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Introduction

Renal cell carcinoma (RCC) is the most common neoplasm in the adult kidney and accounts for 2–3% of malignant diseases in adults. Locally extensive or metastatic RCC, even following complete resection, have worse prognoses compared to organ-confined diseases. Therefore, there is a need to identify novel biomarkers that enable prediction of early metastasis after nephrectomy, and to develop novel targeted therapies.

Cancer develops as a result of multiple genetic and epigenetic alterations. Better knowledge of changes in gene expression that occur during carcinogenesis may lead to improvements in diagnosis, treatment and prevention. Potential biomarkers identifying high-risk patients have been reported, however, assessment methods for these biomarkers often involve RNA-based techniques and require fresh frozen tissues. By contrast, formalin-fixed paraffin-embedded (FFPE) tissue samples have been collected during decades of routine histopathological examination and those are the most widely available materials in clinical use. However, formaldehyde-containing fixatives cause cross-linkage between nucleic acids and proteins, rendering subsequent extraction and quantification of RNA challenging (1). A major obstacle to RNA expression analysis of FFPE tissues has been the uncertainty as to whether or not gene expression analyses from routinely archived tissues accurately reflect the expression levels prior to fixation, since the fixation process is likely to cause a high degree of RNA fragmentation (2). Given that naturally occurring small RNAs are not affected by fragmentation and therefore do not experience loss of quality, targeting miRNAs is more suitable for analysis of RNA extracted from FFPE samples.

miRNAs are 18- to 25-nucleotide, non-coding RNA molecules that regulate the translation of several genes (3). miRNA expression levels are altered in most types of human cancers (4–6). Several RCC studies have examined miRNAs by microarray analysis using a relatively small amount of frozen tissue samples. Various miRNAs that are dysregulated in RCC have been identified (7,8). Several lines of evidence indicate that miR-486 has oncogenic properties. Overexpression of miR-486 has been reported in cutaneous T-cell lymphoma (9). High serum or plasma expression levels of miR-486 have been reported in non-small cell lung (10) and gastric cancers (11). miR-486 has also been reported to target PTEN and FOXO1, which negatively affect PI3K/Akt signaling (12). The Akt-signaling pathway functions as a potent activator of growth and survival signaling (13). However, expression of miR-486 has not been investigated in RCC.

In the present study, we investigated the association of miR-486 expression with RCC patient prognosis (n=150) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) of FFPE samples. In addition, since it has been reported that olfactomedin 4 (OLFM4), which encodes the olfactomedin 4 protein, is one of the direct targets of miR-486 (14), immunohistochemical analysis of olfactomedin 4 was carried out in RCC tissues.

Materials and methods

Tissue samples

In a retrospective study design, 150 primary tumors were collected consecutively from patients diagnosed with RCC who underwent surgery between 2001 and 2008 at the Hiroshima University Hospital (Hiroshima, Japan). Only patients without preoperative radiotherapy or chemotherapy were enrolled in the study. Of 150 patients, 129 were at stages I, II and III, while 21 were at stage IV. The 129 patients with stage I, II and III RCC underwent curative resection. Of the 129 patients with stage I, II and III RCC, 20 received adjuvant therapy (interferon-α) (15). Of the 21 patients with stage IV RCC, 17 received postoperative therapy using interferon-α (15). Postoperative follow-up was scheduled every 1, 2 or 3 months during the first 2 years after surgery, and every 6 months thereafter unless more frequent follow-up was deemed necessary. Chest-abdominal computed tomography scan and serum chemistries were performed during each 6 month visit at least. Patients were monitored by their physician until they succumbed to the disease or the date of the last documented contact. The median follow-up period was 64 months (range, 2–120). Operative mortality was defined as death within 30 days of the patient leaving the hospital, and these patients were omitted from the analysis.

For qRT-PCR analysis, archival FFPE tissues were used. Histological classification was based on the World Health Organization system. RCC cases were classified into clear cell RCC (ccRCC) and non-ccRCC. Tumor staging was performed according to the TNM grouping system. Since written informed consent was not obtained, for reasons of strict privacy protection, identifying information for the samples was removed prior to analysis. This procedure was in accordance with the Ethical Guidelines for Human Genome/Gene Research of the Japanese Government.

RNA extraction and qRT-PCR

FFPE samples were sectioned (10 μm), deparaffinized and stained with hematoxylin and eosin (H&E) to ensure that the sectioned block contained tumor cells. The tumor areas in the adjacent sections were marked under a light microscope without hematoxylin staining. Tumor areas were macrodissected with sterile disposable scalpels and subjected to RNA isolation using the Recover All™ Total Nucleic Acid Isolation kit (Ambion, Austin, TX, USA), according to the manufacturer’s instructions. Expression levels of miR-486 and RNU6B were measured using TaqMan® assays for miRNA (Applied Biosystems, Austin, TX, USA) in such a manner that the identity and clinical outcomes of samples were blinded. Complementary DNA (cDNA) was synthesized using miRNA-specific primers and the TaqMan® MicroRNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. Briefly, 40 ng of RNA was reverse transcribed in a 20 μl reaction with gene-specific RT probes. qRT-PCR was performed using the ABI 7900 Version 2.3 Sequence Detection System (Applied Biosystems). RNU6B was used as an endogenous normalization control for miR-486. The assays were performed in triplicate. Quantification of miR-486 relative expression was calculated using the RQ manager 1.2 (Applied Biosystems).

Immunohistochemistry

FFPE samples were sectioned, deparaffinized and stained with H&E to ensure that the sectioned block contained tumor cells. Adjacent sections were then stained immunohistochemically with the Dako EnVision+ Mouse Peroxidase Detection System (DakoCytomation, Carpinteria, CA, USA). Antigen retrieval was performed by microwave heating in citrate buffer (pH 6.0) for 30 min. After peroxidase activity was blocked with 3% H2O2 methanol for 10 min, sections were incubated with normal goat serum (DakoCytomation) for 20 min to block non-specific antibody binding sites. Sections were incubated with primary antibodies against olfactomedin 4 (1:50 dilution, using an anti-olfactomedin 4 antibody raised in our laboratory) (16) for 1 h at room temperature, followed by incubations with EnVision+ anti-mouse peroxidase for 1 h. Staining was completed with 10-min incubation with the substrate-chromogen solution. Sections were counterstained using 0.1% hematoxylin.

Statistical analysis

Associations between clinicopathological characteristics and miR-486 expression were analyzed using the Fisher’s exact test. To evaluate the associations between clinical covariates and cancer-specific mortality univariate and multivariate Cox regression analysis was used and conducted using SPSS software (SPSS Inc., Chicago, IL, USA). Hazard ratio (HR) and 95% confidence interval (CI) were estimated from the Cox proportional hazard models. For the analyses, age was treated as a categorical variable (65 plus >65 vs. <65 years old). For the final multivariate Cox regression models, the variables that were moderately associated (P<0.10) with cancer-specific mortality were included. Differences in miR-486 expression levels between the two groups were determined by the Mann-Whitney U test using Graphpad Prism 5.0 (Graphpad Software, Inc., San Diego, CA, USA). Kaplan-Meier survival curves were constructed for high- and low-miR-486 patients using the Graphpad Prism 5.0 software. Differences between survival curves were tested for statistical significance using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of miR-486 in RCC and non-neoplastic kidney tissue

The expression of miR-486 and the corresponding non-neoplastic kidney tissue samples were assessed in 86 RCC cases using qRT-PCR. The expression of miR-486 in RCC samples was ∼2.7-fold higher compared to that in corresponding non-neoplastic kidney samples (P<0.0001, Mann-Whitney U-test).

Association between miR-486 expression levels and clinicopathological characteristics

Expression of miR-486 was examined in 64 additional RCC tissue samples using qRT-PCR. We investigated the association between the clinicopathological characteristics and miR-486 expression levels in 150 RCC cases (Table I). These 150 RCC cases were divided into high- and low-miR-486 cases. When low-miR-486 expression was classified according to the lowest quartile, the number of high- and low-miR-486 cases was 112 and 38, respectively. Expression of miR-486 was not associated with clinicopathological characteristics such as gender, T, N and M classifications, tumor stage or venous invasion.

Table I Association between miR-486 expression and clinicopathological characteristics.

Table I

Association between miR-486 expression and clinicopathological characteristics.

	miR-486 expression
Characteristics	High (%)	Low	P-value
Age (years)
<66	48 (69)	22	0.1084
≥66	64 (80)	16
Gender
Male	80 (73)	29	0.5552
Female	32 (78)	9
T classification
1	71 (77)	21	0.5009
2	12 (71)	5
3	27 (69)	12
4	2 (100)	0
N classification
0	107 (76)	34	0.4049
1	2 (50)	2
2	3 (60)	2
M classification
0	98 (75)	32	0.6119
1	14 (70)	6
Tumor stage
I	71 (79)	19	0.1792
II	10 (71)	4
III	16 (67)	8
IV	15 (68)	7
Histological classification
ccRCC	101 (74)	36	0.3649
Non-ccRCC	11 (85)	2
Venous invasion
Positive	35 (74)	12	0.9699
Negative	77 (75)	26
Interferon-α treatment
Received	23 (62)	14	0.0519
Not received	89 (79)	24

[i] ccRCC, clear cell renal cell carcinoma.

Association between miR-486 expression and survival

The association between miR-486 expression levels and cancer-specific mortality was evaluated. The Kaplan-Meier analysis for the 150 RCC cases (stages I–IV) was performed. As shown in Fig. 1A, cancer-specific mortality did not statistically vary in high- and low-miR-486 cases.

Figure 1 (A) Kaplan-Meier curves of miR-486-high or -low renal cell carcinoma (RCC) in stage I–IV patients. Cancer-specific mortality did not statistically vary in high- and low-miR-486 cases. (B) Kaplan-Meier curves of miR-486-high or -low RCC in stage III and IV patients. High-miR-486 expression had significantly worse cancer-specific mortality compared to those with low-miR-486 expression. (C) Kaplan-Meier curves of miR-486-high or -low RCC in stage III and IV patients who did not receive interferon-α treatment. Cancer-specific mortality of patients with high-miR-486 expression tended to be worse compared to that of patients with low-miR-486 expression. (D) Kaplan-Meier curves of miR-486-high or -low RCC in stage III and IV patients who received interferon-α treatment. Expression of miR-486 was not significantly associated with therapeutic outcome.

Patients with RCC at stage I and II have a good rate of survival, whereas it is difficult to predict patient survival with stage III and IV RCC. These patients would benefit greatly from prognostic biomarkers. Therefore, we analyzed the prognostic value of miR-486 expression in patients with stage III and IV RCC (n=46). Using the Kaplan-Meier analysis, we found that RCC cases with high-miR-486 expression had significantly worse cancer-specific mortality compared to those with low-miR-486 expression (P=0.0331, log-rank test, Fig. 1B). Since treatment with interferon-α for patients with stage III and IV RCC may affect patient survival, individuals who did not receive interferon-α treatment (n=14) were assessed. Although cancer-specific mortality among patients with high- and low-miR-486 expression exhibited no statistically significant difference (P=0.0574, log-Rank test, Fig. 1C), cancer-specific mortality of patients with high-miR-486 expression tended to be worse compared to that of patients with low-miR-486 expression.

To evaluate the potential for miR-486 expression as a prognostic predictor in patients with stage III and IV RCC, univariate and multivariate Cox proportional hazards analyses were used to evaluate the association of miR-486 expression with cancer-specific mortality (Table II). Findings of the univariate analysis showed that N (HR, 2.73; 95% CI, 1.10–6.24; P=0.0315) and M classifications (HR, 3.16; 95% CI, 1.46–7.08; P=0.0035), tumor stage (HR, 4.03; 95% CI, 1.82–9.54; P=0.0005) and miR-486 expression (HR, 3.38; 95% CI, 1.18–14.26; P=0.0202) were significantly associated with cancer-specific mortality. According to the multivariate model, which included N and M classifications, tumor stage and miR-486 expression, expression of miR-486 was an independent prognostic classifier of cancer-specific mortality (HR, 4.33; 95% CI, 1.45–18.71; P=0.0064).

Table II Univariate and multivariate Cox regression analysis of miR-486 expression and cancer-specific mortality in stage III and IV RCC.

Table II

Univariate and multivariate Cox regression analysis of miR-486 expression and cancer-specific mortality in stage III and IV RCC.

	Univariate analysis		Multivariate analysis
Characteristics	HR (95% CI)	P-value	HR (95% CI)	P-value
Age (years)
<66	1 (Ref.)	0.5719
≥66	1.24 (0.58–2.66)
Gender
Male	1 (Ref.)	0.5919
Female	0.73 (0.17–2.08)
Histological classification
Non-ccRCC	1 (Ref.)	0.5714
ccRCC	0.64 (0.18–4.00)
Venous invasion
Negative	1 (Ref.)	0.9318
Positive	1.04 (0.42–3.10)
T classification
1/2	1 (Ref.)	0.8861
3/4	1.09 (0.37–4.60)
N classification
0	1 (Ref.)	0.0315	1 (Ref.)	0.9832
1/2	2.73 (1.10– 6.24)		1.01 (0.32–2.89)
M classification
0	1 (Ref.)	0.0035	1 (Ref.)	0.2398
1	3.16 (1.46–7.08)		0.31 (0.05–2.47)
Tumor stage
III	1 (Ref.)	0.0005	1 (Ref.)	0.0386
IV	4.03 (1.82–9.54)		9.72 (1.14–59.82)
Interferon-α treatment
Not received	1 (Ref.)	0.5558
Received	1.28 (0.57–3.28)
miR-486 expression
Low	1 (Ref.)	0.0202	1 (Ref.)	0.0064
High	3.38 (1.18–14.26)		4.33 (1.45–18.71)

[i] RCC, renal cell carcinoma; ccRCC, clear cell RCC; HR, hazard ratio; CI, confidence interval.

Survival analysis was performed using an alternatively defined cut-off point. When low-miR-486 expression was defined on the basis of lower than median levels of expression of miR-486, the number of high- as well as low-miR-486 miR-486 cases was 75. Kaplan-Meier analysis revealed that cancer-specific mortality did not exhibit statistically significant differences in high- and low-miR-486 cases (data not shown). When low-miR-486 expression was classified according to the lowest tertile, the number of high- and low-miR-486 cases was 100 and 50, respectively. Kaplan-Meier analysis revealed that cancer-specific mortality did not exhibit statistically significant differences in high- and low-miR-486 cases (data not shown).

Expression of miR-486 and therapeutic outcomes

Biomarkers that predict therapeutic outcomes may provide tools to allow physicians to better stratify patients to more effective treatments. Therefore, we analyzed the association between miR-486 expression and therapeutic outcomes in stage III and IV RCC patients treated with interferon-α (n=32). However, expression of miR-486 was not significantly associated with therapeutic outcome (P=0.2931, log-rank test, Fig. 1D).

Expression of OLFM4 in RCC

OLFM4, which encodes the OLFM4 protein, has been reported as one of the direct targets of miR-486 (14). Although alteration of OLFM4 expression has been reported in several types of human cancer (16–18), expression of OLFM4 in RCC has not been investigated. Therefore, the expression of OLFM4 was analyzed using immunohistochemistry in 86 RCC cases. In non-neoplastic kidney, OLFM4 staining was observed in uriniferous tubules but not in glomeruli (Fig. 2A). By contrast, OLFM4 staining was not expressed in RCC cells (Fig. 2B). No OLFM4 staining was detectable in the 86 RCC cases.

Figure 2 Immunohistochemical analysis of olfactomedin 4. (A) In non-neoplastic kidney, olfactomedin 4 staining was observed in uriniferous tubule but not glomerulus (original magnification, ×200). (B) In renal cell carcinoma, olfactomedin 4 staining was not present (original magnification, ×400).

Discussion

Several lines of evidence have suggested that miRNAs are useful as biomarkers as well as therapeutic targets for cancer. Prediction of the survival of patients with stage III and IV RCC remains difficult, and these groups would benefit from the detection of prognostic markers that identify individuals for whom adjuvant or post-operative treatment would be advantageous. In the present study, RNA from 150 FFPE RCC tissues was prepared, since biomarkers developed from FFPE samples may be more readily translated into clinical application. Expression of miR-486 in patients with stage III and IV RCC was significantly associated with cancer-specific mortality according to a Kaplan-Meier analysis. The univariate and multivariate Cox proportional hazards analyses demonstrated that expression of miR-486 was an independent prognostic classifier. Furthermore, since treatment with interferon-α for patients with stage III and IV RCC affects patient survival, individuals who did not receive interferon-α treatment were assessed. Although cancer-specific mortality among patients with high- and low-miR-486 expression showed no statistically significant difference, cancer-specific mortality of patients with high-miR-486 expression tended to be worse compared to that of patients with low-miR-486 expression. This finding suggests that miR-486 expression is associated with a more aggressive RCC histology. Taken together, these results indicate that miR-486 is a promising biomarker to identify patients with poor prognosis in stage III and IV RCC.

In the present study, we demonstrated that miR-486 expression was associated with cancer-specific mortality in patients with stage III and stage IV RCC. In addition, miR-486 expression was associated with cancer-specific mortality in stage III and IV RCC patients who were not treated with interferon-α. Therefore, measurement of miR-486 expression may help identify patients with a high risk of disease recurrence, while treatment with interferon-α may be indicative for patients with miR-486-positive RCC. However, it is unclear whether such patients may benefit from interferon-α treatment. To address this issue, we examined whether or not miR-486 expression was able to identify patients for whom interferon-α treatment is beneficial in stage III and IV RCC. However, expression of miR-486 was not significantly associated with therapeutic outcome. These results suggest that even when patients with a high risk of disease recurrence are identified by miR-486 measurement, those patients may not benefit from interferon-α treatment. Novel therapeutic methods may be more effective for these patients.

miR-486 has been reported to have oncogenic properties. Overexpression of miR-486 has been reported in several human malignancies. In the present study, although expression of miR-486 in RCC samples was significantly higher compared to that in corresponding non-neoplastic kidney samples, expression of miR-486 was not associated with tumor stage. Therefore, high-miR-486 expression is likely to be involved in carcinogenesis, but not in tumor progression. Since targets of miR-486 are PTEN and FOXO1 (12), expression of PTEN and FOXO1 may be low in high-miR-486 RCC cases. In addition to PTEN and FOXO1, one of the targets of miR-486 is OLFM4 (14). In the present study, although OLFM4 staining was observed in uriniferous tubules, staining of OLFM4 was not detected in the 86 RCC cases. Previously, we showed that patients with OLFM4-positive gastric cancer had a better survival rate compared to patients with OLFM4-negative gastric cancer (16). Forced expression of OLFM4 has also been reported to decrease cell adhesion and migration (17,18). Taken together, OLFM4 is likely to have tumor suppressive properties in RCC. Given that staining of OLFM4 was not detected in the 86 RCC cases, overexpression of miR-486 is unlikely to have a major role in the loss of OLFM4.

In summary, we showed that a high-miR-486 expression is an independent prognostic classifier in stage III and IV RCC. Therefore, measurement of miR-486 helps identify high-risk patients. In this study, expression of miR-486 from FFPE samples was assessed. Therefore, measurement of miR-486 can be readily translated into clinical applications.

Acknowledgements

This study was supported in part by grants-in-aid for Cancer Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan and in part by a grant-in-aid for the Third Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan. The authors would like to thank Mr. Shinichi Norimura for his excellent technical assistance and advice. This study was conducted in collaboration with the Research Center for Molecular Medicine of the Faculty of Medicine of Hiroshima University. We also thank the Analysis Center of Life Science of Hiroshima University for the use of their facilities.

References