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Immunohistochemical Analysis of Carbonic Anhydrase IX (CA IX) in Renal Cortical Tumors

A subset of renal cortical tumors cannot be accurately classified based on their morphologic features owing to partial overlap in their histologic appearance. Since the prognosis is different and clear cell carcinomas may be treated by the different therapeutic regimens there is a need for accurate classification.

A number of markers have been tested to assist in this task, but even when several markers are used a significant number of tumors remain unclassifiable. Carbonic anhydrase IX (CA IX) plays an important role in maintaining the pH of several tumor types. It is a stable cell surface protein that is amenable for detection by immunohistochemistry. CA IX has been found to be expressed in clear cell renal cell carcinoma (RCC) and type 1 papillary renal cell carcinoma. In this study, we have investigated the expression pattern of CA IX in 44 papillary RCC, 42 clear cell RCC, 37 chromophobe RCC and 28 oncocytomas.

The majority (95%) of clear cell RCC were positive, whereas only two cases (5%) of papillary RCC showed focal positivity. All chromophobe RCC and oncocytoma were negative. Immunohistochemistry for CA IX can be easily performed and positive staining in the absence of papillary features strongly suggest clear cell RCC.


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.
A. Negative tumor (no staining);
B. Focal weak staining (score 2);
C. Diffuse moderate staining (score 5);
D. diffuse strong staining (score 6).

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 Bond 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.

IntensityNo stainingweakmoderatestrong
ExtentNo staining<25%25-50%>50%
Table 1. Scoring criteria for intensity and extent of staining tumor cells
Figure 2. CA IX immunohistochemistry of the positive papillary cell renal cell carcinoma
A. Hematoxylin eosin stained section of tumor #1.
B. Focal strong CA IX staining (score 4) of tumor #1.
C. Hematoxylin eosin stained section of tumor #2.
D. Focal moderate CA IX staining (score 3) of tumor #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).

CAIX Positive
Clear Cell RCC39/41 (95%)
Papillary RCC2/44 (5%)
Chromophobe RCC0/37 (0%)
Oncocytoma0/28 (0%)
Table 2. Results of CA IX immunostaining of clear cell renal cell carcinoma (RCC), Papillary RCC (PRCC), chromophobe renal cell carcinoma (ChRCC) and oncocytoma.

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.

Strong membranous staining is observed in gallbladder (A)
Strong membranous staining in gastric mucosa (B)
Strong membranous staining in intrahepatic bile duct epithelium (C)
Weak-moderate staining of epidermis (D)
Sertoli cells in the testis (E)
Negative normal renal parenchyma (F)


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.


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