Carbonic anhydrase IX is expressed in mesothelioma and metastatic ...

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High immunohistochemical expression of carbonic anhydrase IX (CAIX) is found in clear cell renal cell carcinoma (ccRCC), but no studies have assessed CAIX ...
Virchows Arch (2012) 460:89–93 DOI 10.1007/s00428-011-1178-7

ORIGINAL ARTICLE

Carbonic anhydrase IX is expressed in mesothelioma and metastatic clear cell renal cell carcinoma of the lung Mitchell L. Ramsey & Benjamin J. Yuh & Matthew T. Johnson & Anjana V. Yeldandi & Debra L. Zynger

Received: 12 October 2011 / Revised: 10 November 2011 / Accepted: 22 November 2011 / Published online: 6 December 2011 # Springer-Verlag 2011

Abstract High immunohistochemical expression of carbonic anhydrase IX (CAIX) is found in clear cell renal cell carcinoma (ccRCC), but no studies have assessed CAIX in metastatic ccRCC (mccRCC) of the lung. As 75% of patients with mccRCC show lung involvement, characterization of protein expression in these lesions is warranted. This investigation analyzed CAIX immunohistochemical expression in pulmonary/pleural tumors including mccRCC (n022), mesothelioma (n019), squamous cell carcinoma (n027), small cell carcinoma (n09), and adenocarcinoma (n049), as well as other mesothelial lesions (n04). Membranous immunoreactivity was semiquantitatively evaluated for percent of cells stained and intensity. All cases of mccRCC (1+, 4.5%; 3+, 95.5%) and mesothelioma (2+, 10.5%; 3+, 89.5%) expressed CAIX. Most cases of lung squamous cell carcinoma (0, 11.1%; 1+, 25.9%; 2+, 22.2%; 3+, 40.7%) and small cell carcinoma were reactive (0, 11.1%; 1+, 22.2%; 2+, 33.3%; 3+, 33.3%), while CAIX was detected less frequently in pulmonary adenocarcinoma (0, 61.2%; 1+, 16.3%; 2+, 12.2%; 3+, 10.2%). In addition, CAIX was positive in adenomatoid tumor (3+, 100%) and mesothelial hyperplasia (3+, 100%). M. L. Ramsey : B. J. Yuh : D. L. Zynger (*) Department of Pathology, The Ohio State University Medical Center, 410 W 10th Ave., E401 Doan Hall, Columbus, OH 43210, USA e-mail: [email protected] M. T. Johnson Department of Urology, The Ohio State University Medical Center, Columbus, OH 43210, USA A. V. Yeldandi Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA

We demonstrate that CAIX is sensitive for mccRCC within the lung and a novel immunohistochemical marker for mesothelial proliferations, notably mesothelioma. Variable immunoreactivity is present among primary pulmonary epithelial tumors. Knowledge of expression overlap between these entities may prevent an incorrect interpretation of immunohistochemical results, particularly when limited tissue is available. As new carbonic anhydrase inhibitors are being evaluated, testing additional tumors for CAIX may lead to novel treatment options. Keywords Carbonic anhydrase IX . CAIX . Mesothelioma . Metastatic . Clear cell renal cell carcinoma . Lung

Introduction Carbonic anhydrase IX (CAIX) is a transmembrane, zinc-containing metalloenzyme that catalyzes reversible reactions of the bicarbonate buffer system to regulate pH in hypoxic conditions [1–3]. CAIX is overexpressed in aggressive tumors where rapid growth limits available oxygen [3–5]. However, CAIX is not limited to cancerous cells but is present in gastric mucosa, larger bile ducts, and several other locations [3, 6, 7]. In addition, Northern blot analysis and immunohistochemistry techniques were used to demonstrate expression of CAIX in nonneoplastic mesothelial cells [1, 3, 6]. CAIX has been well described as a diagnostic marker for clear cell renal cell carcinoma (ccRCC) [3, 7–11], with a few studies noting generally high expression in metastatic ccRCC (mccRCC) [3, 7, 8]. Although 85% of ccRCC recurrences present within 3 years, ccRCC is known for late metastases to the lung, liver, bone, and brain [9, 12, 13]. When metastatic, approximately 75% of tumors have lung involvement [14]. However, CAIX expression of mccRCC

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Table 1 CAIX immunohistochemical expression in pulmonary lesions Percentage of cells stained

Intensity (mean)

0 (50%)

Metastatic ccRCC to the lung (n022)

0

1 (4.5%)

0

21 (95.5%)

2.9

Mesothelioma of the pleura (n019) Mesothelial hyperplasia of the pleura (n01)

0 0

0 0

2 (10.5%) 0

17 (89.5%) 1 (100%)

2.8 3.0

Adenomatoid tumor (n03)

0

0

0

3 (100%)

2.7

Squamous cell carcinoma of the lung (n027)

3 (11.1%)

7 (25.9%)

6 (22.2%)

11 (40.7%)

2.3

Small cell carcinoma of the lung (n09)

1 (11.1%)

2 (22.2%)

3 (33.3%)

3 (33.3%)

2.3

Adenocarcinoma of the lung (n049)

30 (61.2%)

8 (16.3%)

6 (12.2%)

5 (10.2%)

0.8

Nonneoplastic lung (bronchial epithelium/pneumocytes) (n024)

24 (100%)

0

0

0

0

mccRCC within the lung and pleural mesothelioma have consistent CAIX expression. Squamous cell carcinoma and small cell carcinoma have variable expression, while adenocarcinoma has infrequent diffuse expression.

has not yet been evaluated in these metastases. Understanding the expression overlap between primary lung tumors and mccRCC to the lung is relevant for diagnosis and potential treatment. Some patients with mccRCC have shown favorable outcomes with treatment by interferon-α monotherapy, interleukin (IL)-2, or combination therapy [14, 15]. Recent advances in the sulfamide, sulfamate, and sulfonamide series may provide alternative treatments through CAIX inhibition [16]. Regarding primary lung tumors, previous articles report variable expression of CAIX in non-small cell lung carcinoma [17, 18]. Expression of CAIX in mesothelioma effusions was identified via reverse transcriptase polymerase chain reaction, but mesothelioma has yet to be tested by immunohistochemical analysis [17]. This study aims to assess CAIX protein expression of mccRCC in the lung, epithelial lung tumors, and pleural mesothelioma via immunohistochemistry. Better understanding of the expression profile will aid in diagnostics and may lead to novel treatment opportunities.

Materials and methods Archived formalin-fixed, paraffin-embedded tissue blocks were obtained from The Ohio State University Medical Center and Northwestern Memorial Hospital. Tissues from 130 tumors were sampled, which contained eight types of neoplasms and nonneoplastic conditions. All diagnoses were based on World Health Organization histologic criteria [19]. Four-micrometer-sections were dried at 55°C for 3 h then subjected to heat-induced epitope retrieval for 25 min at pH 6.0 on a Leica-Bond Autostainer. The slides were incubated with diluted CAIX monoclonal antibody (Leica, NCL-L-CAIX, 1:100) for 30 min. Bond Polymer Refine Detection kit was applied, and the sections were counterstained with hematoxylin.

Immunohistochemical scoring was based on membranous reactivity and was recorded semiquantitatively as follows: negative (0, 50% of cells stained). Staining intensity was averaged across the lesion and graded from 0 to 3, with 3 being the most intense. Non-mccRCC was used as a positive control, and ccRCC lacking the CAIX primary antibody was used as a negative control. When possible, cases were also evaluated for CAIX staining in bronchial epithelium and alveolar pneumocytes.

Results Table 1 depicts the immunohistochemical results for CAIX expression in lung lesions. All mccRCC cases (n022; 12 biopsies, 10 resections) demonstrated positivity, with only 1 case that did not exhibit diffuse expression (1+, 4.5%; 3+, 95.5%) (Fig. 1a and b). Three cases with sarcomatoid differentiation were included, one of which had focal staining (Fig. 1c and d). The mean intensity for mccRCC was high (2.9). Fig. 1 CAIX expression in lung lesions. (a, b) Hematoxylin and eosin b and CAIX immunohistochemical stains of mccRCC with diffuse, membranous reactivity. Alveolar pneumocytes are negative (upper left) (×20). (c, d) mccRCC with sarcomatoid differentiation demonstrating diffuse, moderate expression (×20). (e, f) Epithelioid mesothelioma exhibiting strong and diffuse membranous and cytoplasmic reactivity (×20). (g, h) The sarcomatoid focus in a biphasic mesothelioma expresses CAIX strongly in a membranocytoplasmic pattern (×20). (i, j) Adenomatoid tumor displays strong, diffuse membranous and cytoplasmic reactivity (×20). (k, l) Squamous cell carcinoma of the lung highly expresses CAIX, notably at the periphery of tumor nests (×20). (m, n) Small cell carcinoma with strong, diffuse membranous staining (×20). (o, p) Primary lung adenocarcinoma is negative for membranous CAIX reactivity with weak focal cytoplasmic expression (×20)

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92

All cases of pleural mesothelioma (n019; 13 biopsies, 6 resections) expressed CAIX strongly and diffusely (2+, 10.5%; 3+, 89.5%; mean intensity02.8) (Fig. 1e and f). Mesothelioma included epithelioid (n014) and biphasic subtypes (spindle cell and epithelioid) (n05), with no appreciable difference in reactivity (Fig. 1g and h). Other mesothelial lesions with strong and diffuse reactivity include mesothelial hyperplasia (n01 biopsy) of the pleura (3+, 100%; intensity03.0) and adenomatoid tumor of the testicle (n02 resections) and fallopian tube (n01 resection) (3+, 100%; mean intensity02.7) (Fig. 1i and j). Regarding epithelial lung primary tumors, squamous cell carcinoma (n027 resections) had the highest percentage of diffusely positive cases (0, 11.1%; 1+, 25.9%; 2+, 22.2%; 3+, 40.7%; mean intensity02.3) (Fig. 1k and l). In squamous cell carcinoma, expression was most intense at the periphery of squamous nests. Small cell carcinoma of the lung (n09 resections) had variable reactivity (0, 11.1%; 1+, 22.2%; 2+, 33.3%; 3+, 33.3%; mean intensity02.3) (Fig. 1m and n). Lung adenocarcinoma (n049 resections) was predominately negative with occasional diffuse expression (0, 61.2%, 1+, 16.3%; 2+, 12.2%; 3+, 10.2%; mean intensity00.8) (Fig. 1o and p). Associated nonneoplastic lung tissues were evaluated for CAIX expression (n024). Among these, bronchial epithelium (n015) was consistently negative for membranous expression (0, 100%; mean intensity00), although 53.3% revealed strong and diffuse cytoplasmic staining. Alveolar pneumocytes were negative for CAIX (0, 100%; mean intensity00) (Fig. 1a and b, upper left). Pleural nonneoplastic mesothelium (n03) had strong and diffuse expression (3+, 100%; mean intensity03.0). A single case contained bronchial submucosal glandular tissue, which was negative (0, 100%; mean intensity00).

Discussion CAIX has been identified as a reliable marker for ccRCC, with promising results for mccRCC [3, 7–11]. While it is known that mccRCC commonly involves the lung, there are no reports specifically detailing the expression of CAIX in mccRCC of the lung [14]. This study aims to test lung tumors for CAIX expression to determine the diagnostic utility of this marker as well as potential candidacy for CAIX-related treatments. Nonneoplastic mesothelium has been reported to diffusely express CAIX via immunohistochemistry [6]. Using reverse transcriptase polymerase chain reaction to assay malignant pleural effusions in patients with a history of mesothelioma, researchers detected CAIX gene expression in seven of eight cases [17]. We describe for the first time the immunohistochemical expression of CAIX in pleural mesothelioma.

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Concordant with prior preliminary data, CAIX was detected in all cases of mesothelioma, including biopsies and resections. Membranous expression was strong and diffuse in 89.5% of tumors, with similar expression seen in epithelioid and biphasic subtypes. Other mesothelial proliferations, including mesothelial hyperplasia of the pleura and adenomatoid tumor, were diffusely positive. CAIX appears to be an additional novel mesothelial marker. We demonstrated CAIX immunohistochemical expression in all cases of mccRCC in the lung, including resections and biopsies. Published data reveal high CAIX expression in 69% to 75% of mccRCC, with metastases generally expressing CAIX less than the primary tumor [3, 7, 8, 20, 21]. While lung metastases were likely included in earlier studies, specific data for metastatic sites were not provided [3, 7, 8, 20, 21]. The few cases with sarcomatoid differentiation that we examined had less consistent expression. Similarly, Tickoo et al. [22] found 82% of organ confined sarcomatoid ccRCC to express CAIX compared with 95% of ccRCC without sarcomatoid differentiation. As anticipated, CAIX has a high sensitivity for mccRCC of the lung. Among primary lung tumors, we report CAIX expression in 89% of squamous cell carcinoma, 89% of small cell carcinoma, and 39% of adenocarcinoma. Squamous cell carcinoma and small cell carcinoma varied in percentage of reactive cells. Both tumors did have a substantial subset that was diffusely positive and could mimic the expression seen in mccRCC and mesothelioma. Prior authors observed CAIX expression in 58% to 96% of pulmonary squamous cell carcinoma [23–25]. We found adenocarcinoma to be predominately negative with only 10% of cases diffusely positive, unlike the other neoplasms. A range of CAIX expression of 30%–81% in lung adenocarcinoma has been noted in the literature [23–25]. Kim et al. [23, 24] concluded that CAIX expression is significantly higher in squamous cell carcinoma than in adenocarcinoma, compatible with our data. No former studies have assessed CAIX expression in small cell carcinoma of the lung for comparison. In light of the variable reactivity in primary lung epithelial tumors, CAIX does not seem to be a useful diagnostic marker for these lesions, although diffuse expression of CAIX does not favor a diagnosis of pulmonary adenocarcinoma. The potential roles for CAIX include serum and radiologic diagnostics as well as guiding or predicting therapeutic response [7, 10, 16, 21, 26, 27]. While the usual treatment for primary ccRCC is surgical, targeted therapy has resulted in improved long-term disease-free survival in those with metastatic disease. Higher CAIX expression tends to increase the efficacy of IL-2, and CAIX screening may improve patient selection for higher-dose IL-2 regimens [10, 26, 27]. Numerous publications describe the usage of IL-2 for treatment of mccRCC, and by extension, other CAIX-expressing tumors may be implicated [7, 21, 26]. In addition to IL-2 (and

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interferon-α combination treatments), recent advances in carbonic anhydrase inhibiting drugs may result in treatment options for a variety of CAIX-dependent tumors arising from the sulfamide, sulfonamide, and sulfamate series [16]. In conclusion, CAIX immunohistochemical expression has a high sensitivity for mccRCC and pleural mesothelioma. However, it is important to note the overlap of primary lung tumor reactivity. In the wake of numerous reports of CAIXbased treatment for ccRCC, there may be future applications in diagnostic assessment, treatment, and radiologic imaging of CAIX-expressing lung tumors. Conflict of interest statement of interest.

We declare that we have no conflict

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