establishment of tumor-type specific markers would represent a major advance in this area.

The role of hypoxia in recurrence following resection of Dukes’ B colorectal cancer

Tumour hypoxia has been shown to be a predictor of early distant relapse in node-negative breast and cervical cancer. The purpose of the present study was to determine the role of hypoxia in predicting patients who are at high risk of disease recurrence in Dukes B colorectal cancers. Archival tissue was retrieved from 52 patients who had undergone surgical resection for primary colorectal cancer. Tissue micro-arrays were constructed using tissue from the margin and the centre of the tumour. Hypoxia markers hypoxia-inducible factor (Hif)-1 alpha, vascular endothelial growth factor (VEGF), carbonic anhydrase (CA)-9 and glucose transporter (Glut)-1 were visualised using immunohistochemical detection and quantified using semi-quantitative analysis of the digitised images. Clinical details and outcome data were retrieved by case note review and collated with hypoxia markers data in a statistical database. Primary colorectal cancers with a high Hif-1 alpha expression tended to have a significantly worse disease-free survival (log rank p < 0.001) and overall survival (log rank p = 0.012). VEGF was also a significant predictor of disease recurrence in primary colorectal cancers (p = 0.015). Significant correlations were also noted between Hif-1 alpha and VEGF (Pearson's p = 0.009). Glut-1 and CA-9 did not show a similar pattern with no differences in the expression pattern and no correlation observed with any of the markers. Multivariate analysis of prognostic factors showed vascular invasion (p < 0.001) and Hif-1 alpha at the tumour margin (p < 0.001) to be independent predictors for the development of liver metastases. These results suggest an important role for Hif-1 alpha and VEGF in colorectal cancer progression, with both markers biological mechanisms directly interlinked through the hypoxic pathway. Identification of high-risk patients using the above factors will improve treatment strategies in node-negative disease and help improve patient outcome.

Lactate Dehydrogenase-5 (LDH-5) Expression in Human Gastric Cancer: Association with Hypoxia-Inducible Factor (HIF-1α) Pathway, Angiogenic Factors Production and Poor Prognosis

Lactate-dehydrogenase-5 (LDH-5) is an important isoenzyme converting pyruvate to lactate under hypoxic conditions and might play an important role in the development and progression of malignancies. However, the role of LDH-5 in gastric cancer is still unclear. In this study, we investigated the clinical significance of LDH-5 expression in gastric carcinoma. LDH-5 expression in 152 patients with different grade and stage gastric carcinoma was analyzed by immunohistochemistry. In addition, hypoxia-inducible factor 1alpha (HIF-1alpha) as a marker of tumor hypoxia, as well as vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) as angiogenesis parameters were also assessed in this study. Correlations between the expression of investigated proteins and various clinicopathological factors including survival were determined. There were 94 cases (61.8%) showing high LDH-5 expression, and 95 patients (62.5%) had high HIF-1alpha expression. Positive correlation was found between LDH-5 expression and HIF-1alpha, VEGF, and COX-2. The overexpression of LDH-5 was more prevalent in advanced tumors having positive vessel invasion. Patients with overexpression of LDH-5 showed far lower disease-free (63.5% vs 82.7%) and overall (56.3% vs 78.4%) survival rates compared with patients with low LDH-5 expression. HIF-1alpha expression was shown to have no significance on survival. In multivariate analysis, high LDH-5 expression kept its independence as a negative prognostic indicator. The results of the current study show that LDH-5 expression may be a useful prognostic factor for patients with gastric carcinoma.

The Expression of Hypoxia-Inducible Factor-1α in Hepatitis B Virus-Related Hepatocellular Carcinoma: Correlation with Patients’ Prognosis and Hepatitis B Virus X Protein

Hypoxia inducible factor-1alpha (HIF-1alpha) was well correlated with carcinogenesis and tumor progression in many kinds of cancer. In this study, high expression of HIF-1alpha was found in 37 of the 72 (51.39%) tumor specimens, and significantly correlated with venous invasion and lymphonode invasion. Patients with high expression of HIF-1alpha had a significantly shorter overall survival rate and disease-free survival rate than those with low expression. Multivariate analysis showed high HIF-1alpha expression was a borderline independent factor of overall survival. HIF-1alpha expression was also found to be significantly correlated with the expression of hepatitis B virus X protein (HBx), and over-expressed HBx upregulated HIF-1alpha protein expression in vitro. These results suggested that HIF-1alpha, which was partially regulated by HBx, might be a prognostic marker of HBV-related HCC patients.

Immunohistochemical detection of osteopontin in advanced head-and-neck cancer: Prognostic role and correlation with oxygen electrode measurements, hypoxia-inducible-factor-1α-related markers, and hemoglobin levels

The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma marker of tumor hypoxia. However, the association of immunohistochemical OPN expression in tumor sections with tumor oxygenation parameters (HF5, median pO(2)), the hypoxia-related markers hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase IX (CAIX), or hemoglobin and systemic vascular endothelial growth factor (VEGF) levels has not been investigated. Tumor tissue sections of 34 patients with advanced head-and-neck cancer treated with radiotherapy were assessed by immunochemistry for the expression of OPN, HIF-1alpha, and CA IX. Relationship of OPN expression with tumor oxygenation parameters (HF5, median pO(2)), HIF-1alpha and CA IX expression, hemoglobin and serum VEGF level, and clinical parameters was studied. Bivariate analysis showed a significant correlation of positive OPN staining with low hemoglobin level (p = 0.02), high HIF-1alpha expression (p = 0.02), and high serum vascular endothelial growth factor level (p = 0.02) for advanced head-and-neck cancer. Furthermore, considering the 31 Stage IV patients, the median pO(2) correlated significantly with the OPN expression (p = 0.02). OPN expression alone had only a small impact on prognosis. However, in a univariate Cox proportional hazard regression model, the expression of either OPN or HIF-1alpha or CA IX was associated with a 4.1-fold increased risk of death (p = 0.02) compared with negativity of all three markers. Osteopontin expression detected immunohistochemically is associated with oxygenation parameters in advanced head-and-neck cancer. When the results of OPN, HIF-1alpha, and CA IX immunohistochemistry are combined into a hypoxic profile, a strong and statistically significant impact on overall survival is found.

Expression pattern of angiogenic factors and prognosis after hepatic resection in hepatocellular carcinoma: importance of angiopoietin‐2 and hypoxia‐induced factor‐1a

Hepatocellular carcinoma (HCC) is a hypervascular tumor and angiogenesis plays an important role in its progression. Angiogenesis is regulated by a balance between pro and antiangiogenic molecules. The aim of this study was to investigate the expressions of angiogenic factors and elucidate their roles in angiogenesis in HCC. We investigated immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), hypoxia-induced factor-1alpha (HIF-1alpha) and thrombospondin-1 (TSP-1) in 60 specimens of surgically resected HCC. We investigated the relationship between their expressions and clinicopathological factors or prognosis. Ang-2 staining had a significant correlation with the grade of differentiation of HCC cells (P=0.0082). VEGF and Ang-2 expression correlated positively with microvessel density (MVD) (P=0.0061 and 0.0374, respectively). MVD of well-differentiated HCC were significantly lower than those of moderately and poorly differentiated HCC. The disease-free survival time of patients with high Ang-2 and/or HIF-1alpha expression was significantly shorter than that of the low expression group (P=0.0278 and 0.0374, respectively). Our study showed that the expression of VEGF and Ang-2 correlated with MVD. Strong Ang-2 expression and/or high nuclear expression of HIF-1alpha is a significant predictive factor for recurrence after curative resection in HCC patients.

Hypoxia inducible factor‐1α is a prognostic marker in premenopausal patients with intermediate to highly differentiated breast cancer but not a predictive marker for tamoxifen response

Hypoxia is common in many solid tumours, including breast cancer. Hypoxia triggers the expression of hypoxia inducible factor-1alpha (HIF-1alpha), and HIF-1alpha has been associated with an impaired prognosis in breast cancer and down-regulation of the oestrogen receptor (ER), potentially affecting the treatment efficiency of antioestrogens. The role of HIF-1alpha regarding prognostic and treatment predictive information in breast cancer has not been established and we therefore analyzed HIF-1alpha using immunohistochemistry in a cohort of 377 premenopausal stage II breast cancers arranged in a tissue microarray. The patients were included in a randomized trial with either 2 years of tamoxifen or no adjuvant treatment. The tamoxifen treatment effect could be studied in subgroups of breast cancer and pure prognostic information could be scrutinized for untreated control patients. HIF-1alpha was scored as positive in 24% of the tumours and correlated positively to tumour size, Nottingham histological grade (NHG), Ki-67, Her2 and cyclin E expression and negatively to lymph node status, cyclin D1, ER and PR (progesterone receptor) expression. Surprisingly, there was no difference in tamoxifen response for patients with high or low HIF-1alpha expressing tumours. In lymph node-positive patients as well as NHG 1/2 tumours, high HIF-1alpha protein expression was significantly associated with an impaired recurrence-free survival (p=0.014, 0.018). When analyzing the subgroup of NHG 1/2 tumours, a high HIF-1alpha expression was the only independent significant prognostic marker in multivariate analysis, including standard prognostic markers, suggesting that HIF-1alpha might be a useful prognostic marker in this subgroup of breast cancer, with a rather good but diverse prognosis.

Hypoxia-inducible Factor-1α is Associated with Risk of Aggressive Behavior and Tumor Angiogenesis in Gastrointestinal Stromal Tumor

The objective of this study was to evaluate the relationship of HIF-1alpha expression and tumor angiogenesis, recurrence/distant metastasis, and its value in the prediction of aggressive behavior of gastrointestinal stromal tumor (GIST). Paraffin-embedded specimens from 62 patients with GIST were divided into two groups, low risk (n = 31) and high risk (n = 31) according to the tumor size, mitotic count and proliferating cell nuclear antigen (PCNA) index. We investigated the expression of the HIF-1alpha and analyzed correlation with tumor angiogenesis monitored by expression of vascular endothelial growth factor (VEGF) and tumor microvessel density using immunohistochemical staining. The data were analyzed with chi(2) test or Fisher's exact test and multivariate test. There were statistically significant differences between high risk (29/31; 93.5%) and low risk (8/31; 25.8%) of GIST in high expression of HIF-1alpha (P<0.0001). In addition, the incidence of recurrence/distant metastasis was significantly higher in cases of high HIF-1alpha expression (12/35; 34.3%) than in cases of low HIF-1alpha expression (1/24; 4.2%)(P = 0.009). Moreover, high VEGF expression (37/43; 86.0%) and microvessel density (30/32; 93.8%) were significantly higher in high HIF-1alpha expression tumors than in low-expression tumors (P < 0.0001). Our findings suggest that HIF-1alpha may play an important role in aggressive behavior and tumor angiogenesis in GIST. In addition, high expression of HIF-1alpha was significantly correlated with tumor recurrence/distant metastasis, so it may provide an ancillary prognostic factor for GIST.

The expression of hypoxia inducible factor-1,2 alpha in sporadic clear cell renal cell carcinoma and their relationships to the mutations of von Hippel-Lindau gene

To evaluate the expression of hypoxia inducible factor (HIF)-1alpha, 2alpha in sporadic clear cell renal cell carcinoma and their relationships to the mutations of von Hippel-Lindau (VHL) gene. Mutations of VHL gene, expression of HIF-1alpha and 2alpha were detected by polymerase chain reaction (PCR), direct DNA sequencing and immunohistochemistry in 77 cases of Chinese sporadic clear cell renal cell carcinoma (CCRCC). The stage was pT(1)N(0)M(0)in 55 patients (71%), pT(2)N(0)M(0) in 7 patients (9%), pT(3)N(0)M(0) in 14 patients (18%), and pT(4)N(0)M(0) in 1 patient (1%). The classification according to the tumor nuclear grading system showed 15 carcinomas (19%) of tumor nuclear grade 1, 56 (73%) of tumor nuclear grade 2 and 6 (8%) of tumor nuclear grade 3. None of the VHL gene mutations were found in all the normal tissue specimens. VHL gene mutations were detected in 40 (52%) cases of CCRCC. The positive rate of HIF-2alpha (81%) was higher than that of HIF-1alpha (66%) (chi(2) = 23.310, P < 0.01); The positive rate of HIF-1alpha and HIF-2alpha in the cases of mutations (98% and 93% respectively) was higher than that of them in non-mutations (32% and 68% respectively) (chi(2) = 36.386, 7.617, P < 0.01); The correlation between HIF-1alpha and VHL gene mutations was closer than that between HIF-2alpha and VHL gene mutations (partial correlation coefficiency was 4.481 and 2.027 respectively, P < 0.01). The expression of HIF-1alpha and 2alpha in different pathological grade and stage of CCRCC showed no significant difference (P > 0.05). Our study suggests that VHL gene mutations are frequent in sporadic CCRCC, and the high expression of HIF-1alpha and 2alpha are found in the group of VHL mutations. However, we have not found significant correlation between the expression of HIF-1alpha and 2alpha and pathological grade and stage of CCRCC in our study.

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-1α, HIF-2α 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-1α expression. Poorly differentiated carcinomas were associated with nuclear HIF-1α 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.

Mutation of the von Hippel-Lindau (VHL) gene in human colorectal carcinoma: association with cytoplasmic accumulation of hypoxia-inducible factor (HIF)-1alpha.

We screened for mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and examined the relationship of these mutations with expression of hypoxia-inducible factor (HIF)-1alpha protein in human colorectal carcinomas. DNAs were extracted from 130 paraffin-embedded tumor specimens and subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis followed by direct sequencing. We identified 13 mutations in the coding sequence of VHL, 12 of which were unique events. Three mutations were located in exon 2 and the others in exon 3. These mutations were detected in 10 of 88 (11.4%) tumors tested. Furthermore, seven of the 13 (53.8%) VHL mutants immunohistochemically showed high HIF-1alpha expression. The mean percentage of cells with strong cytoplasmic HIF-1alpha expression was 67.5% in tumors with VHL mutations, and this level was significantly higher than that in tumors without mutations (50.8%, P < 0.05). These findings suggest that mutations in VHL may play a role in colorectal carcinoma via activation of the HIF-related transcriptional cascade.