PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC Sep 1, 2012.
Published in final edited form as:
PMCID: PMC3167031
NIHMSID: NIHMS301958
microRNA-10b Expression Correlates with Response to Neoadjuvant Therapy and Survival in Pancreatic Ductal Adenocarcinoma
Meir Preis,(1) Timothy B. Gardner,(1) Stuart R. Gordon,(1) J. Marc Pipas,(1) Todd A. Mackenzie,(2) Erin E. Klein,(1) Daniel S. Longnecker,(3) Edward J. Gutmann,(3) Lorenzo F. Sempere,*,(1) and Murray Korc*,(1),(2)
(1) Department of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH
(2) Department of Pharmacology and Toxicology Dartmouth Medical School, Hanover, NH
(3) Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, NH
* Corresponding authors: Murray Korc, M.D., Department of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, N.H. 03756, Murray.Korc/at/Dartmouth.EDU, phone: 603-650-7936; fax: 603-650-6122. Lorenzo F. Sempere, Ph. D., Department of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH 03756, Lorenzo.F.Sempere/at/Dartmouth.EDU, phone: (603)653-9936; fax: (603)653-9952
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Diagnosis and management of PDAC are hampered by the absence of sensitive and specific disease biomarkers. MicroRNAs are non-coding regulatory RNAs involved in initiation and progression of human cancers. In this study we sought to determine whether miR-10b could serve as a biomarker for PDAC.
Methods
miRNA expression was characterized by fluorescence-based in situ hybridization (ISH) using Locked Nucleic Acid (LNA)-modified DNA probes against miR-10b, miR-21, miR-155, miR-196a, and miR-210, followed by co-detection of proteins by immunohistochemistry on the same tissue sections. miRNA expression in surgically resected PDAC tissues and in endoscopic ultrasonography (EUS)-guided fine needle aspirate (EUS-FNA) samples was analyzed in cytokeratin 19 (CK19)-positive epithelial cells using optical intensity analysis.
Results
In 10 resected PDAC samples miR-10b was the most frequently and consistently overexpressed miRNA among characterized miRNAs, exhibiting a 4-fold increase in the cancer cells (p=0.012). Given this preferential overexpression of miR-10b, we sought to determine whether miR-10b expression was clinically relevant. Accordingly, miR-10b expression was examined in 106 EUS-FNA samples obtained from pancreatic lesions. miR-10b expression was increased in cancer cells compared to CK19-positive epithelial cells in benign lesions (p=0.0001). In patients with PDAC, lower levels of miR-10b were associated with improved response to multimodality neoadjuvant therapy, likelihood of surgical resection, delayed time to metastasis, and increased survival.
Conclusion
miR-10b is a novel diagnostic biomarker for PDAC when assessing pancreatic lesions. Expression of miR-10b is predictive of response to neoadjuvant therapy and outcome in this disease.
Keywords: Pancreatic cancer, microRNA-10b, FNA, metastasis, neoadjuvant chemotherapy
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer-related deaths in the US with an annual mortality of nearly 38,000, a median survival of 6–7 months and a five-year survival rate of 4 to 5%.(1) PDAC is characterized by aggressive local invasion, early lymphatic and hematogenous dissemination, and chemotherapeutic resistance.(2) Tissue diagnosis is most often obtained by EUS-FNA sampling of the pancreatic mass or other suspicious lesion.(3) Less than 20% of PDAC patients are candidates for surgical resection at diagnosis.(45) Despite improved survival with adjuvant therapy following resection, most patients will eventually die of their disease.(68) The tumor associated antigen CA 19-9, which may be used adjunctively in the management of patients with PDAC following tumor resection to monitor disease recurrence, cannot predict the metastatic potential of tumor or patient survival following resection.(9) Currently, no tissue biomarkers are available to guide therapeutic strategies or predict patient prognosis in PDAC.
MicroRNAs (miRNAs) are short, 18–25 nucleotide, non-coding regulatory RNA that target the 3′-untranslated region of mRNAs by binding to imperfectly matching sites, thereby inducing mRNA degradation and/or repressing translation. These novel mechanisms for regulating gene expression and function have been shown to modulate cell proliferation, migration, differentiation and apoptosis, as well as tissue differentiation and morphogenesis during development, and to participate in cancer development and progression.(10)
The expression of several microRNAs, as determined by quantitative reverse-transcription PCR analysis (qRT-PCR), is altered in PDAC. Among others, miR-21, miR-10b, miR-155, miR-196a, miR-203, miR-210 and miR-221 levels are increased in PDAC by comparison with the normal pancreas, (1115) whereas miR-375 and miR-148 levels are decreased in PDAC.(12, 16) However, some of these studies did not take into account the fact that PDAC is highly desmoplastic, and that the pancreatic tumor mass may include variable amounts of inflammatory cells and mast cells, degenerating acinar cells, proliferating ductal cells, foci of acinar to ductal metaplasia (ADM) and/or pancreatic intraepithelial neoplasia (PanIN), as well as many cancer-associated fibroblasts and stellate cells.(17) It is important, therefore, to assess the spatial expression of miRNAs in PDAC tissue and to determine whether changes in expression of particular miRNAs are specific to the cancer cells, or involve other cell types in the pancreatic tumor mass.
We have previously reported on a highly sensitive fluorescence-based in situ hybridization (ISH) technique to assess miRNA expression within individual cells in formalin-fixed paraffin-embedded (FFPE) clinical specimens of solid tumors.(18) In the present study, we initially characterized by ISH the expression of miR-10b, miR-21, miR-155 miR-196a and miR-210 in 10 resected FFPE PDAC tissue samples and in three non-cacerous pancreatic tissues. Among these miRNAs, miR-10b was most frequently and consistently up-regulated in the cancer cells.
Previously, miR-10b has been reported to be expressed at high levels in metastatic breast cancer cell lines(19) and to promote the migration and invasion of esophageal cancer cells.(20) We reasoned, therefore, that characterization of miR-10b spatial expression by ISH in PDAC, in conjunction with our ability to assess miRNA expression specifically in the cancer cells (18), may yield clinically relevant information. Accordingly, we sought to determine whether miR-10b is differentially expressed between cancer cells and adjoining non-malignant cells in resected PDAC samples, whether its expression can be assessed by ISH in EUS-FNA samples, and whether its levels of expression in pancreatic cancer cells within the FNA samples could serve as a diagnostic and/or prognostic marker in PDAC, thereby potentially guiding therapeutic options. Evidence is provided for a strong positive correlation between increased miR-10b expression in the cancer cells and shorter time to metastasis, decreased overall patient survival and resistance to multimodality neoadjuvant chemotherapy and radiation. Thus, miR-10b could serve as a novel diagnostic and prognostic biomarker in PDAC, and as a tool for predicting response to therapy.
Human tissues
The study was approved by the Dartmouth College Committee for the Protection of Human Subjects. Ten paraffin embedded (FFPE) PDAC tissue blocks and three non-cancerous pancreatic specimens were obtained from the Dartmouth-Hitchcock Medical Center Department of Pathology archival files. The benign pancreatic tissues derived from a gun-shot victim, a patient with nesidioblastosis, and a patient who had a partial pancreatectomy whose pancreas only exhibited mild inflammatory changes. Non-malignant control tissues, deriving from the adjoining normal pancreas that was at least 2 cm away from the resection margin, were available for 8 of the 10 PDAC tissues.
After getting informed consent, EUS-FNA samples were obtained from 155 patients who were initially evaluated for suspicious pancreatic lesions that were mostly detected by CT scan, and in some cases by abdominal ultrasound. FNA samples that were either benign (n=11) or confirmed as PDAC (n=95) were included in this study. The remaining 49 cases, which were not included in this study were diagnosed as intraductal papillary mucinous neoplasm (11 cases), neuroendocrine tumors (20 cases), and other non-pancreatic malignancies (11 cases). In 7 cases the pathological diagnosis was undetermined.
All FNA samples were fixed in 10% formalin or ethanol, embedded in paraffin, cut in 4 μm sections, and mounted on positively-charged barrier frame slides. Clinical outcome data were obtained from medical records. Disease response to treatment was defined according to standard RECIST criteria.(21) Categories of response included: disease progression (DP), stable disease (SD), partial response (PR), complete response (CR).
Combined in situ hybridization and immunohistochemical staining
FFPE sections (4 μm thick) were subjected to combined ISH/IHC, as previously described.(18) For miRNA detection, we used LNA-modified DNA probes directed against the full length of the mature miRNA sequence. miR-10b probe: 5′-CA+CAA+ATT+CGG+TT+CTA+CAG+GGTA-3′; miR-21 probe: 5′- T+CAA+CAT+CA+GT+CTG+ATA+AG+CTA-3′; miR-155 probe: 5′-T+TA+AT+GCT+AAT+CGT+GAT+AG+GG+GT-3′; miR-196 a probe: 5′-CC+CAA+CAA+CAT+GA+AA+CT+AC+CTA-3′; miR-210 probe: 5′-TCA+GCC+GCT+GTC+ACA+CGC+ACAG-3′ (+N Denotes LNA modified nucleotide). FAM moieties were coupled to 5′ and 3′ terminal Ts, not part of the miRNA complementary sequence, as hapten for antibody detection. Tissue sections were also incubated with biotinylated DNA probe against U6 snRNA: 5′-CGTGTCATCCTTGCGCAGGGGCCATGCTAATCTTCTCTGT-3′. Then, expression of CK19 and amylase was assessed by sequential Tyramide Signal Amplification (TSA) reactions, as previously described (18). Briefly, CK19 was detected using mouse anti-CK 19 antibody (1:200, cat no. MU246-UC Biogenex), goat anti-mouse antibody conjugated to HRP (1:500, Cat no. 170-6516, Biorad), and TSA reaction with tyramine-conjugated to Rhodamine or Dylight594, Following heat induced epitope retrieval in buffered citrate, amylase expression was detected using mouse anti-amylase antibody (1:200, Cat no. SC-46657, Santa Cruz), and goat anti-mouse antibody conjugated to HRP (1:500, Cat no. 170-6516, Biorad), and TSA reaction with tyramine-conjugated to Dylight680. Slides were washed with PBST and mounted with anti-fading ProLong Gold solution (Invitrogen, Carlsbad, CA).
Quantitative image analysis
Fluorescent images were captured with a monochrome camera (EXi blue, Q Imaging) mounted on an Olympus BX60 microscope. Exposure time was determined by an automated setting using the ImagePro software (Media Cybernetics, Bethesda, MD) and the same magnification (x200) was used for image acquisition. A monochromatic image was obtained and saved for further analysis. The same field was captured through different filter cubes to record the staining for U6 snRNA (AMCA, Chroma Filter Set 31000), miR-10b (Fluorescein, Olympus Filter Set U-MNIBA), CK19 (rhodamine, Chroma Filter Set SP102V1 or Dylight 594, Chroma Filter Set SP107) and amylase (Dylight680, Chroma Filter Set SP105). Image files were later colorized as displayed in Figures 1, ,22 and and4.4. CK19-positive cells were marked as areas of interest (AOI) using ImagePro software. This AOI was applied on the matched miR-10b images and the fluorescence intensity for miR-10b was calculated using the signal intensity tool in the CK19-positive cells. Background subtraction of auto fluorescence was performed on the same AOI. We used the corrected (following background subtraction) mean fluorescent intensity (in arbitrary units) to present our data.
Figure 1
Figure 1
Spatial characterization of microRNA expression in resected pancreatic tissues
Figure 2
Figure 2
Spatial characterization of miR-10b expression in EUS-FNA smaples from pancreatic lesions
Figure 4
Figure 4
Levels of miR-10b and time to metastasis
Statistics
Kruskal-Wallis, and ANOVA were used as appropriate to compare continuous variables (e.g., age, miR-10b expression) between cancer stages. Wilcoxon-Mann-Whitney was used to compare miR-10b expression between PDAC and benign patients. Kruskal-Wallis, t-tests and ANOVA were used as appropriate to compare continuous characteristics across stages. miR-10b expression in the tissue samples was analyzed based on 3 levels of fluorescence intensity: low <5,000 a.u., intermediate 5,000–7,999 a.u., high ≥ 8,000 a.u. The utility of differential miR-10b expression for predicting response was tested using ordinal logistic regression (proportional odds Wald test), while its ability to predict suitability for surgery following induction chemotherapy was tested using logistic regression. Overall survival and metastatic free survival curves were subjected to Kaplan-Meier analysis and stratified by categories of miR-10b signal intensity. Cox’s model was employed to estimate hazard ratios for the association of miR-10b with survival and metastatic free survival. p<0.05 was taken as statistically significant.
MicroRNA expression in PDAC and normal pancreatic tissue
To determine the predominant cell-type responsible for deregulated expression of PDAC-associated miRNAs, we characterized the spatial distribution of miR-10b, miR-21, miR-155, miR-196a and miR-210 by fluorescence-based ISH assay in PDAC tissues and adjacent normal tissues. The same tissue sections were immunostained for CK19, a ductal cell marker in the normal pancreas and a cancer cell marker in PDAC, and for amylase, an acinar cell marker (Fig. 1A). The fluorescence intensity of each miRNA was determined in the CK19-positive cells in resected PDACs (n=10) and in benign pancreatic tissues (n=3). In the normal pancreas, H&E staining revealed an abundance of amylase-positive acinar cells, and a few CK19-positive ductal cells, and both cell types exhibited a weak miR-10b signal (Fig. 1A). By contrast, in PDAC, there was an abundance of CK19-positive cells cancer cells that expressed high levels of miR-10b (Fig. 1A). Among the 5 tested miRNAs, miR-10b was the most frequently and consistently overexpressed miRNA within cancer cells (Fig. 1B, S1), exhibiting a 4-fold increase in the cancer cells when compared with miR-10b levels in CK19-positive cells from normal pancreatic tissues (p=0.012); miR-21 exhibited the second highest level of selective expression in PDAC (Fig. 1B, S2).
PDACs often harbor chronic-pancreatitis like changes with foci of atrophic acinar cells and proliferating ductal cells, and both cell types may overexpress signaling molecules.(22) We therefore sought to determine whether there was differential expression of miR-10b in the cancer cells by comparison with the adjoining, non-malignant CK19-positive cells (Fig. S3). Accordingly, only samples for which adjoining normal pancreas (8 of the 10 used to generate the heat map) was available were included in this analysis. The mean fluorescence intensity in the CK19-positive cancer cells was significantly higher when compared with CK19-positive cells from the adjoining normal appearing pancreas (3,642±1322 vs. 733±394 a.u.; p=0.006), with only one PDAC sample not exhibiting high miR-10b levels (Fig. 1C).
MiR-10b expression in US guided FNA from suspicious pancreatic lesions
EUS-FNA sampling from a suspicious pancreatic mass is useful for diagnosing PDAC. To investigate whether miR-10b detection by ISH could be informative for guiding therapeutic intervention, EUS-FNA samples were obtained from 155 patients with suspicious pancreatic lesions. PDAC was diagnosed in 95/155 cases by light microscopy and benign cytology was suggested in 11/155 cases. The histopathological diagnosis of the remaining 49 case is detailed in the methods section.
In the benign lesions, the CK19-positive epithelial cells exhibited a weak miR-10b signal, which was also faintly present in the pancreatic acinar cells (Fig. 2A). In the FNA samples obtained from patients with PDAC, CK19-positive cells exhibited a strong and intense miR-10b signal (Fig. 2A). Based on the analysis of miR-10b expression in the CK19-positive cells, miR-10b expression was significantly elevated in the samples that were subsequently diagnosed as PDAC (Fig. 2B), when compared with miR-10b expression in samples from benign lesions (6,291±2,251 vs. 1,249±921 a.u.; p=0.0001).
Clinical characteristics and treatment modality
The clinical characteristics of the patients diagnosed with PDAC are described in Table 1.(23) As expected, patients with stage I disease had the longest median survival, whereas patients with stage IV disease had the shortest median survival. At Dartmouth-Hitchcock Medical Center, patients with PDAC without evidence of metastatic disease are generally offered a multimodality approach with neoadjuvant gemcitabine-based chemo-radiotherapy followed by attempted resection.(8, 2425) Forty-nine patients were treated with a multimodality neoadjuvant approach with the intention to follow with surgery, but only 24 of these patients underwent tumor resection (Table 1). None of the patients had surgery alone as a treatment modality (Table 1), and only 3 patients had surgery followed by adjuvant chemotherapy (Table 1). Among patients with stage I disease that did not have surgical resection, 4 patients elected to have palliative care and 4 patients had disease progression, including detection of previously undetectable metastatic disease or local disease progression. Similarly, of the 16 patients with stage II disease that did not have surgical resection, 4 patients elected to receive palliative care, one was lost to follow-up and 11 were treated initially with multimodality neoadjuvant chemo-radiotherapy. Of these 11 patients, one died of a myocardial infarction, one opted to discontinue therapy due to treatment-related toxicity, five had disease progression and 4 had worsening performance status that precluded surgical intervention. Patients with metastatic disease or with advanced disease that were not candidates for surgical resection were preferentially treated with palliative gemcitabine and/or best supportive care.
Table 1
Table 1
Baseline Characteristics and treatment choices of PDAC patient diagnosed by EUS-guided FNA
Clinical utility of miR-10b measurement in FNA samples
Correlation of miR-10b measurements with clinical outcome was assessed retrospectively following determination of the mean fluorescence intensity for miR-10b. In patients with resectable or locally advanced disease, relatively low miR-10b expression was highly predictive of response to multimodality neoadjuvant, gemcitabine based, chemo-radiotherapy (Fig. 3A, n=44, p=0.0012). By contrast, miR-10b levels did not correlate with response in patients with metastatic disease treated with gemcitabine-based, palliative chemotherapy (Fig. 3B, n=18, p=0.26). By logistic regression, low miR-10b expression (<5000 a.u.) predicted tumor resectability (p=0.0356). Moreover, the mean fluorescence intensity of miR-10b was significantly lower in patients with stage I or II disease that were treated with neoadjuvant chemo-radiation and underwent surgery, when compared with similarly-treated stage I/II patients whose tumors were unresectable due to disease progression (Fig. 3C, 5314±2039 vs. 7561±2061 a.u.; p=0.01). Two patients with locally advanced disease that were unresectable at baseline (stage III) and were treated with multimodality neoadjuvant approach were down-staged and underwent resection. Although both patients had lower levels of miR-10b compared with the 10 patients that remained unresectable, the numbers were too small to meaningfully assess statistical significance (Fig. 3C).
Figure 3
Figure 3
Levels of miR-10b predict response to multimodality neoadjuvant treatment
To assess correlation with metastasis free survival, miR-10b expression was analyzed based on 3 levels of fluorescence intensity: low <5,000 a.u., intermediate 5,000–7,999 a.u., and high ≥ 8,000 a.u. (Fig. 4A). The median time for progression to metastatic disease was significantly shorter in the patient with high miR-10b expression (3.7 months) compared to patients with intermediate (7.1 months) or low (8.1 months) levels of expression (Fig. 4B, p=0.001). The difference was statistically significant in stages I and II (HR, 3.3 for high vs. low miR-10b levels; 95% CI, 1.4–7.78; p=0.0055). All patients with high levels of miR-10b developed metastatic disease within 12 months, whereas approximately one-third of patients with low miR-10b levels were alive and free of metastasis after 24 months.
A comparison of survival between patients with low and high miR-10b levels demonstrated that miR-10b significantly predicted survival (p=0.0003, as a continuous marker). The median survival in the low, intermediate and high miR-10b expression groups was 12.1 months, 10.2 months and 4.04 months, respectively (Fig. 5A). The hazard ratio of miR-10b expression in the high vs. low group was 3.1 (95% CI, 1.66–5.77; p=0.0003). By contrast, there was no significant difference between the intermediate and low miR-10b expressing groups (p=0.11). In patients with stage I or II disease at diagnosis, but not in stage III or IV, high levels of miR-10b were associated with decreased survival compared to the patients with low miR-10b levels (Fig. 5A, HR, 3.86; 95% CI, 1.55–9.65; p=0.0032). Moreover, in patients who received multimodality neoadjuvant treatment followed by complete surgical resection, low miR-10b levels at diagnosis were associated with a statistically significant increase in survival compared with patients who had high miR-10b levels at diagnosis (Fig. 5B, HR, 5.1; 95% CI, 1.25–20.7; p=0.02).
Figure 5
Figure 5
miR-10b levels negatively correlate with overall survival
In patients presenting with localized PDAC, resection is usually followed by adjuvant chemotherapy or chemo-radiotherapy.(26) However, most patients treated in this fashion will eventually die of recurrent disease. For patients with locally advanced disease, no standard of care exists. Consequently, a number of centers have evaluated the usefulness of with neoadjuvant therapy to attempt to downsize/downstage tumors, improve rates of complete resection, and reduce local relapse rates.(27) This approach provides the potential benefit of early treatment of local disease and allows for the identification of those patients with rapidly progressive metastatic disease (thereby avoiding futile surgery). A recent large population-based comparison demonstrated a survival benefit for the neoadjuvant approach.(28) However, prospective randomized data comparing primary resection with neoadjuvant therapy are lacking.
In this study we initially evaluated the spatial expression of miR-10b, miR-21, miR-155, miR-196a and miR-210 in 10 resected PDAC samples, and determined that miR-10b was the most frequently overexpressed miRNA in the cancer cells within the tumor mass. We also demonstrated for the first time the spatial expression of miRNAs in EUS-FNA samples by ISH. The co-localization of miR-10b with CK19 in the same FNA samples enabled us to reproducibly assess miR-10b expression in the cancer cells within the tumor mass and the ductal cells in the normal pancreas. We were thus able to demonstrate that miR-10b expression was increased in the pancreatic cancer cells within the FNA samples by comparison with miR-10b levels in pancreatic ductal cells within benign lesions. Moreover, we determined that lower levels of miR-10b in the cancer cells were associated with response to neoadjuvant gemcitabine-based chemo-radiotherapy, surgical resectability, time to metastasis and overall survival.
The 5 year survival of PDAC patients treated with either upfront surgery or neoadjuvant chemo-radiotherapy is less than 30% despite complete resection.(4, 28) Although our study focused principally on patients who underwent neoadjuvant chemo-radiotherapy, it demonstrates for the first time that miR-10b expression in FNA samples can be used to delineate a subgroup of patients that will truly benefit from subsequent surgery. Thus, those patients whose cancers express low levels of miR-10b are predicted to have greater than 50% survival after 2 years. By contrast, high levels of miR-10b project poor outcome and early disease progression even after surgical resection. Our study design does not allow us to assess the potential role of characterizing miR-10b expression in patients who undergo primary surgical resection, but raises the possibility that such an analysis would also be useful in this group of patients.
It is noteworthy that miR-10b expression is elevated in several malignancies, including leukemia,(29) hepatocellular carcinoma,(30) melanoma,(31) and malignant glioma,(32) underscoring the potential importance of this miRNA in human cancers. Interestingly, the significance of miRNAs in tumor metastasis was first described by Ma et al. who demonstrated high expression of miR-10b in the metastatic breast cancer cell lines.(19) Moreover, silencing of miR-10b led to more than a 10-fold reduction in the invasive properties of these cells, whereas miR-10b overexpression induced a 4–5 fold increase in cell motility and invasiveness.(19) In vivo, miR-10b overexpression triggered a significant desmoplastic reaction and enhanced cell invasion. (33) Conversely, the administration of a miR-10b antagonist to orthotopic breast cancer cell implantation in mice markedly suppressed the formation of distant lung metastases.(33) Taken together, these reports underscore the importance of miR-10b in human malignancies and its potential to promote metastatic disease.
PDAC is a highly metastatic malignancy, and miR-10b has been linked to the pro-metastatic protein Twist1. (18) miR-10b also exerts post-transcriptional inhibition of the target gene HOXD10, (19) and represses the expression of genes that have significant role in modulating cell migration and remodeling the extracellular matrix.(19) Recently, a cascade of activation has been described whereby the extracellular matrix component hyaluronan binds to CD44 and promotes c-Src kinase activation, which then activates the transcription factor Twist.(34) Further analyses reveal that miR-10b is controlled by Twist and this process results in the reduction of a tumor suppressor protein HOXD10 leading to RhoA/RhoC up-regulation and Rho-kinase (ROK) activation promoting breast tumor cell invasion.(34) CD44 was also found to be expressed in tumor stem cells that have the unique ability to act as tumor initiating cells. (35) Additional targets for miR-10b were identified in esophageal cancer cells where miR-10b directly targets Kruppel-like factor 4(KLF4), promoting cell migration and invasion initiated by miR-10b.(20) By contrast, Tiam1 (T lymphoma invasion and metastasis 1) was identified as an additional target gene for miR-10b causing inhibition of breast cancer cell migration.(36) This finding underscores the importance of context specific expression of miR-10b in cancer cells. It will be important, therefore, to conduct studies with pancreatic cancer cell lines to examine the mechanisms of action of miR-10b in PDAC, which may provide additional evidence for considering miR-10b as a potential therapeutic target in PDAC.
EUS-FNA is commonly used for evaluating a pancreatic mass lesion, with 80% to 90% diagnostic sensitivity.(37) However, false negative and equivocal results may diminish the utility of this procedure. Previous studies that have analyzed miRNA expression in FNA samples have used qRT-PCR, which does not provide information regarding the cell type present in the sample and the specific site of expression of a particular miRNA. For example, let-7a-1 precursor RNA expression was consistently detected at lower levels in PDAC FNA samples compared to normal pancreatic tissue, but the small number of patients studied did not allow for the assessment of its diagnostic or prognostic value.(38) Similarly, the ratio of miR-196a to miR-217, as determined by qRT-PCR, was proposed to distinguish PDAC FNA samples from chronic pancreatitis and benign lesions.(13) However, it is not apparent from either of these studies whether a minimum content of cancer cells in these FNA samples was required to obtain an accurate diagnosis. This is of particular importance in light of recent findings that indicate that the accuracy of prognostic value of EGFR and gemcitabine resistance-related genes in FNA samples from PDAC patients who received gemcitabine-based therapy was greatly improved when laser capture microdissection (LCM) was performed to enrich for cancer cell content prior to qRT-PCR analysis.(3940) Thus, the use of a combined ISH/IHC assay enables rapid and direct evaluation of changes of miRNA expression within the suspicious cells highlighted by CK19 staining and provides an innovative alternative approach to the use of the technically demanding LCM coupled with qRT-PCR analysis.
Our findings must be interpreted with caution due to the limitations common to retrospective studies. Nonetheless, the present data suggest that assessing miR-10b expression by this combined ISH/IHC assay in FNA samples from pancreatic lesions could serve as a diagnostic maker that could help distinguish malignant from benign pancreatic tissue, and that miR-10b expression could potentially serve as a prognostic marker in PDAC. Analysis of additional miRNAs and proteins in the same FNA sample may further enhance the diagnostic accuracy of the EUS-FNA procedure and inform therapeutic options. Moreover, the same technology could be used in assessing biopsy material from a variety of solid tumors. Therefore, prospective studies are needed to determine whether this approach may ultimately serve as important tool for clinicians to guide clinical decision making regarding neoadjuvant treatment and surgery.
Translational Relevance
Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal malignancy, with few biomarkers for guiding therapeutic options. MicroRNAs have been implicated as having a role in PDAC. In the present study we evaluated the expression of miR-10b by in situ hybridization (ISH), using endoscopic ultrasonography (EUS)-guided fine needle aspirate (EUS-FNA) samples that were obtained from suspicious pancreatic lesions. We determined that miR-10b expression was increased in cancer cells compared with cells from benign lesions. Lower levels of miR-10b were associated with improved response to multimodality neoadjuvant therapy, longer time to metastasis and improved overall survival. Our findings indicate that miR-10b is a novel marker for assessing suspicious pancreatic lesions, that miR-10b may also serve as a biomarker for response to gemcitabine-based neoadjuvant therapy, and that it may be a predictor of early metastasis formation. Thus, a miR-10b-based clinical assay could potentially improve management and treatment of PDAC patients.
Supplementary Material
Figure S1
Figure S2
Figure S3
Acknowledgments
We thank: Carol Hart, Rebecca O’Meara and Mary Schwab at PTRL, Dartmouth Hitchcock Medical Center for their invaluable help.
Grant Support: This work was supported, in part, by National Cancer Institute (NCI) grants R21 CA113715 (M.K.) and CA- R37-075059 (M.K), and by an AACR-PanCAN Laurie and Paul MacCaskill Career Development Award for Pancreatic Cancer Research grant 08-20-25-SEMP (L.F.S.)
Footnotes
Conflict of interest: None
1. Bilimoria KY, Bentrem DJ, Ko CY, Ritchey J, Stewart AK, Winchester DP, et al. Validation of the 6th edition AJCC Pancreatic Cancer Staging System: report from the National Cancer Database. Cancer. 2007 Aug 15;110(4):738–44. [PubMed]
2. Preis M, Korc M. Kinase signaling pathways as targets for intervention in pancreatic cancer. Cancer Biol Ther. 2010 May 10;9(10) [PubMed]
3. Kahl S, Malfertheiner P. Role of endoscopic ultrasound in the diagnosis of patients with solid pancreatic masses. Dig Dis. 2004;22(1):26–31. [PubMed]
4. Sener SF, Fremgen A, Menck HR, Winchester DP. Pancreatic cancer: a report of treatment and survival trends for 100,313 patients diagnosed from 1985–1995, using the National Cancer Database. J Am Coll Surg. 1999 Jul;189(1):1–7. [PubMed]
5. Lim JE, Chien MW, Earle CC. Prognostic factors following curative resection for pancreatic adenocarcinoma: a population-based, linked database analysis of 396 patients. Ann Surg. 2003 Jan;237(1):74–85. [PubMed]
6. Herman JM, Swartz MJ, Hsu CC, Winter J, Pawlik TM, Sugar E, et al. Analysis of fluorouracil-based adjuvant chemotherapy and radiation after pancreaticoduodenectomy for ductal adenocarcinoma of the pancreas: results of a large, prospectively collected database at the Johns Hopkins Hospital. J Clin Oncol. 2008 Jul 20;26(21):3503–10. [PMC free article] [PubMed]
7. Corsini MM, Miller RC, Haddock MG, Donohue JH, Farnell MB, Nagorney DM, et al. Adjuvant radiotherapy and chemotherapy for pancreatic carcinoma: the Mayo Clinic experience (1975–2005) J Clin Oncol. 2008 Jul 20;26(21):3511–6. [PubMed]
8. Pipas JM, Mitchell SE, Barth RJ, Jr, Vera-Gimon R, Rathmann J, Meyer LP, et al. Phase I study of twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas. Int J Radiat Oncol Biol Phys. 2001 Aug 1;50(5):1317–22. [PubMed]
9. Locker GY, Hamilton S, Harris J, Jessup JM, Kemeny N, Macdonald JS, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol. 2006 Nov 20;24(33):5313–27. [PubMed]
10. Sempere Lorenzo F, Kauppinen Sakari. Translational Implications of MicroRNAs in Clinical Diagnostics and Therapeutics. In: Bradshaw Ralph A, Dennis Edward A., editors. Handbook of Cell Signaling. 2. Oxford: Academic Press; 2009. pp. 2965–81.
11. Greither T, Grochola LF, Udelnow A, Lautenschlager C, Wurl P, Taubert H. Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival. Int J Cancer. 2010 Jan 1;126(1):73–80. [PubMed]
12. Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, et al. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA. 2007 May 2;297(17):1901–8. [PubMed]
13. Szafranska AE, Doleshal M, Edmunds HS, Gordon S, Luttges J, Munding JB, et al. Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues. Clin Chem. 2008 Oct;54(10):1716–24. [PubMed]
14. Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL, et al. Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer. 2007 Mar 1;120(5):1046–54. [PMC free article] [PubMed]
15. du Rieu MC, Torrisani J, Selves J, Al Saati T, Souque A, Dufresne M, et al. MicroRNA-21 is induced early in pancreatic ductal adenocarcinoma precursor lesions. Clin Chem. 2010 Apr;56(4):603–12. [PubMed]
16. Hanoun N, Delpu Y, Suriawinata AA, Bournet B, Bureau C, Selves J, et al. The silencing of microRNA 148a production by DNA hypermethylation is an early event in pancreatic carcinogenesis. Clin Chem. 2010 Jul;56(7):1107–18. [PubMed]
17. Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho RA. Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev. 2006 May 15;20(10):1218–49. [PubMed]
18. Sempere LF, Preis M, Yezefski T, Ouyang H, Suriawinata AA, Silahtaroglu A, et al. Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res. 2010 Aug 15;16(16):4246–55. [PMC free article] [PubMed]
19. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007 Oct 11;449(7163):682–8. [PubMed]
20. Tian Y, Luo A, Cai Y, Su Q, Ding F, Chen H, et al. MicroRNA-10b promotes migration and invasion through KLF4 in human esophageal cancer cell lines. J Biol Chem. 2010 Mar 12;285(11):7986–94. [PubMed]
21. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1) Eur J Cancer. 2009 Jan;45(2):228–47. [PubMed]
22. Kloppel G, Adsay NV. Chronic pancreatitis and the differential diagnosis versus pancreatic cancer. Arch Pathol Lab Med. 2009 Mar;133(3):382–7. [PubMed]
23. Callery MP, Chang KJ, Fishman EK, Talamonti MS, William Traverso L, Linehan DC. Pretreatment assessment of resectable and borderline resectable pancreatic cancer: expert consensus statement. Ann Surg Oncol. 2009 Jul;16(7):1727–33. [PubMed]
24. Pipas JM, Barth RJ, Jr, Zaki B, Tsapakos MJ, Suriawinata AA, Bettmann MA, et al. Docetaxel/Gemcitabine followed by gemcitabine and external beam radiotherapy in patients with pancreatic adenocarcinoma. Ann Surg Oncol. 2005 Dec;12(12):995–1004. [PubMed]
25. Pipas JM, Zaki B, Suriawinata AA, Tsapakos MJ, Ripple GH, Colacchio TA, et al. Cetuximab, Intensity-Modulated Radiotherapy (IMRT), and Twice-Weekly Gemcitabine for Pancreatic Adenocarcinoma. Proceedings American Society of Clinical Oncology; 2006.
26. Network NCC. NCCN clinical practice guidelines in oncology: pancreatic adenocarcinoma. 2010 http://www.nccn.org/professionals/physician.gls/f_guidelines.asp.
27. Gillen S, Schuster T, Meyer Zum Buschenfelde C, Friess H, Kleeff J. Preoperative/neoadjuvant therapy in pancreatic cancer: a systematic review and meta-analysis of response and resection percentages. PLoS Med. 2010;7(4):e1000267. [PMC free article] [PubMed]
28. Artinyan A, Anaya DA, McKenzie S, Ellenhorn JD, Kim J. Neoadjuvant therapy is associated with improved survival in resectable pancreatic adenocarcinoma. Cancer. 2010 Nov 18; [PubMed]
29. Garzon R, Garofalo M, Martelli MP, Briesewitz R, Wang L, Fernandez-Cymering C, et al. Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3945–50. [PubMed]
30. Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S, et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology. 2008 Jun;47(6):1955–63. [PubMed]
31. Jukic DM, Rao UN, Kelly L, Skaf JS, Drogowski LM, Kirkwood JM, et al. Microrna profiling analysis of differences between the melanoma of young adults and older adults. J Transl Med. 2010;8:27. [PMC free article] [PubMed]
32. Sasayama T, Nishihara M, Kondoh T, Hosoda K, Kohmura E. MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC. Int J Cancer. 2009 Sep 15;125(6):1407–13. [PubMed]
33. Ma L, Reinhardt F, Pan E, Soutschek J, Bhat B, Marcusson EG, et al. Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nat Biotechnol. 2010 Apr;28(4):341–7. [PMC free article] [PubMed]
34. Bourguignon LY, Wong G, Earle C, Krueger K, Spevak CC. Hyaluronan-CD44 interaction promotes c-Src-mediated twist signaling, microRNA-10b expression, and RhoA/RhoC up-regulation, leading to Rho-kinase-associated cytoskeleton activation and breast tumor cell invasion. J Biol Chem. 2010 Nov 19;285(47):36721–35. [PubMed]
35. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3983–8. [PubMed]
36. Moriarty CH, Pursell B, Mercurio AM. miR-10b targets Tiam1: implications for Rac activation and carcinoma migration. J Biol Chem. 2010 Jul 2;285(27):20541–6. [PubMed]
37. Eloubeidi MA, Chen VK, Eltoum IA, Jhala D, Chhieng DC, Jhala N, et al. Endoscopic ultrasound-guided fine needle aspiration biopsy of patients with suspected pancreatic cancer: diagnostic accuracy and acute and 30-day complications. Am J Gastroenterol. 2003 Dec;98(12):2663–8. [PubMed]
38. Torrisani J, Bournet B, du Rieu MC, Bouisson M, Souque A, Escourrou J, et al. let-7 MicroRNA transfer in pancreatic cancer-derived cells inhibits in vitro cell proliferation but fails to alter tumor progression. Hum Gene Ther. 2009 Aug;20(8):831–44. [PubMed]
39. Fujita H, Ohuchida K, Mizumoto K, Itaba S, Ito T, Nakata K, et al. High EGFR mRNA expression is a prognostic factor for reduced survival in pancreatic cancer after gemcitabine-based adjuvant chemotherapy. Int J Oncol. 2011 Mar;38(3):629–41. [PubMed]
40. Fujita H, Ohuchida K, Mizumoto K, Itaba S, Ito T, Nakata K, et al. Gene expression levels as predictive markers of outcome in pancreatic cancer after gemcitabine-based adjuvant chemotherapy. Neoplasia. 2010 Oct;12(10):807–17. [PMC free article] [PubMed]