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Article Type : Original Article
Expression and diagnostic implications of carbonic anhydrase IX in several tumours with predominantly clear cell morphology
Xu-Yong Lin,1,2 Heng Zhang,1,2 Na Tang,1,2 Xiu-Peng Zhang,1,2 Wei Zhang,3 En-Hua Wang1,2 & Yu-Chen Han1,2
1
Department of Pathology, the First Affiliated Hospital and College of Basic Medical Sciences,
2
Institute of Pathology and Pathophysiology, China Medical University, Shenyang, China, and
3
Department of Pathology, Fushun Mining Bureau General Hospital, Fushun, China
Address for correspondence: Professor Yu-Chen Han, Department of Pathology, the First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang 110001, China. e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/his.12536 This article is protected by copyright. All rights reserved.
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Keywords: CA IX, clear cell renal cell carcinoma, clear cell hidradenoma, clear cell tumours, haemangioblastoma
Aims: High expression of carbonic anhydrase IX (CA IX) has been reported in clear cell renal cell carcinoma (ccRCC); few studies have reported CA IX expression in other tumours with predominantly clear cell morphology. The aim of study was to examine the expression and diagnostic implications of CA IX in these latter tumours.
Methods and results: An immunohistochemical study was performed of 159 tumours with predominantly clear cell morphology. The results showed that, in addition to primary (25/25) and metastatic (10/11) ccRCC, CA IX was also expressed in breast (2/2), pulmonary (3/5) and hepatic (1/4) clear cell carcinoma, urothelial carcinoma with clear cell change (3/6), clear cell meningioma (4/6) and ependymoma (2/3), haemangioblastoma (10/10), and clear cell hidradenoma (5/6). However, while strong and diffuse positivity for CA IX was observed in ccRCC, clear cell breast carcinoma, haemangioblastoma, and clear cell hidradenoma, the other cases showed predominantly focal positivity for CA IX. In particular, CA IX staining was often seen at the periphery of necrotic areas.
Conclusions: Our results indicate that strong and diffuse CA IX expression may be useful for differentiating ccRCC from several clear cell tumours, with the exception of clear cell breast carcinoma, haemangioblastoma, and clear cell hidradenoma.
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Introduction Carbonic anhydrase IX (CA IX) is a membrane-associated carbonic anhydrase that was first detected in the HeLa cancer cell line.1 In addition to regulating pH and being involved of cell adhesion, CA IX also functions in the growth of tumour cells under hypoxic conditions. CA IX is a downstream effector of the von Hippel–Lindau hypoxia-inducible pathway.2 Inactivation of the von Hippel–Lindau tumour suppressor gene (VHL), which has been found in approximately 60–80% of clear cell renal cell carcinoma (ccRCC) cases, can lead to the up-regulation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The gene encoding CA IX is one of the target genes of HIF-1α, and can be up-regulated through stabilization of HIF-1α.3 Because of the high frequency of VHL inactivation in ccRCCs, high expression of CA IX has been detected in ccRCC. Consequently, CA IX was considered to be a useful diagnostic marker for ccRCC.4,5 ccRCC accounts for the majority of RCCs.6 It can metastasize to almost any body site, and this poses a great challenge in distinguishing metastatic ccRCC from primary clear cell tumours. Although it has been reported that CAIX is also expressed in a variety of other malignancies,7–9 there are few reports on CA IX expression in other common clear cell tumours or tumours with predominantly clear cell morphology. We therefore performed an immunohistochemical study to investigate CAIX expression in tumours with predominantly clear cell morphology, including tumours in the lung, liver, thyroid, breast, prostate, parotid, adrenal gland, and brain.
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Materials and methods PATIENTS AND SPECIMENS
We collected paraffin-embedded samples of clear cell tumours from the First Affiliated Hospital of China Medical University, dating from 2009 to 2013. The total of 159 tumour tissues included: primary ccRCC (25), metastatic ccRCC (11), urothelial carcinoma with clear cell change (six), clear cell breast carcinoma (two), adrenocortical adenoma (eight), adrenocortical carcinoma (two), parathyroid adenoma (six), prostatic acinar cell carcinoma (12), pulmonary clear cell carcinoma (five), pulmonary perivascular epithelioid cell tumour (PEComa) (three), uterine clear cell carcinoma (seven), ovarian clear cell carcinoma (eight), hepatic clear cell carcinoma (four), clear cell sarcoma of soft tissue (10), clear cell meningioma (six), clear cell ependymoma (three), haemangioblastoma (10), parotid oncocytoma, clear cell type (one), parotid acinar cell carcinoma (eight), parotid clear cell carcinoma (six), cutaneous clear cell hidradenoma (six), clear cell thyroid carcinoma (four), and clear cell thyroid adenoma (six).
All specimens were re-evaluated for diagnosis by two independent pathologists according to the World Health Organization (WHO) criteria for classification of tumours. This study was conducted according to the regulations of the institutional review boards (China Medical University).
IMMUNOHISTOCHEMICAL STAINING
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All of the resected specimens had been fixed in 10% neutral buffered formalin and embedded in paraffin blocks. Sections were cut at 4-μm thickness, deparaffinized in xylene, rehydrated with graded alcohols, and immunostained using anti-CA IX (NB100-417, rabbit polyclonal antibody; Novus, Littleton, CO, USA) and a streptavidin–peroxidase system (KIT-9710, Ultrasensitive TM S-P; Maixin, Fuzhou， China); the chromogen used was diaminobenzidine tetrahydrochloride substrate (DAB kit; Maixin). Sections were lightly counterstained with haematoxylin, dehydrated, and mounted. For negative controls, the primary antibody was replaced with PBS. Five high-power fields of each section were examined for membranous staining, and 500 tumour cells were counted in each field. Membranous immunoreactivity was evaluated semiquantitatively as negative (0, 50% of cells stained).
Results The results of immunohistochemical staining for CA IX are summarized in Table 1. Using Fuhrman nuclear grading, the selected 25 ccRCCs included four grade 1, eight grade 2, seven grade 3, and six grade 4. Twelve grade 1 and grade 2 ccRCCs showed uniform and diffuse positivity for CA IX (Figure 1A,B). One grade 3 ccRCC and two grade 4 ccRCCs showed focally weak expression or lack of expression (Figure 1C,D). The adjacent normal kidney tissue was uniformly negative for CA IX. The 11 metastatic ccRCCs included lung (four), subcutaneous soft tissue (one), bone (one), liver (one), thyroid (two) and ovary (one) metastases. Except for a case
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of bone metastatic ccRCC, the metastatic ccRCCs showed strong and diffuse positivity for CA IX (Figure 3B,D,F).
OTHER UROGENITAL TUMOURS
All prostate acinar cell carcinomas (12), uterine clear cell carcinomas (seven) and ovarian clear cell carcinomas (seven) were uniformly negative for CA IX (Figure 3E). The adjacent normal tissue was negative in all cases. In addition, we examined CA IX expression in six urothelial carcinomas with predominantly clear cell change, and found that three of them showed positivity: ++ in one, and + in two (Figure 1E,F).
CLEAR CELL BREAST CARCINOMA
Both clear cell breast carcinomas were diffusely positive for CA IX. In contrast, the normal ductal epithelium was negative (Figure 2A,B).
PULMONARY CLEAR CELL CARCINOMA
Three of the five pulmonary clear cell carcinomas showed mild to moderate positivity for CA IX (Figure 3A). However, the expression lacked the diffuse staining seen in metastatic ccRCC of lung (Figure 3B), and the staining was more easily seen at the periphery of areas of necrosis.
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PULMONARY PERIVASCULAR EPITHELIOID CELL TUMOUR (PECOMA)
Three PEComas showed uniformly negative staining for CA IX (Figure 2C,D). In contrast, ciliated columnar epithelial cells of the bronchiole showed focal strong membranous staining.
HEPATIC CLEAR CELL CARCINOMA
One of the four hepatic clear cell carcinomas showed mild positivity for CA IX (Figure 3C), in contrast to the strong expression in metastatic ccRCC of liver (Figure 3D); the other three cases were negative. Normal hepatocytes were negative for CA IX.
TUMOURS OF ENDOCRINE/EXOCRINE GLANDS
We failed to detect CA IX expression in the eight adrenocortical adenomas and two adrenocortical carcinomas. The six thyroid clear cell adenomas, four thyroid clear cell carcinomas and six parathyroid adenomas were uniformly negative for CA IX, as were normal tissues.
The selected parotid tumours (one oncocytoma, eight acinar cell carcinomas, and six clear cell carcinomas) were all negative for CA IX.
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NERVOUS SYSTEM TUMOURS
Four of the six clear cell meningiomas and two of the three clear cell ependymomas showed focal positivity for CA IX (Figure 4A,B). In contrast, all 10 haemangioblastomas were diffusely and strongly positive for CA IX (Figure 4C,D).
CLEAR CELL SARCOMA AND CLEAR CELL HIDRADENOMA
All clear cell sarcomas of subcutaneous soft tissue were negative for CA IX. In contrast, five of the six clear cell hidradenomas of skin were positive, with three showing diffuse and strong positivity (Figure 5).
Discussion With advances in radiological technology and the widespread use of routine radiological imaging, an increasing number of RCCs are being incidentally detected. ccRCC accounts for the majority of all RCCs.6 Approximately one-third of patients may present with metastatic ccRCC, with spread to almost any body site, but commonly involving bone, lung, liver, and brain.10,11 Histologically, metastatic ccRCCs can show great overlap with primary clear cell tumours of the target organ. Moreover, in many patients, metastasis of ccRCCs may be their initial manifestation. Thus, it can be a great challenge to distinguish metastatic ccRCC from primary clear cell tumour.
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CA IX is an important downstream effector of the von Hippel–Lindau hypoxia-inducible pathway. Because of the high frequency of VHL inactivation and up-regulation of the hypoxia-inducible pathway in ccRCC, high expression of CA IX is detected in the majority of ccRCCs. Thus, CA IX was considered to be a useful diagnostic marker of ccRCC.12 However there have been few reports on CA IX expression in other clear cell tumours.13–16 .
In our immunohistochemical study, we investigated the usefulness of CA IX expression in the differential diagnosis of a wide variety of clear cell tumours and tumours with predominantly clear cell morphology. CA IX was found to be expressed in all 25 primary ccRCCs. In the majority of cases, the staining was diffuse and strong. Among 11 metastatic ccRCCs, CA IX expression was found in 10 cases, the exception being a case of bone metastatic ccRCC. Our findings were in agreement with other studies of CAIX expression in ccRCCs.4,5 Genega et al. reported that CA IX expression was associated with nuclear grade,17 and we found that CA IX could show focally weak or absent membranous expression in grade 3 or grade 4 ccRCC. We also examined CA IX staining in six urothelial carcinomas of the kidney with clear cell change, which can be easily confused with ccRCC. Unfortunately, three cases showed positivity for CA IX: ++ on one, and + in two. Therefore, it is challenging to differentiate high-grade ccRCC from urothelial carcinoma with clear cell change using this marker.
The selected metastatic ccRCCs involved lung, subcutaneous soft tissue, bone, liver, thyroid, and ovary. Primary clear cell tumour can also occur in lung, liver, thyroid, and ovary.
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Histologically, it may be difficult to differentiate primary and metastatic clear cell tumours. Therefore, immunostaining may be a useful method for differential diagnosis. Our results indicated that pulmonary PEComa, thyroid clear cell tumour (including adenoma and adenocarcinoma), ovarian clear cell carcinoma and clear cell sarcoma of soft tissue were uniformly negative for CA IX; pulmonary clear cell carcinoma and hepatic clear cell carcinoma were focally positive for this antigen. These results demonstrate that the diffuse CA IX expression in ccRCC may be related to VHL inactivation, whereas the focal staining in lung cancer and liver cancer appears to be related to focal tumour necrosis and hypoxia. Although published studies have reported CA IX expression in carcinoma of lung, ovary, and liver,7,14 in our experience the majority of these lack the diffuse staining that can be seen in ccRCC. Consequently, strong and diffuse CA IX staining may be potentially useful for distinguishing metastatic ccRCC from primary clear cell tumour in the above organs. However, it also should be noted that strong and diffuse CA IX staining may be lacking in high-grade ccRCC. Rarely, ccRCC can also metastasize to the adrenal gland, mimicking adrenocortical adenoma, and the lack of CA IX expression in adrenocortical adenoma indicates that this antigen may be of help in this differential diagnosis. Additionally, lack of CAIX expression was found in parathyroid adenoma and tumours of the parotid.
We also evaluated CA IX expression in clear cell meningioma and clear cell ependymoma. Three of six clear cell meningiomas and two of three clear cell ependymomas showed mild and focal positivity for CA IX; one clear cell meningioma showed moderate positivity. The staining of
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these two tumour types was predominantly focal and mild, and the lack of diffuse and strong CA IX expression may also be of help in differential diagnosis.
Haemangioblastoma is also related to VHL disorder and VHL protein inactivation, and has a similar histological structure to ccRCC, with abundant capillaries.18 As expected, haemangioblastoma also showed diffuse membranous positivity for CA IX, suggesting that diffuse expression was probably related to VHL protein inactivation.
We also examined CA IX expression in two primary clear cell carcinomas of the breast, which are rare carcinomas. Unexpectedly, diffuse and strong expression was found in both cases, in contrast to the lack of expression in normal ductal epithelium. Published studies have also reported CA IX expression in breast carcinoma, although the reported expression rate was lower, and there was usually a lack of diffuse staining.19–21 Moreover, CA IX expression was more often seen in high-grade or triple-negative breast cancer.20,21 In contrast, the present two cases were both grade 2, and both showed ER and PR expression. The significance of diffuse expression of CA IX in clear cell breast carcinoma remains unclear.
Across all tumours, we found that CA IX was predominantly expressed in those that were malignant. In addition to ccRCC, CA IX can also be expressed in carcinoma of the lung, liver, and breast, although the staining may be focal and mild. This indicates that CA This article is protected by copyright. All rights reserved.
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IX expression is more likely to be related to hypoxia in malignant tumours. In particular, we found that staining was seen particularly in areas of necrosis, which further indicates that necrosis and hypoxia may lead to CA IX expression. However, the benign tumour clear cell hidradenoma showed diffuse and strong positivity for CAIX, which is to our knowledge the first report of such expression, the significance of which remains uncertain.
In conclusion, in the present study we evaluated CA IX expression in a wide range of clear cell tumours. In addition to ccRCC, strong and diffuse expression was observed in clear cell breast carcinoma and clear cell hidradenoma, and focal expression in urothelial carcinoma, pulmonary clear cell carcinoma, hepatic clear cell carcinoma, clear cell meningioma, and clear cell ependymoma. Our immunohistochemical results indicate that strong and diffuse CA IX expression may be useful for recognizing ccRCC and haemangioblastoma related to VHL inactivation. The immunoreactivity for CA IX in hepatic and pulmonary clear cell carcinomas and clear cell meningiomas and ependymomas is more probably related to focal tissue necrosis and hypoxia. Interpretation of CA IX expression in tumours with clear cell morphology requires knowledge of the clinical setting and the results from a panel of other antibody markers.
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Acknowledgements The study was conducted according to the regulations of the institutional review boards at China Medical University. This work was supported by the National natural Science Foundation of China (grants 30971137 and 81272605).
Conflicts of interest The authors declare that they have no competing financial interests. There are no financial or other relationships that might lead to a conflict of interest of this paper.
Author contributions Xu-Yong Lin and Yu-Chen Han participated in the histopathological evaluation, performed the literature review, acquired photomicrographs, and drafted the manuscript. Heng Zhang and Na Tang performed immunohistochemical staining. Xiu-Peng Zhang and Wei Zhang conceived and designed the study. En-Hua Wang revised the manuscript. All authors read and approved the final manuscript.
References 1.
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2.
Linehan WM, Lerman MI, Zbar B. Identification of the von Hippel–Lindau (VHL) gene: its role in renal disease. JAMA 1995; 273; 564–570.
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Kaelin WG Jr. The von Hippel–Lindau tumor suppressor gene and kidney cancer. Clin. Cancer Res. 2004; 10; 6290–6295.
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Gupta R, Balzer B, Picken M et al. Diagnostic implications of transcription factor Pax2 protein and transmembrane enzyme complex carbonic anhydrase IX immunoreactivity in adult renal epithelial neoplasms. Am. J. Surg. Pathol. 2009; 33; 241–247.
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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. 1997; 57; 2827–2831.
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Eble JN, Sauter G, Epstein JI et al. eds. World Health Organization classification of tumours: pathology and genetics of tumours of the urinary system and male genital organs. Lyon: IARC Press, 2004.
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Ramsey ML, Yuh BJ, Johnson MT et al. Carbonic anhydrase IX is expressed in mesothelioma and metastatic clear cell renal cell carcinoma of the lung. Virchows Arch. 2012; 460; 89–93.
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Kaya AO, Gunel N, Benekli M et al. Hypoxia inducible factor-1 alpha and carbonic anhydrase IX overexpression are associated with poor survival in breast cancer patients. J. BUON. 2012; 17; 663–668.
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Hynninen P, Vaskivuo L, Saarnio J et al. Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours. Histopathology 2006; 49; 594–602.
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10. Azam F, Abubakerr M, Gollins S. Tongue metastasis as an initial presentation of renal cell carcinoma: a case report and literature review. J. Med. Case Rep. 2008; 2; 249.
11. Hoffmann NE, Gillett MD, Cheville JC et al. Differences in organ system of distant metastasis by renal cell carcinoma subtype. J. Urol. 2008; 179; 474–477.
12. Giménez-Bachs JM, Salinas-Sánchez AS, Serrano-Oviedo L et al. Carbonic anhydrase IX as a specific biomarker for clear cell renal cell carcinoma: comparative study of Western blot and immunohistochemistry and implications for diagnosis. Scand. J. Urol. Nephrol. 2012; 46; 358–364.
13. Donato DP, Johnson MT, Yang XJ et al. Expression of carbonic anhydrase IX in genitourinary and adrenal tumours. Histopathology 2011; 59; 1229–1239.
14. He H, Zhou GX, Zhou M et al. The distinction of clear cell carcinoma of the female genital tract, clear cell renal cell carcinoma, and translocation-associated renal cell carcinoma: an immunohistochemical study using tissue microarray. Int. J. Gynecol. Pathol. 2011; 30; 425–430.
15. Cimino-Mathews A, Sharma R, Netto GJ. Diagnostic use of PAX8, CAIX, TTF-1, and TGB in metastatic renal cell carcinoma of the thyroid. Am. J. Surg. Pathol. 2011; 35; 757–761.
16. Prayson RA, Chamberlain WA, Angelov L. Clear cell meningioma: a clinicopathologic study of 18 tumors and examination of the use of CD10, CA9, and RCC antibodies to distinguish between clear cell meningioma and metastatic clear cell renal cell carcinoma. Appl. Immunohistochem. Mol. Morphol. 2010; 18; 422–428.
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17. Genega EM, Ghebremichael M, Najarian R et al. Carbonic anhydrase IX expression in renal neoplasms: correlation with tumor type and grade. Am. J. Clin. Pathol. 2010; 134; 873–879.
18. Doyle LA, Fletcher CD. Peripheral hemangioblastoma: clinicopathologic characterization in a series of 22 cases. Am. J. Surg. Pathol. 2013; 38;119-127.
19. Pinheiro C, Sousa B, Albergaria A et al. GLUT1 and CAIX expression profiles in breast cancer correlate with adverse prognostic factors and MCT1 overexpression. Histol. Histopathol. 2011; 26; 1279–1286.
20. Chen CL, Chu JS, Su WC et al. Hypoxia and metabolic phenotypes during breast carcinogenesis: expression of HIF-1alpha, GLUT1, and CAIX. Virchows Arch. 2010; 457; 53–61.
21. Neumeister VM, Sullivan CA, Lindner R et al. Hypoxia-induced protein CAIX is associated with somatic loss of BRCA1 protein and pathway activity in triple negative breast cancer. Breast Cancer Res. Treat. 2012; 136; 67–75.
Figure 1. A,B, Clear cell renal cell carcinoma (ccRCC) showing diffuse and strong positivity for CA IX, in contrast to the lack of expression of this marker in normal tubular epithelium. C,D, High-grade ccRCC showed focal weak staining (C) or lack of CA IX expression (D). E, One case of urothelial carcinoma with clear cell change showed strong membranous positivity for CA IX. F, A urothelial carcinoma with clear cell change negative for CA IX.
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Figure 2. A,B, This clear cell breast carcinoma was diffusely positive for CA IX, whereas normal ductal epithelium was negative. C,D, Pulmonary PEComa tumour cells showing uniformly negative staining for CA IX, but ciliated columnar epithelial cells of bronchioles showed focal strong membranous staining.
Figure 3. A, This pulmonary clear cell carcinoma was focally positive for CA IX in the area peripheral to tumour necrosis. C, Hepatic clear cell carcinoma showing mild positivity for CA IX. B,D, These pulmonary and hepatic metastatic ccRCCs were diffusely and strongly positive for CA IX. E,F, Ovarian clear cell carcinoma was negative for CA IX (E), in contrast to the strong expression in ovarian metastatic ccRCC (F).
Figure 4. Clear cell meningioma (A) and clear cell ependymoma (B) showing focal positivity for CA IX. C,D, Haemangioblastomas showed diffuse positivity for CA IX.
Figure 5. Clear cell hidradenoma showing strong positivity for CA IX.
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Table 1. Immunohistochemical staining for CA IX in 159 tumours with predominantly clear cell morphology
CA IX staining Tumour type
n
0
1+
2+
3+ 22(four
2 (one Primary ccRCC
25
0
1 (Grade
Grade 4
4)
and one Grade 3)
Grade 1, eight Grade 2, six Grade 3 and four Grade 4.)
Metastatic ccRCC
11
1
0
0
10
6
3
2
1
0
Adrenocortical adenoma
8
8
0
0
0
Adrenocortical carcinoma
2
2
0
0
0
Prostatic acinar cell carcinoma
12
12
0
0
0
Breast clear cell carcinoma
2
0
0
0
2
Uterine clear cell carcinoma
7
7
0
0
0
Ovarian clear cell carcinoma
8
8
0
0
0
Pulmonary PEComa
3
3
0
0
0
Pulmonary clear cell carcinoma
5
2
2
1
0
Hepatic clear cell carcinoma
4
3
1
0
0
Thyroid clear cell adenoma
6
6
0
0
0
Thyroid clear cell carcinoma
4
4
0
0
0
Parathyroid adenoma
6
6
0
0
0
Parotid oncocytoma, clear cell type 1 rights1 reserved.0 This article is protected by copyright. All
0
0
Urothelial carcinoma with clear cell change
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Parotid acinar cell carcinoma
8
8
0
0
0
Parotid clear cell carcinoma
6
6
0
0
0
Clear cell meningioma
6
2
3
1
0
Clear cell ependymoma
3
1
2
0
0
Haemangioblastoma
10
0
0
0
10
Clear cell sarcoma of soft tissue
10
10
0
0
0
Cutaneous clear cell hidradenoma
6
1
1
1
3
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Article Type : Original Article
Expression and diagnostic implications of carbonic anhydrase IX in several tumours with predominantly clear cell morphology
Xu-Yong Lin,1,2 Heng Zhang,1,2 Na Tang,1,2 Xiu-Peng Zhang,1,2 Wei Zhang,3 En-Hua Wang1,2 & Yu-Chen Han1,2
1
Department of Pathology, the First Affiliated Hospital and College of Basic Medical Sciences,
2
Institute of Pathology and Pathophysiology, China Medical University, Shenyang, China, and
3
Department of Pathology, Fushun Mining Bureau General Hospital, Fushun, China
Address for correspondence: Professor Yu-Chen Han, Department of Pathology, the First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang 110001, China. e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/his.12536 This article is protected by copyright. All rights reserved.
Accepted Article
Keywords: CA IX, clear cell renal cell carcinoma, clear cell hidradenoma, clear cell tumours, haemangioblastoma
Aims: High expression of carbonic anhydrase IX (CA IX) has been reported in clear cell renal cell carcinoma (ccRCC); few studies have reported CA IX expression in other tumours with predominantly clear cell morphology. The aim of study was to examine the expression and diagnostic implications of CA IX in these latter tumours.
Methods and results: An immunohistochemical study was performed of 159 tumours with predominantly clear cell morphology. The results showed that, in addition to primary (25/25) and metastatic (10/11) ccRCC, CA IX was also expressed in breast (2/2), pulmonary (3/5) and hepatic (1/4) clear cell carcinoma, urothelial carcinoma with clear cell change (3/6), clear cell meningioma (4/6) and ependymoma (2/3), haemangioblastoma (10/10), and clear cell hidradenoma (5/6). However, while strong and diffuse positivity for CA IX was observed in ccRCC, clear cell breast carcinoma, haemangioblastoma, and clear cell hidradenoma, the other cases showed predominantly focal positivity for CA IX. In particular, CA IX staining was often seen at the periphery of necrotic areas.
Conclusions: Our results indicate that strong and diffuse CA IX expression may be useful for differentiating ccRCC from several clear cell tumours, with the exception of clear cell breast carcinoma, haemangioblastoma, and clear cell hidradenoma.
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Introduction Carbonic anhydrase IX (CA IX) is a membrane-associated carbonic anhydrase that was first detected in the HeLa cancer cell line.1 In addition to regulating pH and being involved of cell adhesion, CA IX also functions in the growth of tumour cells under hypoxic conditions. CA IX is a downstream effector of the von Hippel–Lindau hypoxia-inducible pathway.2 Inactivation of the von Hippel–Lindau tumour suppressor gene (VHL), which has been found in approximately 60–80% of clear cell renal cell carcinoma (ccRCC) cases, can lead to the up-regulation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The gene encoding CA IX is one of the target genes of HIF-1α, and can be up-regulated through stabilization of HIF-1α.3 Because of the high frequency of VHL inactivation in ccRCCs, high expression of CA IX has been detected in ccRCC. Consequently, CA IX was considered to be a useful diagnostic marker for ccRCC.4,5 ccRCC accounts for the majority of RCCs.6 It can metastasize to almost any body site, and this poses a great challenge in distinguishing metastatic ccRCC from primary clear cell tumours. Although it has been reported that CAIX is also expressed in a variety of other malignancies,7–9 there are few reports on CA IX expression in other common clear cell tumours or tumours with predominantly clear cell morphology. We therefore performed an immunohistochemical study to investigate CAIX expression in tumours with predominantly clear cell morphology, including tumours in the lung, liver, thyroid, breast, prostate, parotid, adrenal gland, and brain.
This article is protected by copyright. All rights reserved.
Accepted Article
Materials and methods PATIENTS AND SPECIMENS
We collected paraffin-embedded samples of clear cell tumours from the First Affiliated Hospital of China Medical University, dating from 2009 to 2013. The total of 159 tumour tissues included: primary ccRCC (25), metastatic ccRCC (11), urothelial carcinoma with clear cell change (six), clear cell breast carcinoma (two), adrenocortical adenoma (eight), adrenocortical carcinoma (two), parathyroid adenoma (six), prostatic acinar cell carcinoma (12), pulmonary clear cell carcinoma (five), pulmonary perivascular epithelioid cell tumour (PEComa) (three), uterine clear cell carcinoma (seven), ovarian clear cell carcinoma (eight), hepatic clear cell carcinoma (four), clear cell sarcoma of soft tissue (10), clear cell meningioma (six), clear cell ependymoma (three), haemangioblastoma (10), parotid oncocytoma, clear cell type (one), parotid acinar cell carcinoma (eight), parotid clear cell carcinoma (six), cutaneous clear cell hidradenoma (six), clear cell thyroid carcinoma (four), and clear cell thyroid adenoma (six).
All specimens were re-evaluated for diagnosis by two independent pathologists according to the World Health Organization (WHO) criteria for classification of tumours. This study was conducted according to the regulations of the institutional review boards (China Medical University).
IMMUNOHISTOCHEMICAL STAINING
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All of the resected specimens had been fixed in 10% neutral buffered formalin and embedded in paraffin blocks. Sections were cut at 4-μm thickness, deparaffinized in xylene, rehydrated with graded alcohols, and immunostained using anti-CA IX (NB100-417, rabbit polyclonal antibody; Novus, Littleton, CO, USA) and a streptavidin–peroxidase system (KIT-9710, Ultrasensitive TM S-P; Maixin, Fuzhou， China); the chromogen used was diaminobenzidine tetrahydrochloride substrate (DAB kit; Maixin). Sections were lightly counterstained with haematoxylin, dehydrated, and mounted. For negative controls, the primary antibody was replaced with PBS. Five high-power fields of each section were examined for membranous staining, and 500 tumour cells were counted in each field. Membranous immunoreactivity was evaluated semiquantitatively as negative (0, 50% of cells stained).
Results The results of immunohistochemical staining for CA IX are summarized in Table 1. Using Fuhrman nuclear grading, the selected 25 ccRCCs included four grade 1, eight grade 2, seven grade 3, and six grade 4. Twelve grade 1 and grade 2 ccRCCs showed uniform and diffuse positivity for CA IX (Figure 1A,B). One grade 3 ccRCC and two grade 4 ccRCCs showed focally weak expression or lack of expression (Figure 1C,D). The adjacent normal kidney tissue was uniformly negative for CA IX. The 11 metastatic ccRCCs included lung (four), subcutaneous soft tissue (one), bone (one), liver (one), thyroid (two) and ovary (one) metastases. Except for a case
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of bone metastatic ccRCC, the metastatic ccRCCs showed strong and diffuse positivity for CA IX (Figure 3B,D,F).
OTHER UROGENITAL TUMOURS
All prostate acinar cell carcinomas (12), uterine clear cell carcinomas (seven) and ovarian clear cell carcinomas (seven) were uniformly negative for CA IX (Figure 3E). The adjacent normal tissue was negative in all cases. In addition, we examined CA IX expression in six urothelial carcinomas with predominantly clear cell change, and found that three of them showed positivity: ++ in one, and + in two (Figure 1E,F).
CLEAR CELL BREAST CARCINOMA
Both clear cell breast carcinomas were diffusely positive for CA IX. In contrast, the normal ductal epithelium was negative (Figure 2A,B).
PULMONARY CLEAR CELL CARCINOMA
Three of the five pulmonary clear cell carcinomas showed mild to moderate positivity for CA IX (Figure 3A). However, the expression lacked the diffuse staining seen in metastatic ccRCC of lung (Figure 3B), and the staining was more easily seen at the periphery of areas of necrosis.
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PULMONARY PERIVASCULAR EPITHELIOID CELL TUMOUR (PECOMA)
Three PEComas showed uniformly negative staining for CA IX (Figure 2C,D). In contrast, ciliated columnar epithelial cells of the bronchiole showed focal strong membranous staining.
HEPATIC CLEAR CELL CARCINOMA
One of the four hepatic clear cell carcinomas showed mild positivity for CA IX (Figure 3C), in contrast to the strong expression in metastatic ccRCC of liver (Figure 3D); the other three cases were negative. Normal hepatocytes were negative for CA IX.
TUMOURS OF ENDOCRINE/EXOCRINE GLANDS
We failed to detect CA IX expression in the eight adrenocortical adenomas and two adrenocortical carcinomas. The six thyroid clear cell adenomas, four thyroid clear cell carcinomas and six parathyroid adenomas were uniformly negative for CA IX, as were normal tissues.
The selected parotid tumours (one oncocytoma, eight acinar cell carcinomas, and six clear cell carcinomas) were all negative for CA IX.
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NERVOUS SYSTEM TUMOURS
Four of the six clear cell meningiomas and two of the three clear cell ependymomas showed focal positivity for CA IX (Figure 4A,B). In contrast, all 10 haemangioblastomas were diffusely and strongly positive for CA IX (Figure 4C,D).
CLEAR CELL SARCOMA AND CLEAR CELL HIDRADENOMA
All clear cell sarcomas of subcutaneous soft tissue were negative for CA IX. In contrast, five of the six clear cell hidradenomas of skin were positive, with three showing diffuse and strong positivity (Figure 5).
Discussion With advances in radiological technology and the widespread use of routine radiological imaging, an increasing number of RCCs are being incidentally detected. ccRCC accounts for the majority of all RCCs.6 Approximately one-third of patients may present with metastatic ccRCC, with spread to almost any body site, but commonly involving bone, lung, liver, and brain.10,11 Histologically, metastatic ccRCCs can show great overlap with primary clear cell tumours of the target organ. Moreover, in many patients, metastasis of ccRCCs may be their initial manifestation. Thus, it can be a great challenge to distinguish metastatic ccRCC from primary clear cell tumour.
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CA IX is an important downstream effector of the von Hippel–Lindau hypoxia-inducible pathway. Because of the high frequency of VHL inactivation and up-regulation of the hypoxia-inducible pathway in ccRCC, high expression of CA IX is detected in the majority of ccRCCs. Thus, CA IX was considered to be a useful diagnostic marker of ccRCC.12 However there have been few reports on CA IX expression in other clear cell tumours.13–16 .
In our immunohistochemical study, we investigated the usefulness of CA IX expression in the differential diagnosis of a wide variety of clear cell tumours and tumours with predominantly clear cell morphology. CA IX was found to be expressed in all 25 primary ccRCCs. In the majority of cases, the staining was diffuse and strong. Among 11 metastatic ccRCCs, CA IX expression was found in 10 cases, the exception being a case of bone metastatic ccRCC. Our findings were in agreement with other studies of CAIX expression in ccRCCs.4,5 Genega et al. reported that CA IX expression was associated with nuclear grade,17 and we found that CA IX could show focally weak or absent membranous expression in grade 3 or grade 4 ccRCC. We also examined CA IX staining in six urothelial carcinomas of the kidney with clear cell change, which can be easily confused with ccRCC. Unfortunately, three cases showed positivity for CA IX: ++ on one, and + in two. Therefore, it is challenging to differentiate high-grade ccRCC from urothelial carcinoma with clear cell change using this marker.
The selected metastatic ccRCCs involved lung, subcutaneous soft tissue, bone, liver, thyroid, and ovary. Primary clear cell tumour can also occur in lung, liver, thyroid, and ovary.
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Histologically, it may be difficult to differentiate primary and metastatic clear cell tumours. Therefore, immunostaining may be a useful method for differential diagnosis. Our results indicated that pulmonary PEComa, thyroid clear cell tumour (including adenoma and adenocarcinoma), ovarian clear cell carcinoma and clear cell sarcoma of soft tissue were uniformly negative for CA IX; pulmonary clear cell carcinoma and hepatic clear cell carcinoma were focally positive for this antigen. These results demonstrate that the diffuse CA IX expression in ccRCC may be related to VHL inactivation, whereas the focal staining in lung cancer and liver cancer appears to be related to focal tumour necrosis and hypoxia. Although published studies have reported CA IX expression in carcinoma of lung, ovary, and liver,7,14 in our experience the majority of these lack the diffuse staining that can be seen in ccRCC. Consequently, strong and diffuse CA IX staining may be potentially useful for distinguishing metastatic ccRCC from primary clear cell tumour in the above organs. However, it also should be noted that strong and diffuse CA IX staining may be lacking in high-grade ccRCC. Rarely, ccRCC can also metastasize to the adrenal gland, mimicking adrenocortical adenoma, and the lack of CA IX expression in adrenocortical adenoma indicates that this antigen may be of help in this differential diagnosis. Additionally, lack of CAIX expression was found in parathyroid adenoma and tumours of the parotid.
We also evaluated CA IX expression in clear cell meningioma and clear cell ependymoma. Three of six clear cell meningiomas and two of three clear cell ependymomas showed mild and focal positivity for CA IX; one clear cell meningioma showed moderate positivity. The staining of
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these two tumour types was predominantly focal and mild, and the lack of diffuse and strong CA IX expression may also be of help in differential diagnosis.
Haemangioblastoma is also related to VHL disorder and VHL protein inactivation, and has a similar histological structure to ccRCC, with abundant capillaries.18 As expected, haemangioblastoma also showed diffuse membranous positivity for CA IX, suggesting that diffuse expression was probably related to VHL protein inactivation.
We also examined CA IX expression in two primary clear cell carcinomas of the breast, which are rare carcinomas. Unexpectedly, diffuse and strong expression was found in both cases, in contrast to the lack of expression in normal ductal epithelium. Published studies have also reported CA IX expression in breast carcinoma, although the reported expression rate was lower, and there was usually a lack of diffuse staining.19–21 Moreover, CA IX expression was more often seen in high-grade or triple-negative breast cancer.20,21 In contrast, the present two cases were both grade 2, and both showed ER and PR expression. The significance of diffuse expression of CA IX in clear cell breast carcinoma remains unclear.
Across all tumours, we found that CA IX was predominantly expressed in those that were malignant. In addition to ccRCC, CA IX can also be expressed in carcinoma of the lung, liver, and breast, although the staining may be focal and mild. This indicates that CA This article is protected by copyright. All rights reserved.
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IX expression is more likely to be related to hypoxia in malignant tumours. In particular, we found that staining was seen particularly in areas of necrosis, which further indicates that necrosis and hypoxia may lead to CA IX expression. However, the benign tumour clear cell hidradenoma showed diffuse and strong positivity for CAIX, which is to our knowledge the first report of such expression, the significance of which remains uncertain.
In conclusion, in the present study we evaluated CA IX expression in a wide range of clear cell tumours. In addition to ccRCC, strong and diffuse expression was observed in clear cell breast carcinoma and clear cell hidradenoma, and focal expression in urothelial carcinoma, pulmonary clear cell carcinoma, hepatic clear cell carcinoma, clear cell meningioma, and clear cell ependymoma. Our immunohistochemical results indicate that strong and diffuse CA IX expression may be useful for recognizing ccRCC and haemangioblastoma related to VHL inactivation. The immunoreactivity for CA IX in hepatic and pulmonary clear cell carcinomas and clear cell meningiomas and ependymomas is more probably related to focal tissue necrosis and hypoxia. Interpretation of CA IX expression in tumours with clear cell morphology requires knowledge of the clinical setting and the results from a panel of other antibody markers.
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Acknowledgements The study was conducted according to the regulations of the institutional review boards at China Medical University. This work was supported by the National natural Science Foundation of China (grants 30971137 and 81272605).
Conflicts of interest The authors declare that they have no competing financial interests. There are no financial or other relationships that might lead to a conflict of interest of this paper.
Author contributions Xu-Yong Lin and Yu-Chen Han participated in the histopathological evaluation, performed the literature review, acquired photomicrographs, and drafted the manuscript. Heng Zhang and Na Tang performed immunohistochemical staining. Xiu-Peng Zhang and Wei Zhang conceived and designed the study. En-Hua Wang revised the manuscript. All authors read and approved the final manuscript.
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Figure 1. A,B, Clear cell renal cell carcinoma (ccRCC) showing diffuse and strong positivity for CA IX, in contrast to the lack of expression of this marker in normal tubular epithelium. C,D, High-grade ccRCC showed focal weak staining (C) or lack of CA IX expression (D). E, One case of urothelial carcinoma with clear cell change showed strong membranous positivity for CA IX. F, A urothelial carcinoma with clear cell change negative for CA IX.
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Figure 2. A,B, This clear cell breast carcinoma was diffusely positive for CA IX, whereas normal ductal epithelium was negative. C,D, Pulmonary PEComa tumour cells showing uniformly negative staining for CA IX, but ciliated columnar epithelial cells of bronchioles showed focal strong membranous staining.
Figure 3. A, This pulmonary clear cell carcinoma was focally positive for CA IX in the area peripheral to tumour necrosis. C, Hepatic clear cell carcinoma showing mild positivity for CA IX. B,D, These pulmonary and hepatic metastatic ccRCCs were diffusely and strongly positive for CA IX. E,F, Ovarian clear cell carcinoma was negative for CA IX (E), in contrast to the strong expression in ovarian metastatic ccRCC (F).
Figure 4. Clear cell meningioma (A) and clear cell ependymoma (B) showing focal positivity for CA IX. C,D, Haemangioblastomas showed diffuse positivity for CA IX.
Figure 5. Clear cell hidradenoma showing strong positivity for CA IX.
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Table 1. Immunohistochemical staining for CA IX in 159 tumours with predominantly clear cell morphology
CA IX staining Tumour type
n
0
1+
2+
3+ 22(four
2 (one Primary ccRCC
25
0
1 (Grade
Grade 4
4)
and one Grade 3)
Grade 1, eight Grade 2, six Grade 3 and four Grade 4.)
Metastatic ccRCC
11
1
0
0
10
6
3
2
1
0
Adrenocortical adenoma
8
8
0
0
0
Adrenocortical carcinoma
2
2
0
0
0
Prostatic acinar cell carcinoma
12
12
0
0
0
Breast clear cell carcinoma
2
0
0
0
2
Uterine clear cell carcinoma
7
7
0
0
0
Ovarian clear cell carcinoma
8
8
0
0
0
Pulmonary PEComa
3
3
0
0
0
Pulmonary clear cell carcinoma
5
2
2
1
0
Hepatic clear cell carcinoma
4
3
1
0
0
Thyroid clear cell adenoma
6
6
0
0
0
Thyroid clear cell carcinoma
4
4
0
0
0
Parathyroid adenoma
6
6
0
0
0
Parotid oncocytoma, clear cell type 1 rights1 reserved.0 This article is protected by copyright. All
0
0
Urothelial carcinoma with clear cell change
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Parotid acinar cell carcinoma
8
8
0
0
0
Parotid clear cell carcinoma
6
6
0
0
0
Clear cell meningioma
6
2
3
1
0
Clear cell ependymoma
3
1
2
0
0
Haemangioblastoma
10
0
0
0
10
Clear cell sarcoma of soft tissue
10
10
0
0
0
Cutaneous clear cell hidradenoma
6
1
1
1
3
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