invade. Furthermore, CA IX has also been proposed to diminish the intracellular pH gradient in the hypoxic core of three-dimensional tumor spheroids grown from cancer-derived cell lines (Swietach et al.,2008).
Another potential mechanism for the function of CA IX was proposed by the finding that CA IX modulates E-cadherin mediated cell adhesion by decreasing the binding of this cell adhesion molecule to beta-catenin (Pastorekova et al.,2004,2006). The disruption of the link between these adhesion molecules would possibly promote cell motility and invasion. Acetazolamide, a potent CA inhibitor, suppresses invasion of renal can- cer cells in vitro. However, when the inhibition of CA IX in RNAi-treated breast cancer cells was studied, the invasion capacity of the cells appeared to be slightly reduced compared with the control, but this difference was not considered significant (Robertson et al.,2004).
Immunohistochemical and Analytical
Fig. 7.1 CA IX immunostaining of a glioblastoma. Neoplastic cells surrounding capillaries (arrows) are negative, whereas tumor cells in border of necrotic area are strongly immunopos- itive (asterisk). Moderate immunopositivity is seen in some non-necrotic cells (arrow heads). (a) Magnification ×100;
(b) Magnification×200; haematoxylin counter staining
in the gliomas with higher differentiation. The CA IX cytoplasmic immunoreactivity showed a strong asso- ciation with increasing tumor WHO grade. In grade 2 astrocytomas, 65% of the tumors were identified as CA IX-positive cases, whereas 73% of grade 3 astro- cytomas and 82% of grade 4 astrocytomas (glioblas- tomas) were positive. When CA IX intensity was compared to important clinicopathologic and molec- ular factors, CA IX did not correlate to p53 expres- sion, epidermal growth factor receptor amplification, apoptosis, or cell proliferation by MIB-1. However, others have described an association between CA IX expression and proliferation (Proescholdt et al.,2005;
Korkolopoulou et al.,2007).
CA IX intensity had significant prognostic value in univariate analysis, and the immunopositive patients had a worse prognosis. The survival difference was also significant when grade 3 and grade 4 astrocy- tomas were evaluated separately. Most importantly, Cox multivariate survival analysis revealed that CA IX intensity, along with patient age and WHO tumor grade, was an independent prognostic factor.
Our finding on the prognostic significance of CA IX in astrocytic tumors has been confirmed by Korkolopoulou et al. (2007). Increasing CA IX immunopositivity was associated with shortened sur- vival in the entire cohort of 84 adult patients in univariate analysis. Furthermore, multivariate analysis revealed that CA IX with the tumor grade and patient age were the only parameters independently affecting the survival. CA IX expression predicted significantly the survival of patients with grades 2 and 3 tumors.
They also demonstrated a perinecrotic distribution of CA IX immunostaining which increased in parallel with the extent of necrosis and histological grade.
Sathornsumetee et al. (2008) reported that high expression of CA IX was associated with poor survival outcome in a patient cohort treated with bevacizumab and irinotecan. Additionally, when both CA IX and HIF-2alpha were simultaneously included in a Cox model as two separate factors, only CA IX remained as a statistically significant factor.
Hypoxia-regulated protein expression, including CA IX, has been recently studied in a cohort of glioblastomas (Flynn et al.,2008). The authors found no correlation between CA IX expression and patient survival nor did they report correlation between CA IX expression and tumor grade when low-grade astro- cytomas were included in the analysis. In fact, the patients with CA IX-positive tumors seemed to have worse prognosis, although this finding was not statis- tically significant. The reported discrepancy between this and the other studies may be related to differ- ent immunostaining methods and to a smaller number of patients investigated. The M75 antibody, used e.g., by us and Korkolopoulou et al. (2007) against human CA IX has been characterized for specificity, and it has shown no cross-reactivity with other CAs (Saarnio et al., 1998b), representing the most reliable CA IX antibody thus far.
The study of Ivanov et al. (2001) showed high expression of CA IX in a large series of cancer cell lines, and also in human tumor tissues. Strong CA IX
immunoreactivity was reported in glioblastomas, espe- cially in necrotic/hypoxic regions, whereas low-grade gliomas were negative. The results of Proescholdt et al. (2005) were comparable to the study described above. The strongest and most consistent staining was observed in glioblastomas and predominantly near necrotic areas. The statistical analyses exhibited a pos- itive correlation between the CA IX expression and increasing tumor grade. In some cases, the staining was seen in most tumor cells, including those located near the blood vessels. This finding led the authors to suggest that CA IX induction in gliomas may involve hypoxia-independent mechanisms. Interestingly, it has been shown that acidosis also induces CA IX indepen- dently of pericellular hypoxia in glioblastoma cell lines (Ihnatko et al.,2006).
Because CA IX is a hypoxia/necrosis marker, it can be used also as a diagnostic tool in the grading of astrocytomas. Of these tumors, necrotic neoplasms correspond to WHO grade 4 (glioblastoma). Small tumor biopsies, which contain only a small amount of tissue for diagnosis, are especially good targets for the evaluation of necrosis by CA IX immunostaining.
In conclusion, CA IX is expressed abundantly in astrocytic tumors and especially in high-grade astro- cytomas. This might be due to both hypoxia and high cell density, because glioblastomas represent a cate- gory of highly hypoxic and cellular tumors. CA IX may increase extracellular acidification and promote the survival and migration of malignant cells. As a result, CA IX has been shown to be a useful biomarker for predicting poor prognosis of astrocytomas and the immunostaining can also be used in the diagnostic evaluation. Furthermore, CA IX could be used as a target molecule for therapeutic interventions.
References
Beasley NJ, Wykoff CC, Watson PH, Leek R, Turley H, Gatter K, Pastorek J, Cox GJ, Ratcliffe P, Harris AL (2001) Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis, and microvessel density.
Cancer Res 61:5262–5267
Chen J, Röcken C, Hoffmann J, Krüger S, Lendeckel U, Rocco A, Pastorekova S, Malfertheiner P, Ebert MP (2005) Expression of carbonic anhydrase 9 at the invasion front of gastric cancers. Gut 54:920–927
Flynn JR, Wang L, Gillespie DL, Stoddard GJ, Reid JK, Owens J, Ellsworth GB, Salzman KL, Kinney AY, Jensen RL (2008) Hypoxia-regulated protein expression, patient characteris- tics, and preoperative imaging as predictors of survival in adults with glioblastoma multiforme. Cancer 113:1032–1042 Giatromanolaki A, Koukourakis MI, Sivridis E, Pastorek J, Wykoff CC, Gatter KC, Harris AL (2001) Expression of hypoxia-inducible carbonic anhydrase-9 relates to angio- genic pathways and independently to poor outcome in non- small cell lung cancer. Cancer Res 61:7992–7998
Haapasalo J, Nordfors K, Hilvo M, Rantala I, Soini Y, Parkkila A-K, Pastorekova S, Pastorek J, Parkkila S, Haapasalo H (2006) Expression of carbonic anhydrase IX in astrocytic tumors predicts poor prognosis. Clin Cancer Res 12:473–477 Hilvo M, Baranauskiene L, Salzano AM, Scaloni A, Matulis D, Innocenti A, Scozzafava A, Monti SM, Di Fiore A, De Simone G, Lindfors M, Jänis J, Valjakka J, Pastoreková S, Pastorek J, Kulomaa MS, Nordlund HR, Supuran CT, Parkkila S (2008) Biochemical characterization of CA IX:
one of the most active carbonic anhydrase isozymes.J Biol Chem 283:2799–2809
Hilvo M, Rafajova M, Pastorekova S, Pastorek J, Parkkila S (2004) Expression of carbonic anhydrase IX in mouse tissues.J Histochem Cytochem 52:1313–1322
Ihnatko R, Kubes M, Takacova M, Sedlakova O, Sedlak J, Pastorek J, Kopacek J, Pastorekova S (2006) Extracellular acidosis elevates carbonic anhydrase IX in human glioblas- toma cells via transcriptional modulation that does not depend on hypoxia. Int J Oncol 29:1025–1033
Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, Merrill MJ, Proescholdt MA, Oldfield EH, Lee J, Zavada J, Waheed A, Sly W, Lerman MI, Stanbridge EJ (2001) Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol 158:905–911
Kim SJ, Rabbani ZN, Dewhirst MW, Vujaskovic Z, Vollmer RT, Schreiber EG, Oosterwijk E, Kelley MJ (2005) Expression of HIF-1alpha, CAIX, VEGF, and MMP-9 in surgically resected non-small cell lung cancer. Lung Cancer 49:
325–335
Korkolopoulou P, Perdiki M, Thymara I, Boviatsis E, Agrogiannis G, Kotsiakis X, Angelidakis D, Rologis D, Diamantopoulou K, Thomas-Tsagli E, Kaklamanis L, Gatter K, Patsouris E (2007) Expression of hypoxia-related tissue factors in astrocytic gliomas. A multivariate survival study with emphasis upon carbonic anhydrase IX. Hum Pathol 38:629–638
Loncaster JA, Harris AL, Davidson SE, Logue JP, Hunter RD, Wycoff CC, Pastorek J, Ratcliffe PJ, Stratford IJ, West CM (2001) Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res 61:6394–6399 McKiernan JM, Buttyan R, Bander NH, Stifelman MD, Katz
AE, Chen MW, Olsson CA, Sawczuk IS (1997) Expression of the tumor-associated gene MN: a potential biomarker for human renal cell carcinoma. Cancer Res 57:2362–2365 Niemelä AM, Hynninen P, Mecklin J-P, Kuopio T, Kokko
A, Aaltonen L, Parkkila A-K, Pastorekova S, Pastorek J, Waheed A, Sly WS, Ørntoftm TF, Kruhøfferm M, Haapasalo H, Parkkila S, Kivelä AJ (2007) Carbonic anhydrase IX
is highly expressed in hereditary non-polyposis colorectal cancer.Cancer Epidemiol Biomarkers Prev 16:1760–1766 Pastorek J, Pastoreková S, Callebaut I, Mornon JP, Zelník V, Opavsky R, Zat’ovicová M, Liao S, Portetelle D, Stanbridge EJ, Závada J, Burny A, Kettman R (1994) Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.Oncogene 9:2877–2888
Pastoreková S, Parkkila S, Parkkila A-K, Opavský R, Zelnik V, Saarnio J, Pastorek J (1997) Carbonic anhydrase IX, MN/CA IX: Analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.
Gastroenterology 112:398–408
Pastoreková S, Parkkila S, Pastorek J, Supuran CT (2004) Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem 19:199–229
Pastoreková S, Parkkila S, Zavada J (2006) Tumor-associated carbonic anhydrases and their clinical significance. Adv Clin Chem 42:167–216
Pastoreková S, Závadová Z, Kost’ál M, Babusiková O, Závada J (1992) A novel quasi-viral agent, MaTu, is a two-component system. Virology 187:620–626
Proescholdt MA, Mayer C, Kubitza M, Schubert T, Liao SY, Stanbridge EJ, Ivanov S, Oldfield EH, Brawanski A, Merrill MJ (2005) Expression of hypoxia-inducible carbonic anhy- drases in brain tumors. Neuro Oncol 7:465–475
Robertson N, Potter C, Harris AL (2004) Role of carbonic anhy- drase IX in human tumor cell growth, survival, and invasion.
Cancer Res 64:6160–5
Saarnio J, Parkkila S, Parkkila A-K, Haukipuro K, Pastoreková S, Pastorek J, Kairaluoma MI, Karttunen TJ (1998a) Immunohistochemical study of colorectal tumors for expres- sion of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am J Pathol 153:279–285
Saarnio J, Parkkila S, Parkkila A-K, Pastoreková S, Haukipuro K, Pastorek J, Juvonen T, Karttunen TJ (2001) Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours. J Hepatol 35:643–649
Saarnio J, Parkkila S, Parkkila A-K, Waheed A, Casey MC, Zhou ZY, Pastoreková S, Pastorek J, Karttunen T, Haukipuro K, Kairaluoma MI, Sly WS (1998b) Immunohistochemistry of carbonic anhydrase isozyme IX (MN/CAIX) in human gut reveals polarized expression in the epithelial cells with the highest proliferative capacity. J Histochem Cytochem 46:497–504
Said HM, Staab A, Hagemann C, Vince GH, Katzer A, Flentje M, Vordermark D (2007) Distinct patterns of hypoxic expres- sion of carbonic anhydrase IX (CA IX) in human malignant glioma cell lines. J Neurooncol 81:27–38
Sandlund J, Oosterwijk E, Grankvist K, Oosterwijk-Wakka J, Ljungberg B, Rasmuson T (2007) Prognostic impact of carbonic anhydrase IX expression in human renal cell car- cinoma. BJU Int 100:556–60
Sathornsumetee S, Cao Y, Marcello JE, Herndon JE 2nd, McLendon RE, Desjardins A, Friedman HS, Dewhirst MW, Vredenburgh JJ, Rich JN (2008) Tumor angiogenic and hypoxic profiles predict radiographic response and survival in malignant astrocytoma patients treated with bevacizumab and irinotecan. J Clin Oncol 26:271–8
Span PN, Bussink J, De Mulder PH, Sweep FC (2007) Carbonic anhydrase IX expression is more predictive than prognostic in breast cancer. Br J Cancer 96:1309
Swietach P, Wigfield S, Supuran CT, Harris AL, Vaughan-Jones RD (2008) Cancer-associated, hypoxia-inducible carbonic anhydrase IX facilitates CO2diffusion. BJU Int 101:22–4 Turner JR, Odze RD, Crum CP, Resnick MB (1997) MN antigen
expression in normal, preneoplastic, and neoplastic esopha- gus: a clinicopathological study of a new cancer-associated biomarker. Hum Pathol 28:740–744