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CA9

carbonic anhydrase IX;CA9;carbonic anhydrase IX

CA-IX|P54/58N|RCC-associated antigen G250|RCC-associated protein G250|carbonate dehydratase IX|carbonic anhydrase 9|carbonic dehydratase|membrane antigen MN|pMW1|renal cell carcinoma-associated antigen G250

9p13.3

protein-coding

INTRODUCTION: Circulating tumor cells (CTCs) reflect aggressive tumor behavior by hematogenous tumor cell dissemination. The tissue inhibitor of metalloproteinase 1 (TIMP-1) plays a role in tissue invasion and is also involved in angiogenesis, abrogation of apoptosis and in chemoresistance. Carbonic anhydrase IX (CAIX) is a metalloenzyme involved in cell adhesion, growth and survival of tumor cells. The aim of the study was to investigate whether serum concentrations of TIMP-1 and CAIX are associated with the detection of CTC in metastatic breast cancer. METHODS: Blood was obtained in a prospective multicenter setting from 253 patients with metastatic breast cancer at the time of disease progression. Serum TIMP-1 and CAIX were determined using commercial ELISA-kits (Oncogene Science). CTC were detected with the CellSearch system (Veridex). RESULTS: Five or more CTCs were detected in 122 patients out of 245 evaluable patients (49.8%). Out of 253 metastatic patients 70 (28%) had serum TIMP-1 levels above 454 ng/mL. Serum CAIX was elevated above 506 ng/mL in 90 (35%) patients. Both serum markers had prognostic significance. Median progression free survival (PFS) was 7.2 months with elevated TIMP-1 vs. 11.4 months with non-elevated levels (p < 0.01). OS was 11.5 vs. 19.1 months (p < 0.01). Median PFS was 7.5 months with elevated CAIX vs. 11.7 months with non-elevated levels (p < 0.01), overall survival (OS) was 13.4 months vs. 19.1 months (p < 0.01). In patients with five or more CTCs, serum levels were above the cut-off for CAIX in 47% vs. 25% in those with less than five CTCs (p = 0.01). For TIMP-1, 37% patients with five or more CTCs had elevated serum levels and 17% of patients with less than five CTCs (p = 0.01). Including TIMP-1, CAIX, CTC and established prognostic factors in the multivariate analysis, the presence of CTCs, the therapy line and elevated CAIX remained independent predictors of OS. CONCLUSIONS: Elevated serum levels of the invasion markers TIMP-1 and CAIX in metastatic breast cancer are prognostic markers and are associated with the presence of CTCs. Whether increased secretion of TIMP-1 and/or CAIX might directly contribute to tumor cell dissemination remains to be elucidated in further investigations. TRIAL REGISTRATION: Current Controlled Trials: ISRCTN59722891.

PURPOSE: Carbonic anhydrase IX (CA9) has emerged as an important surrogate marker for hypoxia in solid tumors. CA12 shares a role with CA9 in acidification of micromilieu but it is less strictly regulated by hypoxia than CA9. In this study, we investigated expression of CA9 and CA12 mRNA in primary cervical cancer. We also examined whether CA9 expression can be an indicator of reoxygenation of tumor by measuring its mRNA expression during fractionated radiotherapy. METHODS: tumor tissues were obtained from 59 patients with uterine cervical cancer who underwent radiotherapy, and a second biopsy was taken after patients had received either 10 or 20 Gy of radiation. The follow-up period ranged from 2.4 to 75 months (median=23 months). The ratio of CA9 and beta-actin mRNA expression was determined both pre- and during radiation treatment by RT-PCR. RESULTS: CA9 and CA12 mRNA expression was detected in 62.7 and 88.1% of tumors (i.e. patients), respectively, and co-expression was observed in 61% of patients. Multivariate analysis revealed that CA9 expression was the most significant factor associated with metastasis-free survival (P=0.008, hazard ratio 34.8), whereas CA12 mRNA expression was linked to a lower risk of metastasis (P=0.007, hazard ratio of 0.07). tumor CA9 expression was not altered following either 10 or 20 Gy of radiotherapy. CONCLUSION: The strong correlation between CA9 expression and metastasis suggests that CA9 expression might be an important indicator for identifying patients who require more aggressive systemic therapy.

This study aimed to investigate whether the expression of carbonic anhydrase IX (CAIX) is associated with the expression of vascular endothelial growth factor (VEGF) and whether the co-expression of the two correlates with survival outcome in clear cell renal cell carcinoma (ccRCC). The expression of CAIX and VEGF was evaluated immunohistochemically in 122 paraffin-embedded ccRCC specimens. The clinical significance of these markers in relation to disease-specific survival (DSS) was analyzed. Patients with a low expression of CAIX had a significantly worse prognoses than those with a high expression (p=0.0005). Inversely, patients with a high expression of VEGF had a significantly worse prognoses than the patients with a low expression (p=0.0030). Furthermore, CAIX expression significantly stratified the DSS of patients with high-stage (p=0.0001), high-grade (p=0.0392), low-grade (p=0.0273), metastasis (p=0.0034), no metastasis (p=0.0303) and ECOG-PS=0 (p=0.0003). VEGF expression significantly predicted the survival of patients with low-grade (p=0.0003), high-stage (p=0.0401) and ECOG-PS=0 (p=0.0063). A multivariate Cox regression analysis showed that tumor stage (p=0.0054), metastasis (p=0.0193), ECOG-PS (p=0.0065) and CAIX expression (p=0.0001) were independent prognostic factors of DSS. Since CAIX and VEGF expression correlated inversely (p=0.0032), the prognostic value of the co-expression of CAIX-VEGF was evaluated. Multivariate analysis revealed that the co-expression was an independent prognostic factor of DSS (p=0.0002). In addition, the co-expression was able to stratify DSS into three risk groups: high-risk, intermediate-risk and low-risk (p<0.0001). In patients with ccRCC, CAIX and VEGF expression correlated inversely. Independent expression of CAIX and a co-expression of CAIX-VEGF were found to be independent predictors of DSS. Furthermore, the co-expression data for CAIX-VEGF provide more accurate prognostic information than the individual data. This information may be useful for survival prediction and risk stratification of patients with ccRCC.

PURPOSE: We assessed CA9 single nucleotide polymorphisms and mutations, and their association with CAIX protein expression, overall survival and response to interleukin-2 in white patients with metastatic clear cell renal cell carcinoma. MATERIALS METHODS: Genomic DNA was extracted from frozen tumor samples of 54 metastatic clear cell renal cell carcinomas. The CA9 gene exons and flanking regions were amplified by polymerase chain reaction and sequenced. The monoclonal antibody M75 was used to evaluate CAIX protein expression in the primary tumor by immunohistochemistry. RESULTS: CA9 reference single nucleotide polymorphisms rs2071676, rs12553173, rs3829078 and rs1048638 were found in 59%, 15%, 11% and 33% of patients, respectively. The deletion c.376del393 was observed in 2 patients. CAIX expression was greater than 85% in 65% of patients. No single nucleotide polymorphisms were significantly associated with CAIX expression. Patients with the C allele variant of rs12553173 had improved median survival (27.3 vs 13.6 months, p = 0.0431) and a greater likelihood of an interleukin-2 response (57% vs 22%, p = 0.081) Likewise high CAIX expression was associated with longer median survival (25.5 vs 8.5 months, p <0.0001) and a greater interleukin-2 response rate (37% vs 8%, p = 0.070). In a multivariate Cox model the C allele variant of CA9 single nucleotide polymorphism rs12553173 and CAIX expression were retained as independent prognostic factors. CONCLUSIONS: CA9 single nucleotide polymorphisms are common in patients with metastatic clear cell renal cell carcinoma. The synonymous C allele variant of rs12553173 may be associated with improved overall survival and a greater likelihood of a response to interleukin-2. CA9 rs12553173 and CAIX are independent prognostic factors of overall survival and complementary for predicting the prognosis of metastatic clear cell renal cell carcinoma.

BACKGROUND: Carbonic anhydrase 9 (CA9) is a marker for hypoxia and acidosis, which is linked to a poor prognosis in human tumors. The purpose of this comparative analysis was to evaluate whether CA9 and VEGF expression are associated with survival outcomes in patients with metastatic colorectal cancer (mCRC) after treatment with bevacizumab as second or later line treatment. METHODS: Thirty-one mCRC patients who were treated with bevacizumab-containing chemotherapy as second or later line treatment and who had analyzable tumor paraffin blocks were selected for this study. The planned dose of bevacizumab was 5 mg/kg/2-week. Immunohistochemical (IHC) staining of CA9 and VEGF was performed and their expression was scored by the intensity multiplied by percentage of stained area. RESULTS: The overall response rate was 19.4% and the disease control rate (DCR) was 61.3% with 6 partial responses and 13 cases of stable disease. The DCR was significantly higher in patients with a lower CA9 expression score compared to those with a higher score (80.0% vs. 27.3%, respectively, P = 0.004). The patients with a low CA9 expression score also showed better outcomes with regard to the median progression-free survival (P = 0.028) and overall survival (P = 0.026). However, VEGF expression was not associated with the DCR and survival. CONCLUSION: Lower degree of CA9 expression was associated with better clinical outcomes in patients with mCRC treated with lower dose bevacizumab-based chemotherapy. Prospective studies are now needed to determine the correlation between CA9 expression and clinical outcomes after bevacizumab treatment, at different doses and in varied settings.

Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme that is overexpressed by cancer cells from many tumor types, and is a component of the pH regulatory system invoked by these cells to combat the deleterious effects of a high rate of glycolytic metabolism. CAIX functions to help produce and maintain an intracellular pH (pHi) favorable for tumor cell growth and survival, while at the same time participating in the generation of an increasingly acidic extracellular space, facilitating tumor cell invasiveness. Pharmacologic interference of CAIX catalytic activity using monoclonal antibodies or CAIX-specific small molecule inhibitors, consequently disrupting pH regulation by cancer cells, has been shown recently to impair primary tumor growth and metastasis. Many of these agents are in preclinical or clinical development and constitute a novel, targeted strategy of cancer therapy.

About 30-40% of patients with renal cell carcinoma (RCC) will develop metastasis after curative nephrectomy. There is a strong need to identify the early metastasis with conventional and molecular risk factors. The present study aimed to test if analysis of the CA9 gene can provide useful information to predict early metastasis after nephrectomy. This study included 63 patients with a conventional RCC. Ten tumors were N+ or/and M+ at diagnosis. The mean follow-up was 43 months (range, 4-67 months). About 11 M0N0 patients were found to have a metastasis during the follow-up. Quantitative RT-PCR of CA9 gene expression was performed. The metastasis-free survival curve was established according to the Kaplan-Meier method with comparison by the Log-Rank test. At diagnosis, the average of CA9 gene expression was significantly lower (p = 0.004) in metastatic tumors (N+ or/and M+) than in non-metastatic tumors (N0M0). For the follow-up of M0N0 patients, the metastasis-free survival rate was significantly higher (p = 0.005) in the high CA9 group than in the low-CA9 group. When combined with CA9, the metastasis-free survival rates, in terms of stage (p = 0.015) or grade (p = 0.010) were significantly different. When the stage, grade, and CA9 were combined, there was a significant difference (p = 0.004) in metastasis-free survival rates (T1T2 + G1G2 + high expression of CA9 versus T3 + G3G4 + low expression of CA9). Finally, the multivariate regression analysis identified CA9 expression (p = 0.036) as an independent predictor of early metastasis. Our study confirms that the expression level of CA9 gene in conventional RCC is related to metastasis. CA9 may be a potential marker for the prediction of early metastasis after nephrectomy and to guide post-operative follow-up and treatment.

OBJECTIVE: The aim of this study was to determine reliable predictive biomarkers for patients with metastatic renal cell carcinomas (RCCs) who had received cytokine therapy. METHODS: Tissue specimens were obtained from 62 patients with metastatic RCCs between 1995 and 2006. Paraffin wax embedded tissues were immunostained for carbonic anhydrase IX (CAIX), cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF). RESULTS: Fifty-two specimens (84%) were assessed as clear cell type, with 5, 3, and 2 tumors showing sarcomatoid, papillary, and undifferentiated features, respectively. With a median 54 months of follow-up, 15/18 responding patients (83%) exhibited high CAIX staining compared with only 24/44 (55%) nonresponding patients (odds ratio, OR, 4.1; 95% confidence interval,

The majority of clear-cell renal cell carcinomas (ccRCC) show high and homogeneous expression levels of the tumor associated antigen (TAA) carbonic anhydrase IX (CAIX), and treatment with interleukin-2 (IL-2) based immunotherapy can lead to cure in patients with metastatic renal cell carcinoma (mRCC). However, the involvement of CAIX specific CD8+ T cells and/or NK cells in the tumor eradication is unknown. We investigated T cell and antibody reactivity against overlapping 15-mer CAIX-peptides as well as HLA haplotype frequency and NK cell cytotoxicity in 11 patients with no evidence of disease (NED) following treatment with IL-2 based immunotherapy, and thus potentially cured. Immune reactivity in these patients was compared with samples from patients with dramatic tumor response obtained immediately at the cessation of therapy, samples from patients that experienced progressive disease during treatment and samples from healthy controls. We observed more focused but only weak and not consistent CAIX specific T-cells in the late observation and early observation response groups compared with the healthy control group. An increased frequency of the class II alleles HLA-DRB4 01:01, HLA-DPB 01:01 and HLA-DPB 03:01 was noted in the NED patients. In contrast, NK cytotoxicity was low even in the late observation response group as compared with controls. In particular, a HLA-B*40:01 restricted CD8+ T cell response recognizing the CAIX- derived peptide SEEEGSLKL was identified. This may have interest in future cancer vaccines, but more studies are needed to elucidate the immunological mechanisms of action in potentially cured patients treated with an immunotherapeutic agent.

OBJECTIVES: To examine carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma that recently has been described in malignant effusions. METHODS: Pleural and peritoneal fluids with the following diagnoses-reactive (n = 23), carcinoma (n = 17), and "suspicious for mesothelioma" (n = 4)-were immunostained for CAIX, calretinin, Ber-EP4, and MOC31. A tissue microarray of epithelioid (n = 27) and sarcomatoid (n = 8) mesotheliomas and three cases of benign mesothelium were also immunostained for CAIX. RESULTS: Mesothelial cells in both reactive (18/23) and malignant effusions (18/21) were positive for CAIX (P > .05). In carcinomatous effusions, CAIX expression was restricted to the mesothelial cells. Agreement between CAIX and calretinin expression was present in 89% of cases. In tissues, CAIX was positive in 100% of benign and 91% of malignant mesothelium. CONCLUSIONS: CAIX can be a useful ancillary marker for identifying mesothelial cells. There is no difference in CAIX expression between benign and malignant mesothelium. Caution should be exercised while evaluating for metastasis from renal cell carcinoma.