This article is dedicated to the memory of Professor Annemarie Poustka, who was the founder and head of the Division Molecular Genome Analysis at the DKFZ. She was an inspiring scientist and a wonderful person.
Circulating miRNAs have recently been indicated as practicable and promising biomarkers for noninvasive diagnosis in various tumor entities. However, cell-free miRNAs have not been found to correlate with clinicopathological variables in epithelial carcinomas. To learn more about the potential clinical relevance of circulating miRNAs in prostate cancer, we screened 667 miRNAs in serum samples from patients with metastatic (n = 7) and localized prostate cancer (n = 14). Various miRNAs were highly abundant in the sera of patients with metastatic disease, and five upregulated miRNAs (miRNA-375, miRNA-9*, miRNA-141, miRNA-200b and miRNA-516a-3p) were selected for further validation. In the first validation study (n = 45), selected miRNAs were analyzed in a prospectively collected serum set taken from different prostate cancer risk groups. Most of the selected miRNAs were significantly correlated with adverse risk factors when different clinicopathological variables were analyzed. Circulating miRNA-375 and miRNA-141 turned out to be the most pronounced markers for high-risk tumors. Their levels also correlated with high Gleason score or lymph-node positive status in a second independent validation study (n = 71). In addition, the expression levels of miRNA-375 and miRNA-141 were monitored in 72 prostate tissue samples (36 tumor vs. 36 benign). Both miRNAs were highly expressed in all samples and significantly upregulated in the tumors compared to normal tissues. Overall, our observations suggest that miRNA-375 and miRNA-141 expression is enhanced in prostate cancer specimens and their release into the blood is further associated with advanced cancer disease.
Prostate cancer is the most frequently diagnosed malignancy and second most cause of cancer related death among western males.1 As development and progression of cancer is driven by molecular alterations, the analysis of molecular features may enable a better prediction of the behavior of individual cancers. Since tissues are heterogeneous, alterations on the serum level would be especially suited as diagnostic and prognostic markers in prostate cancer.2
miRNAs are short RNA molecules (19–26 nucleotides), which have been suggested to be important regulators of biological functions.3, 4 Alterations in miRNA expression can affect important cellular processes like cell cycle, proliferation or apoptosis, thus providing a direct link to cancer development and progression.5
The possibility to extract and measure stable free miRNA in serum has recently been shown. Thus, serum represents a rich resource for potential biomarkers. Lawrie et al. were the first to show the presence of miRNAs in body fluids of patients suffering from B-cell lymphoma.6 Using a mouse model, Mitchell et al. demonstrated that tumor-derived miRNAs enter the circulation even when originated from epithelial cancer types.7 They also showed that circulating miRNA-141 was elevated in metastatic prostate cancer when compared to healthy controls.7
Different levels of circulating miRNAs have been reported to occur between tumor patients and healthy controls in various other cancer diseases.8–12 This lead to the suggestion that circulating miRNAs are useful as novel noninvasive biomarkers. However, different levels of cell-free miRNAs have not been found between various stages in epithelial carcinomas as yet.10, 12 Since miRNA profiles in tumor cells have already been shown to have a prognostic relevance for some cancer patients,13, 14 it is conceivable that similar profiles also exist in serum. Therefore, we performed a screening study to identify circulating miRNAs correlating with prostate tumor progression by monitoring 667 human miRNAs. Of several miRNAs identified, circulating miRNA-375 and miRNA-141 turned out to be the most pronounced markers for tumor progression in two independent validation studies.
Material and Methods
Whole blood samples of prostate cancer patients were collected at the Martini-Clinic, Prostate Cancer Center (University Medical Center Hamburg-Eppendorf). All samples were taken from prostate cancer patients between 2002 and 2008 (Supporting Information Table 1) following written patient consent. The samples were allowed to stand at room temperature for 30 min. Blood was centrifuged at 2000g for 10 min to separate serum and cellular fractions. The serum was stored at −80°C. For initial screening, serum samples were taken from seven patients with primarily metastasized (bone-metastasis) tumors (median age: 69 ± 6; median PSA level: 308 ng/ml) and 14 patients with surgically treated localized prostate cancer one day prior to radical prostatectomy (median age: 63 ± 6; median PSA level: 4 ng/ml). As a selection criterion for this group, >5-year biochemical recurrence-free follow-up was applied. Since treatment has been shown to influence the level of circulating miRNAs,15 only serum samples from patients who received no hormone ablative or cytotoxic therapy before blood sampling, were used.
For our first validation study, 45 serum samples were prospectively collected from 42 prostate cancer patients one day prior to radical prostatectomy (IGPSV 1-30 and IGPSV 34-40) and three patients with primarily metastasized tumors (IGPSV 31-33) between March and June 2009 (Supporting Information Table 2).
For our second validation study, we selectively collected 71 serum samples from patients with high-risk tumors (Gleason ≥ 8 or N1) as well as intermediate risk tumors (Gleason 7 or N0). All samples were taken from prostate cancer patients between January 2008 and January 2009 (Supporting Information Table 3).
Preparation of tissue sections.
Radical prostatectomy specimens were obtained from 72 patients (Supporting Information Table 4). None of the patients had been treated with radio- or chemotherapy or ablative hormonal therapy. After surgical removal of the prostate, tissue samples were immediately taken with a 6-mm punch biopsy instrument (Biopsy Punch, Stiefel, Wächtersburg, Germany) from areas that were suspected to contain tumor foci based on information obtained from the preoperative systematic 10-location biopsies. Each tissue punch biopsy was immediately immersed in RNAlater (Qiagen, Hilden, Germany), stored overnight at ambient temperature and frozen at −20°C until preparation of tissue sections: First, the specimen was thawed at room temperature. To elute most of the RNAlater from the tissue, two subsequent washing steps of 5 min each in 10 ml ice-cold sterile PBS-buffer were performed. After fixation using Tissue-Tek® (Sakura, Netherlands) and thorough freezing in a cryo-microtome, cryo-sections were taken, stained with hematoxylin and eosin (H&E) and analyzed by an uropathologist. Tissues were only enrolled into the validation if at least 70% of cells were epithelial prostate tumor cells. Then, 10–15 subsequent unstained sections were transferred to a cryo-tube and the last section was again H&E stained and analyzed by an uropathologist. Normal prostate tissues were collected from tumor free areas and processed as described.
Isolation of circulating RNA from serum samples.
A combination of phenol/guanidine-based lysis and silicamembrane-based purification was used for the isolation of cell-free RNA from serum samples. Briefly, 5.4 ml Tri Reagent BD (Sigma, Munich, Germany) was added to 1.8 ml serum. To ensure complete dissociation of nucleoprotein complexes, the mixtures were incubated 5 min at room temperature. Then, a pool of miRNA mimics from C. elegans (cel-miRNA-39, cel-miRNA-54, cel-miRNA-238) (Qiagen) was added as a spike-in control for purification efficiency.7, 16 A total of 1.44 ml pure chloroform was added; the mix was shaken vigorously and allowed to stand for 5 min at room temperature. After 45 min of centrifugation (4000g; 4°C), 3.5 ml of the upper aqueous phase were transferred to a fresh tube. Purification of extracted total RNA was performed with miRNeasy (Qiagen) according to the manufacturer's instructions. RNA was eluted in a final volume of 30 μl RNase free water.
For the validation studies, a small-scale version of the same extraction protocol was employed. Phase separation was performed twice with 200 μl serum. After 15 min of centrifugation (6000g, 4°C), two aliquots of 300 μl aqueous phase were combined, and RNA was purified as described above. miRNeasy 96-well plates were applied due to the manufacturer's recommendations. RNA was eluted in a final volume of 50 μl RNase free water.
Screening and validation of tumor associated cell-free miRNA.
Total RNA was reverse transcribed to cDNA by priming with a mixture of looped primers and preamplified according to the manufacturer's instructions (Megaplex Pools with Preamplification, Applied Biosystems, Foster City, CA). Subsequently, quantitative real-time PCR amplification of miRNAs using low-density Taqman arrays v2.0 was performed using the Applied Biosystems 7900 Sequence Detection System. For validation and spike-in normalization, single miRNA assays were employed to quantify the mature miRNAs. Each miRNA assay was run in triplicate.
miRNA analysis in prostate tissue sections.
Total RNA including miRNAs was extracted using the AllPrep DNA/RNA Mini kit (Qiagen) according to the manufacturer's instructions. Tissue sections were homogenized in 1 ml RLT Plus buffer using TissueLyser (Qiagen). After DNA separation, 1.5 volumes of 100% ethanol were added to the total RNA and the mixture was purified. The cDNA equivalent to 100 ng total RNA was analyzed by low-density Taqman arrays and the protocols used for the serum samples (see above). In our study, we focused on the abundance of miRNA-375 and miRNA-141.
Analysis of the miRNA screening study: The Ct-values of the TaqMan arrays were analyzed for differential occurrences of miRNAs using the statistical computational environment R (http://www.r-project.org/). Ct-values not detected in the measurable range (Ct = 40) were considered as undetermined. After sample outlier removal, a filtering step was applied, wherein Ct values ≥35 were considered as nonspecific.17 miRNAs, for which >70% of all values were nonspecific, were excluded from the analysis.
Normalization was performed using C. elegans spike-in miRNAs as previously described.7, 16 After normalization, Limma analysis18 was used to identify differentially abundant circulating miRNAs. In this analysis, undetermined values were given lower weights (factor 0.1). Multiple testing19 adjusted false discovery rates (fdr) <0.05 were considered as significant.
To select additional miRNA for the validation study, the nonadjusted p-values were used as a ranking criterion. From the ranked list, miRNA with a log fold-change <−3 and an average Ct value ≤35 were included in the validation study.
Analysis of the validation and tissue study: The normalization for the Ct values of the validation study was performed as described for the screening study. In the tissue study, miRNA Ct values between samples were normalized using quantile normalization. To assess the significant differences of single miRNAs between measurements of different sample groups, a one-sided Wilcoxon test was used. A p-value <0.05 was considered as significant.
Serum screening study.
Circulating miRNAs are significantly upregulated in metastatic prostate cancer
To screen for circulating miRNAs associated with prostate cancer progression, 21 serum samples (Supporting Information Table 1) were analyzed. RNA was purified and 667 miRNAs were measured with low-density Taq-Man arrays. One sample (IGPS-20) was discarded as an outlier due to the large number of undetermined values (610 of 768). The overall abundances of circulating miRNAs revealed a significantly higher level of cell-free miRNAs in serum samples taken from patients with metastasized tumors when compared to those with primary prostate cancer (p = 0.02, Wilcoxon test; Supporting Information Figure 1). Limma analysis identified 69 miRNAs with higher abundance in this group (p < 0.05, without adjusting for multiple testing; Fig. 1a). miRNA-375 was the top marker and the only circulating miRNA which was significantly higher abundant in the sera of patients with distant metastases (fdr = 0.036; Fig. 1b). The log fold-change between sera of patients with metastatic prostate cancer and patients with primary cancers was considerably high (∼4 Ct values). All samples were within the measurable range (Ct < 40; Fig. 1).
Serum validation studies.
Circulating miRNAs are correlated with clinicopathological endpoints
Five miRNAs (miRNA-375, miRNA-9*, miRNA-141, miRNA-200b and miRNA-516a-3p) were chosen from the screening study (Table 1) for validation in 45 serum samples of an independent patient cohort (Supporting Information Table 2). The hierarchical clustering analysis demonstrated that five patients exhibited high levels of selected circulating miRNAs (Fig. 2). The tumors of these patients were characterized by at least one adverse risk factor (high Gleason score, lymph-node involvement or distant metastasis). In contrast, patients with lower risk profiles (low-grade tumors, organ confined tumors) showed low levels of the five selected miRNAs. miRNA-141 and miRNA-200b were highly correlated (Pearson; r = 0.8; 95% CI = 0.66) in the sample set.
Table 1. miRNAs in serum of metastatic prostate cancer patients identified by miRNA profiling
Top results of Limma analysis ranked by p-value. Markers for follow-up studies are bolded. Three miRNAs (e.g., miRNA-429, miRNA-618 and miRNA-502) were defined as unreliable, since most values were undetermined (average expression value: Ct > 36).
For the analysis, all serum samples were grouped according to the Gleason scores, lymph-node and pT status of the patients. Serum samples from patients with distant metastases were included in the group of Gleason score ≥8 tumors and lymph-node positive patients. Statistical analysis was performed for the comparison of pathological stage, Gleason scores and lymph-node status. Three of the selected miRNAs (miRNA-375, miRNA-141 and miRNA-200b) were significantly correlated with adverse risk factors in different comparisons and were further analyzed (Table 2). First, all three miRNAs were analyzed in comparison to the patients' PSA levels. Comparable to PSA, circulating miRNAs showed significant results when patients with a low-risk prostate cancer were compared to patients with an intermediate risk tumor (Table 2). However, PSA levels and circulating miRNA-200b did not distinguish between Gleason score 7 and Gleason score ≥8/M+ tumors or lymph-node status. An additional ANOVA analysis demonstrated improved prediction accuracy of high-risk tumors (Gleason score >8 or M+) when circulating miRNA-375 or miRNA-141 were introduced as additional parameters into a model containing PSA (Supporting Information Table 5).
Table 2. Circulating miRNAs are correlated with clinicopathological endpoints
Circulating miRNA-375, miRNA-141, miRNA-200b and PSA were analyzed for their association with different clinicopathological parameters in a prospective validation set (n = 45). Statistical analysis was done using one-sided Wilcoxon test (trend: p < 0.1; *p < 0.05; **p < 0.01; ***p < 0.001).
The number of high-risk prostate cancer samples (6 Gleason >8, 3 M+ and 9 N1) was limited in the first prospectively collected cohort. Additionally, the three patients with bone metastases were among those samples with the highest abundance of circulating miRNAs in the serum (data not shown). Therefore, we collected a larger independent serum sample set (n = 71; Supporting Information Table 3) from primary prostate cancer patients with a high-risk tumor disease (N1 or Gleason ≥ 8) to further analyze the serum level of the most promising circulating miRNAs (miRNA-375, miRNA-141 and miRNA-200b) in comparison to tumors with an intermediate risk (N0 or Gleason 7). Comparable to the first validation study, circulating miRNA-200b showed no significant differences in serum abundance when high-risk tumors were compared with intermediate risk prostate cancer (data not shown). However, higher levels of circulating miRNA-375 and miRNA-141 were again detected in patients with high-risk prostate cancer. miRNA-375 and miRNA-141 were considerably higher abundant in the sera of patients with lymph-node positive prostate cancer, whereas only a significant difference was observed for miRNA-141 in the comparison of Gleason 7 and 8 tumors (Fig. 3).
miRNA-375 and miRNA-141 are also upregulated in prostate tumor tissues
The levels of miRNA-375 and miRNA-141 were monitored in prostate tissue samples. Thirty-six tumor samples were compared with 36 benign prostate tissue samples (Supporting Information Table 4). Both miRNAs were significantly upregulated in tumor specimens (Fig. 4).
One of the major challenges in cancer research is the identification of novel biomarkers, which can be routinely measured in surrogates. An emerging field is the analysis of circulating miRNAs, which have recently been shown to be promising markers for the detection of common human cancer diseases.20
Here, we show for the first time that circulating miRNAs correlate with clinicopathological endpoints in prostate cancer. We screened the serum miRNA levels in two different clinical risk groups. The levels of circulating miRNAs were considerably higher in serum samples from patients with a metastasized tumor when compared to those with primary tumors. In line with this, Lodes et al. also recently reported a relative upregulation of serum miRNAs in prostate cancer when compared to sera from healthy donors.21
Our analysis identified 69 miRNAs to be present at higher levels in patients with malignant tumors compared to those with low risk. miRNA-375, miRNA-141 and miRNA-200b turned out to be the markers with the highest correlation with clinical parameters in the first prospectively collected sample set. In comparison with PSA, miRNA-375 and miRNA-141 indicated a better performance for the discrimination of patients with high-risk tumors (Gleason score ≥8 or metastases) from those with Gleason score 7 prostate cancers. ANOVA analysis indicated that circulating miRNAs might be additional noninvasive markers, which could improve clinical decisions.
Because of the timeframe of the prospective sample collection, the number of highly aggressive tumors was limited in our validation set. The interpretation of the results appeared to be further influenced by the fact that we had three serum samples from patients with metastases in our high-risk group. Therefore, we collected an independent second validation set consisting of serum samples from patients with high-risk tumors and compared the circulating miRNA levels with patients suffering from intermediate risk tumors.
miRNA-375 was the top marker in the screening study (metastatic vs. localized prostate cancer) and turned out to be the most pronounced marker in the first prospective validation set. In our second validation set, miRNA-375 was considerably associated with the lymph-node status of the prostate cancer patients, but there was no significant difference in the serum levels of patients with Gleason score 8 tumors and Gleason score 7 tumors. Therefore, it seems that the association of circulating miRNA-375 is more related to a systemic prostate cancer disease (lymph-node involvement or metastases) rather than to the grading of the primary prostate cancer. However, miRNA-375 expression was also monitored in prostate cancer tissues. Here, we observed higher expression of miRNA-375 in prostate tumor tissue compared to normal epithelium. This observation is in line with previous results22, 23 and might point to a potential link between tumor and serum status in prostate cancer patients. miRNA-375 has been shown to regulate a cluster of genes controlling cellular growth and proliferation in the pancreas, and its function is essential for glucose-induced insulin secretion.24, 25 However, the role of miRNA-375 in prostate tissue and tumor progression is unknown and should be further examined in functional studies.
Circulating miRNA-141 has recently been shown to distinguish between patients with metastatic prostate cancer and healthy controls.7 In agreement with this, we also identified higher abundance of miRNA-141 in serum of patients with high-grade tumors when compared to those with intermediate and low-grade tumors. miRNA-141 and miRNA-200b (another member of the same miRNA family) were reported as the two most overexpressed miRNAs in prostate epithelial cells relative to prostate stromal cells.7 Both miRNAs belong to the top markers identified in this screening study, and their serum levels were also correlated in the first prospective validation sample set. In contrast to miRNA-200b, circulating miRNA-141 turned out to be considerably higher abundant in patients with high-risk tumors when compared to intermediate risk samples in both independent validation sets. In the tissues, miRNA-141 was also found to be significantly upregulated in the tumor samples.
The mechanism of release and the roles of circulating miRNAs are still largely unclear. Recently, it was shown that the interaction between cells via mRNA and miRNA can be accomplished by microvesicle transfer.26 Circulating miRNAs are proposed to be released from tumor cells in microvesicles to ensure communication with recipient cells in the surrounding microenvironment: Skog et al. could show that glioblastoma-derived RNA contained in microvesicles is functional and processed in surrounding cells.27 Because circulating miRNAs might manipulate target cells, cell-free miRNAs might be not only serum markers for cancer and disease progression but also functionally relevant and therefore potential targets for novel therapy approaches.
In conclusion, our study indicates that circulating miRNAs offer promise as noninvasive biomarkers for tumor progression in the future. However, the prognostic relevance of particular markers like miRNA-375 and miRNA-141 has to be further corroborated. To this end, large-scale clinical studies for the detection of miRNA levels in serial samples after surgery or treatment are required.
We thank Annika Bittmann, Sabrina Balaguer-Puig, Thorsten Kühlwein and Marcello Schifani for excellent technical assistance. T.B. was supported by the DFG Clinical Research Group 179.