Soluble Serum αKlotho Is a Potential Predictive Marker of Disease Progression in Clear Cell Renal Cell Carcinoma

Margherita Gigante, Giuseppe Lucarelli, Chiara Divella, Giuseppe Stefano Netti, Paola Pontrelli, Cesira Cafiero, Giuseppe Grandaliano, Giuseppe Castellano, Monica Rutigliano, Giovanni Stallone, Carlo Bettocchi, Pasquale Ditonno, Loreto Gesualdo, Michele Battaglia, Elena Ranieri, Margherita Gigante, Giuseppe Lucarelli, Chiara Divella, Giuseppe Stefano Netti, Paola Pontrelli, Cesira Cafiero, Giuseppe Grandaliano, Giuseppe Castellano, Monica Rutigliano, Giovanni Stallone, Carlo Bettocchi, Pasquale Ditonno, Loreto Gesualdo, Michele Battaglia, Elena Ranieri

Abstract

Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies, and clear cell RCC (ccRCC), that has a high metastatic index and high relapse rate, is the most common histological subtype. The identification of new biomarkers in ccRCC is fundamental for stratifying patients into prognostic risk groups and to guide therapy. The renoprotective antiaging gene, αKlotho, has recently been found to work as a tumor suppressor in different human cancers. Here, we evaluated αKlotho expression in tissue and serum of ccRCC patients and correlated it with disease progression. Tissue αKlotho expression was studied by quantitative RT-PCR and immunohistochemistry. In addition, soluble serum αKlotho levels were preoperatively measured in 160 patients who underwent nephrectomy for RCC with ELISA. Estimates of cancer-specific (CSS) and progression-free survival (PFS) were calculated according to the Kaplan-Meier method. Multivariate analysis was performed to identify the most significant variables for predicting CSS and PFS. αKlotho protein levels were significantly decreased in RCC tissues compared with normal tissues (P < 0.01) and the more advanced the disease, the more evident the down-regulation. This trend was also observed in serum samples. Statistically significant differences resulted between serum αKlotho levels and tumor size (P = 0.003), Fuhrman grade (P = 0.007), and clinical stage (P = 0.0004). CSS and PFS were significantly shorter in patients with lower levels of αKlotho (P < 0.0001 and P = 0.0004, respectively). At multivariate analysis low serum levels of αKlotho were independent adverse prognostic factors for CSS (HR = 2.11; P = 0.03) and PFS (HR = 2.18; P = 0.03).These results indicate that a decreased αKlotho expression is correlated with RCC progression, and suggest a key role of declining αKlotho in the onset of cancer metastasis.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

FIGURE 1
FIGURE 1
Klotho gene expression levels evaluated by qRT–PCR. Histogram represents mean ± SD of normalized Klotho gene expression levels by quantitative real-time PCR in 18 ccRCC samples (T) compared with their matched-NT (∗P < 0.001).
FIGURE 2
FIGURE 2
In vivo analysis for Klotho expression using immunohistochemistry. Renal Klotho expression was evaluated in RCC tumor tissues (T) and matched nontumoral (NT) kidney portions from patients with different grades of RCC (G1–G2 and G3–G4) and patients with metastases (M1). Klotho was expressed at tubular level in NT portion of G1–G2 RCC patients and significantly decreased in G3–G4 and M1 patients. In T tissue, αKlotho was slightly expressed in patients with G1–G2 and patients with G3–G4 and M1 showed a very low or undetectable expression. Klotho staining was quantified as described in the “Methods” section and was expressed as mean ± SD. All images are from a single patient and are representative of all 8 patients for G1–G2 and G3–G4 groups and 4 patients for M1. The positive control is a normal kidney tissue and negative control is obtained as described in the “Methods” section.
FIGURE 3
FIGURE 3
Median αKlotho levels in 20 healthy subjects compared with 45 patients with metastatic (M1) and 115 nonmetastatic (M0) ccRCC.
FIGURE 4
FIGURE 4
Serum αKlotho levels stratified according to tumor size (≤7 cm vs >7 cm; panel A) and nuclear grade (Fuhrman 1–2 vs 3–4, panel B).
FIGURE 5
FIGURE 5
Spearmann correlation coefficient (rs) and linear regression line between αKlotho tissue signal intensity and serum values from 20 ccRCC patients.
FIGURE 6
FIGURE 6
Comparison of αKlotho and CA 15-3 receiver-operating characteristic (ROC) curves for cancer-specific survival (panel A), and progression-free survival (panel B).
FIGURE 7
FIGURE 7
Kaplan–Meier cancer-specific survival (CSS) curves, stratified by αKlotho serum levels.
FIGURE 8
FIGURE 8
Kaplan–Meier progression-free survival (PFS) curves, stratified by αKlotho serum levels.

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Source: PubMed

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