Figures
Abstract
Introduction
Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.
Materials and Methods
Total RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.
Results
All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA.
Citation: Link A, Becker V, Goel A, Wex T, Malfertheiner P (2012) Feasibility of Fecal MicroRNAs as Novel Biomarkers for Pancreatic Cancer. PLoS ONE 7(8): e42933. https://doi.org/10.1371/journal.pone.0042933
Editor: Jörg D. Hoheisel, Deutsches Krebsforschungszentrum, Germany
Received: January 9, 2012; Accepted: July 16, 2012; Published: August 8, 2012
Copyright: © Link et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Pancreatic cancer (PCA) is the second leading cause of gastrointestinal cancer-related deaths in Europe and USA [1], [2]. Despite intensive basic and clinical research the median survival of patients with PCA remains approximately 6–10 months after diagnosis, which highlights the aggressive tumor biology of this disease [3]. The majority of patients with PCA (>80%) present with regional or distant tumor dissemination at diagnosis that renders them ineligible for surgical tumor removal, which is at present the only potential curative therapy for this malignancy [1], [3]. Besides potential cancer-preventive lifestyle modifications (diet, physical exercise, cessation of smoking etc.), screening and detection of early PCA is considered as most promising strategy for reducing PCA-associated mortality [4]–[6]. Current clinical screening and diagnostic management of PCA patients is based on imaging techniques such as endoluminal ultrasound (EUS) or contrast-enhanced multi-detector CT scan. However, due to their invasive nature, use of radiation, and low sensitivity/specificity, such techniques at best may only be used for the screening in high-risk patients (e.g. hereditary PCA, Peutz-Jeghers syndrome etc.) [6], [7]. Therefore, there is an immediate need for identification and development of novel biomarkers, preferably non-invasive diagnostic technologies that could predict the development of PCA or its diagnosis at earliest stages.
The importance of such non-invasive biomarkers has long been recognized. Consequently, blood, stool or other body fluids are currently believed to be the best avenues for biomarker research that incorporates a broad spectrum of genetic and/or epigenetic alterations associated with cancer [6], [8]. Based upon this paradigm, multiple biomarkers such as hTERT, CA72-4, Osteopontin, REG4, MIC-1, K-ras, p-53 or aberrant methylation of the CpG islands of the tumor suppressor genes are currently under evaluation [3], [9]–[11]. Nevertheless, till date, only carbohydrate antigen 19-9 (CA19-9) has found its clinical usefulness in the early detection of recurrent disease, following surgical treatment of PCA patients [12]. However, the use of CA19-9 for the PCA-screening is not recommended due to low sensitivity and specificity [13].
MicroRNAs (miRNAs), the small non-coding transcripts of ∼22 nucleotides, have recently been identified as a new class of cellular molecules with significant diagnostic, prognostic and therapeutic implications [14]. MiRNAs play an important role in a wide range of physiological and pathological processes [15]. By virtue of regulating gene expression, miRNAs are involved at the earliest steps in cancer pathogenesis of multiple human cancers [15], [16]. In addition, existence of unique miRNA expression patterns permit identification of different types and subtypes of cancers [17]–[19]. One of the most exciting features of miRNAs is their biological stability compared to DNA or mRNA. MiRNAs are remarkably protected from endogenous and exogenous degradation due to their small size and exosomal presence in body fluids [20], [21]. Based on this paradigm, several research groups have independently shown the potential of miRNA as blood-based biomarkers for PCA [21]–[23]. While blood-based approach is still a matter of the intensive research, it has also been suggested that gastrointestinal cancer-related genetic and epigenetic alterations may potentially be detected in feces [8], [24]. Considering the high volume (∼1.5 l) of pancreatic juice production and excretion into the bowel, it has been hypothesized that precancerous or early cancer-related molecular changes may be detectable in feces and blood of pancreatic cancer patients, providing a strong rationale for fecal biomarker research [8].
Taking into account that PCA is associated with unique alteration pattern of miRNA expression, we hypothesized that miRNA expression signature may be identifiable in feces of patients with pancreatic neoplasia. Accordingly, in the present study, we evaluated the potential of fecal miRNA expression analyses for identifying PCA. As a proof of principal, we demonstrate that several fecal miRNAs are differentially expressed in patients with pancreatic disease. Pilot analyses of stool specimens from patients with chronic pancreatitis and PCA suggest a potential role for fecal miRNAs as novel biomarkers in the early detection of PCA.
Materials and Methods
Clinical samples
The study was performed on the archived fecal specimens from consecutive patients that have been collected for clinical analyses and prospectively collected feces from healthy subjects. All prospectively recruited healthy volunteers provided written informed consent and the protocol was approved by the Institutional Review Board at Baylor University Medical Center [24]. The archived fecal samples were de-identified, since the written informed consent could not be obtained due to patient's migration or death, and the protocol was approved by the Institutional Review Board of Otto-von-Guericke University Magdeburg (Magdeburg, Germany). A total of 45 stool specimens, including 15 controls, 15 patients with chronic pancreatitis and 15 PCA patients were included in the study. Control samples were randomly selected from the stool samples collected for detection of Helicobacter pylori in feces from patients with non-malignant GI-disorders such as reflux or gastritis. Samples from patients with chronic pancreatitis and PCA were selected from consecutive patients with available frozen stool samples, which were collected for analyzing fecal elastase levels as a routine diagnostic test for exocrine pancreas insufficiency. Clinical and demographical data of the patients are presented in Table 1.
Stool samples collection
All samples were collected and processed uniformly by using feces sample collection tubes (Sarstedt AG, Nümbrecht, Germany) as practiced in the routine clinical settings. Ambulatory patients were asked to collect the feces at home and bring the feces during the next presentation in the department. Fecal sample from stationary patients have been collected in similar way. Prior to reaching the laboratory the samples were either stored at +4°C or at room temperature. Once the samples reached the laboratory, the samples were aliquoted and stored continuously at −30°C until further use.
RNA isolation
Total RNA (including miRNAs) was extracted from stool specimens using Qiagen's miRNAeasy Mini Kits (Qiagen) as described previously [24]. Briefly, approximately 30–50 µl of frozen aliquoted stool was homogenized with RNase-free water to a total 150 µl and promptly lysed with 800 µl of QIAzol lysis reagent (Qiagen, Hilden, Germany). Precipitation was performed with chloroform and the aqueous phase was mixed with 1.5 volumes of 100% ethanol. Following elution, RNA quality was assessed using A260/A280 ratios using UV-spectroscopy and the samples were stored at −80°C until further analysis.
MicroRNA quantification by real-time PCR
Quantitative miRNA expression analyses were performed using either TaqMan miRNA Assays (Applied Biosystems, CA, USA) or SYBRgreen method according to the manufacturers' instructions as described previously [24]. After reverse transcription, samples were run using iCycler IQ® detection system (Bio-Rad, CA, USA). To avoid an inter-plate bias/variability, miRNA expression analyses were performed using a single 96-well plate in duplicates. Differences between the groups are presented as ΔCt, which represent differences between the mean Ct value of the miRNA of interest and the mean Ct value of the normalizer miRNA. We used miR-16 for the fecal miRNA normalization in all samples as it has been shown to be most consistently expressed in different fecal samples including samples from colorectal cancer patients as described previously [24]. Selection of microRNAs was performed using following criteria: a) reported as pancreas-specific microRNA (miR-375) [25]; b) previously reported for potential implication in pancreatic carcinogenesis (miR-21, -196a, -210, -143, -155, -375, -216a) [26]–[28]; c) differential expression of miRNAs has been reported for blood (miR-21, -155, -196a, -210) [21]–[23] and/or pancreatic fluid (miR-21, -155; Table 2) [29]. Primer sequences for the RT-PCR assays are listed in the Table S1.
MicroRNA microarray expression profiling and data analysis
MicroRNA microarray expression profiling was performed with the aim to evaluate the presence of selected miRNAs in feces as previously described [24]. Briefly, total RNA was extracted from a single stool sample from a healthy subject using Qiagen's miRNAeasy Mini Kit as described above. Amplification and hybridization was performed according to the manufacturer's instructions (Illumina, Inc., San Diego, CA). Microarray data processing and analysis were performed using Illumina's BeadStudio software. Normalization has been performed using the Lumi Bioconductor software package creating log miRNA expression values [30]. Following conservative probe filtering step, which excluded probes that did not reach a detection value of P<0.05 the analyses resulted in the reliable detection for 630 probes.
Statistical analysis
Data analyses were performed with GraphPad Prism 4.0 software (San Diego, CA, USA) or SPSS 17.0 (IBM Deutschland GmbH, Munich, Germany). The differences between two groups were analyzed using Student's t-test. The differences between more than two groups were analyzed using Kruskal-Wallis method with appropriate Dunn's multiple comparison post hoc test. Correlation analyses were performed using Pearson's test. Wherever appropriate, logarithmic regression was used to calculate the R2 and to create the equation of the slope. A two sided p-value of <0.05 was regarded as significant.
Results
Selection of fecal miRNAs
To investigate whether PCA patients have detectable fecal miRNA expression alterations, we first performed systematic analyses of the literature with the aim to select several miRNAs with highest evidence for miRNA deregulation in PCA patients. Based on the differential miRNA expression in pancreatic tumor tissues [18], [26]–[28], alterations in pancreatic juice [29] and in blood [21]–[23], as described above. We subsequently selected 5 upregulated (miR-21, -210, -143, -155, -196a) and 2 downregulated miRNAs (miR-216a and -375) for further analyses. Table 2 provides a global overview of PCA-related miRNA expression alterations. To gain a global overview for the detectability of the selected miRNAs in feces in normal healthy subjects, fecal miRNA expression profiling was performed using miRNA microarrays using a single stool sample from the healthy volunteer. As shown in Figure 1A, all of the selected miRNAs were present in feces at detectable concentrations.
(A) MiRNA microarray expression analyses were performed using Illumina microarray to evaluate the presence of selected miRNAs (miR-16, -375, -196a, -216a, -21, -143, -155 and -210) in a single stool sample of the healthy subject. Fecal miRNA expression was converted to log-expression values following Lumi Bioconductor normalization. (B & C) Expression of selected miRNA was confirmed in all 45 samples including controls, chronic pancreatitis and pancreatic cancer patients. Figure (B) represents the raw miRNA expression Ct-values for qualitative assessment. (C) Normalization was performed using standard ΔCt-method using miR-16 as internal fecal normalizer.
Variation and normalization of fecal miRNAs
Next, we evaluated the miRNA expression in all samples simultaneously. Overall, all fecal miRNAs analyzed showed high degree of variation (over 10 Ct's) for the raw values (Figure 1B). To normalize the miRNA expression, ΔCt-values were calculated using miR-16 as an internal endogenous normalizer as described previously [24]. Following normalization, we discovered that the expression levels of miR-375, -21 and -210 demonstrated low (standard deviation (±SD) 0.76, 0.71, 0.78, respectively), and those of miR-216a, -143 and -155 (±SD: 3.31, 1.97, 2.15, respectively) showed high inter-individual variation across all samples (Figure 1C).
Long-term stability of fecal miRNAs
It has been previously shown that fecal miRNAs are stable in freshly collected samples or FOBT-kits [24], but it is not known whether miRNAs are stable during long-term storage. To test the stability of miRNAs under the long-term storage conditions, we analyzed fecal miRNA samples from an additional group of 30 healthy subjects. Among these, fifteen fecal samples (1–15) were collected between 2004 and 2006 and fifteen samples (16–30) between 2009 and 2010. As shown on the Figure 2A, all analyzed miRNAs demonstrated similar expression levels for both miR-16 and miR-196a. Mean±SD for miR-16 (Figure 2B) was 30.53±2.21 for samples after long-term storage compared to 29.59±1.39 for freshly collected samples (p = 0.172), and that of miR-196a was 33.41±2.84 vs. 32.75±1.43 (p = 0.426), respectively (Figure 2C). After normalization with miR-16, mean ΔCt-value for miR-196a was -2.88±1.03 for samples collected from 2004–2006 and −3.16±0.93 (p = 0.441) for samples collected during 2009–2010 (Figure 2D).
To evaluate the long-term stability of the samples, we performed miRNA analyses in the fecal samples collected at different time points. Samples 1–15 were collected between 2004 and 2006 and samples 16–30 between 2009 and 2010 from healthy subjects. All samples were stored and processed in similar conditions. Figure (A) shows variations in miR-16 and miR-196a expression among all feces samples. (B) miR-16 and (C) miR-196a expression in subgroup analyses showed similar expression (p>0.1). (D) Normalized miR-196a expression is comparable in long- and short-term stored samples (p = 0.441). (E) Since miR-216a was present in feces at lowest concentrations, and its expression was analyzed by two independent quantitative RT-PCR runs to evaluate the reproducibility of the analysis (p<0.0001). Normalization was performed with using miR-16 as internal normalizer.
Reproducibility of results for the miRNAs with low expression
Among all analyzed miRNAs, miR-216a was present in feces at lowest concentration (Figure 1C). To investigate if even miRNA with low expression can be reliably and reproducibly analyzed in feces, we performed two independent expression measurements. As demonstrated in Figure 2E, miR-216a expression was reproducibly detected in feces from all samples despite its low concentration (R2 = 0.761, p<0.0001).
Differential expression of fecal miRNAs in PCA patients
To evaluate whether fecal miRNA expression analysis approach might be useful in PCA diagnosis, 45 fecal samples were evaluated. The clinical characteristics of the patients are shown in the Table 1. Fifteen fecal samples from controls were compared with fifteen each from patients with PCA and with chronic pancreatitis. All 7 tested miRNAs that are abundantly dysregulated in pancreatic cancer, were present in feces, although with specific variations in miRNA expression. Four of 7 miRNAs (miR-196a, -216a, -143 and -155) were expressed at significantly lower levels in stool samples from patients with PCA compared to controls (Kruskal-Wallis with Dunn's post test p<0.05 for miR-196a and miR-216a and p<0.001 for miR-143 and -155). Among these, only miR-143 and -155 reveal significant differences between patients with chronic pancreatitis and controls. Interestingly, subgroup analyses among the three groups revealed gradual decline in the median ΔCt for miRNA expression in fecal samples from controls to lower expression in patients with chronic pancreatitis and lowest in patients with PCA (miR-196a: −2.50 vs. −3.00 vs. −3.65; miR-216a: −5.30 vs. −9.10 vs. −10.80; miR-143: −5.20 vs. −6.00 vs. −7.35 and miR-155: 0.15 vs. −1.95 vs. −2.55 for controls vs. chronic pancreatitis vs. PCA, respectively) as shown in Figure 3. It is worth mentioning that in spite of the lower miRNA expression in feces from patients with PCA and chronic pancreatitis, miRNA expression differences were not statistically significant. Notably, miR-21, -375 and -210, which are frequently dysregulated in pancreatic cancers, were found to be unchanged in feces from controls and patients with PCA and chronic pancreatitis (Figure 3).
Figures (A) to (G) represent different miRNAs that were selected for the study based on the alterations in PCA tissues. *represents p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75th and 25th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90th and 10th percentiles, respectively.
A combination of miRNAs as diagnostic tool for PCA
It was previously reported that expression pattern of several miRNAs rather than single miRNA would likely result in higher sensitivity and specificity for the identification of human cancers [28]. Based on this assumption we hypothesized that combination of 4 differentially expressed miRNAs would provide better discrimination of patients with PCA compared to controls. Since all 4 miRNAs showed gradual decrease in miRNA expression, we summarized ΔCt-values of miR-196a, -216a, -143 and -155 for each sample. As shown in Figure 4, the median ∑ΔCt-Values were: for controls −12.78 vs. −21.45 for chronic pancreatitis vs. −23.25 for PCA (Kruskal-Wallis test p<0.0002, Dunn's post test controls vs. chronic pancreatitis or PCA p<0.05 and 0.001, respectively). Using this combination, we achieved median ΔCt-Value difference of 10.47 (or 1418 fold) between PCA and controls, while ΔCt-value difference for miR-143 alone was 2.15 (or 4.4 fold).
Summation of the ΔCt-values of miR-196a, -216a, -143 and -155 was performed to calculate the Σ ΔCt-value. *- p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75th and 25th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90th and 10th percentiles, respectively.
Discussion
In this proof-of-principle study, we evaluated feasibility of fecal miRNAs as potential biomarkers for the detection of PCA. Using a subset of miRNAs that are frequently dysregulated in PCA, we found that miR-196a, -216a, -143 and -155 are present at lower levels in fecal samples from patients with PCA compared to controls. In addition, we show that changes in fecal miRNA expression can also be detected in patients with chronic pancreatitis. Lastly, we demonstrate that a combination of a subset of miRNAs may provide a superior diagnostic value with a higher sensitivity and specificity for the identification of pancreatic neoplasia.
Apart from imaging techniques, there is a clear need for alternative modalities for the early detection of PCA. For example, the invasive collection of pancreatic juice or pancreatic duct brushing may seem potentially promising; this can only be performed in a limited number of patients due to its invasiveness and lack of suitability for screening purposes. Blood-based biomarker research has always been the focus, as it has tremendous potential for developing non-invasive screening approaches. However, feces have also been suggested as promising alternative, specifically for GI-related malignancies, due to constant shedding of exfoliated cells and production of pancreatic fluid [8], [24]. It is believed that fecal markers may even have higher diagnostic yield, since molecular cancer-related alterations in DNA, mRNA and miRNAs can be detected much earlier in feces. Recently, we and others have shown that detection of fecal miRNAs is a feasible approach that has a high degree of reproducibility, and that patients with colon neoplasia often show higher expression of miR-21 and -106a compared to controls [24], [31].
Currently, compelling evidence exists for tissue-specific alterations in miRNA expression in PCA [21], [26]–[28]. Those miRNAs have been the focus of multiple studies demonstrating complex interactions with key pathways and targets in pancreatic carcinogenesis. Based on existing knowledge for miRNA expression patterns in PCA, in the present study we selected 7 PCA-associated miRNAs that are either up- (miR-21, -155, -143, -210 and – 196a) or down-regulated (miR-375, -216a) for miRNA expression analyses in feces. In a pivotal study, Bloomston et al. demonstrated that PCA have distinct miRNA expression pattern with specific alterations of 25 miRNAs vs. benign pancreatic tissues and 21 miRNAs when comparing to chronic pancreatitis tissues. Using this miRNA expression pattern the authors could distinguish PCA tissues from benign and chronic pancreatitis tissues with accuracy of more than 90% [26]. Subsequently, it was shown that expression of miR-21 and -196a was associated with high proliferation (Ki67-index), presence of liver metastasis and prediction of poor survival/prognosis [19], [26]. Our data confirm previous reports demonstrating remarkable stability of fecal miRNAs in feces, as well as their high concentrations present in fecal samples [24], [32], [33]. Using target-based approach, we found differential miRNA expression of 4 out of 7 miRNAs (miR-196a, -216a, -143 and -155) in feces from patients with PCA in comparison to controls, while 3 miRNAs (miR-21, -375 and -210) were expressed at similar levels among various healthy subjects and patients. In this proof-of-principle study it was somewhat surprising to note that four of the differentially expressed miRNAs in our study showed decreased miRNA expression in feces of patients with PCA, while previous studies have demonstrated upregulation of miR-196a, -143 and -155 in pancreatic tumor tissues of patients with pancreatic cancer and down-regulation of miR-216a [26]–[28]. We hypothesized that the reason for the change in fecal vs. tissues expression of these miRNAs may be because of the alterations in pancreatic fluid outflow, which is frequently pathological in patients with pancreatic cancer and might be partially responsible for this discrepant observation. Because of the retrospective nature of the study, we were unable to gather data for the status of bile/pancreatic duct obstruction or perform paired tumor and fecal miRNA analyses. However, our hypothesis gains support from several another studies. First, it was previously shown that selected miRNAs (miR-21, -210, -155, -196a) were upregulated in plasma from patients with PCA, suggesting their mechanistic involvement in pancreatic cancers [21], [22]. Second, an independent study has demonstrated increased expression of miR-155 in pancreatic fluid of PCA patients [29], further supporting our observation. Third, our own observation for the gradual gradual decrease in the expression of selected miRNAs in feces of patients with chronic pancreatitis compared to controls and PCA patients support this argument. Nevertheless, we did not find any significant differences in fecal miRNA expression between PCA and patients with chronic pancreatitis that may reflect either the high risk for PCA development in patients with CP from one hand side or may potentially suggest a relationship to exocrine or endocrine pancreatic insufficiency.
In this study, miRNA profiling was solely used to gain global overview for the identification of miRNA expression patterns in fecal samples from pancreatic cancer patients. Since available samples were retrospectively collected and stored for long-term, we purposely avoided profiling of these tumor tissues in order to avoid potential selection bias using a high-throughput microarray-based technique. Even though selected miRNAs showed insignificant differences between CP and PCA patients, we believe that our results are thought provoking for PCA biomarker research, and provide a compelling springboard for undertaking additional prospective studies in the future to systematically evaluate potential differences in miRNAs that are unique to these patient subsets with regards to pancreatic and bile duct status. Results of our study highlight the need for further research on the biological significance of deregulated miRNAs in pancreatic neoplasia. As shown in the Table 2, most of the miRNAs analyzed in our study have multiple, cancer-related validated gene targets in human cancers. For example, Gironella et al. have shown that miR-155 targets Tumor protein 53-induced nuclear protein 1 (TP53INP1) disrupting its anti-tumoral activity in pancreatic cancer cells [34]. Several groups have shown that miR-21 targets PTEN, RECK, PDCD4, which may be responsible of oncogenic role of miR-21 [35], [36]. Likewise, miR-143, which is frequently deregulated in pancreatic cancers, has been shown to interact with KRAS [37] and restitution of tumor suppressor miRNA may inhibit tumor growth in xenograft pancreatic cancer model using nanovector delivery of miR-143/145 [38]. Additionally, miR-196a and miR-210 have been shown to play an oncogenic role by targeting several tumor suppressor genes (ANXA1, KRT5, RAD52) affecting proliferation and hypoxic stress [39]–[42], while miR-375 and -216a, which are frequently downregulated in pancreatic cancer, have been implicated in insulin secretion and in regulation of cell survival and proliferation by targeting several oncogenes such as PDK1, JAK2, ATG7 [25], [43]–[46]. This data are appealing for further intensive research of biological significance of miRNAs as biological effect of exosomal miRNA may be beyond the cellular or local tumoral effects [47], [48].
Current data implicate that miRNA expression patterns, rather than a single miRNA might be necessary for better utility for a miRNA-based diagnostic approach. For example, in our study, we performed a simple summation of the ΔCt values that allowed us to achieve a better discrimination of median ΔCt-values patients with PCA compared to controls. Such an approach may be of importance if fecal miRNA expression patterns are used for the screening of other GI malignancies. This is particularly relevant since fecal analysis is widely implemented in the clinical practice e.g. fecal occult blood tests for colorectal cancer screening and for the evaluation of pancreatic elastase in stool as a biomarker for exocrine pancreatic insufficiency. If validated in future, the vision of fecal miRNA-based approach for the identification of GI tumors could be easily translated into the diagnostics, an approach that will undoubtedly have a superior patient compliance compared to the currently available invasive diagnostic tests.
Although promising, fecal miRNA-based approach needs further intensive investigation prior to clinical application. As acknowledged previously, due to our ability to include only limited number of samples in this proof-of-principle study, the statistical significance of our results provides limited evidence for clinical applicability and potential of fecal miRNA for diagnosis of PCA patients in immediate future. Nonetheless, we believe that our findings are novel and future studies including prospective collection of the samples with various tumor stages with complete biochemical and clinical annotation for pancreatic/bile duct obstruction are needed because of potential impact of the pancreatic duct obstruction on fecal miRNA expression levels. Additionally, systematic analyses of feces using microarray or ultra-high throughput sequencing will help in uncovering novel fecal miRNAs and allow a methodological selection of potential biomarkers and their specificity for various diseases and conditions. Taking into consideration recent publication, where the slight increase of miR-16 has been described in KRASG12D transgenic animal model, high-throughput approaches will allow independent validation of specific microRNA as internal normalizers for PCA [49]. In light of these facts, ongoing research will have to elucidate the benefits of fecal miRNA not only in sporadic PCA but also in the high-risk populations that included patients with familial history.
In summary, we demonstrate that evaluation of fecal miRNAs can be effectively deployed in the PCA biomarker research. Differential expression of miRNAs in stool of patients with PCA suggests that fecal miRNAs can be attractive biomarkers for detection of PCA. Alterations in fecal miRNA expression levels offer the novel tool for further biomarker research for PCA screening in larger prospective studies.
Acknowledgments
We thank Ursula Stolz, Simone Philipsen, Marion Holley and Ingrid Bierwirth for their technical assistance. The authors thank Francesc Balaguer and Juan José Lozano for the help with microarray data expression analysis.
Author Contributions
Conceived and designed the experiments: AL TW PM. Performed the experiments: AL VB. Analyzed the data: AL VB AG. Contributed reagents/materials/analysis tools: AL AG TW. Wrote the paper: AL AG TW PM.
References
- 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2009) Cancer statistics, 2009. CA Cancer J Clin 59: 225–249.
- 2. La Vecchia C, Bosetti C, Lucchini F, Bertuccio P, Negri E, et al. (2010) Cancer mortality in Europe, 2000–2004, and an overview of trends since 1975. Ann Oncol 21: 1323–1360.
- 3. Ghaneh P, Costello E, Neoptolemos JP (2008) Biology and management of pancreatic cancer. Postgrad Med J 84: 478–497.
- 4. Stan SD, Singh SV, Brand RE (2010) Chemoprevention strategies for pancreatic cancer. Nat Rev Gastroenterol Hepatol 7: 347–356.
- 5. Furukawa H, Okada S, Saisho H, Ariyama J, Karasawa E, et al. (1996) Clinicopathologic features of small pancreatic adenocarcinoma. A collective study. Cancer 78: 986–990.
- 6. Greenhalf W, Neoptolemos JP (2006) Increasing survival rates of patients with pancreatic cancer by earlier identification. Nat Clin Pract Oncol 3: 346–347.
- 7. Canto MI (2007) Strategies for screening for pancreatic adenocarcinoma in high-risk patients. Semin Oncol 34: 295–302.
- 8. Haug U, Wente MN, Seiler CM, Jesenofsky R, Brenner H (2008) Stool testing for the early detection of pancreatic cancer: rationale and current evidence. Expert Rev Mol Diagn 8: 753–759.
- 9. Harsha HC, Kandasamy K, Ranganathan P, Rani S, Ramabadran S, et al. (2009) A compendium of potential biomarkers of pancreatic cancer. PLoS Med 6: e1000046.
- 10. Buchholz M, Kestler H, Gress TM (2008) Differential diagnosis of pancreatic tumors by molecular analysis of clinical specimens. Pancreatology 8: 551–557.
- 11. Melnikov AA, Scholtens D, Talamonti MS, Bentrem DJ, Levenson VV (2009) Methylation profile of circulating plasma DNA in patients with pancreatic cancer. J Surg Oncol 99: 119–122.
- 12. Hidalgo M (2010) Pancreatic cancer. N Engl J Med 362: 1605–1617.
- 13. Greenhalf W, Grocock C, Harcus M, Neoptolemos J (2009) Screening of high-risk families for pancreatic cancer. Pancreatology 9: 215–222.
- 14. Ambros V (2004) The functions of animal microRNAs. Nature 431: 350–355.
- 15. Calin GA, Croce CM (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 6: 857–866.
- 16. Balaguer F, Link A, Lozano JJ, Cuatrecasas M, Nagasaka T, et al. (2010) Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis. Cancer Res 70: 6609–6618.
- 17. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435: 834–838.
- 18. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, et al. (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 103: 2257–2261.
- 19. Roldo C, Missiaglia E, Hagan JP, Falconi M, Capelli P, et al. (2006) MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J Clin Oncol 24: 4677–4684.
- 20. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105: 10513–10518.
- 21. Wang J, Chen J, Chang P, LeBlanc A, Li D, et al. (2009) MicroRNAs in plasma of pancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of disease. Cancer Prev Res (Phila) 2: 807–813.
- 22. Kong X, Du Y, Wang G, Gao J, Gong Y, et al. (2011) Detection of differentially expressed microRNAs in serum of pancreatic ductal adenocarcinoma patients: miR-196a could be a potential marker for poor prognosis. Dig Dis Sci 56: 602–609.
- 23. Ali S, Almhanna K, Chen W, Philip PA, Sarkar FH (2010) Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer. Am J Transl Res 3: 28–47.
- 24. Link A, Balaguer F, Shen Y, Nagasaka T, Lozano JJ, et al. (2010) Fecal MicroRNAs as novel biomarkers for colon cancer screening. Cancer Epidemiol Biomarkers Prev 19: 1766–1774.
- 25. Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, et al. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432: 226–230.
- 26. Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, et al. (2007) MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 297: 1901–1908.
- 27. Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, et al. (2007) Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer 120: 1046–1054.
- 28. Szafranska AE, Davison TS, John J, Cannon T, Sipos B, et al. (2007) MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene 26: 4442–4452.
- 29. Sadakari Y, Ohtsuka T, Ohuchida K, Tsutsumi K, Takahata S, et al. (2010) MicroRNA expression analyses in preoperative pancreatic juice samples of pancreatic ductal adenocarcinoma. JOP 11: 587–592.
- 30. Du P, Kibbe WA, Lin SM (2008) lumi: a pipeline for processing Illumina microarray. Bioinformatics 24: 1547–1548.
- 31. Ahmed FE, Jeffries CD, Vos PW, Flake G, Nuovo GJ, et al. (2009) Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue. Cancer Genomics Proteomics 6: 281–295.
- 32. Wu CW, Ng SS, Dong YJ, Ng SC, Leung WW, et al. (2012) Detection of miR-92a and miR-21 in stool samples as potential screening biomarkers for colorectal cancer and polyps. Gut 61: 739–745.
- 33. Kalimutho M, Del Vecchio BG, Di Cecilia S, Sileri P, Cretella M, et al. (2011) Differential expression of miR-144* as a novel fecal-based diagnostic marker for colorectal cancer. J Gastroenterol 46: 1391–1402.
- 34. Gironella M, Seux M, Xie MJ, Cano C, Tomasini R, et al. (2007) Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development. Proc Natl Acad Sci U S A 104: 16170–16175.
- 35. Bhatti I, Lee A, James V, Hall RI, Lund JN, et al. (2011) Knockdown of microRNA-21 inhibits proliferation and increases cell death by targeting programmed cell death 4 (PDCD4) in pancreatic ductal adenocarcinoma. J Gastrointest Surg 15: 199–208.
- 36. Park JK, Lee EJ, Esau C, Schmittgen TD (2009) Antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. Pancreas 38: e190–e199.
- 37. Kent OA, Chivukula RR, Mullendore M, Wentzel EA, Feldmann G, et al. (2010) Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway. Genes Dev 24: 2754–2759.
- 38. Pramanik D, Campbell NR, Karikari C, Chivukula R, Kent OA, et al. (2011) Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice. Mol Cancer Ther 10: 1470–1480.
- 39. Crosby ME, Kulshreshtha R, Ivan M, Glazer PM (2009) MicroRNA regulation of DNA repair gene expression in hypoxic stress. Cancer Res 69: 1221–1229.
- 40. Luthra R, Singh RR, Luthra MG, Li YX, Hannah C, et al. (2008) MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers. Oncogene 27: 6667–6678.
- 41. Maru DM, Singh RR, Hannah C, Albarracin CT, Li YX, et al. (2009) MicroRNA-196a is a potential marker of progression during Barrett's metaplasia-dysplasia-invasive adenocarcinoma sequence in esophagus. Am J Pathol 174: 1940–1948.
- 42. Tsuchiya S, Fujiwara T, Sato F, Shimada Y, Tanaka E, et al. (2011) MicroRNA-210 regulates cancer cell proliferation through targeting fibroblast growth factor receptor-like 1 (FGFRL1). J Biol Chem 286: 420–428.
- 43. Chang Y, Yan W, He X, Zhang L, Li C, et al. (2012) miR-375 Inhibits Autophagy and Reduces Viability of Hepatocellular Carcinoma Cells Under Hypoxic Conditions. Gastroenterology
- 44. Ding L, Xu Y, Zhang W, Deng Y, Si M, et al. (2010) MiR-375 frequently downregulated in gastric cancer inhibits cell proliferation by targeting JAK2. Cell Res 20: 784–793.
- 45. Kato M, Wang L, Putta S, Wang M, Yuan H, et al. (2010) Post-transcriptional up-regulation of Tsc-22 by Ybx1, a target of miR-216a, mediates TGF-{beta}-induced collagen expression in kidney cells. J Biol Chem 285: 34004–34015.
- 46. Tsukamoto Y, Nakada C, Noguchi T, Tanigawa M, Nguyen LT, et al. (2010) MicroRNA-375 is downregulated in gastric carcinomas and regulates cell survival by targeting PDK1 and 14-3-3zeta. Cancer Res 70: 2339–2349.
- 47. Kosaka N, Iguchi H, Yoshioka Y, Hagiwara K, Takeshita F, et al. (2012) Competitive interactions of cancer cells and normal cells via secretory microRNAs. J Biol Chem 287: 1397–1405.
- 48. Zhang HG, Grizzle WE (2011) Exosomes and cancer: a newly described pathway of immune suppression. Clin Cancer Res 17: 959–964.
- 49. LaConti JJ, Shivapurkar N, Preet A, Deslattes MA, Peran I, et al. (2011) Tissue and serum microRNAs in the Kras(G12D) transgenic animal model and in patients with pancreatic cancer. PLoS One 6: e20687.