Carbonic anhydrase IX expression is associated with tumor progression and a poor prognosis of lung adenocarcinoma

Carbonic anhydrase (CA) IX catalyzes the hydration of carbon dioxide into carbonic acid and participates in a variety of physiological and biological processes. The aim of this study was to evaluate the prognostic significance of CA IX expression in patients with lung adenocarcinoma. Standard immunohistochemical techniques were used to study CA IX expression in 134 patients who underwent curative resection for adenocarcinoma of the lung at our hospital between January 1995 and December 1996. We evaluated the correlations between CA IX expression levels on cancer cells and clinicopathological factors. CA IX expression was not observed in normal lung tissue or specimens from non-invasive adenocarcinomas. CA IX immunostaining was detected in 33 (24.6%) invasive adenocarcinoma cases. Poor differentiated histological phenotype (p=0.0015), pathological stage (p=0.0400), vascular invasion (p=0.0009) and lymphatic permeation (p=0.0050) were significantly related to CA IX expression. On univariate analysis, CA IX positive cases showed significantly shorter overall survival (p=0.0083) and disease-free survival (p=0.0122). In particular, the overall and disease-free survivals in stages I+II were significantly shorter in the CA IX positive than in the CA IX negative cases (p=0.0269 and 0.0011, respectively). Our results suggest that CA IX expression is strongly associated with tumor progression and indicates a poor prognosis for patients with stages I+II lung adenocarcinoma.     

Carbonic anhydrase IX as a marker for poor prognosis in soft tissue sarcoma.

PURPOSE: Hypoxia is associated with malignant progression and poor outcome in several human tumors, including soft tissue sarcoma. Recent studies have suggested that carbonic anhydrase (CA) IX is an intrinsic marker of hypoxia, and that CA IX correlates with poor prognosis in several types of carcinoma. The aim of this study was to quantify the extent of CA IX expression and to investigate whether CA IX is a marker for poor prognosis in soft tissue sarcoma patients at high risk of developing metastasis. EXPERIMENTAL DESIGN: Archival paraffin-embedded blocks were retrieved from 47 patients with deep, large, high-grade soft tissue sarcoma. Sections from two separate and representative tumor areas were immunostained for CA IX, and the CA IX-positive area fraction was quantified by image analysis, excluding areas of normal stroma and necrosis that were identified from serial H&E-stained sections. Patients were then subject to survival analysis. RESULTS: CA IX-positive area fractions of viable tumor tissue varied significantly between tumors (range, 0-0.23; median, 0.004), with positive membranous CA IX staining in 66% (31 of 47) of the tumors. Patients with CA IX-positive tumors had a significantly lower disease-specific and overall survival than patients with CA IX-negative tumors (P = 0.033 and P = 0.044, respectively). CONCLUSIONS: These data suggest that CA IX, a potential intrinsic marker of hypoxia, predicts for poor prognosis in patients with deep, large, high-grade soft tissue sarcoma. Larger studies are required to determine whether CA IX has independent prognostic value in this group of tumors.     

Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas.

PURPOSE: Hypoxia is associated with adverse outcome for a number of solid tumors, including cervical carcinomas. Direct pO(2) measurement requires specialized equipment and expertise that is not generally available. Immunohistochemical measurement of intrinsic tissue markers of hypoxia is an alternative approach. Recent studies suggest that carbonic anhydrase IX (CA IX), which is regulated via hypoxia-inducible factor 1, is a useful intrinsic marker of tumor hypoxia. EXPERIMENTAL DESIGN: Biopsies were obtained from 110 patients with locally advanced cervical carcinoma treated with radiotherapy or chemoradiotherapy. Tissue sections were labeled using an immunofluorescence technique and CA IX expression in the viable tumor area measured using a semiautomated fluorescence image analysis technique. Results were compared with direct pO(2) values obtained using an Eppendorf probe and to patient outcome. Intratumoral heterogeneity of CA IX expression was examined in a subgroup of patient who underwent multiple biopsies. RESULTS: The median percentage of tumor area staining for CA IX was 3.56 (range, 0.01-58.85). CA IX staining did not correlate with the Eppendorf pO(2) measurements. Whereas the latter values were predictive of patient outcome, the CA IX levels were not. Measurement of CA IX in multiple biopsies indicated that intratumoral heterogeneity accounted for 41% of the total variance in the data set. CONCLUSIONS: In contrast to some recent studies, we did not find significant associations between CA IX expression and tumor pO(2) levels or patient outcome in locally advanced carcinomas of the cervix. Probable explanations relate to the problems of sampling error using single biopsies and the existence of biological factors other than hypoxia that influence CA IX levels.     

Carbonic anhydrase IX in early-stage non-small cell lung cancer.

PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might allow tumors to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation. We hypothesized that CA IX expression is related to tumor cell proliferation and poor disease-free survival in patients with early-stage non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: CA IX expression was measured in 75 resected NSCLC tumors to assess prognostic implications for disease-free survival. The relationship of CA IX expression with microvessel density (MVD) and proliferation (Ki-67) index was assessed via colocalization analysis. RESULTS: All patients had operable NSCLC (stage I, 58; stage II, 17). CA IX expression was present in 54 (72%) of 75 patients and was associated with tumor necrosis (P < 0.05). CA IX-positive tumor areas showed greater cell proliferation as measured by Ki-67 index (P < 0.05) and less MVD (P < 0.05) than did CA IX-negative areas in colocalization analysis. The percentage of CA IX-positive tumor cells was significantly related to postoperative recurrence and poor disease-free survival (P < 0.05). Ki-67 index and pathologic stage were also independent prognostic factors for worse disease-free survival (P < 0.05). CONCLUSIONS: CA IX expression of tumor cells may be an indicator for poor disease-free survival in early-stage NSCLC.     

Hypoxia-regulated carbonic anhydrase IX expression is associated with poor survival in patients with invasive breast cancer

Tumour hypoxia is a microenvironmental factor related to poor response to radiation, chemotherapy, genetic instability, selection for resistance to apoptosis, and increased risk of invasion and metastasis. Hypoxia-regulated carbonic anhydrase IX (CA IX) has been studied in various tumour sites and its expression has been correlated with the clinical outcome. The purpose of this study was to investigate the correlation of CA IX expression with outcome in patients with invasive breast cancer. We conducted a retrospective study examining the effects of carbonic anhydrase IX (CA IX) on survival in patients with breast cancer. To facilitate the screening of multiple tissue blocks from each patient, tissue microarrays were prepared containing between two and five representative samples of tumour per patient. Immunohistochemistry was used to examine expression of CA IX in patients with breast cancer. The study includes a cohort of 144 unselected patients with early invasive breast cancer who underwent surgery, and had CA IX expression and follow-up data available for analysis. At the time of analysis, there were 28 deaths and median follow-up of 48 months with 96% of patients having at least 2 years of follow-up. CA IX was negative for 107 patients (17 deaths) and positive for 37 patients (11 deaths). Kaplan-Meier survival curves show that survival was superior in the CA IX-negative group with a 2-year survival of 97% for negatives and 83% for positives (log-rank test P=0.01). Allowing for potential prognostic variables in a Cox regression analysis, CA IX remained a significant independent predictor of survival (P=0.035). This study showed in both univariate and multivariate analysis that survival is significantly inferior in patients with tumour expressing CA IX. Prospective studies are underway to investigate this correlation in clinical trial setting.     

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.     

Low Wilms' tumor gene expression in tumor tissues predicts poor prognosis in patients with non-small-cell lung cancer

We elucidated the relationship between prognosis of non-small-cell lung cancer (NSCLC) and Wilms' tumor gene (WT1) mRNA expression in tumor tissue. The WT1 mRNA expression levels of the fatal cases were lower as compared with those of the survival cases. Overall survival (OS) and disease-free survival (DFS) of the high WT1 expression group were longer than of the low expression group. As for squamous cell lung cancer (SQLC), low WT1 expression was significantly associated with lymph node metastasis. Cox analysis revealed that the gene level was a significant prognostic factor in OS and DFS. Low WT1 expression predicted poor prognosis in patients with NSCLC.     

c-erbB-2 expression in small-cell lung cancer is associated with poor prognosis

Small-cell lung cancer (SCLC) carries a bad prognosis despite good initial response to chemotherapy. It is therefore important to identify molecular markers that influence survival as potential new therapeutic targets. In our study, expression of the tyrosine kinase c-erbB-2 (HER2/neu) receptor in tumor tissues of 107 consecutive newly diagnosed patients with primary SCLC was quantified using a monoclonal antibody directed against the c-terminal domain of c-erbB-2. A clear-cut positive expression of c-erbB-2 was observed in 13% of patients. Surprisingly, c-erbB-2 was an independent prognostic factor (RR = 2.16; p = 0.014) when a proportional-hazard model was adjusted to stage (limited vs. extensive disease) and performance status (WHO I-IV), the most relevant clinical parameters. Similarly, a significant association between c-erbB-2 and survival was obtained if a larger number of clinical parameters were included into the analysis, namely response to chemotherapy, TNM stage, lactate dehydrogenase (LDH), neuron-specific enolase (NSE), gender and age (p = 0.033). Interestingly, c-erbB-2 expression was more relevant for patients with advanced tumors. In the subgroup of patients with bad performance status (WHO II-IV), median survival of patients with undetectable c-erbB-2 expression was 274 days compared with only 23 days for patients with clear-cut positive c-erbB-2 immunohistochemistry (p = 0.0031; log-rank test). Similar results were obtained for patients with extensive disease (p = 0.028) and high TNM stages (T>2 or N>1 or M1; p < 0.068, all comparisons). In contrast, c-erbB-2 expression was not associated with survival in patients with limited disease (p = 0.97), low TNM stages (p > 0.56, all comparisons) and good performance status (p = 0.97). In conclusion, c-erbB-2 is expressed in more than 10% of SCLC. Expression of c-erbB-2 is an independent prognostic factor of survival. The effect of c-erbB-2 expression seems to become more important in advanced stages of the disease. Since c-erbB-2 is a therapeutical target in other types of cancer, further studies to identify the role of c-erbB-2 in SCLC are clearly warranted.     

MCAM expression is associated with poor prognosis in non-small cell lung cancer

Background

MCAM has been recently identified as a biomarker for epithelial–mesenchymal transition (EMT) and is potentially involved in metastasis of cancer. The current study aimed at investigating the expression of MCAM in non-small-cell lung cancer (NSCLC) and its clinico-pathological significance.

Methods

A follow-up analysis was performed on 118 patients with NSCLC resected by lobectomy or pneumectomy with systematic lymph node dissection. All patients were followed for 6–60 months. Immunostaining of tissue sections from primary tumors and their lymph node metastasis was performed and evaluated using monoclonal antibody against MCAM, E-cadherin, and vimentin. Correlations were investigated between MCAM immunostaining in primary tumors and E-cadherin, vimentin immunostaining, lymph node metastasis, and survival.

Results

MCAM protein expression was found in 46.61 % of squamous cell carcinomas and 37.47 % of adenocarcinomas; MCAM expression positively correlated with vimentin, but inversely with E-cadherin (both P values <0.05). There were significant correlations between the MCAM immunostaining score in primary tumors and in their lymph node metastasis (P = 0.03). According to the Kaplan–Meier survival estimate, the level of MCAM expression in primary tumors was a statistically significant prognostic factor (P < 0.05).

Conclusions

MCAM expression in surgically treated NSCLC is clearly associated with lymph node metastasis and poor prognosis.     

Carbonic anhydrase IX, a marker of hypoxia: correlation with clinical outcome in transitional cell carcinoma of the bladder

The purpose of this study was to investigate the effects of expression of CA IX, a marker of hypoxia, on outcome in bladder cancer. Immunohistochemistry was used to examine expression of CA IX in archival tissues of patients included in various therapeutic trials for transitional cell carcinoma (TCC) of the bladder. Data were collected on 57 patients who first presented with either invasive or superficial bladder cancer. Median follow-up for the 19 alive patients was 110 months (range 12 months to 14 years and 7 months). Median survival was 48 months (95% CI 35-110 months). Median survivals based on CA IX expression were as follows: there was a non-significant trend towards shorter survival for tumour expressing CA IX weakly (median 45 vs. 70 months, p=0.21). The hazard ratio for this variable is 1.5 (95% CI 0.8-3.0). Significantly more superficial bladder cancers than invasive cancers strongly expressed CA IX (82% vs. 52%; p=0.03). For non-invasive bladder cancer (n=28), CA IX positive patients had a median survival of 111 months while the median has not yet been achieved for CA IX negative patients (p=0.9). For invasive bladder cancer (n=29), CA IX positive patients had a median survival of 18 months as compared to 26 months for CA IX negative patients (p=0.94). There was a trend towards longer survival for tumour expressing CA IX strongly. This is likely to be a reflection of the significantly higher rate of strong CA IX expression in non-invasive cancers influencing these survival data.     

Coexpression of epidermal growth factor receptor with related factors is associated with a poor prognosis in non-small-cell lung cancer

The epidermal growth factor receptor (EGFR) is commonly expressed in non-small-cell lung cancer (NSCLC) and promotes a host of mechanisms involved in tumorigenesis. However, EGFR expression does not reliably predict prognosis or response to EGFR-targeted therapies. The data from two previous studies of a series of 181 consecutive surgically resected stage I-IIIA NSCLC patients who had survived in excess of 60 days were explored. Of these patients, tissue was available for evaluation of EGFR in 179 patients, carbonic anhydrase (CA) IX in 177 patients and matrix metalloproteinase-9 (MMP-9) in 169 patients. We have previously reported an association between EGFR expression and MMP-9 expression. We have also reported that MMP-9 (P=0.001) and perinuclear (p)CA IX (P=0.03) but not EGFR expression were associated with a poor prognosis. Perinuclear CA IX expression was also associated with EGFR expression (P<0.001). Multivariate analysis demonstrated that coexpression of MMP-9 with EGFR conferred a worse prognosis than the expression of MMP-9 alone (P<0.001) and coexpression of EGFR and pCA IX conferred a worse prognosis than pCA IX alone (P=0.05). A model was then developed where the study population was divided into three groups: group 1 had expression of EGFR without coexpression of MMP-9 or pCA IX (number=21); group 2 had no expression of EGFR (number=75); and group 3 had coexpression of EGFR with pCA IX or MMP-9 or both (number=70). Group 3 had a worse prognosis than either groups 1 or 2 (P=0.0003 and 0.027, respectively) and group 1 had a better prognosis than group 2 (P=0.036). These data identify two cohorts of EGFR-positive patients with diametrically opposite prognoses. The group expressing either EGFR and or both MMP-9 and pCA IX may identify a group of patients with activated EGFR, which is of clinical relevance with the advent of EGFR-targeted therapies.     

Elevated RABEX-5 expression predicts poor prognosis in non-small-cell lung cancer

RABEX-5 has been studied in various solid tumors, but its role in non-small-cell lung cancer (NSCLC) remains unknown. This study is aimed to investigate the expression, the potential relevance to clinicopathological characters and prognostic significance of RABEX-5 in patients with NSCLC. A total of 120 NSCLC patients who underwent radical surgery between 2005 and 2010 were enrolled in the study. The clinicopathological data and survival time were reviewed. The mRNA and protein expression of RABEX-5 from the paired tumor specimens and adjacent normal tissues were determined, and its relationship with clinicopathological variables and prognosis was analyzed. Univariate and multivariate analyses were performed to investigate the prognostic significance of RABEX-5 for NSCLC. We found the mRNA and protein expression levels of RABEX-5 were significantly elevated in NSCLC tissues. The increased RABEX-5 expression was correlated strongly with tumor recurrence (P=0.005). The 5-year median OS and DFS were significantly shorter in the higher RABEX-5 expression group compared to that in the lower RABEX-5 expression group. Multivariate Cox analysis indicated that high RABEX-5 expression was an independent prognostic factor for OS and DFS (P<0.001). This data suggests that RABEX-5 is a potentially useful indicator for a poor prognosis for NSCLC.     

Carbonic anhydrase XII is a marker of good prognosis in invasive breast carcinoma

Hypoxia and pH influence gene expression in tumours, and it is becoming increasingly clear that the pattern of genes expressed by a tumour determines its growth and survival characteristics. Hypoxia-inducible factor-1 (HIF-1) is a key mediator of the cellular response to hypoxia and high HIF-1 expression has been identified as a poor prognostic factor in tumours. Recently, we identified the tumour-associated carbonic anhydrases (CA), CA9 and CA12 as hypoxia-inducible in tumour cell lines. Furthermore, we identified CA IX to be a poor prognostic factor in breast cancer. The aim of this study was to assess the prognostic significance of CA XII. CA XII expression was studied by immunohistochemistry in a series of 103 cases of invasive breast cancer and any association with recognised prognostic factors or relation with the outcome was examined. CA XII expression was present in 77 out of 103 (75%) cases and was associated with lower grade (P=0.001), positive estrogen receptor status (P<0.001), and negative epidermal growth factor receptor status (P<0.001). Furthermore, although CA XII expression was associated with an absence of necrosis (P<0.001), expression of CA XII in some high-grade tumours was induced in regions directly adjacent to morphological necrosis. Additionally, using univariate analysis, CA XII positive tumours were associated with a lower relapse rate (P=0.04) and a better overall survival (P=0.01). In conclusion, CA XII expression is influenced both by factors related to differentiation and hypoxia in breast cancer in vivo and CA XII expression is associated with a better prognosis in an unselected series of invasive breast carcinoma patients.     

Carbonic anhydrase IX is highly expressed in hereditary nonpolyposis colorectal cancer.

Carbonic anhydrase (CA) II, CA IX, and CA XII are expressed in various neoplasias and have been linked to tumorigenesis. We examined their expression in three different groups of colorectal cancer [i.e., microsatellite stable (MSS), microsatellite instable (MSI), and hereditary nonpolyposis colorectal cancer (HNPCC)]. First, we analyzed gene expression profiles of 113 specimens by a microarray method to study the expression of various CA isozymes in the subgroups of colorectal cancer. The results indicated that mRNAs for CA II and CA XII are down-regulated and CA IX mRNA is up-regulated in all three tumor categories when compared with the normal tissue. The up-regulation of CA IX was greatest in the HNPCC group. For more information, 77 specimens were immunohistochemically stained to study the levels of CA II, CA IX, and CA XII. Immunohistochemical analyses further confirmed that the subgroups express CA II, CA IX, and CA XII differentially, and the HNPCC tumors express high levels of CA IX. Expression of these CAs did not correlate to Dukes stage or grade of differentiation. Our results show that CAs are differentially expressed in the subgroups of colorectal cancer, and CA IX expression seems to be very high in most cases of HNPCC. CA IX could be a potential diagnostic and therapeutic target in HNPCC.     

Alphav integrin expression is a novel marker of poor prognosis in advanced-stage ovarian carcinoma.

PURPOSE: To analyze the possible correlation between expression of the alphav and beta1 integrin chains and survival in advanced-stage ovarian carcinomas, studying two patient groups with extremely different disease outcome. EXPERIMENTAL DESIGN: Sections from 56 primary ovarian carcinomas and metastatic lesions from 34 patients diagnosed with advanced-stage ovarian carcinoma (Fédération Internationale des Gynaecologistes et Obstetristes stages III-IV), divided into long-term (16) and short-term (18) survivors, were evaluated for expression of alphav and beta1 integrin chains using mRNA in situ hybridization. Protein expression was additionally studied in 52 specimens using immunohistochemistry. RESULTS: The mean values for disease-free survival and overall survival were 115 and 132 months for long-term survivors, as compared with 4 and 23 months for short-term survivors, respectively. Expression of alphav integrin mRNA was observed in carcinoma (18 of 56; 32%) and stromal (17 of 56; 30%) cells. beta1 integrin mRNA was similarly detected in carcinoma (25 of 56; 47%) and stromal (19 of 56; 34%) cells. No significant differences were observed when primary and metastatic lesions were compared (P > 0.05). Alphav integrin mRNA was present more often in carcinoma cells in tumors of short-term survivors (P = 0.017 for carcinoma cells). In univariate survival analysis for all cases, alphav integrin mRNA expression in tumor cells correlated with poor survival (P = 0.012). This finding retained its predictive power in a multivariate survival analysis, in which all of the molecules studied previously in this patient cohort were included (P = 0.031). Immunohistochemistry confirmed the differences in alphav integrin expression in tumor cells of short-term as compared with long-term survivors, whereas beta1 integrin protein expression was comparable in the two groups. CONCLUSIONS: To our best knowledge, this is the first evidence associating integrin expression with poor survival in ovarian carcinoma. Alphav integrin is, thus, a novel prognostic marker in advanced-stage ovarian carcinoma.     

Erlotinib in patients with advanced non-small-cell lung cancer: impact of dose reductions and a novel surrogate marker

Erlotinib is a relatively well-tolerated treatment option for patients with advanced non-small-cell lung cancer (NSCLC). Some patients suffer from severe skin toxicity or diarrhea, making dose reductions or even treatment cessation necessary. Recent clinical trials usually defined a 100 mg daily dose as the lowest acceptable dose, whereas little is known about the efficacy with lower doses. We retrospectively reviewed the files of all patients with advanced non-small-cell lung cancer (NSCLC) treated with erlotinib. We assessed demographic, disease- and treatment-related information. We tried to correlate tolerability with clinical efficacy. EGF receptor exon 18/19/21 mutations were analyzed in selected patients. Fifty-three patients with advanced non-small-cell lung cancer were treated with erlotinib. In nine patients (17%), the doses had to be reduced to 75 or 50 mg daily due to toxicity. We observed several sustained disease stabilizations in this subgroup. Patients suffering from paronychia with erlotinib had a significantly longer time to progression than did subjects without nail toxicity (P = 0.04). If patients were free from any toxicity, they were at high risk for early tumor progression (P = 0.001) and death. In patients with disease stabilization for 6 months or longer, we observed EGFR 18/19/21 wild type, exon 19 and exon 21 mutations. In conclusion, several patients required dose reductions during treatment with erlotinib. However, in tumors with sensitivity to erlotinib, even daily doses of 50–75 mg can result in sustained disease control. Paronychia represents a favorable surrogate marker for efficacy.     

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.