Tumor Biol. DOI 10.1007/s13277-014-2584-7
RESEARCH ARTICLE
Circulating miRNAs 21 and 221 as biomarkers for early diagnosis of prostate cancer Sameh Kotb & Ashraf Mosharafa & Mona Essawi & Heba Hassan & Alaa Meshref & Ahmed Morsy
Received: 17 May 2014 / Accepted: 29 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract To compare the expression of two promising circulating micro-ribonucleic acids (miRNAs 21 and 221) in patients with prostate cancer to subjects without cancer and to evaluate their potential role as specific noninvasive molecular biomarkers for prostate cancer diagnosis, circulating miRNAs 21 and 221 expression profiles were analyzed in 20 men aged 50–75 years, presenting with lower urinary tract symptoms (LUTSs) and undergoing transrectal ultrasound (TRUS)-guided prostate biopsy based on either elevated serum prostatespecific antigen (PSA) (>4.0 ng/ml) or suspicious digital rectal examination (DRE). The performance of miRNAs 21 and 221 in differentiating prostate cancer from nonmalignant cases was evaluated and compared to DRE and elevated PSA. miRNA 21 was overexpressed in 90 % of group A vs. 10 % of group B, while miRNA 221 was overexpressed in 80 % of group A vs. 20 % of group B (p=0.001). MiRNA 21 overexpression had the highest performance as a diagnostic test with a sensitivity of 90 % and a specificity 90 % (p=0.02). No correlations were noted between Gleason score of prostate cancer cases and relative quantity (RQ) 21 (r=−0.355, p = 0.292) or RQ 221 (r= −0.044, p= 0.892). Our study showed that serum miRNAs 21 and 221 expression profiling tests may be used as specific noninvasive molecular biomarkers for prostate cancer diagnosis due to their higher sensitivity and specificity with a high negative predictive value leading to a decrease in the biopsies taken for patients with elevated serum PSA values.
S. Kotb : A. Mosharafa (*) : A. Meshref : A. Morsy Urology Department, Cairo University, Cairo, Egypt e-mail: [email protected] M. Essawi : H. Hassan Medical Molecular Genetics Department, National Research Centre, Cairo, Egypt
Keywords miRNAs . Prostate neoplasms . Diagnosis . Biological markers
Introduction Since the publication of the first systematic profiling report detailing micro-ribonucleic acid (miRNA) expression in prostate cancer in 2007 [1], several studies have investigated miRNAs as possible diagnostic or prognostic biomarkers for prostate cancer. miRNAs are small (19–23 nucleotides) noncoding RNAs that have been shown to be involved in important biological processes including initiation of carcinogenesis or driving tumor progression [2]. There is growing evidence suggesting a role for certain miRNAs in prostate carcinogenesis and linking altered miRNA expression to androgen signaling and prostate cancer aggressiveness [3, 4]. In the current study, the authors compare the expression of two promising circulating miRNAs (miRNAs 21 and 221) in patients with prostate cancer to subjects without cancer and evaluate their potential role as specific noninvasive molecular biomarkers for prostate cancer diagnosis.
Patients and methods Patient selection Following approval by the institutional review board, patient recruitment was started on January 2012 until April 2013. Patients who met the following inclusion criteria were prospectively enrolled from the outpatient clinic of our institution—consecutive men aged 50–75 years—presenting with lower urinary tract symptoms (LUTSs), and indicated for transrectal ultrasound (TRUS) guided biopsy based on either elevated serum prostate-specific antigen (PSA) (>4.0 ng/ml)
Tumor Biol.
or suspicious digital rectal examination (DRE). Patients were excluded from the study if they had any contraindication for prostate biopsy or if they were diagnosed with other malignancies. Patients entered in the study underwent evaluation with complete history and physical examination, PSA, molecular analysis of miRNA as detailed below, and TRUS-guided prostate biopsy. A group of aged-matched men with no lower urinary tract symptoms and no indications for TRUS were used as controls for the miRNA analysis. Transrectal ultrasound-guided prostate biopsy Patients scheduled for TRUS-guided biopsies received a prophylactic fluoroquinolone antibiotic for 5 days and underwent the procedure with periprostatic nerve block. Routine ten-core prostate biopsies were obtained and sent for histopathologic examination. miRNA extraction and quantification Isolation of miRNAs from patients and controls followed the protocol for miRNeasy RNA isolation kit (Qiagen, Germany). Separation of serum took place immediately within 2 h from blood sample collection. Five volumes QIAzol lysis reagent were added to 200 μl of the serum, and then 1 volume of chloroform was added. After centrifugation, three phases are separated and a 1.5 volume of 100 % ethanol was added to the upper colorless aqueous phase and was then transferred to RNeasy mini spin column. This was followed by the washing steps and elution step producing a 35 μl of total RNA including miRNA. Quantitative reverse transcription (RT) polymerase chain reaction (PCR) assays were performed using TaqMan® MicroRNA assay and kit in accordance with the manufacturer’s recommended protocol with minor modifications. TaqMan endogenous control SNORD47 was used to normalize the expression levels of target genes by correcting differences in the amount of cDNA loaded into PCR reactions. Relative quantity (RQ) of miRNAs 21 and 221 was calculated by the formula (RQ=2−ΔΔCt), where Ct is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. Evaluating the role of miRNAs 21 and 221 as potential diagnostic markers Based on the results of histopathologic examination, patients were classified into group A (prostate cancer) or group B (nonmalignant prostate). Clinical, imaging, and PSA characteristics of patients in the two groups were compared. Expression levels of miRNAs 21 and 221 were compared to
suspicious DRE and elevated PSA as predictors of prostate cancer. The relation between miRNA expression and Gleason score in prostate cancer patients was also evaluated.
Statistical methods Data management and analysis were performed using SigmaStat program version 3.5 (Systat Software, Inc., USA). The graphs were done using Microsoft Excel 2007. The numerical data were statistically presented in terms of range, mean, standard deviation, median, and interquartile range (IQR). Categorical data were summarized as percentages. Comparisons between numerical variables of two groups were done by unpaired Student’s t test for parametric data or MannWhitney rank sum test for nonparametric data. One-way analysis of variance (ANOVA) test was used for comparing numerical variables of three groups. Comparing categorical variables was done by chi-square test or Fisher’s exact test for small sample size. Pearson product-moment correlation was used for testing association between different numerical variables. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratio (LR) of suspicious DRE, serum PSA at a cutoff value ≥10 ng/ml, overexpressed RQ 21, and overexpressed RQ 221 in detection of prostate cancer were calculated. p values less than 0.05 were considered significant.
Results Twenty patients were enrolled in the study. Age ranged from 53 to 75 years (mean±SD 64.5±6.0). Patients presented with LUTS, including two patients who presented with acute urinary retention. Digital rectal examination was suspicious in four (20 %) patients. Mean (±SD) estimated prostate weight of the prostate was 80.6 (±38.2)g, while mean (±SD) estimated weight of the adenoma was 55.8 (±32.6)g. The median PSA was 30 ng/ml (range 6.2 to 241 ng/ml). RQ of miRNAs 21 and 221 as calculated by the formula (RQ=2−ΔΔCt) revealed that RQ 21 ranged from 0.002 to 11.4 with a mean (±SD) of 2.9 (±3.8), and ten patients were showing overexpression. RQ 221 ranged from 0.004 to 13.16 with a mean (±SD) of 4.0 (±4.6), and ten patients were showing overexpression. Ten (50 %) patients were diagnosed with prostate adenocarcinoma (group A); details of the pathology are shown in Table 1. Table 2 details the differences in clinical, imaging, PSA, and miRNA expression characteristics between prostate cancer patients (group A) and non-prostate cancer patients (group B). Age, prostate size, and adenoma size were comparable in both groups. DRE was suspicious in 4 patients in group A, while it was not suspicious in the remaining 16
Tumor Biol. Table 1 Details of pathology results for prostate cancer patients Patient
Gleason score
Number of positive cores (% involved)
1 2 3 4 5 6 7 8 9 10
3+4 3+3 4+3 3+5 5+5 3+4 3+3 3+3 3+4 3+4
2 cores (80 and 100 %) 1 core 3 cores (30–40 %) All cores 5 cores (50–80 %) 5 cores (20–80 %) 1 core (10 %) 1 core All cores 6 cores (30–100 %)
patients. Median serum PSA in group A (50.5 ng/ml) was significantly higher than group B (18 ng/ml) (p=0.01). Table 2 Comparison of clinical, imaging, PSA, and miRNA expression characteristics of groups A and B Variable
Group A (prostate Ca), n=10
Group B (BPH), n=10
p value
Mean age±SD Suspicious DRE (N of patients) Prostate size (g) Range Mean±SD Median IQR (25–75 %) Adenoma size (g) Range Mean±SD Median IQR (25–75 %) Serum PSA (ng/ml) Range Mean±SD
64.9±6.5 4
64.1±5.7 0
0.8
25–113 68.7±29.6 66.15 45–93
23.5–170 92.6±43.4 79.5 69–124
13–90 42.3±24.8 30 25.5–57.5
15.2–126 67.8±35.2 59 43–105
6.2–241 78.9±72.8
10.3–34.9 20.9±9.7
Median IQR (25–75 %) RQ 21
50.5 27–101
18 13–33
Range Mean±SD Median IQR (25–75 %) RQ 221 Range Mean±SD Median IQR (25–75 %)
0.6–10.6 4.5±3.6 3.1 1.3–8.0
0.002–11.4 1.3±3.6 0.175 0.12–0.4
0.5–13.2 6.9±4.6 7.4 1.2–9.7
0.004–8.2 1.2±2.5 0.4 0.03–0.9
a
Statistically significant
0.169
0.089
0.011a
0.003a
a
0.004
RQ 21 and RQ 221 were calculated for groups A and B, and their comparison revealed that mean and median RQ21 and RQ 221 in prostate cancer patients were significantly higher than in BPH patients (p < 0.05). miRNA 21 was overexpressed (RQ>1) in nine (90 %) group A patients vs. one (10 %) patient in group B, and this difference was statistically significant (p = 0.001). miRNA 221 was overexpressed in eight (80 %) group A patients vs. two (20 %) in group B (p=0.02). The sensitivity, specificity, positive predictive value, and negative predictive value of suspicious DRE; serum PSA at cutoff value ≥10 ng/ml; overexpressed RQ 21; and overexpressed RQ 221 as clinical and laboratory predictors of malignant prostate biopsy were evaluated, and the results are shown in Table 3. miRNA 21 overexpression had the highest performance as a diagnostic test with a sensitivity of 90 %, a specificity of 90 %, and a likelihood ratio of 9. No correlation was noted between Gleason score of prostate cancer cases and RQ 21 (r=−0.355, p=0.292) or RQ 221 (r=−0.044, p=0.892).
Discussion The role of miRNAs in the genesis and progression of cancer was first suggested by Calin et al., who found that a genomic region at 13q14 containing two miRNAs (miR-15a and miR16-1) is frequently deleted in leukemia [5]. Altered miRNA expression has been since reported in many malignancies and can occur through genetic or epigenetic mechanisms. Dysregulated miRNAs can impact key pathways involved in cellular proliferation, metastasis, apoptosis, and angiogenesis [6]. miRNA 21 has been shown to exert an antiapoptotic effect mediated through the p53 network, specifically by targeting programmed cell death 4 (PDCD4) and the phosphatase and tensin homologue (PTEN) mRNAs [7, 8]. In addition, overexpression of miR-21 in a prostate cancer cell line (DU145) increased expression of HIF-1α and VEGF [9]. miRNA 221 has been shown to be upregulated in PC3 prostate cancer cells, and it is believed to inhibit the cell cycle by targeting p27 (kip1) to induce cell proliferation. Overexpression of miR-221 strongly affected the growth potential of LNCaP cells by inducing a G1 to S shift in the cell cycle and was sufficient Table 3 Clinical and laboratory predictors of malignant prostate biopsy
Suspicious DRE PSA ≥10 ng/ml miRNA 21 (overexpression) miRNA 221 (overexpression)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
40 90 90 80
100 0 90 80
100 47 90 80
62.5 0 90 80
PPV positive predictive value, NPV negative predictive value
Tumor Biol.
to induce a powerful enhancement of their colony-forming potential in soft agar [10]. Both miRNAs 21 and 221 are also involved in modulating androgen pathways and are implicated in androgen-independent growth of prostate cancer cells [3, 11]. miRNAs have been described in biological fluids (plasma, serum, urine, and others), and these extracellular miRNAs are relatively stable and resistant to physical degradation [12]. The use of circulating miRNA, therefore, has received a lot of attention as potential diagnostic or prognostic markers. Brase et al. analyzed serum samples in a group of patients with localized and metastatic prostate cancer, screening for 667 different miRNAs, and reported that miRNAs 141 and 375 correlated with the diagnosis and possibly the prognosis of prostate cancer [13]. Other studies identified panels of miRNAs that were able to discriminate prostate cancer patients from normal individuals and BPH patients [14–18]. In a study by Zhang et al., miRNA 21 was found to correlate significantly with the diagnosis of metastatic prostate cancer, PSA, and possibly docetaxel resistance [19]. Agaoglu et al. [20] reported that the expression of both miRNAs 21 and 221 in 51 patients with prostate cancer (median 1.51 and 0.71, respectively) was significantly higher than 20 controls (0.039 and 0.04, respectively). Our findings are consistent with these studies, with a clear pattern of overexpression of miRNAs 21 and 221 in prostate cancer patients (nine of ten and eight of ten patients, respectively) as compared to noncancer patients (one of ten and two of ten subjects, respectively). The ability of miRNAs 21 and 221 to differentiate between localized and metastatic prostate cancer was investigated by a number of authors. In the study by Agaoglu et al. [20], levels of miRNAs 21 and 221 were compared in patients with localized/locally advanced vs. metastatic prostate cancer, and the authors reported that patients with metastatic PCa had significantly higher levels for both miRNAs [1.15 for miRNA 21 in localized/locally advanced PCa and 2.64 in metastatic PCa (p=0.035); miRNA 221 levels were 0.23 and 1.18, respectively (p=0.01)]. Shen et al. evaluated the possible role of miRNAs in predicting aggressiveness of prostate cancer in a cohort of 82 patients. They reported that miRNA 21 was significantly upregulated in patients with intermediate and high CAPRA scores compared to those with low score. The expression of miRNA 21 was also significantly higher in patients with intermediate D’Amico scores than those with low scores with a sensitivity of 38.1 % and a specificity of 94.2 % [21]. Shen et al. did not find, however, an independent ability for miRNA 221 in predicting the aggressiveness of prostate cancer in their cohort. The relationship between miRNA 221 expression and prostate cancer aggressiveness is controversial, with some studies noting downregulation with aggressive prostate cancer and clinical recurrence [22, 23], others noting upregulation in
castration-resistant prostate cancer cell lines [11], while others finding no correlation between miRNA 221 expression and Gleason score [24]. In our group of patients, we could not find an association between overexpressed miRNA 21 or overexpressed miRNA 221 and Gleason score. The small number of patients in different Gleason score categories (e.g., only two patients with high Gleason score of 8–10) may have precluded the finding of significant associations. In our small cohort of patients, we found that circulating miRNAs 21 and 221 appeared to be promising biomarkers for the prediction of malignant prostate biopsy. Their performance as a diagnostic test was clearly superior to DRE and PSA. Whether this performance will hold true for other groups of patients will need further evaluation. The authors believe that miRNAs 21 and 221 can be included in a small panel of miRNAs to be tested as a potential diagnostic panel, especially in areas of clinical relevance such as patients with elevated PSA and prior negative biopsy or patients with “gray zone” PSA. The possible role of these miRNAs as targets for therapy is even more exciting, and further research in this area may prove very rewarding.
Conclusion Circulating miRNAs 21 and 221 expression profiles appear to be promising as potential noninvasive molecular biomarkers for the diagnosis of prostate cancer. In our cohort, overexpressed miRNAs 21 and 221 were able to predict malignant prostate biopsy with a sensitivity of 90 and 80 % and a specificity of 90 and 80 %, respectively.
References 1. Porkka KP, Pfeiffer MJ, Waltering KK, et al. MicroRNA expression profiling in prostate cancer. Cancer Res. 2007;67:6130–5. 2. Huang X, Liang M, Dittmar R, Wang L. Extracellular microRNAs in urologic malignancies: chances and challenges. Int J Mol Sci. 2013;14:14785–99. 3. Ribas J, Ni X, Haffner M, et al. miR-21: an androgen receptorregulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer growth. Cancer Res. 2009;69:7165–9. 4. Waltering K, Porkka K, Jalava S, Urbanucci A, Kohonen P, Latonen L, et al. Androgen regulation of micro-RNAs in prostate cancer. Prostate. 2011;71:604–14. 5. Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A. 2004;101:11755–60. 6. Ma R, Jiang T, Kang X. Circulating microRNAs in cancer: origin, function and application. J Exp Clin Cancer Res. 2012;31:38. 7. Papagiannakopoulos T, Shapiro A, Kosik KS. MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Res. 2008;68:8164–72.
Tumor Biol. 8. Lu Z, Liu M, Stribinskis V, et al. MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene. Oncogene. 2008;27:4373–9. 9. Liu LZ, Li C, Chen Q, Jing Y, Carpenter R, Jiang Y, et al. MiR-21 induced angiogenesis through AKT and ERK activation and HIF-1α expression. PLoS ONE. 2011;6(4):e19139. doi:10.1371/journal. pone.0019139. 10. Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafrè SA, et al. miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1. J Biol Chem. 2007;282(32):23716–24. 11. Sun T, Wang Q, Balk S, Brown M, Lee GS, Kantoff P. The role of microRNA-221 and microRNA-222 in androgen-independent prostate cancer cell lines. Cancer Res. 2009;69:3356–63. 12. Reid G, Kirschner MB, van Zandwijk N. Circulating microRNAs: association with disease and potential use as biomarkers. Crit Rev Oncol Hematol. 2011;80:193–208. 13. Brase JC, Johannes M, Schlomm T, et al. Circulating miRNAs are correlated with tumor progression in prostate cancer. lnt J Cancer. 2011;128:608–16. 14. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105:10513–8. 15. Moltzahn F, Olshen AB, Baehner L, et al. Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients. Cancer Res. 2011;71:550. 16. Lodes MJ, Caraballo M, Suciu D, Munro S, Kumar A, Anderson B. Detection of cancer with serum miRNAs on an oligonucleotide microarray. PLoS ONE. 2009;4:e6229.
17. Chen ZH, Zhang GL, Li HR, Luo JD, Li ZX, Chen GM, et al. A panel of five circulating microRNAs as potential biomarkers for prostate cancer. Prostate. 2012;72:1443–52. 18. Mahn R, Heukamp LC, Rogenhofer S, von Ruecker A, Muller SC, Ellinger J. Circulating microRNAs (miRNA) in serum of patients with prostate cancer. Urology. 2011;77:1265.e9–16. 19. Zhang HL, Yang LF, Zhu Y, et al. Serum miRNA-21: elevated levels in patients with metastatic hormone-refractory prostate cancer and potential predictive factor for the efficacy of docetaxel-based chemotherapy. Prostate. 2011;71:326–31. 20. Agaoglu FY, Kovancilar M, Dizdar Y, Darendeliler E, Holdenrieder S, Dalay N, et al. Investigation of miR-21, miR-141, and miR-221 in blood circulation of patients with prostate cancer. Tumor Biol. 2011;32:583–8. 21. Shen J, Hruby GW, McKiernan JM, Gurvich I, Lipsky MJ, Benson MC, et al. Dysregulation of circulating microRNAs and prediction of aggressive prostate cancer. Prostate. 2012;72: 1469–77. 22. Spahn M, Kneitz S, Scholz CJ, Stenger N, Rudiger T, Strobel P, et al. Expression of microRNA-221 is progressively reduced in aggressive prostate cancer and metastasis and predicts clinical recurrence. Int J Cancer. 2010;127(2):394–403. 23. Schaefer A, Jung M, Mollenkopf HJ, Wagner I, Stephan C, Jentzmik F, et al. Diagnostic and prognostic implications of microRNA profiling in prostate carcinoma. Int J Cancer. 2010;126(5):1166–76. 24. Kang SG, Ha YR, Kim SJ, et al. Do microRNA 96, 145 and 221 expressions really aid in the prognosis of prostate carcinoma. Asian J Androl. 2012;14:752–7.
RESEARCH ARTICLE
Circulating miRNAs 21 and 221 as biomarkers for early diagnosis of prostate cancer Sameh Kotb & Ashraf Mosharafa & Mona Essawi & Heba Hassan & Alaa Meshref & Ahmed Morsy
Received: 17 May 2014 / Accepted: 29 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract To compare the expression of two promising circulating micro-ribonucleic acids (miRNAs 21 and 221) in patients with prostate cancer to subjects without cancer and to evaluate their potential role as specific noninvasive molecular biomarkers for prostate cancer diagnosis, circulating miRNAs 21 and 221 expression profiles were analyzed in 20 men aged 50–75 years, presenting with lower urinary tract symptoms (LUTSs) and undergoing transrectal ultrasound (TRUS)-guided prostate biopsy based on either elevated serum prostatespecific antigen (PSA) (>4.0 ng/ml) or suspicious digital rectal examination (DRE). The performance of miRNAs 21 and 221 in differentiating prostate cancer from nonmalignant cases was evaluated and compared to DRE and elevated PSA. miRNA 21 was overexpressed in 90 % of group A vs. 10 % of group B, while miRNA 221 was overexpressed in 80 % of group A vs. 20 % of group B (p=0.001). MiRNA 21 overexpression had the highest performance as a diagnostic test with a sensitivity of 90 % and a specificity 90 % (p=0.02). No correlations were noted between Gleason score of prostate cancer cases and relative quantity (RQ) 21 (r=−0.355, p = 0.292) or RQ 221 (r= −0.044, p= 0.892). Our study showed that serum miRNAs 21 and 221 expression profiling tests may be used as specific noninvasive molecular biomarkers for prostate cancer diagnosis due to their higher sensitivity and specificity with a high negative predictive value leading to a decrease in the biopsies taken for patients with elevated serum PSA values.
S. Kotb : A. Mosharafa (*) : A. Meshref : A. Morsy Urology Department, Cairo University, Cairo, Egypt e-mail: [email protected] M. Essawi : H. Hassan Medical Molecular Genetics Department, National Research Centre, Cairo, Egypt
Keywords miRNAs . Prostate neoplasms . Diagnosis . Biological markers
Introduction Since the publication of the first systematic profiling report detailing micro-ribonucleic acid (miRNA) expression in prostate cancer in 2007 [1], several studies have investigated miRNAs as possible diagnostic or prognostic biomarkers for prostate cancer. miRNAs are small (19–23 nucleotides) noncoding RNAs that have been shown to be involved in important biological processes including initiation of carcinogenesis or driving tumor progression [2]. There is growing evidence suggesting a role for certain miRNAs in prostate carcinogenesis and linking altered miRNA expression to androgen signaling and prostate cancer aggressiveness [3, 4]. In the current study, the authors compare the expression of two promising circulating miRNAs (miRNAs 21 and 221) in patients with prostate cancer to subjects without cancer and evaluate their potential role as specific noninvasive molecular biomarkers for prostate cancer diagnosis.
Patients and methods Patient selection Following approval by the institutional review board, patient recruitment was started on January 2012 until April 2013. Patients who met the following inclusion criteria were prospectively enrolled from the outpatient clinic of our institution—consecutive men aged 50–75 years—presenting with lower urinary tract symptoms (LUTSs), and indicated for transrectal ultrasound (TRUS) guided biopsy based on either elevated serum prostate-specific antigen (PSA) (>4.0 ng/ml)
Tumor Biol.
or suspicious digital rectal examination (DRE). Patients were excluded from the study if they had any contraindication for prostate biopsy or if they were diagnosed with other malignancies. Patients entered in the study underwent evaluation with complete history and physical examination, PSA, molecular analysis of miRNA as detailed below, and TRUS-guided prostate biopsy. A group of aged-matched men with no lower urinary tract symptoms and no indications for TRUS were used as controls for the miRNA analysis. Transrectal ultrasound-guided prostate biopsy Patients scheduled for TRUS-guided biopsies received a prophylactic fluoroquinolone antibiotic for 5 days and underwent the procedure with periprostatic nerve block. Routine ten-core prostate biopsies were obtained and sent for histopathologic examination. miRNA extraction and quantification Isolation of miRNAs from patients and controls followed the protocol for miRNeasy RNA isolation kit (Qiagen, Germany). Separation of serum took place immediately within 2 h from blood sample collection. Five volumes QIAzol lysis reagent were added to 200 μl of the serum, and then 1 volume of chloroform was added. After centrifugation, three phases are separated and a 1.5 volume of 100 % ethanol was added to the upper colorless aqueous phase and was then transferred to RNeasy mini spin column. This was followed by the washing steps and elution step producing a 35 μl of total RNA including miRNA. Quantitative reverse transcription (RT) polymerase chain reaction (PCR) assays were performed using TaqMan® MicroRNA assay and kit in accordance with the manufacturer’s recommended protocol with minor modifications. TaqMan endogenous control SNORD47 was used to normalize the expression levels of target genes by correcting differences in the amount of cDNA loaded into PCR reactions. Relative quantity (RQ) of miRNAs 21 and 221 was calculated by the formula (RQ=2−ΔΔCt), where Ct is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. Evaluating the role of miRNAs 21 and 221 as potential diagnostic markers Based on the results of histopathologic examination, patients were classified into group A (prostate cancer) or group B (nonmalignant prostate). Clinical, imaging, and PSA characteristics of patients in the two groups were compared. Expression levels of miRNAs 21 and 221 were compared to
suspicious DRE and elevated PSA as predictors of prostate cancer. The relation between miRNA expression and Gleason score in prostate cancer patients was also evaluated.
Statistical methods Data management and analysis were performed using SigmaStat program version 3.5 (Systat Software, Inc., USA). The graphs were done using Microsoft Excel 2007. The numerical data were statistically presented in terms of range, mean, standard deviation, median, and interquartile range (IQR). Categorical data were summarized as percentages. Comparisons between numerical variables of two groups were done by unpaired Student’s t test for parametric data or MannWhitney rank sum test for nonparametric data. One-way analysis of variance (ANOVA) test was used for comparing numerical variables of three groups. Comparing categorical variables was done by chi-square test or Fisher’s exact test for small sample size. Pearson product-moment correlation was used for testing association between different numerical variables. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratio (LR) of suspicious DRE, serum PSA at a cutoff value ≥10 ng/ml, overexpressed RQ 21, and overexpressed RQ 221 in detection of prostate cancer were calculated. p values less than 0.05 were considered significant.
Results Twenty patients were enrolled in the study. Age ranged from 53 to 75 years (mean±SD 64.5±6.0). Patients presented with LUTS, including two patients who presented with acute urinary retention. Digital rectal examination was suspicious in four (20 %) patients. Mean (±SD) estimated prostate weight of the prostate was 80.6 (±38.2)g, while mean (±SD) estimated weight of the adenoma was 55.8 (±32.6)g. The median PSA was 30 ng/ml (range 6.2 to 241 ng/ml). RQ of miRNAs 21 and 221 as calculated by the formula (RQ=2−ΔΔCt) revealed that RQ 21 ranged from 0.002 to 11.4 with a mean (±SD) of 2.9 (±3.8), and ten patients were showing overexpression. RQ 221 ranged from 0.004 to 13.16 with a mean (±SD) of 4.0 (±4.6), and ten patients were showing overexpression. Ten (50 %) patients were diagnosed with prostate adenocarcinoma (group A); details of the pathology are shown in Table 1. Table 2 details the differences in clinical, imaging, PSA, and miRNA expression characteristics between prostate cancer patients (group A) and non-prostate cancer patients (group B). Age, prostate size, and adenoma size were comparable in both groups. DRE was suspicious in 4 patients in group A, while it was not suspicious in the remaining 16
Tumor Biol. Table 1 Details of pathology results for prostate cancer patients Patient
Gleason score
Number of positive cores (% involved)
1 2 3 4 5 6 7 8 9 10
3+4 3+3 4+3 3+5 5+5 3+4 3+3 3+3 3+4 3+4
2 cores (80 and 100 %) 1 core 3 cores (30–40 %) All cores 5 cores (50–80 %) 5 cores (20–80 %) 1 core (10 %) 1 core All cores 6 cores (30–100 %)
patients. Median serum PSA in group A (50.5 ng/ml) was significantly higher than group B (18 ng/ml) (p=0.01). Table 2 Comparison of clinical, imaging, PSA, and miRNA expression characteristics of groups A and B Variable
Group A (prostate Ca), n=10
Group B (BPH), n=10
p value
Mean age±SD Suspicious DRE (N of patients) Prostate size (g) Range Mean±SD Median IQR (25–75 %) Adenoma size (g) Range Mean±SD Median IQR (25–75 %) Serum PSA (ng/ml) Range Mean±SD
64.9±6.5 4
64.1±5.7 0
0.8
25–113 68.7±29.6 66.15 45–93
23.5–170 92.6±43.4 79.5 69–124
13–90 42.3±24.8 30 25.5–57.5
15.2–126 67.8±35.2 59 43–105
6.2–241 78.9±72.8
10.3–34.9 20.9±9.7
Median IQR (25–75 %) RQ 21
50.5 27–101
18 13–33
Range Mean±SD Median IQR (25–75 %) RQ 221 Range Mean±SD Median IQR (25–75 %)
0.6–10.6 4.5±3.6 3.1 1.3–8.0
0.002–11.4 1.3±3.6 0.175 0.12–0.4
0.5–13.2 6.9±4.6 7.4 1.2–9.7
0.004–8.2 1.2±2.5 0.4 0.03–0.9
a
Statistically significant
0.169
0.089
0.011a
0.003a
a
0.004
RQ 21 and RQ 221 were calculated for groups A and B, and their comparison revealed that mean and median RQ21 and RQ 221 in prostate cancer patients were significantly higher than in BPH patients (p < 0.05). miRNA 21 was overexpressed (RQ>1) in nine (90 %) group A patients vs. one (10 %) patient in group B, and this difference was statistically significant (p = 0.001). miRNA 221 was overexpressed in eight (80 %) group A patients vs. two (20 %) in group B (p=0.02). The sensitivity, specificity, positive predictive value, and negative predictive value of suspicious DRE; serum PSA at cutoff value ≥10 ng/ml; overexpressed RQ 21; and overexpressed RQ 221 as clinical and laboratory predictors of malignant prostate biopsy were evaluated, and the results are shown in Table 3. miRNA 21 overexpression had the highest performance as a diagnostic test with a sensitivity of 90 %, a specificity of 90 %, and a likelihood ratio of 9. No correlation was noted between Gleason score of prostate cancer cases and RQ 21 (r=−0.355, p=0.292) or RQ 221 (r=−0.044, p=0.892).
Discussion The role of miRNAs in the genesis and progression of cancer was first suggested by Calin et al., who found that a genomic region at 13q14 containing two miRNAs (miR-15a and miR16-1) is frequently deleted in leukemia [5]. Altered miRNA expression has been since reported in many malignancies and can occur through genetic or epigenetic mechanisms. Dysregulated miRNAs can impact key pathways involved in cellular proliferation, metastasis, apoptosis, and angiogenesis [6]. miRNA 21 has been shown to exert an antiapoptotic effect mediated through the p53 network, specifically by targeting programmed cell death 4 (PDCD4) and the phosphatase and tensin homologue (PTEN) mRNAs [7, 8]. In addition, overexpression of miR-21 in a prostate cancer cell line (DU145) increased expression of HIF-1α and VEGF [9]. miRNA 221 has been shown to be upregulated in PC3 prostate cancer cells, and it is believed to inhibit the cell cycle by targeting p27 (kip1) to induce cell proliferation. Overexpression of miR-221 strongly affected the growth potential of LNCaP cells by inducing a G1 to S shift in the cell cycle and was sufficient Table 3 Clinical and laboratory predictors of malignant prostate biopsy
Suspicious DRE PSA ≥10 ng/ml miRNA 21 (overexpression) miRNA 221 (overexpression)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
40 90 90 80
100 0 90 80
100 47 90 80
62.5 0 90 80
PPV positive predictive value, NPV negative predictive value
Tumor Biol.
to induce a powerful enhancement of their colony-forming potential in soft agar [10]. Both miRNAs 21 and 221 are also involved in modulating androgen pathways and are implicated in androgen-independent growth of prostate cancer cells [3, 11]. miRNAs have been described in biological fluids (plasma, serum, urine, and others), and these extracellular miRNAs are relatively stable and resistant to physical degradation [12]. The use of circulating miRNA, therefore, has received a lot of attention as potential diagnostic or prognostic markers. Brase et al. analyzed serum samples in a group of patients with localized and metastatic prostate cancer, screening for 667 different miRNAs, and reported that miRNAs 141 and 375 correlated with the diagnosis and possibly the prognosis of prostate cancer [13]. Other studies identified panels of miRNAs that were able to discriminate prostate cancer patients from normal individuals and BPH patients [14–18]. In a study by Zhang et al., miRNA 21 was found to correlate significantly with the diagnosis of metastatic prostate cancer, PSA, and possibly docetaxel resistance [19]. Agaoglu et al. [20] reported that the expression of both miRNAs 21 and 221 in 51 patients with prostate cancer (median 1.51 and 0.71, respectively) was significantly higher than 20 controls (0.039 and 0.04, respectively). Our findings are consistent with these studies, with a clear pattern of overexpression of miRNAs 21 and 221 in prostate cancer patients (nine of ten and eight of ten patients, respectively) as compared to noncancer patients (one of ten and two of ten subjects, respectively). The ability of miRNAs 21 and 221 to differentiate between localized and metastatic prostate cancer was investigated by a number of authors. In the study by Agaoglu et al. [20], levels of miRNAs 21 and 221 were compared in patients with localized/locally advanced vs. metastatic prostate cancer, and the authors reported that patients with metastatic PCa had significantly higher levels for both miRNAs [1.15 for miRNA 21 in localized/locally advanced PCa and 2.64 in metastatic PCa (p=0.035); miRNA 221 levels were 0.23 and 1.18, respectively (p=0.01)]. Shen et al. evaluated the possible role of miRNAs in predicting aggressiveness of prostate cancer in a cohort of 82 patients. They reported that miRNA 21 was significantly upregulated in patients with intermediate and high CAPRA scores compared to those with low score. The expression of miRNA 21 was also significantly higher in patients with intermediate D’Amico scores than those with low scores with a sensitivity of 38.1 % and a specificity of 94.2 % [21]. Shen et al. did not find, however, an independent ability for miRNA 221 in predicting the aggressiveness of prostate cancer in their cohort. The relationship between miRNA 221 expression and prostate cancer aggressiveness is controversial, with some studies noting downregulation with aggressive prostate cancer and clinical recurrence [22, 23], others noting upregulation in
castration-resistant prostate cancer cell lines [11], while others finding no correlation between miRNA 221 expression and Gleason score [24]. In our group of patients, we could not find an association between overexpressed miRNA 21 or overexpressed miRNA 221 and Gleason score. The small number of patients in different Gleason score categories (e.g., only two patients with high Gleason score of 8–10) may have precluded the finding of significant associations. In our small cohort of patients, we found that circulating miRNAs 21 and 221 appeared to be promising biomarkers for the prediction of malignant prostate biopsy. Their performance as a diagnostic test was clearly superior to DRE and PSA. Whether this performance will hold true for other groups of patients will need further evaluation. The authors believe that miRNAs 21 and 221 can be included in a small panel of miRNAs to be tested as a potential diagnostic panel, especially in areas of clinical relevance such as patients with elevated PSA and prior negative biopsy or patients with “gray zone” PSA. The possible role of these miRNAs as targets for therapy is even more exciting, and further research in this area may prove very rewarding.
Conclusion Circulating miRNAs 21 and 221 expression profiles appear to be promising as potential noninvasive molecular biomarkers for the diagnosis of prostate cancer. In our cohort, overexpressed miRNAs 21 and 221 were able to predict malignant prostate biopsy with a sensitivity of 90 and 80 % and a specificity of 90 and 80 %, respectively.
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