Renal cortical tumors can be classified according to their histologic appearance and/or genetic abnormalities.1, 2 A minority (5%) of tumors cannot be typed owing to overlap in their morphologic features and to a lesser degree also in their genetic alterations.2, 3 Accurate classification is important since different tumors have different prognosis and clear cell carcinomas may be treated differently.
Furthermore, the increased use of core biopsy to diagnose small tumors that are ablated at the time the biopsy is performed and tumors of patients who are poor surgical candidates increases the need for accurate typing on limited amounts of tissue. Commonly used antibodies including cytokeratin AE1/AE3, Pax-2, PAX-8, CK7, CK20, RCC, CD10, and vimentin can be helpful but do not provide definitive answer in a subset of cases.4–7
Recently, a new antibody became available for studying carbonic anhydrase IX (CA IX) expression in formalin fixed paraffin embedded tissue sections. Carbonic anhydrases are widely expressed in living organisms including in mammalian cells with 15 recognized isoenzymes.8–11 They catalyze reversible hydration of carbon dioxide (H2O + CO2 = H+ + HCO3) that results in a net extrusion of H+ and an increase in intracellular pH. The maintenance of pH during hypoxia is a key protective mechanism to prevent hypoxia induced cell death. Hypoxia upregulates the activity of a number of genes through hypoxia inducible factor-1alpha (HIF-1alpha) including two carbonic anhydrases (CA IX and CA XII).8–11 CA IX is a 54/58 KDa transmembrane glycoprotein that has a cell surface enzyme activity and can be detected in normal gastrointestinal mucosa and a number of different tumors.
In the kidney, CA IX overexpression has been described in clear cell renal cell carcinoma and in type 1 papillary renal cell carcinoma but not in normal renal tissue.10–11 In this study, our aim was to investigate the expression patterns of CA IX in the most common types of renal cortical neoplasms and to determine whether CA IX expression can be helpful for differentiating these tumor types.
Figure 1. CA IX immunohistochemistry of Clear cell renal cell carcinoma.
Materials and Methods
Five micron sections of previously constructed tissue microarrays (TMA) of 44 papillary RCC, 42 clear cell renal cell RCC, 37 chromophobe RCC and 28 oncocytomas were subjected to immunohistochemistry using a mouse monoclonal antibody for CA IX (Leica Biosystems, Newcastle, UK) on a Leica BondTM automated stainer (Leica Microsystems, Bannockburn, IL). The TMA also included samples of benign brain, pancreas, kidney, thyroid, testis, lung, smooth muscle, liver, tonsil, thymus, skin, small intestine, ovary and fibroconnective tissue.
The staining was evaluated according to criteria listed in Table 1. The scores of both intensity and extent were added and a stain was considered positive with a combined score of greater than 2.
The results are summarized in Table 2. The majority (95%) of clear cell RCC were positive for CA IX with most cases showing moderate (2+) to strong (3+) staining in over 50% of the tumor cells (Figure 1).
Clear Cell RCC
The papillary RCC TMA contained both type 1 and type 2 tumors. The two positive cases (5%) were both type 1 and showed moderate to strong membranous staining in less than 25% of the tumor cells (Figure 2). All chromophobe RCC and oncocytomas were negative for CA IX.
Of the benign tissues strong membranous staining was noted in gastric, small bowel, and gallbladder mucosa, and in intrahepatic bile duct epithelium (Figure 3).
Diffuse weak to moderate staining was noted in Sertoli cells and focal weak to moderate staining was seen in the epidermis (Figure 3). All other benign tissues including kidney were negative (Figure 3).
Figure 3. CA IX immunohistochemistry of benign tissues.
Since a subset of renal cortical tumors cannot be classified even when using current ancillary techniques there is a need for additional markers that can assist in this effort. CA IX is present in a number of tumors and in some benign tissues and is one of the most uniformly induced genes in hypoxia. Since CA IX is a stable cell surface protein it is a good target for immunohistochemical detection. Under normal physiologic conditions CA IX expression is largely limited to the GI tract mucosa. CA IX is also expressed in a number of tumors which include some of the most aggressive types of cancer. Some authors have recommended using CA IX expression by immunohistochemistry as a marker of hypoxia12, moreover CA IX has also been investigated as target for therapy.13–16
In our study, we found CA IX expression in the large majority of clear cell RCC, but not in chromophobe RCC or oncocytomas. There were only two papillary renal RCC (both type 1) that were positive and both cases showed less extensive staining of the tumor. As expected normal renal cortex and medulla were negative and strong staining was noted in mucosa from the upper gastrointestinal tract and intrahepatic bile duct epithelium.
Immunohistochemistry for CA IX can be easily performed on paraffin-embedded formalin-fixed tissue. CA IX immunostains can be used to differentiate clear cell RCC from chromophobe RCC and oncocytoma. Focal staining can be seen in a small subset of type 1 papillary RCC where careful examination of the histologic features should be performed and the use of additional markers should be considered. Furthermore, since CA IX plays an important role in maintaining the tumor cell homeostasis and in preventing cell death induced by hypoxia it is a potential candidate for targeted therapy.
- Pathology and genetics: Tumors of the urinary system and male genital organs. World Health Organization classification of tumors. IARC Press 2006.
- Kovacs G, Akhtar M, Beckwith BJ, et al. The Heidelberg classification of renal cell tumours, J Pathol 1997; 183: 131–133.
- Barocas DA, Rojan SM, Kao J et al. Diagnosis of renal tumors on needle biopsy specimens by histological and molecular analysis. The J Urol 2006; 176: 1957–1962.
- Skinnider BF, Amin MB. An immunohistochemical approach to the differential diagnosis of renal tumors. Semin Diagn Pathol. 2005; 22: 51–68.
- Every AK, Beckstead J, Renshaw A, et al. Use of antibodies to RCC and CD10 in the differential diagnosis of renal neoplasms. Am J Surg Pathol 2000; 24: 203–210.
- Stopyra G, Warhol M, Multhaupt HAB. Cytokeratin 20 immunoreactivity in renal oncocytomas. J Histochem Cytochem 2001; 49: 919–920.
- Delahunt B, Eble JN. Papillary renal cell carcinoma: A clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol 1997; 10: 537–544.
- Parkkila S. An overview of the distribution and function of carbonic anhydrases in mammals. In the carbonic anhydrases: New horizons. Chegwidden WR, Carter N and Edwards Y; Eds. Birkhauser Verlag, Basel, Switzerland, 2000, pp 76–93.
- Pastorekova S, Parkkila S, Pastorek J, et al. Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem. 2004: 19: 199–229.
- Hilvo M, Tolvanen M, Clark A, et al. Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase. Biochem J 2005: 392: 83–92.
- Potter C, Harris AL. Hypoxia inducible carbonic anhydrase IX, Marker of tumor hypoxia, survival pathway and therapy target. Cell Cycle 2004; 3: 164–167.
- Dorai T, Sawczuk I, Pastorek J, et al. Role of carbonic anhydrases in the progression of renal cell carcinoma subtypes: Proposal of a united hypothesis.
- Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004; 4: 891–899.
- Graeber TG, Osmanian C, Jacks T, et al. Hypoxia mediated selection of cells with diminished apoptotic potential in solid tumors. Nature 1996; 329: 88–91.
- Martinez-Zaguilan R, Seftor EA, Sefor RE, et al. Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis 1996; 14: 176–186.
- Brand K. Aerobic glycolysis by proliferating cells: protection against oxidative stress at the expense of energy yield. J Bioener Biomemb 1997; 29: 355–364.