Carbonic Anhydrase 9 Expression Research Articles

Expression of a Hypoxia-Associated Protein, Carbonic Anhydrase-9, Correlates with Malignant Phenotypes of Gastric Carcinoma

Background/Aims: Gastric cancers are characterized by a heterogeneously hypoxic environment. Hypoxia might stimulate the malignant potential of cancer cells. The purpose of our study was to clarify the significance of hypoxia in gastric carcinoma by evaluating the expression of a hypoxic marker, namely carbonic anhydrase-9 (CA-9). Methods: A total of 265 patients who had undergone a resection of the primary tumor and were confirmed histologically to have sporadic gastric cancer were enrolled in this study. We evaluated the immunohistochemical expression of CA-9 in the paraffin-embedded specimens of 265 gastric adenocarcinomas. Results: The CA-9 expression was positive in 88 (33%) of 265 gastric carcinomas. The CA-9 expression level was significantly high in cases of type 4 carcinoma (60%, p < 0.001) and diffuse type carcinoma (41%, p < 0.001), and significantly correlated with the invasion depth (p < 0.001), lymph node metastasis (p < 0.001) and lymphatic invasion p = 0.002). The prognosis for CA-9-positive patients was significantly poorer than that of CA-9-negative patients (p = 0.003, log-rank). Conclusion: Hypoxia might be associated with aggressive tumor phenotypes of gastric carcinomas. The hypoxic marker CA-9 may be a useful prognostic indicator.

The expressions of carbonic anhydrase 9 and vascular endothelial growth factor in astrocytic tumors predict a poor prognosis

Hypoxia in the tumor microenvironment triggers a variety of genetic and adoptive responses that regulate tumor growth. Tumor hypoxia is often associated with a malignant phenotype, resistance to therapy, and poor survival. The objectives of this study were to evaluate the expressions of carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) in astrocytic gliomas and to relate patterns of expression with prognosis, that is with histological grade and survival. We investigated 78 World Health Organization (WHO) grade II, III, and IV astrocytic gliomas. CA9 expression was examined in paraffin-embedded sections by immunohistochemistry. Fourteen tumors were grade II, 30 were grade III, and 34 were grade IV. It was found that CA9 expression was significantly associated with a higher-grade histology (p<0.001). There were 3 CA9 positive tumors in grade II (21.4%), 10 in grade III (33.3%), and 27 in grade IV (79.4%). For all tumors and WHO grade II, overall survival was found to be significantly dependent on CA9 expression (p=0.004, p=0.01). Furthermore, VEGF expression was found to be significantly related to tumor grade (p=0.02) and tended to be related to overall survival (p=0.1). However, no relation was found between the expression of CA9 and VEGF (p=0.17). Nevertheless, the expressions of CA9 and VEGF were found to be associated with tumor grade and possibly with survival. Further studies on a larger patient population are needed to determine the correlation between the expressions of CA9, and VEGF in astrocytic gliomas and clinical outcome.

Role of the HBx oncoprotein in carbonic anhydrase 9 induction

Carbonic anhydrase 9 (CA9), as one of the most hypoxia-responsive genes, has been associated almost exclusively with hypoxic tumors. Its principal role is in pH regulation which helps tumor cells overcome intracellular acidosis and survive extended periods of time with low oxygen. Hypoxia-inducible factor 1 (HIF-1) is the main transcriptional activator of CA9. Hepatitis B virus X protein (HBx) has been shown to increase the transcriptional activity of HIF-1. HBx is often expressed from the gene integrated in the hepatocytes infected persistently and contributes significantly to alterations in host gene expression that can lead to the development of hepatocellular carcinoma (HCC) associated with Hepatitis B virus (HBV). The aim of this study was to determine the effect of HBx on expression of CA9. Transient transfection of HBx led to an increase in the expression of CA9 as assessed by RT-PCR and Western blotting. HBx was able to increase CA9 promoter activity significantly in several cell lines. The effect was mediated via HIF-1 and a functional HRE element located -10/-3 bp upstream of the CA9 transcription initiation site. These data suggest that CA9 may be involved in the development of HCC by contributing to the survival of hepatocytes infected with HBV in liver tissue with fibrosis.

VHL inactivation is an important pathway for the development of malignant sporadic pancreatic endocrine tumors

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel-Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1alpha (HIF1-alpha), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-alpha downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1-alpha (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

The Role of Carbonic Anhydrase 9 in Regulating Extracellular and Intracellular pH in Three-dimensional Tumor Cell Growths

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of approximately 300 microm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH(i)) and pH in the restricted extracellular spaces (pH(e)). With no CA9 expression, spheroids developed very low pH(i) (approximately 6.3) and reduced pH(e) (approximately 6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH(i) = approximately 6.6), whereas extracellular acidity was enhanced (pH(e) = approximately 6.6), so that radial pH(i) gradients were smaller and radial pH(e) gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH(e) indicates the enzyme is facilitating CO(2) excretion from cells (by converting vented CO(2) to extracellular H(+)), rather than facilitating membrane H(+) transport (such as H(+) associated with metabolically generated lactic acid). This latter process requires titration of exported H(+) ions with extracellular HCO(3)(-), which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH(e) will depend on the cellular CO(2)/lactic acid emission ratio (set by local oxygenation and membrane HCO(3)(-) uptake). Our results suggest that CO(2)-producing tumors may express CA9 to facilitate CO(2) excretion, thus raising pH(i) and reducing pH(e), which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

Pancreatic Endocrine Microadenomatosis in Patients With von Hippel-Lindau Disease

Von Hippel-Lindau (VHL) disease is an inherited syndrome caused by germline mutation in the VHL tumor suppressor gene predisposing to pancreatic endocrine tumors (PET). Whether these tumors derive from preexisting endocrine microadenomatosis as in multiple endocrine neoplasia type 1 (MEN1) is yet unknown. pVHL regulates hypoxia-inducible factor (HIF) that causes transcription activity of target genes like carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), and cyclin D1. Our aim was to look for overexpression of these molecules to identify precursor endocrine lesions in the pancreas of VHL patients. Nontumoral pancreas of 18 VHL patients operated on for PET, was examined for microadenomatosis (<or=5 mm) and compared with pancreatic specimen obtained from non-VHL patients or MEN1 patients. The immunohistochemical expression of chromogranin, insulin, glucagon, HIF-1alpha, HIF-2alpha, VEGF, CA9, cyclin D1, and CD34 was assessed. In addition to 39 macrotumors (1 to 5/patient), chromogranin-positive endocrine microadenomas were found in 13 (72%) patients located within acini or close to ducts or islets. Strong coexpression of HIF-1alpha, cyclin D1, CA9, and VEGF and lack of expression of insulin and glucagon allowed distinction with normal or hyperplastic islets. CD34 identified a high microvessel density in these nodules. Expression of HIF-1alpha and CA9 was not found in islets of controls and in MEN1 microadenomas. Pancreatic endocrine microadenomas are present in >70% of VHL patients operated on for PET. These results demonstrate that the pVHL/HIF pathway is involved very early in pancreatic endocrine tumorigenesis in this disease.

An Exploratory Study Into the Role of Dynamic Contrast-Enhanced Magnetic Resonance Imaging or Perfusion Computed Tomography for Detection of Intratumoral Hypoxia in Head-and-Neck Cancer

Hypoxia in patients with head-and-neck cancer (HNC) is well established and known to cause radiation resistance and treatment failure in the management of HNC. This study examines the role of parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and perfusion computed tomography (CT) as surrogate markers of intratumoral hypoxia, defined by using the exogenous marker of hypoxia pimonidazole and the endogenous marker carbonic anhydrase 9 (CA9). Patients with HNC underwent preoperative DCE-MRI, perfusion CT, and pimonidazole infusion. Imaging parameters were correlated with pimonidazole and CA9 staining. The strength of correlations was tested by using a two-tailed Spearman's rank correlation coefficient. Twenty-three regions of interest were analyzed from the 7 patients who completed the DCE-MRI studies. A number of statistically significant correlations were seen between DCE-MRI parameters (volume transfer between blood plasma and extracellular extravascular space [EES], volume of EES, rate constant between EES and blood plasma, time at arrival of contrast inflow, time to peak, average gradient, and time to onset) and areas with a pimonidazole score of 4. In the case of CA9 staining, only a weak correlation was shown with wash-in rate. There were no significant correlations between perfusion CT parameters and pimonidazole staining or CA9 expression. Intratumoral hypoxia in patients with HNC may be predicted by using DCE-MRI; however, perfusion CT requires further investigation.

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

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

Expression of carbonic anhydrase 9, a potential intrinsic marker of hypoxia, is associated with poor prognosis in oesophageal squamous cell carcinoma

Carbonic anhydrase 9 (CA9) is a protein to be upregulated under exposure to hypoxic conditions. Hypoxic conditions are known to be associated with resistance to chemotherapy and radiotherapy, and with poor cancer prognosis. We examined CA9 expression in surgical specimens from oesophageal squamous cell carcinoma (ESCC) patients (n=127) using immunohistochemistry and real-time RT–PCR. We also examined CA9 expression and cell proliferation in ESCC cell lines (TE-2, TE-8 and TE-15) and an immortalised human oesophageal cell line (CHEK-1) using real-time RT–PCR, Western blotting, ELISA and MTT assay. Immunohistochemistry, high expression of CA9 was found in 63 of the 127 primary tumour specimens and was correlated with poor outcome (P=0.0003) and more aggressive/less favourable clinicopathological parameters (tumour size (P=0.0235), tumour depth (P<0.0001), regional lymph node metastasis (P=0.0031), distant lymph node metastasis (P=0.0077), stage (P<0.0001) and blood vessel invasion (P=0.006)). In vitro, CA9 expression in cultured cells and culture medium was also induced by hypoxia (P<0.01). CA9 is correlated with poor prognosis and malignant phenotype in patients with ESCC, and was upregulated by hypoxia. It is suggested that control of CA9 expression might improve the effectiveness of chemotherapy and radiotherapy in ESCC.

Tumor Angiogenic and Hypoxic Profiles Predict Radiographic Response and Survival in Malignant Astrocytoma Patients Treated With Bevacizumab and Irinotecan

The combination of a vascular endothelial growth factor (VEGF) -neutralizing antibody, bevacizumab, and irinotecan is associated with high radiographic response rates and improved survival outcomes in patients with recurrent malignant gliomas. The aim of these retrospective studies was to evaluate tumor vascularity and expression of components of the VEGF pathway and hypoxic responses as predictive markers for radiographic response and survival benefit from the bevacizumab and irinotecan therapy. In a phase II trial, 60 patients with recurrent malignant astrocytomas were treated with bevacizumab and irinotecan. Tumor specimens collected at the time of diagnosis were available for further pathologic studies in 45 patients (75%). VEGF, VEGF receptor-2, CD31, hypoxia-inducible carbonic anhydrase 9 (CA9), and hypoxia-inducible factor-2alpha were semiquantitatively assessed by immunohistochemistry. Radiographic response and survival outcomes were correlated with these angiogenic and hypoxic markers. Of 45 patients, 27 patients had glioblastoma multiforme, and 18 patients had anaplastic astrocytoma. Twenty-six patients (58%) had at least partial radiographic response. High VEGF expression was associated with increased likelihood of radiographic response (P = .024) but not survival benefit. Survival analysis revealed that high CA9 expression was associated with poor survival outcome (P = .016). In this patient cohort, tumor expression levels of VEGF, the molecular target of bevacizumab, were associated with radiographic response, and the upstream promoter of angiogenesis, hypoxia, determined survival outcome, as measured from treatment initiation. Validation in a larger clinical trial is warranted.

Hypoxia inducible factor (HIF1α and HIF2α) and carbonic anhydrase 9 (CA9) expression and response of head-neck cancer to hypofractionated and accelerated radiotherapy

Purpose: Tumor hypoxia and low intrinsic radiosensitivity may counteract the efficacy of standard radiotherapy for locally advanced head and neck cancer (HNC). We investigated the involvement of hypoxia-regulated proteins (Hypoxia inducible factors HIF1α, HIF2α and carbonic anhydrase CA9) in HNC resistance to accelerated and hypofractionated radiotherapy.Materials and methods: Thirty-nine patients with locally advanced HNC received 15 daily fractions of 3.4 Gy amounting to a total tumor dose of 51 Gy (equivalent to 63 Gy in four weeks – one week split); this was combined with platinum chemotherapy and amifostine cytoprotection administered subcutaneously. Immunohistochemical analysis of hypoxia-regulated proteins, namely HIF1α, HIF2α and CA9, was performed in formalin-fixed paraffin-embedded tissues obtained prior to radio-chemotherapy.Results: HIF1α and HIF2α were expressed in the nuclei and cytoplasm of cancer cells, while CA9 had a membrane reactivity. A high expression of HIF1α, HIF2α and CA9 was noted in 21/39 (53.8%), 20/39 (51.3%) and 23/39 (58.9%) cases, respectively. Complete response was obtained in 85.2% of patients and HIF1α was marginally related with persistent disease after RT (p = 0.05). HIF1α was significantly associated with poor local relapse free survival (LRFS) (p = 0.006) and overall survival (p = 0.008), whilst HIF2α was not. A significant association of CA9 expression with poor LRFS was noted (p = 0.01).Conclusion: In accord with previously reported studies, high levels of the hypoxia regulated proteins HIF1α and CA9 in HNC predict resistance to platinum based radio-chemotherapy. Whether HIF2α expressing tumors are more sensitive to larger radiotherapy fractions, compared to standard radiotherapy fractionation, is an issue that deserves further investigation.

Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells

BackgroundHypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells.MethodsOptimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O2, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h).ResultsChetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 ± 7.2% and 39.6 ± 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.ConclusionHIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

Carbonic anhydrase 9 (CA9) expression in tumor cells enhances sensitivity to tirapazamine

Carbonic anhydrase 9 (CA9) is over-expressed in many human solid tumors under conditions of low oxygen concentration and can be associated with a low probability of survival. In this study, stable CA9-expressing cell lines were established using the CA9 gene-defective human C33a cell line and the HeLa cell line to investigate the role of CA9 in response to ionizing radiation and hypoxia-selective cytotoxin, Tirapazamine (TPZ). Human CA9 cDNA or an empty vector was transfected into the C33a and HeLa cell lines and C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines were produced accordingly. Sensitivity of the C33a-vector/C33a-CA9 cells to ionizing radiation and TPZ was measured using clonogenic assays. The alkaline comet assay was used to measure single strand DNA breaks caused by TPZ in the C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines. Radiation sensitivity, as determined with clonogenic survival assays, of C33a-vector/C33a-CA9 cells did not differ under either normoxic or hypoxic conditions. However, increased clonogenic sensitivity to TPZ was observed in C33a-CA9 cells under the hypoxic condition by 26% (95% CI 14-39%, P = 0.02 in comparison to the C33a-vector cells). The comet assay showed significantly greater DNA damage in the C33a-CA9 cells compared with that of the C33a-vector cells with the same treatment under hypoxic conditions, supporting the results of the clonogenic survival data. Because this difference in the amount of DNA damage was not observed for the hypoxic HeLa-CA9/HeLa-vector cell lines, both of which have induced CA9 expression by hypoxia, the enhanced sensitivity of C33a-CA9 cells to TPZ is considered to be due to the specific condition of CA9 over-expression. Our results suggest the possibility that CA9 over-expression in tumors might be exploited to increase the treatment effects of TPZ.

Expression of the Hypoxia Marker Carbonic Anhydrase 9 Is Associated with Anaplastic Phenotypes in Meningiomas

Purpose: Hypoxia in the tumor microenvironment triggers a variety of genetic and adaptive responses that regulate tumor growth. Tumor hypoxia is often associated with more malignant phenotypes, resistance to therapy, and poor survival. The purpose of this study was to evaluate the prevalence of hypoxia in meningiomas using the endogenous hypoxia marker carbonic anhydrase 9 (CA9) and to relate the expression of CA9 to tumor vascularity, histopathologic grade, and clinical variables, such as recurrent tumor status. Experimental Design: Expression of CA9 and CD34, an endothelial cell marker, was examined in serial paraffin-embedded sections by immunohistochemistry in 25 grade 1, 17 grade 2, and 20 grade 3 meningiomas. Areas of immunoreactivity were semiquantitatively scored and correlated to clinical variables using Statistical Analysis System statistical software. Results: Approximately 50% (29 of 62) of all meningiomas contained regions of hypoxia as judged by expression of CA9, and this expression was significantly associated with higher-grade histology ( P = 0.001). In contrast, vascularity, as assessed by the percentage of vascular hotspots, was inversely associated with tumor grade ( P = 0.023) and was not associated with CA9 expression. Among lower-grade meningiomas, CA9 expression tended to be more common in recurrent tumors. Conclusions: Tumor hypoxia is an endogenous feature of meningiomas, and therapeutic regimens should include strategies to target the subpopulation of hypoxic as well as the normoxic cells within the tumor. Hypoxia in meningiomas is associated with an aggressive phenotype. Further studies to define the contribution of hypoxia to meningioma pathophysiology are warranted.

Expression of the Hypoxia Marker Carbonic Anhydrase 9 Is Associated with Anaplastic Phenotypes in Meningiomas

Hypoxia in the tumor microenvironment triggers a variety of genetic and adaptive responses that regulate tumor growth. Tumor hypoxia is often associated with more malignant phenotypes, resistance to therapy, and poor survival. The purpose of this study was to evaluate the prevalence of hypoxia in meningiomas using the endogenous hypoxia marker carbonic anhydrase 9 (CA9) and to relate the expression of CA9 to tumor vascularity, histopathologic grade, and clinical variables, such as recurrent tumor status. Expression of CA9 and CD34, an endothelial cell marker, was examined in serial paraffin-embedded sections by immunohistochemistry in 25 grade 1, 17 grade 2, and 20 grade 3 meningiomas. Areas of immunoreactivity were semiquantitatively scored and correlated to clinical variables using Statistical Analysis System statistical software. Approximately 50% (29 of 62) of all meningiomas contained regions of hypoxia as judged by expression of CA9, and this expression was significantly associated with higher-grade histology (P = 0.001). In contrast, vascularity, as assessed by the percentage of vascular hotspots, was inversely associated with tumor grade (P = 0.023) and was not associated with CA9 expression. Among lower-grade meningiomas, CA9 expression tended to be more common in recurrent tumors. Tumor hypoxia is an endogenous feature of meningiomas, and therapeutic regimens should include strategies to target the subpopulation of hypoxic as well as the normoxic cells within the tumor. Hypoxia in meningiomas is associated with an aggressive phenotype. Further studies to define the contribution of hypoxia to meningioma pathophysiology are warranted.

Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas

Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1) in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis.

Detection of carbonic anhydrase-9 gene expression in peripheral blood cells predicts risk of disease recurrence in patients with renal cortical tumors

Objectives To present extended follow-up on a cohort of patients with renal cortical tumors treated with partial or radical nephrectomy and preoperatively assess for carbonic anhydrase 9 tumor marker expression in the peripheral blood. Methods All patients were originally enrolled in an institutional review board-approved study assessing the role of a reverse-transcriptase polymerase chain reaction peripheral blood assay designed to detect the tumor-specific gene carbonic anhydrase-9 (CA9). A total of 41 patients with renal cortical tumors confined to the kidney were enrolled at a single institution and assessed preoperatively with peripheral blood test for CA9 expression before undergoing partial or radical nephrectomy. A Kaplan-Meier estimated survival analysis and log-rank test were performed to determine whether detection of peripheral blood cells expressing the CA9 gene influences disease-free and disease-specific survival. Results The median follow-up was 4.3 years. The median age was 71 years. Of the 41 patients, 26 were men and 15 were women. The estimated 5-year disease-free survival for patients with detectible expression of the CA9 gene in the peripheral blood was 39.5% compared with 88.1% for patients without detection of the CA9 gene ( P = 0.048). On bivariate analysis, disease-free survival correlated with histologic type, tumor diameter, and tumor grade. Conclusions The expression of the tumor-specific marker CA9 in the peripheral blood is associated with decreased disease-free survival in patients with renal cortical tumors. This is the first study reporting on the prognostic value of this peripheral blood-based tumor marker for kidney tumors.

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-1α (Hif-1α) 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-1α expression were correlated (primary tumours P=0.005; lymph node metastases P<0.001). In primary tumours, CA9 and Hif-1α 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-1α 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-1α expression (ECP% r=0.51, P<0.001; TCP r=0.77, P<0.001; CA9 and Hif-1α 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.

DEC1 expression in 1p-aberrant oligodendroglial neoplasms.

Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.

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

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