Clinical Significance of Soluble Major Histocompatibility Complex Class I Chain-related A in Renal Cell Carcinoma Patients

Renal cell carcinoma (RCC) is the most common type (>80%) of renal malignancies and leads to more than 100,000 deaths per year worldwide (Parkin et al., 2002). The clear-cell type is the major histological type of RCC, accounting for about 75-80% of all cases (Banumathy and Cairns, 2010). Despite advances in the understanding and treatment of RCC, the prognosis is still poor due to delayed diagnosis. Approximately one third of RCC patients present with distant metastasis at diagnosis (Lam et al., 2005). Median overall survival for patients with metastatic disease is only 12 months (Motzer and Russo, 2000). The prevalence of various imaging modalities such as intravenous pyelography, ultrasonography, and computed tomography scanning have allowed the detection of earlystage renal cancer in asymptomatic patients. Due to the noninvasive nature, numerous serum markers (e.g. serum amino acid levels and circulating microRNAs) have been explored for the early detection and surveillance of RCC (Mustafa et al., 2011; Redova et al., 2012). However, there is currently no reliable serum biomarker available for the


Introduction
Renal cell carcinoma (RCC) is the most common type (>80%) of renal malignancies and leads to more than 100,000 deaths per year worldwide (Parkin et al., 2002).The clear-cell type is the major histological type of RCC, accounting for about 75-80% of all cases (Banumathy and Cairns, 2010).Despite advances in the understanding and treatment of RCC, the prognosis is still poor due to delayed diagnosis.Approximately one third of RCC patients present with distant metastasis at diagnosis (Lam et al., 2005).Median overall survival for patients with metastatic disease is only 12 months (Motzer and Russo, 2000).The prevalence of various imaging modalities such as intravenous pyelography, ultrasonography, and computed tomography scanning have allowed the detection of earlystage renal cancer in asymptomatic patients.Due to the noninvasive nature, numerous serum markers (e.g.serum amino acid levels and circulating microRNAs) have been explored for the early detection and surveillance of RCC (Mustafa et al., 2011;Redova et al., 2012).However, there is currently no reliable serum biomarker available for the MHC class I polypeptide-related chain A (MICA) is a stress-inducible glycoprotein and frequently expressed in epithelial tumors (Kohga et al., 2010;Zhao et al., 2012).It acts a natural ligand for NKG2D to activate antitumor effects of natural killer (NK) cells and T cells.Accumulating evidence suggests that shedding of MICA from tumor cells represents an important means of evading antitumor immunity (Kohga et al., 2009;Kohga et al., 2010].MICA expression and preoperative soluble MICA (sMICA) levels were found to be independent prognostic factors in resected pancreatic cancer (Duan et al., 2011).In oral squamous cell carcinoma, serum levels of sMICA are useful in the diagnosis of stage IV disease and as an indicator of regional lymph node metastasis (Tamaki et al., 2008).MICA expression has been documented in carcinomas of the kidney (Groh et al., 1999).However, relatively little is known about the expression and clinical significance of MICA in renal cancer.Therefore, in this study we sought to determine tissue expression and serum concentrations of MICA in RCC patients and examine the clinical relevance of sMICA in this disease.

Subjects and tissue samples
A total of 48 patients with RCC and 12 patients with benign renal tumors who were admitted to our hospital between June and August 2012 were enrolled in this study.Patients were excluded due to complicated with other malignancies, any prior anti-cancer therapy, and incomplete clinical data.RCC and adjacent normal renal tissues were obtained during tumor resection.Freshly resected tissues were snap frozen in liquid nitrogen and stored at -80°C until analysis.To evaluate the diagnostic implication of serum MICA, 20 healthy controls were also enrolled.A 2-ml peripheral blood sample was withdrawn from each subject prior to surgery and centrifuged for 10 min at 3,000 r/min at 4°C.Serum was collected, aliquoted, and stored at -20°C until further analysis.All procedures were conducted in accordance with the Helsinki declaration, and with approval from the Harbin Medical University (Harbin, China).Written informed consent was obtained from all participants.

Immunohistochemistry
Tissue specimens were fixed in 10 % formalin, embedded in paraffin, and cut into 4-μm-thick sections.The tissue sections were deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide to deplete endogenous peroxidase.After blocking in 5% bovine serum albumin, goat anti-human MICA polyclonal antibody (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added and incubated overnight at 4°C.After washing, the sections were incubated with horseradish peroxidaseconjugated secondary antibody for 1 h.Peroxidase activity was visualized with 3,3'-diaminobenzidine as a chromogen.Sections were counterstained with hematoxylin.The negative control was included without the addition of primary antibody.The stained sections were blindly scored and the immunohistological results were expressed as low (less than 10% stained cells), moderate (10% to 30% stained cells), and high (greater than 30% stained cells).

Enzyme-linked immunosorbent assay (ELISA)
sMICA levels in the serum were measured using the Human MICA ELISA Kit (Abcam, Cambridge, UK) following the manufacturer's instruction.Briefly, 100 μL of serum samples or standards were added to a 96-well ELISA plates.After incubation for 2 h at 37°C, the wells were washed three times.Each well was added with the detection antibody and incubated for 1 h at 37°C.After washing, 100 μL of the working dilution of horseradish peroxidase-labeled streptavidin was added to each well and incubated for 20 min at room temperature.The substrate solution was then added and incubated for another 20 min.The absorbance was measured at 450 nm with a microtiter plate reader (Thermo Scientific, Waltham, MA, USA) after 100 μL of stop solution was added to each well.Each assay was performed in triplicate and repeated three times.Results were expressed as picograms per milliliter

Statistical analysis
Continuous data were expressed as mean ± standard deviation.Differences in sMICA levels among multiple groups were analyzed using the Kruskal Wallis H test. Student's t-test was used to compare MICA mRNA abundance between RCC and adjacent normal renal tissues.A P value less than 0.05 was considered statistically significant.

Patient characteristics
This study included 48 RCCs and 12 benign renal    tumors (10 renal hamartomas and 2 renal oncocytomas), with a median age of 45 years (34-76 years).There were 37 men and 23 women.Patient characteristics are given in Table 1.Thirty-four RCC patients presented with T1 or T2 disease and 14 with T3 or T4 disease.Vascular invasion, lymph node metastasis, and distant metastasis were detected in 5, 11, and 4 RCC patients, respectively.Thirty-two RCC patients had G1 or G2 tumors and 16 had G3 or G4 tumors.Forty-two RCC patients presented with clear RCC and 6 with other types of RCC.

Elevated expression of MICA in RCC versus adjacent normal tissue
Immunohistological analysis of MICA expression revealed that MICA showed a mixed cytoplasmic/ membranous staining pattern in RCC (Figure 1a-c).Thirty-five of the 48 RCC specimens (72.9%) showed moderate to high expression of MICA and absent or low immunoreactivity was detected in 13 cases (27.1%).In contrast, the majority of adjacent normal renal tissues (91.7%) displayed absence or low expression of MICA (Figure 1d).
We next examined the mRNA expression changes of MICA in RCC.Real-time PCR analysis revealed that MICA mRNA abundance was significantly higher in RCC than in adjacent normal tissues (2.03 ± 0.35 vs. 0.77 ± 0.21, P < 0.05; Figure 2).

Increased serum concentrations of sMICA in patients with RCC
As shown in Table 2, ELISA results demonstrated that serum concentrations of sMICA were significantly greater in patients with RCC (348.5 ± 32.5 pg/ml) or benign renal tumors (289.3 ± 30.4 pg/ml) than in healthy controls (168.4 ± 43.2 pg/ml).

Correlation of serum MICA with clinicopathological features of RCC
sMICA concentrations were tested for possible correlations with various clinicopathological variables.As shown in Table 3, serum levels of sMICA were significantly associated with advanced tumor stage (P = 0.023), presence of lymph node metastasis (P = 0.045), distant metastasis (P = 0.017), vascular invasion (P = 0.014), and higher histological grade (P = 0.016).

Diagnostic performance of serum MICA concentration
Table 4 shows the diagnostic sensitivity specificity of sMICA at different cut-off levels.When the optimal cutoff point was accepted as 250 pg/ml, the specificity and sensitivity of sMICA in distinguishing between RCC and benign renal tumors was 63.2% and respectively

Discussion
MICA is able to bind to the immunoreceptor NKG2D and mediate the cyotoxicity NK and T cells against tumor cells (Oppenheim et al., 2005), thus playing a key role in tumor immunosurveillance.MICA is regarded as a stress-inducible molecule.Infection with vesicular stomatitis virus leads to a robust induction of MICA mRNA expression in cancer cells (Jensen et al., 2011).It has been reported that MICA is rare or absent on the cell surface of healthy cells, but can be upregulated in actively growing epithelial tumors (Groh et al., 1996;Bauer et al., 1999).Groh et al (1999) have revealed the expression of MICA in a broad range of tumors, including carcinomas of the lung, breast, kidney, ovary, prostate, and colon.In line with these findings, our data showed that MICA expression was elevated in RCC compared to adjacent normal renal tissues, at both the protein and mRNA levels.The molecular mechanisms involved in the induction of MICA are still poorly understood.Several lines of evidence suggest that its upregulation may be the result of a DNA damage response (Gasser et al., 2005).Madjd et al (2007) revealed significant relationships between MICA expression and histological grade, lymph node stage, Nottingham Prognostic Index, the presence of vascular invasion, and tumor type in operable breast carcinoma.Wang et al (2012) demonstrated that the MICA mRNA level is higher in oral squamous cell carcinoma than in adjacent noncancerous tissues, and significantly correlated with regional lymph node status and disease stage.In colorectal cancer patients, expression of the MICA serves as an indicator of good prognosis (Watson et al., 2006).The clinical significance of serum MICA is also explored in several malignancies.Weber et al (2004) reported that serum MICA levels are significantly increased in pancreatic cancer patients, and correlate with extent of tumor burden.Serum levels of sMICA are found to be an independent prognostic factor for advanced hepatocellular carcinoma (Li et al., 2013).Our data revealed that the serum level of sMICA was significantly higher in RCC patients versus patients with benign renal tumors or healthy controls.Moreover, sMICA concentrations were significantly associated with advanced tumor stage, presence of lymph node metastasis, distant metastasis, vascular invasion, and higher histological grade.Our data collectively indicate that the release of sMICA is associated with tumor progression and metastasis in RCC.Shedding of NKG2D ligands, in particular MICA, from the cell surface represents a mechanism by which tumors escape NKG2D-mediated immunosurveillance (Kohga et al., 2012;Yamanegi et al., 2012).The production of sMICA has been found to impair NKG2D expression and NK cytotoxicity (Raffaghello et al., 2004).These studies provide a biological explanation for the significant associations between sMICA levels and tumor progression.Accordingly, either blockade of MICA release or neutralization of shed sMICA would be a useful addition to immunologic approaches for cancer therapy.
The utilization of circulating biomarkers has obvious advantages over tissue-based methods in disease assessment, since it is non-invasive, relatively low cost, and easily repeatable.Increased levels of sMICA have been documented in the sera of patients suffering from various types of cancer, including gastrointestinal malignancies, breast and lung tumors, melanoma, prostate cancer, pancreatic carcinomas, hepatocellular cancer, and leukemia (Groh et al., 2002;Salih et al., 2008).Holdenrieder et al. (2006) found that the sMICA in sera of patients with various malignancies was significantly higher than that in healthy individuals.Patients with benign diseases exhibited intermediate sMICA levels.In agreement with these observations, our data demonstrated that sMICA concentrations were significantly greater in the sera from RCC patients than from patients with benign renal disease or healthy controls.It has been suggested that the serum levels of sMICA can be used as diagnostic markers for cancer detection (Salih et al., 2008).Our present data also support the diagnostic value of sMICA in human cancers.When the optimal cut-off point of 250 pg/ml was used, sMICA levels had the specificity and sensitivity of 63.2% and 75.6%, respectively, in differentiating RCC from benign renal tumors.Although the discriminating power of sMICA alone was relatively lower than the expectations of clinical utility, its combination with other biomarkers would improve predictive accuracy and robustness.
Some limitations of this study should be noted.First, no information is available for the relationship between tumoral expression of MICA and sMICA concentrations in sera.Second, the sample size is relatively low.Finally, data on the prognostic relevance of sMICA in RCC are lacking.
In conclusion, we demonstrate that MICA expression is elevated in RCC compared to adjacent normal renal tissues and increased levels of sMICA are associated with aggressive tumor behavior.Serum sMICA alone has limited potential in distinguishing RCC from benign renal tumors.These observations suggest that the release of sMICA has important impacts on RCC progression and metastasis, thus providing a novel therapeutic target for this disease.
Zhao & , Gang-Jun Yuan, Qing-Guo Zhu* diagnosis and monitoring of patients with RCC.

Figure 1 .Figure 2 .
Figure 1.Immunohistochemical Staining for MICA in Paired RCC and Adjacent Normal Renal Tissues.(a-c)MICA displayed a mixed cytoplasmic/membranous staining pattern in RCC, with low (a), moderate (b), and high (c) expression levels.(d) Adjacent normal tissues showing absence of low expression of MICA.Magnification, ×40

Table 1 . Demographic and Clinical Data of the Patients and Healthy Controls Included in This Study
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.10.5651Major Histocompatibility Complex Class I Chain-related A in RCC Patients