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Expression of Galectin-3 in renal neoplasms: a diagnostic, possible prognostic marker.

Renal cell carcinoma (RCC) accounts for 2% to 3% of all adult malignancies, and its incidence rate is increasing. (1) Renal cell carcinoma is clinicopathologically heterogenous and was recently subdivided into clear cell, papillary, chromophobe, collecting duct, and unclassified types by the World Health Organization. (2) Although significant progression has been made in understanding the cytogenetic and molecular changes of RCC, its pathogenetic mechanism, particularly for the sporadic tumors, remains unclear.

Galectin-3 (Gal-3), a protein member of the lectinfamily, binds to [beta]-galactosides and is widely expressed in epithelial and immune cells. Galectin-3 interacts with glycoprotein receptors on the cell membrane and controls several cellular functions. (3,4) Galectin-3 expression was recently shown to correlate with the attenuation of drug-induced apoptosis and anoikis (apoptosis induced by the loss of cell anchorage) that contribute to cell survival, aggressiveness, and metastasis in cancer. (5-7)

In normal rat kidney, Gal-3 is expressed in distal tubular epithelial cells. (8) Galectin-3 expression has also been recently identified in some RCCs by complementary DNA microarray studies in human renal tumors. (9) However, its diagnostic or prognostic significance in RCCs remain unclear because of the few studies and relatively small number of cases. Here, we sought to investigate the value of Gal-3 as a diagnostic and prognostic marker using immunohistochemistry and tissue microarrays of a relatively large series of well-characterized subtypes of renal neoplasms.


Clinicopathologic Parameters

The tissue samples were obtained from 217 renal neoplasms from patients who had undergone curative, initial resections, without any preoperative therapy, at the Methodist Hospital, Houston, Texas, from 1990 through 2004. For each case, the following pathologic features were studied: (1) histologic type, according to the World Health Organization histologic classification of kidney tumors (2); (2) nuclear grade (Fuhrman 1-4); and (3) tumor stage according to the AJCC Cancer Staging Handbook of the American Joint Committee on Cancer. (10) The 217 evaluated renal neoplasms included 137 clear cell RCCs, 32 papillary RCCs, 23 oncocytomas, 21 chromophobe RCCs, 3 unclassified RCCs, and 1 collecting duct carcinoma. Of the 137 clear cell RCCs, the Fuhrman nuclear grade distribution was as follows: grade 1, 13 cases (9.5%); grade 2, 75 cases (54.7%); grade 3, 35 cases (25.5%); and grade 4,14 cases (10.2%). Their tumor stages were I (69 cases; 50.4%), II (17 cases; 12.4%), III (41 cases; 29.9%), and IV (10 cases; 7.3%). The follow-up time ranged from 0.4 to 174 months (mean, 61 months) for clear cell RCC.

Tissue Microarray and Immunohistochemistry

Four tissue microarrays were constructed using triplicate cores (1.0 mm) of tumor tissue from each case. One representative core from 21 normal kidney tissues was also included in the tissue microarrays. Conventional full sections included in our study consisted of 10 clear cell RCCs, 6 papillary RCCs, 4 oncocytomas, and 4 chromophobe RCCs.

Immunohistochemistry was performed using a modification of the avidin-biotin peroxidase complex technique with a monoclonal antibody against Gal-3 (dilution 1:250; Novocastra/Vector Laboratories, Burlingame, California). Sections of tissue micro-arrays and regular formalin-fixed, paraffin-embedded, 4-[micro]m-thick tissue sections were immunostained using BenchMark XT (Ventana Medical Systems, Inc, Tucson, Arizona). The sections were deparaffinized in xylene and sequentially washed in graded ethanol. Heat-induced epitope retrieval was performed with Ventana Cell Conditioning 1 (Tris ethylenediaminetetra-acetic acid buffer, pH 8.0) for 30 minutes on the instrument. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 4 minutes. Slides were incubated with primary antibody for 32 minutes at room temperature. The subsequent reactions were performed using components from UltraView DAB Detection Kit (Ventana), including incubation with universal (rabbit and mouse) secondary antibody for 8 minutes, a biotin-free, horseradish-peroxidase enzyme-labeled polymer, and a signal visualization by 3,3'-diaminobenzidine hydrochloride. Sections were then counterstained with hematoxylin before being mounted and examined by light microscopy. Data obtained from microarray sections were compared with those from regular tissue sections.

Evaluation of Galectin-3 Expression and Clinicopathologic Correlation

Immunoreactivity was evaluated independently by 2 observers (J.Y.D. and S.S.S.). Staining was tabulated as either cytoplasmic or nuclear and either positive or negative. The total score was calculated from the sum of the proportion score and intensity score. The proportion score is allocated to represent the estimated percentage of tumor cells staining positive: 0, 0%; 1, 1% to 15%; 2, 16% to 50%; 3, 51% to 75%; 4, 76% to 100%. The intensity score is allocated to represent the average intensity of the positive tumor cells: 0, negative; 1, weak; 2, intermediate; 3, strong. A positive result was defined as the sum of the proportion score and intensity score that equaled at least 5. (11)

The Gal-3 expression in tumors was correlated with histologic types, tumor stage, and nuclear grade. The prognostic significance of Gal-3 expression in clear cell RCCs was evaluated. Of 137 cases of clear cell RCC included in this study, 2 (1.5%) were lost to follow-up, leaving 135 cases (98.5%) for statistical analysis of the survival rate. Analysis was not performed for other histologic subtypes because of the small number of cases.

Statistical Analysis

The differences of categoric variables between groups were tested for significance by the [chi square] test or the Fisher exact test, using P < .05 as the cutoff. The patients' survival was calculated by the Kaplan-Meier method, and the difference was determined by a log-rank test. Statistical analyses were performed using the program SPSS, Version 13.0 (SPSS Inc, Chicago, Illinois).


Expression of Galectin-3 in Normal Kidney

In 21 normal renal tissues, Gal-3 was expressed in collecting ducts, intermediate thin ducts, and distal tubules (Figure 1, A). Diffuse strong cytoplasmic expression of Gal-3 was observed in the normal collecting ducts. The distal tubules demonstrated diffuse or patchy positive cytoplasmic staining for Gal-3. The thin intermediate tubules showed patchy positive cytoplasmic staining for Gal-3, and it was generally less intense than that of collecting ducts. There was no or very weak expression of Gal-3 in proximal tubules and glomeruli.

Expression of Galectin-3 in Renal Neoplasms: Correlation With Histologic Subtypes

Overall, positive expression of Gal-3 was observed in 92 of 217 renal neoplasms (42.4%), but the frequencies varied widely among histologic subtypes (Table 1). Figure 2 displays the variable expression of Gal-3 in different types of renal neoplasms.

Twenty-two of 23 oncocytomas (96%) demonstrated strong expression of Gal-3 (Figure 1, B). The tumor cells had a diffuse homogeneous cytoplasmic and frequent nuclear staining in more than 90% of the tissue cores with a mean staining score of 6.6 (SD 0.77).

Nineteen of 21 chromophobe RCCs (90%) expressed Gal-3 (Figure 1, C). Characteristically, the cells with abundant reticulated cytoplasm showed more frequent membranous Gal-3 staining, whereas the smaller eosinophilic cells demonstrate diffuse dense cytoplasmic expression of Gal-3 with a mean score of 6.8 (SD 1.59).

Only 4 of 32 papillary RCCs (12.5%) showed positive cytoplasmic Gal-3 expression (Figure 1, D). The tumor cells demonstrated weak to moderate cytoplasmic reaction, with a mean score of 3.8 (SD 1.27). Typically, Gal-3 expression was stronger in the apical portion of the cytoplasm in tumor cells (Figure 1, E). The foamy macrophages in the papillary RCC revealed strong, positive Gal-3 immunostains (Figure 1, F).

Forty-seven of 137 clear cell RCCs (34.3%) demonstrated positive expression of Gal-3, with mean score 4.1 [+ or -] 1.67. Expression pattern of Gal-3 in conventional RCC was variable. Tumor cells demonstrated frequent membranous or cytoplasmic and also nuclear staining in positive cases. Sarcomatoid component showed strong cytoplasmic or nuclear staining for Gal-3 (Figure 1, G).

Conventional full sections, which included 10 clear cell RCCs, 6 papillary RCCs, 4 oncocytomas, and 4 chromophobe RCCs, showed similar results. Four of 10 clear cell RCCs (40%), 1 of 6 papillary RCCs (17%), and all oncocytomas (4 of 4; 100%) and chromophobe (4 of 4; 100%) RCCs showed cytoplasmic and/or nuclear staining for Gal-3.

Prognostic Significance of Gal-3 Expression in Clear Cell RCC

To determine the significance of Gal-3 expression in clear cell RCC, we first correlated expression of Gal-3 with nuclear grade (Table 2). Twenty-seven of 49 clear cell RCCs (55.1%) of high-grade (Fuhrman nuclear grades 3 and 4) showed positive Gal-3 expression (Figure 1, H). In contrast, 20 of 88 low-grade (Fuhrman nuclear grades 1 and 2) clear cell RCC (22.7%) expressed Gal-3 (Figure 1, I; P < .001). Kaplan-Meier survival analysis stratified by Gal-3 expression status is shown in Figure 3. The mean and median survivals for Gal-32 clear cell RCC group were 108 and 113 months, respectively. Whereas, the mean and median survival for [Gal-3.sup.+] clear cell RCC group were 87 and 68 months, respectively. There was a strong trend toward an unfavorable prognosis for patients with clear cell RCCs that expressed Gal-3; however, the trend did not reach statistical significance (P = .11).



In this study, we analyzed Gal-3 expression in different histologic types of renal cell neoplasm by immunohistochemistry in a tissue microarray platform to examine the possible utility of Gal-3 as a diagnostic and/or prognostic marker. (12)

In healthy renal tissue, Gal-3 was expressed in collecting ducts, intermediate ducts, and partially distal ducts. Galectin-3 has been detected in the apical domains of ureteric bud branches in a mice embryo study. (13) It has been suggested that Gal-3 has a pivotal role in growth of the ureteric bud, including collecting ducts and connecting segments of distal tubules. (14) Intense expression of Gal-3 in fetal medullary and papillary collecting duct epithelium, especially a-intercalated cells, has been reported. (13,14) These observations are in agreement with those from a study of messenger RNA profiles of renal neoplasm by Young et al. (9)

Strong and homogeneously diffuse expression of Gal-3 was observed in almost all oncocytomas and chromophobe RCCs in our study. These observations are also consonant with increased expression of Gal-3 in distal tubular cells and support the traditional idea that oncocytoma and chromophobe RCC originate from distal renal tubular epithelium. (8,9)

Compared with chromophobe RCCs and oncocytomas, only 4 of 32 papillary RCCs (12.5%) showed positive Gal-3 expression. Interestingly, the foamy macrophages in the papillary RCC showed frequent Gal-3 expression. In the renal inflammatory disease model, Gal-3 expression had been found in the macrophages in the early and late stages. (8) It has been suggested that the intercellular, antiadhesive interaction is the effect of Gal-3 in the injured ischemic proximal tubules in the rat model study. (15) The study is limited, and the functional role of Gal-3 expression in macrophages in RCC is not clear. Continued study is needed to further define the physiologic role of strong Gal-3 expression in macrophages of papillary RCCs.

Forty-seven of 137 clear cell RCCs (34%) demonstrated expression of Gal-3. For Gal-3 expressions in clear cell RCCs, our study demonstrates that Gal-3 expression rates are correlated with Fuhrman nuclear grade. Recent studies revealed that intracellular Gal-3 suppresses the drug-induced apoptosis and anoikis that contribute to cell survival. (7) The findings presented in a previous report revealed a molecular interaction among Gal-3 with [beta]-catenin/TCF complex, and the similarities in the pleiotropic functions of both [beta]-catenin and Gal-3 in the Wnt signaling pathway. (16) It has been reported that the Wnt signal pathway can induce progression of RCC by the inhibition of apoptosis. (17)

Altered expression of certain members of the Frizzled family, and their downstream targets, could provide alternative mechanisms leading to activation of the Wnt signaling pathway in renal carcinogenesis. (18) It has been shown that [beta]-catenin stimulation of cyclin D1 and c-myc expression is Gal-3 dependent. (19)

Galectin-3 has been reported to be negatively and positively correlated with some tumor progression. Furthermore, this overexpression has been shown to associate with tumor invasion and metastasis in several types of human cancers. (12,20-23) Although it was not statistically significant, there was a strong trend toward a less-favorable survival rate in the clear RCC group that expresses Gal-3 in our study. These results are similar with the findings of the Francois et al study on Gal-3 staining in 73 cases of RCCs. (12)

Several previously reported analyses of the histopathologic data led to the conclusion that Gal-3 expression has been positively correlated with aggressiveness of certain tumors, such as mammary carcinomas and colon carcinomas, (21,23) whereas it is inversely correlated with tumor progression in certain other tumors, such as uterine and prostate carcinomas. (24,25)

The molecular mechanism that regulates these functions is not fully deciphered. Galectins are known as a family of low molecular weight, mannose-binding lectins and also have functions in cell growth, cell activation, and cell-cell and cell-matrix adhesion through binding to carcinoembryonic antigens, laminin, and metalloproteinase. Up-regulation in Gal-3 has been reported to allow enhanced interactions with stromal cells and greater adhesion to target organ endothelial cells. In a mammary tumor study, Gal-3 expression has been associated with specific morphologic precursor subtypes of breast cancer and undergoes a transitional shift in expression from luminal to peripheral cells as tumors progressed to comedo-ductal carcinoma in situ or invasive carcinomas. (23) Such a localized expression of Gal-3 in cancer cells proximal to the stroma could lead to increased invasive potential by inducing novel or better interactions with the stromal counterparts. Further study might be needed to localize expression of Gal-3 in RCC. Galectin-3, a pleiotropic protein, has been reported as an important regulator of tumor metastasis, which like [beta]-catenin, shuttles between the nucleus and the cytosol in a phosphorylation-dependent manner.


In conclusion, this study confirms that Gal-3 is frequently overexpressed in renal cell neoplasms, especially of distal tubular differentiation suggesting a possible diagnostic role for differential diagnosis of renal neoplasms with oncocytic or granular cells. A strong association of Gal-3 overexpression and high nuclear grade in clear cell RCC is observed, suggesting a possible role for Gal-3 as a differentiation and prognostic factor in clear cell RCC.


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Jane Y. Dancer, MD; Luan D. Truong, MD; Qihui Zhai, MD; Steven S. Shen, MD, PhD

Accepted for publication April 10, 2009.

From Department of Pathology, The Methodist Hospital and Research Institute and Weill Medical College of Cornell University, Houston, Texas (Drs Troung, Zhai, and Shen);and the Department of Pathology, M. D. Anderson Cancer Center, Houston, Texas (Dr Dancer).

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the annual meeting of the United States and Canadian Academy of Pathology, Atlanta, Georgia, February 11-17, 2006.

Reprints: Steven S. Shen, MD, PhD, Department of Pathology, The Methodist Hospital, M 227, 6565 Fannin St, Houston, TX 77030 (e-mail:
Table 1. Expression of Galectin-3 by Different Histologic Types

Histologic Type (No.) Cases No. (%)

Clear Cell RCC (137) 47 (34.3)
Papillary RCC (32) 4 (12.5)
Chromophobe RCC (21) 19 (90.5)
Oncocytoma (23) 22 (95.7)
Unclassified RCC (3) 0 (0)
Collecting duct carcinoma (1) 0 (0)
Total (217) 92 (42.4)

Abbreviation: RCC, renal cell carcinoma.

Table 2. Galectin-3 Expression of Clear Cell Renal
Cell Carcinoma Stratified by Fuhrman Nuclear Grade

 Grade Cases, No. Positive, No. (%)

1 13 4 (30.7)
2 75 16 (21.3)
3 35 18 (51.4)
4 14 9 (64.2)
Total 137 47 (34.3)

Figure 2. Expression of Galectin-3 in renal

Subtype of renal neoplasm

Clear cell 4.1
Papillary 3.9
Chromophobe 6.8
Oncocytoma 6.6

Note: Table made from bar graph.
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Author:Dancer, Jane Y.; Truong, Luan D.; Zhai, Qihui; Shen, Steven S.
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Clinical report
Geographic Code:1USA
Date:Jan 1, 2010
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