Carbonic Anhydrase IX Expression in Clear Cell ...

Correspondence

9. Bleumer I, Knuth A, Oosterwijk E, et al: A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients. Br J Cancer 90:985-990, 2004 10. Bleumer I, Oosterwijk E, Oosterwijk-Wakka JC, et al: A clinical trial with chimeric monoclonal antibody WX-G250 and low dose interleukin-2 pulsing scheme for advanced renal cell carcinoma. J Urol 175:57-62, 2006 11. Steffens MG, Boerman OC, de Mulder PH, et al: Phase I radioimmunotherapy of metastatic renal cell carcinoma with 131I-labeled chimeric monoclonal antibody G250. Clin Cancer Res 5:3268s–3274s, 1999 (suppl) 12. Brouwers AH, Mulders PF, de Mulder PH, et al: Lack of efficacy of two consecutive treatments of radioimmunotherapy with 131I-cG250 in patients

with metastasized clear cell renal cell carcinoma. J Clin Oncol 23:6540-6548, 2005 13. Brouwers AH, Dorr U, Lang O, et al: 131I-cG250 monoclonal antibody immunoscintigraphy versus [18F]FDG-PET imaging in patients with metastatic renal cell carcinoma: A comparative study. Nucl Med Commun 23:229-236, 2002 14. Brouwers AH, Buijs WCAM, Oosterwijk E, et al: Targeting of metastatic renal cell carcinoma with the chimeric monoclonal antibody G250 labeled with 131 I or 111In: An intrapatient comparison. Clin Cancer Res 9:3953s-3960s, 2003 (suppl)

DOI: 10.1200/JCO.2008.17.6073

■ ■ ■

Carbonic Anhydrase IX Expression in Clear Cell Renal Cell Carcinomas Negatively Correlates With the Proportion of the Granular Cell Component TO THE EDITOR: We read with great interest an article by Leibovich et al1 addressing the expression of carbonic anhydrase IX (CAIX) in clear cell renal cell carcinomas (ccRCC). CAIX is a catalytically active cell membrane isoform of carbonic anhydrase, which was unexpectedly discovered during the virologic research2 and may be ectopically expressed by various tumor cells. The authors evaluated its use as a prognostic marker in ccRCC and presented crucial facts for the interpretation of CAIX immunohistochemistry with the mouse monoclonal antibody M75. Importantly, they identified the correlation among the low level of CAIX expression and several histopathologic indicators of poor prognosis, including advanced nuclear grade, as was confirmed recently using a polyclonal rabbit anti-CAIX antibody NB100-417.3 Although Leibovich et al observed heterogeneity of the immunostaining for CAIX in the majority of ccRCC, the detailed histologic features of neoplastic cells with variable or absent expression of CAIX were not reported in their article. In our opinion, some cases of heterogeneous expression of CAIX could be explained by an admixture of tumor cells with finely granular eosinophilic cytoplasm, which harbor high numbers of mitochondria.4 The variable immunoreactivity for CAIX in this variant of ccRCC was observed previously,5 it was confirmed by reverse transcription polymerase

chain reaction assays,6,7 but it has not been systematically analyzed to date. The granular cell component occurs commonly in ccRCC,8 usually manifesting a higher nuclear grade than the clear-cell part of the tumor,8,9 and therefore it could be interpreted as a sign of tumor dedifferentiation,9 with a possible impact on the patient’s prognosis. This could be the reason why, in the study by Leibovich et al, the low immunoreactivity for CAIX failed to independently predict an increased risk of a patient’s death after adjusting for prognostically relevant histopathologic features including the nuclear grade, although it correlated univariately with a shorter survival time. Similar neoplastic cells with eosinophilic cytoplasm occur frequently in papillary8 and chromophobe4,8 RCCs and their presence is a basic sign for the diagnosis of renal oncocytoma.8 All of these tumors are known to be negative or inconsistently positive for CAIX.1,5,6,10 In our pilot study of CAIX expression in a spectrum of renal tumors (Table 1), we detected CAIX by means of immunohistochemistry in formalin-fixed, paraffin-embedded tissue sections using the mouse monoclonal antibody clone M75 diluted 1:100. Only membranous staining of tumor cells was considered positive for the evaluation of CAIX expression. The proportion of CAIXpositive neoplastic cells was estimated and the intensity of reaction was semiquantitatively scaled from absent to weak to strong. The typical staining patterns for CAIX in particular renal tumors were subsequently identified (Table 1). The granular cells in ccRCC characteristically showed negativity or only a weak expression of CAIX (Fig 1). Based on these data, the variable extent and intensity of immunostaining for CAIX in ccRCC seems to be associated with the proportion of the granular cell component, and it may be dependent on the numbers of mitochondria in the cytoplasm of neoplastic cells.

Table 1. CAIX Expression Detected Immunohistochemically in a Spectrum of Renal Tumors Tumors Expressing CAIX %

Proportion of Tumor Cells Expressing CAIX (%)

Intensity of Immunostaining for CAIX

9 6

90.0 100.0

⬎ 90 30-90

5 0 0

83.3 0 0

1-30 NA NA

Strong Variable (absent, weak, strong) Weak NA NA

Histologic Type of Renal Tumor

No. of Patients

No.

Clear cell RCC (composed exclusively of clear cells) Clear cell RCC (with the granular cell component)

10 6 6 2 3

Papillary RCC Chromophobe RCC Oncocytoma

Typical Immunostaining Pattern for CAIX Diffuse strong positivity Focal variable positivity Focal weak positivity Negativity Negativity

Abbreviations: CAIX, carbonic anhydrase IX; RCC, renal cell carcinoma; NA, not applicable.

www.jco.org

© 2008 by American Society of Clinical Oncology

Downloaded from jco.ascopubs.org on December 7, 2014. For personal use only. No other uses without permission. Copyright © 2008 American Society of Clinical Oncology. All rights reserved.

3809

Correspondence

A

B

C

Fig 1. Example of the immunohistochemical profile of the granular cell component (upper half of the microphotographs) in the clear cell renal cell carcinoma (magnification 200⫻). (A) Hematoxylin and eosin staining; (B) strong immunostaining with antimitochondrial antibody (clone 113-1); (C) weak or absent immunostaining for carbonic anhydrase IX with M75 antibody.

Leibovich et al pointed out the influence of the heterogeneous CAIX staining on the interpretation of results of prior studies, in which tissue microarray (TMA) technologies were employed.10-12 An important observation was recently made in this respect. CAIX was used as a prototypic marker of tumor hypoxia in cervical carcinoma,13 but after the correlation with the direct partial pressure of oxygen measurement in tissue sections and simulation of TMA technique, the distributional heterogeneity of CAIX produced a high sampling error. The size of the tissue sample was therefore recognized to be the major factor affecting accuracy of the observation. In the case of ccRCC, the presence of tumor cells with granular cytoplasm in the tissue sample obtained by TMA technique could probably affect results of the study significantly. Therefore, we cannot fully agree with Leibovich et al that CAIX could be used as a complementary marker for the histopathologic evaluation of small renal mass biopsies. The authors propose that patients with more than 30% CAIX expression in their renal tumors harbor ccRCC rather than papillary RCC or chromophobe RCC. By analogy with the TMA technique, this could be misleading in small, poorly representative tissue specimens taken from ccRCC with an increased number of granular neoplastic cells. Because of a high risk of sampling error, the inclusion of CAIX immunohistochemistry in the diagnostic algorithm of renal tumors composed of cells with granular eosinophilic cytoplasm should be avoided. Given that these cells may be present in all common types of RCC (clear cell, papillary, and chromophobe) as well as in renal oncocytomas, the significance of CAIX immunohistochemistry for the differential diagnosis of renal tumors remains limited. 3810

© 2008 by American Society of Clinical Oncology

Petr Skapa Department of Pathology and Molecular Medicine, Charles University, Second Medical School, University Hospital Motol, Prague, Czech Republic

Lubomir Hyrsl Department of Urology, Charles University, Second Medical School, University Hospital Motol, Prague, Czech Republic

Jan Zavada Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Jan Soukup and Josef Zamecnik Department of Pathology and Molecular Medicine, Charles University, Second Medical School, University Hospital Motol, Prague, Czech Republic

ACKNOWLEDGMENT

Supported by the research project of the Ministry of Health of the Czech Republic VZ FNM 00000064203.

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author(s) indicated no potential conflicts of interest.

REFERENCES 1. Leibovich BC, Sheinin Y, Lohse CM, et al: Carbonic anhydrase IX is not an independent predictor of outcome for patients with clear cell renal cell carcinoma. J Clin Oncol 25:4757-4764, 2007 2. Za´vada J, Zavadova Z, Pastorekova S, et al: Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int J Cancer 54:268-274, 1993 3. Al-Ahmadie HA, Alden D, Qin LX, et al: Carbonic anhydrase IX expression in clear cell renal cell carcinoma: An immunohistochemical study comparing 2 antibodies. Am J Surg Pathol 32:377-382, 2008 JOURNAL OF CLINICAL ONCOLOGY


Correspondence

4. Tickoo SK, Lee MW, Eble JN, et al: Ultrastructural observations on mitochondria and microvesicles in renal oncocytoma, chromophobe renal cell carcinoma, and eosinophilic variant of conventional (clear cell) renal cell carcinoma. Am J Surg Pathol 24:1247-1256, 2000 5. Liao SY, Aurelio ON, Jan K, et al: Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res 57:2827-2831, 1997 6. Murakami Y, Kanda K, Tsuji M, et al: MN/CA9 gene expression as a potential biomarker in renal cell carcinoma. BJU Int 83:743-747, 1999 7. McKiernan JM, Buttyan R, Bander NH, et al: Expression of the tumorassociated gene MN: A potential biomarker for human renal cell carcinoma. Cancer Res 57:2362-2365, 1997 8. Murphy WM, Grignon DJ, Perlman EJ: Tumors of the kidney, bladder, and related urinary structures (AFIP Atlas of Tumor Pathology, 4th Series). Washington, DC, American Registry of Pathology 2004, pp 109-136

9. Hes O, Michal M, Sulc M, et al: Glassy hyaline globules in granular cell carcinoma, chromophobe cell carcinoma, and oncocytoma of the kidney. Ann Diagn Pathol 2:12-18, 1998 10. Sandlund J, Oosterwijk E, Grankvist K, et al: Prognostic impact of carbonic anhydrase IX expression in human renal cell carcinoma. BJU Int 100:556-560, 2007 11. Bui MH, Seligson D, Han KR, et al: Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: Implications for prognosis and therapy. Clin Cancer Res 9:802-811, 2003 12. Bui MH, Visapaa H, Seligson D, et al: Prognostic value of carbonic anhydrase IX and KI67 as predictors of survival for renal clear cell carcinoma. J Urol 171:2461-2466, 2004 13. Iakovlev VV, Pintilie M, Morrison A, et al: Effect of distributional heterogeneity on the analysis of tumor hypoxia based on carbonic anhydrase IX. Lab Invest 87:1206-1217, 2007

DOI: 10.1200/JCO.2008.17.6511

■ ■ ■

IN REPLY: We are grateful for the attention that our article has generated from respected authorities in the field, and appreciate the opportunity to discuss the issue further. The correspondence from Brouwers et al raises several issues pertaining to the potential clinical use of carbonic anhydrase IX (CAIX). As stated in our article,1 our intent was to determine the role of CAIX as a potential independent predictor of survival for clear cell renal cell carcinoma (RCC). However, the lack of utility as an independent prognostic marker rightfully raises questions about the utility of CAIX for other clinical applications. As mentioned by Brouwers et al—and acknowledged in our article—several groups have previously surveyed normal tissues for the presence of CAIX.1-4 We reported our survey of CAIX expression in normal tissues specifically because such information has not been published in the context of targeting CAIX for the immunotherapeutic treatment of RCC or, in fact, for any other cancer. We did not compare intensity of staining between tissues from patients with RCC and patients with normal tissues. As such, Brouwers et al incorrectly suggest that our article discusses levels of staining in normal tissues, stating, “according to the authors, the CAIX expression levels in various normal tissues is high.” However, we made no mention of the level of staining in normal tissues in our article, rather, we simply stated, “CAIX expression was present in gastric mucosa, pancreatobiliary epithelium, and small intestine crypt base. Also, CAIX was seen in two specimens containing mesothelial cells, ovarian surface epithelium, and fetal rete testis.” Furthermore, we have been careful with regard to our statements pertaining to the clinical utility of CAIX as a potential therapeutic target. We state in the discussion of our article, “Unquestionably, targeting CAIX for therapy is attractive because it is highly expressed by ccRCC. To date, clinical trials of CAIX-targeted therapy have not caused any significant toxicity. Given that CAIX is expressed in multiple normal human organs and tissues, however, expectations of anti-CAIX based therapy may need to be tempered. Specifically, it may prove difficult to evoke clinically meaningful RCC tumor regression while maintaining a low toxicity profile, especially if the antibodies tested in the clinical setting recognize the same epitope as the M75 antibody used to survey CAIX expression in RCC tissues and normal organs.” Thus, our intention was to corroborate the presence of CAIX in normal tissues and to provide readers with a balanced understanding of potential concerns pertaining to therapeutic targeting of CAIX for the treatment of cancer. We remain hopeful that www.jco.org

current clinical trials will prove as efficacious as they have proven minimally toxic. We remain concerned that the lack of toxicity may be correlated with lack of efficacy, but hope to be proven wrong in this regard. In the interim, we feel it is prudent when planning future trials to point out the potential limitations of CAIX as a target. With regard to the use of radiolabeled CAIX as a potential imaging modality, Brouwers et al refer to additional studies that merit discussion, which were not originally referenced in our article.5-7 The correspondence from Brouwers et al states: “. . . our experience in other studies with radiolabeled cG250 showed mostly excellent visualization of known metastatic lesions and also frequently, new lesions were detected in patients.” The three additional references provided in the correspondence by Brouwers et al collectively report a total of 46 patients.5-7 None were designed to evaluate the sensitivity or specificity of CAIX as an imaging epitope. The most recent trial of the three by Brouwers et al examined the use of radiolabeled CAIX in 29 patients with metastatic RCC.5 Patients were required to have computed tomography (CT) imaging within 30 days of enrollment and all 29 patients had progressive metastatic cancer at the time of enrollment. Their findings included new lesions not seen on the CT, and they state, “The newly detected lesions were usually located outside the field of view of the chest/abdomen, but also some small (sub)cutaneous lesions and supraclavicular and axillary lymph node lesions were not noted on the CT. Conversely, some metastases visualized on CT were not detected scintigraphically: several lung metastases were not visualized on the scintigraphic images after a diagnostic or high-dose 131I-cG250 infusion. In only five of 25 patients with (numerous) pulmonary metastases were lung metastases clearly visualized after the diagnostic 131I-cG250 infusion.” Therefore, we stand by our assertion that there are currently few data to support the use of radiolabeled CAIX. Brouwers et al have previously reported that [18F]fluorodeoxyglucose-positron emission tomography was superior to 131I-cG250 radioimmunoscintigraphy.8 We are aware of a current large phase III study of CAIX radioimmunoscintigraphy, and remain hopeful that with continued development we may see an improvement in sensitivity. We are grateful to Skapa et al for their additional discussion and corroboration of our findings pertaining to the heterogeneity of CAIX expression in RCC. Though we agree that the heterogeneous staining of CAIX must be taken into account when assaying RCC tissues, we © 2008 by American Society of Clinical Oncology


3811