Methods for the Diagnosis of Pancreatic Cancer

Abstract

The present invention relates to the diagnosis of pancreatic cancer, in particular to a salivary mi RNA for use in the diagnosis of pancreatic cancer.

Claims

1. A method of identifying a subject having or at risk of having or developing pancreatic cancer, comprising a step of measuring in a saliva sample obtained from said subject an expression level of at least one miRNA selected from the group consisting of miR-21, miR-23a and miR-23b.

2. The method according to claim 1 further comprising a step of comparing the expression level of the at least one miRNA in the saliva sample with a reference value, wherein detecting a differential between the expression level of the miRNA in the saliva sample and the reference value is indicative of the subject having or being at risk of having or developing a pancreatic cancer.

3. A method of preventing or treating pancreatic cancer in a subject in need thereof comprising the steps of: i) identifying a subject having or at risk of having or developing pancreatic cancer by performing the method according to claim 1, and ii) treating said subject having or at risk of having or developing a pancreatic cancer with a pancreatic cancer treatment.

4. A method for monitoring treatment of a subject affected with a pancreatic cancer comprising the steps of: i) diagnosing pancreatic cancer before said treatment by performing the method according to claim 1, ii) assessing the status of the pancreatic cancer after said treatment by performing the method according to claim 1, and iii) comparing the results determined at step with the results determined at step ii) wherein a difference between the results determined at step i) and the results determined at step ii) is indicative of the effectiveness of the treatment.

Description

FIGURES

[0082] FIG. 1: Salivary miRNA profiles in pancreatic cancer patients.

[0083] Analysis of miRNA profiles in saliva samples from patients with pancreatic cancer (n=7) or cancer-free patients (n=4). Results are mean±S.D. of ΔCq: (Cq of miRNA of interest−Cq of miR-92a) done in triplicate. *, P<0.05

[0084] FIG. 2: Salivary miRNA profiles in pancreatic cancer and pancreatitis patients.

[0085] Analysis of miRNA profiles in saliva samples from patients with pancreatic cancer (n=7) or pancreatitis (n=4). Results are mean±S.D. of ΔCq: (Cq of miRNA of interest−Cq of miR-92a) done in triplicate.

[0086] FIG. 3: Salivary miRNA profiles in an experimental model of pancreatic cancer.

[0087] Analysis of salivary hsa-miR-21, hsa-miR-23a, hsa-miR-23b levels and Lucia blood levels in mice xenografted with Mia PACA-2 Lucia cells at the time indicated following tumour induction. Results are mean±S.D. of 6 biological replicates done in experimental triplicates. miRNA levels are expressed in Cq, Lucia levels are expressed in relative light units (r.l.u.). The grey zone corresponds to tumour burden monitoring using secreted Lucia.

EXAMPLES

Example 1

[0088] Material & Methods

[0089] Patients

[0090] This protocol was approved by the Ethical Committee (Comité de Protection des Personnes Sud-Ouest et Outre Mer N.sup.o 1, number 1-10-21). To avoid blood contamination, patients were asked not to brush their teeth within 45 minutes prior to sample collection. Saliva was collected using sterile tips and micropipettes during endoscopic examination under general anesthesia with propofol. Saliva was immediately placed in pre-chilled 1.5-ml microcentrifuge tubes containing and equal volume of Saliva protect reagent (Qiagen) and stored at −80° C. until ready for use. In the present invention, the inventors included patients aged >18 years who had given their written informed consent. Other criteria for inclusion were no contraindications for general anesthesia or for endoscopic ultrasound. Fine needle aspiration material was used for histological, cytological and molecular (KRAS activating mutation analysis (12)) diagnosis of pancreatitis or pancreatic cancer. Twenty-one patients were included in this study; 7 were diagnosed with pancreatic cancer, 4 were diagnosed with pancreatitis (either acute or chronic) and 4 had unrelated digestive diseases (control group) (Table 1). Patients with no diagnosis (n=2), diagnosed with other digestive cancers (n=2) or intraductal papillary mucinous neoplasia (n=2) were excluded from the study.

TABLE-US-00003 TABLE 1 Patients' characteristics Patient # Age Diagnostic KRAS status Group: Control 13 64 colon polyps 14 81 Gallstones 15 70 colon polyps 16 66 hypochondriac mean 70 (64-81) Group: Pancreatitis 4 54 Chronic pancreatitis 17 39 Acute pancreatits 18 51 Acute pancreatits 19 54 Chronic pancreatitis mean 50 (39-54) Group: Cancer 3 59 Pancreatic adenocarcinoma positive 6 66 Pancreatic adenocarcinoma positive 7 66 Pancreatic adenocarcinoma negative 8 68 Pancreatic adenocarcinoma positive 9 68 Pancreatic adenocarcinoma positive 10 67 Pancreatic adenocarcinoma positive 12 74 Pancreatic adenocarcinoma positive mean 66 (59-74)

Experimental Protocol

[0091] All animals experiments were conducted according to the national ethical guidelines for experimental research and protocol were approved by the regional ethical committee for animal experimentation and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health). Human pancreatic cancer-derived Mia PACA-2 cells expressing secreted Lucia luciferase (13, 14) are grown in RPMI medium supplemented with 10% fetal calf serum, L-glutamine, an antibiotic, an antimycotic cocktail (Life Technologies), and Plasmocin® (InvivoGen) in a humidified incubator at 37° C. in 5% CO.sub.2. Six two-week-old female nu/nu mice were anesthetized by intraperitoneal injection of pentobarbital (80 mg/kg) diluted in 0.9% NaCl, supplemented with oral anaesthesia using oxygen/isofluorane (2.5 mixture) and Mia PACA-2 Lucia cells were implanted in the tail of pancreas as previously described (13, 14). Saliva secretion was not stimulated by pilocarpine. Saliva was obtained from the oral cavity by micropipette and immediately placed in pre-chilled 1.5-ml microcentrifuge tubes containing and equal volume of Saliva protect reagent (Qiagen). Collection was completed in 20 minutes and samples were stored at −80° C. until analyzed. For non-invasive tracking of tumour growth, blood was sampled by retro-orbital collection and centrifuged at 1000×g for 10 min in microcentrifuge tubes treated with EDTA. Lucia production was measured in 5 μl of plasma using coelenterazine (50 μM) as a substrate. For miRNA quantification studies, tumours were frozen in liquid nitrogen and stored at −80° C. until use.

[0092] RNA Extraction

[0093] Before saliva samples were used, they were defrosted on ice and centrifuged for 15 minutes at 2600×g at 4° C. The cell free supernatant was collected from the pellet and used immediately in the next step. Total RNA was isolated from 250 μL saliva supernatant and from tumours using Trizol LS reagent (Life technologies) and miRNAeasy extraction kit (Qiagen), respectively. DNase I treatment (DNase I, Qiagen) was used to remove contaminating DNA during RNA extraction. The concentration of total RNA was measured using Nanodrop N-100.

[0094] miRNA Quantification

[0095] Total salivary, cellular or tumour RNA (20 ng) was reverse transcribed and pre-amplified using the Universal cDNA synthesis kit (Exiqon), followed by Specific Target Amplification (STA) using TaqMan® PreAmp Master Mix (Life technologies) and pooled 94 microRNA LNA™ PCR primer sets (Exiqon). Following 15 pre-amplification cycles, STA reactions were diluted 1:10 in nuclease free water. qPCR Assay Mix consisted of TaqMan® Gene Expression Master Mix (Life technologies), DNA Binding Dye Sample Loading Reagent (Fluidigm), EvaGreen (Biorad), Forward and Reverse primer mix (Exiqon) and Assay Loading Reagent, and prepared as per the manufacturer's recommendations. Samples and sample mix was loaded on a Fluidigm chip (Fluidgm) and quantitative real time PCR reaction was run at 95° C. for 10 minutes, followed by 30 cycles at 95° C. for 10 seconds and 60° C. for 1 minute on the Fluidigm platform (Fluidgm). The quantification cycle (Cq) value is defined as the cycle number in the fluorescence emission, which exceeds that of a fixed threshold. A Cq of 15 to 30 was considered high expression and a Cq of 35 is considered low expression. A Cq value more than 40 was considered as undetectable miRNA. For miRNA quantitative PCR (qPCR) experiments, has-miR-92a was used as the reference gene in human saliva samples. The inventors calculated ΔCq by subtracting the Cq value of the reference miRNA (has-miR-92a) from the Cq value of each candidate miRNA biomarker. Data normalization was conducted using RQ manager 1.2.1 and Data Assist v3.0 from Applied Biosystems.

[0096] Statistical Analysis

[0097] The qPCR-based gene expression values between the different groups were compared using the nonparametric Wilcoxon rank-sum test. Candidate biomarker miRNAs were then selected based on P<0.05.

[0098] Results

[0099] Identification of Pancreatic Cancer-Specific Salivary miRNAs

[0100] In the present invention, 94 miRNAs were selected from the literature as follow: previously reported biomarkers for cancer, previously reported biomarkers for pancreatic cancer, detected in blood of patients with cancer or detected in saliva of patients with cancer. Expression of candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm in patients with pancreatic cancer (n=7), benign pancreatitis (n=4) or without cancer (n=4).

[0101] Of the 94 miRNAs, 23 miRNAs were undetectable in all samples tested. On the other hand, hsa-miR-23a, hsa-miR-223, hsa-miR-23b, hsa-miR-92a, hsa-miR-21, hsa-miR-205 and hsa-miR-127-5p were expressed at high levels in the saliva of pancreatic cancer and control patients. Using Genorm software, the inventors selected hsa-miR-92a as reference miRNA for this study. The inventors found that 3 miRNAs (has-miR-21, has-miR23a and has-miR-23b) were differently expressed in saliva from patients with pancreatic cancer (n=7) compared with control patients (n=4; Wilcoxon test, P<0.05) (Table 2 and FIG. 1). The expression of candidate miRNAs was higher (mean 3844 fold) in saliva samples from patients with tumours compared with expression in saliva samples from cancer-free patients (Table 2). Of importance, hsa-miR-21 and hsa-miR-23a were strictly specific for pancreatic cancer (100%) with excellent sensitivity (71.4% and 85.7%, respectively). hsa-miR-20a and hsa-miR-210 showed a trend as discriminatory miRNAs between pancreatic cancer and benign pancreatitis patients (0.09>P>0.05) (Table 3). Taken together, the invention demonstrates that salivary hsa-miR-21, hsa-miR-23a and hsa-miR-23b are novel non-invasive biomarkers for pancreatic cancer diagnosis.

TABLE-US-00004 TABLE 2 Average ΔCq values. Cancer Control mean mean ΔCq SD ΔCq SD p ΔΔCq fold increase hsa-miR-23a 2.00 3.00 15.00 0.33 0.011 −13.00 8192 hsa-miR-223 0.86 3.00 −2.00 0.58 0.293 hsa-miR-23b 0.54 0.65 11.00 4.00 0.002 −10.46 1411 hsa-miR-21 4.00 3.00 15.00 0.33 0.022 −11.00 2048 hsa-miR-205 6.00 3.00 4.00 5.00 0.36 hsa-miR- 9.00 3.00 5.00 4.00 0.23 127-5p

[0102] The expression of miRNAs in whole saliva from patients with pancreatic cancer (n=7) were compared to the expression of miRNAs in whole saliva from patients without cancer (n=4).

TABLE-US-00005 TABLE 3 Average ΔCq values. Cancer Pancreatitis mean ΔCq SD mean ΔCq SD p hsa-miR-23a 2.00 3.00 5.00 4.00 0.341 hsa-miR-223 0.86 3.00 4.00 4.00 0.293 hsa-miR-23b 0.54 0.65 5.00 4.00 0.154 hsa-miR-21 4.17 3.00 10.73 4.00 0.164 hsa-miR-20a 7.00 3.00 0.68 1.00 0.128 hsa-miR-210 8.00 3.00 3.00 1.00 0.186 hsa-miR-127-5p 8.00 3.00 7.00 3.00 0.440

[0103] The expression of miRNAs in whole saliva from patients with pancreatic cancer (n=7) were compared to the expression of miRNAs in whole saliva from patients with pancreatitis (n=4).

[0104] Salivary miRNAs Precede Tumour Burden in Experimental Models of Pancreatic Cancer

[0105] The inventors next investigated the kinetic of salivary miRNA detection in experimental model of pancreatic cancer. Mia PACA-2 human-derived pancreatic cancer cells were implanted in the pancreas of athymic mice (n=6). The inventors found that these cells and resulting xenografts express high levels of hsa-miR-21, hsa-miR-23a, hsa-miR-23b and has-miR-29c. As these cells were engineered to express secreted luciferase for non-invasive tumour monitoring (13, 14), pancreatic cancer tumours were detected 25 days following tumour cell engraftment and before they became palpable (FIG. 3).

[0106] Interestingly, hsa-miR-21 was readily detected in saliva from tumour-bearing mice, as soon as 14 days following tumour induction (FIG. 3), while undetectable in the saliva of tumour-free animals. In addition, salivary hsa-miR-21 expression remained elevated during the course of the experiment (FIG. 3). On the other hand, salivary hsa-miR-23a, hsa-miR-23b and has-miR-29c were detected at low levels (FIG. 3). Thus, the inventors validate hsa-miR-21 has a salivary biomarker in this experimental model of pancreatic cancer; in addition our results strongly demonstrate that salivary miRNA can be used for the early diagnosis of pancreatic cancer.

Example 2

[0107] The inventors explored in the present invention the differences in salivary microRNA profiles between patients with pancreatic tumors that are not eligible for surgery (unrespectable pancreatic cancer), precancerous lesions (Intraductal papillary mucinous neoplasms (IPMNs)), inflammatory disease (pancreatitis) or cancer-free patients as an early diagnostic tool.

[0108] In addition to the patients described in the table 1, the inventors included in the study patients diagnosed with intraductal papillary mucinous neoplasia (IPMN) (n=2) (Table 4).

TABLE-US-00006 TABLE 4 Patients' characteristics Group: Benign pancreatic masses Patient # Age Diagnostic 21 52 IPMN (secondary branch ducts) 22 83 IPMN (mixed)

[0109] Results

[0110] In the present invention, 94 miRNAs were selected. Expression of candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm in patients with pancreatic cancer (n=7), pancreatitis (n=4), intraductal papillary mucinous neoplasia (IPMN, n=2) or without cancer (n=4) (Table 1 and Table 4).

[0111] Of the 94 miRNAs, 23 miRNAs were undetectable in all samples tested. The inventors found that 4 miRNAs (hsa-miR-21, hsa-miR23a, hsa-miR-23b and hsa-miR-29c) were significantly expressed in saliva from patients with pancreatic cancer (n=7), while undetectable in the saliva of control patients (n=4; Wilcoxon test, 0.001<p<0.03) (Table 5). The expression of the candidate miRNAs was strictly specific of pancreatic cancer (100%) with excellent sensitivity (ranging from 57% to 86%, Table 5). The has-miR-23a and hsa-miR-23b were detected in the saliva of patients diagnosed with IPMN, a well characterized precursor lesion of PDAC.

TABLE-US-00007 TABLE 5 Average Cq values, sensitivity and specificity of the candidate microRNAs. Cancer Control mean mean Cq SD Cq SD p specificity Sensitivity hsa-miR-21 28.00 3.10 40.00 0.00 0.012 100% 71% hsa-miR-23a 24.90 2.63 40.00 0.00 0.001 100% 86% hsa-miR-23b 25.97 2.55 36.75 3.25 0.014 100% 86% hsa-miR-29c 31.76 2.92 40.00 0.00 0.03 100% 57%

[0112] The expression of miRNAs in whole saliva from patients with PDAC (n=7) were compared to the expression of miRNAs in whole saliva from patients without cancer (n=4). The p value (nonparametric Wilcoxon rank-sum test) is indicated.

REFERENCES

[0113] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0114] 1. Cancer Facts & FIGS. 2013 [Internet]. [cited 2014 Feb. 10]; Available from: http://www.cancer.org/research/cancerfactsfigures/cancerfactsfigures/cancer-facts-figures-2013 [0115] 2. Burris H A 3rd, Moore M J, Andersen J, Green M R, Rothenberg M L, Modiano M R, Cripps M C, Portenoy R K, Storniolo A M, Tarassoff P, Nelson R, Don F A, Stephens C D, Von Hoff D D. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol Off J Am Soc Clin Oncol 1997; 15:2403-13 [PMID: 9196156] [0116] 3. Pancreatic Cancer Action Network [Internet]. Available from: http://www.pancan.org/4. [0117] 4. Humeau M, Torrisani J, Cordelier P. miRNA in clinical practice: Pancreatic cancer. Clin Biochem 2013; [PMID: 23570860 DOI: 10.1016/j.clinbiochem.2013.03.019] [0118] 5. Xiao Y-F, Yong X, Fan Y-H, Lü M-H, Yang S-M, Hu C-J. microRNA detection in feces, sputum, pleural effusion and urine: novel tools for cancer screening (Review). Oncol Rep 2013; 30:535-44 [PMID: 23754129 DOI: 10.3892/or.2013.2525] [0119] 6. Allegra A, Alonci A, Campo S, Penna G, Petrungaro A, Gerace D, Musolino C. Circulating microRNAs: new biomarkers in diagnosis, prognosis and treatment of cancer (review). Int J Oncol 2012; 41:1897-912 [PMID: 23026890 DOI: 10.3892/ijo.2012.1647] [0120] 7. Cheng Y-S L, Rees T, Wright J. A review of research on salivary biomarkers for oral cancer detection. Clin Transl Med 2014; 3:3 [PMID: 24564868 DOI: 10.1186/2001-1326-3-3] [0121] 8. Yoshizawa J M, Wong D T W. Salivary microRNAs and oral cancer detection. Methods Mol Biol Clifton N.J. 2013; 936:313-24 [PMID: 23007518 DOI: 10.1007/978-1-62703-083-0_24] [0122] 9. Matse J H, Yoshizawa J, Wang X, Elashoff D, Bolscher J G M, Veerman E C I, Bloemena E, Wong D T W. Discovery and prevalidation of salivary extracellular microRNA biomarkers panel for the noninvasive detection of benign and malignant parotid gland tumors. Clin Cancer Res Off J Am Assoc Cancer Res 2013; 19:3032-8 [PMID: 23575476 DOI: 10.1158/1078-0432.CCR-12-3505] [0123] 10. Xie Z, Chen G, Zhang X, Li D, Huang J, Yang C, Zhang P, Qin Y, Duan Y, Gong B, Li Z. Salivary microRNAs as promising biomarkers for detection of esophageal cancer. PloS One 2013; 8:e57502 [PMID: 23560033 DOI: 10.1371/journal.pone.0057502] [0124] 11. Brinkmann O, Wong D T W. Salivary transcriptome biomarkers in oral squamous cell cancer detection. Adv Clin Chem 2011; 55:21-34 [PMID: 22126022] [0125] 12. Bourret B, Selves J, Grand D, Danjoux M, Hanoun N, Cordelier P, Buscail L. Endoscopic Ultrasound-guided Fine-Needle Aspiration Biopsy Coupled With a KRAS Mutation Assay Using Allelic Discrimination Improves the Diagnosis of Pancreatic Cancer. J Clin Gastroenterol 2014; [PMID: 24798941 DOI: 10.1097/MCG.0000000000000053] [0126] 13. Delpu Y, Lulka H, Sicard F, Saint-Laurent N, Lopez F, Hanoun N, Buscail L, Cordelier P, Torrisani J. The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool. PLoS ONE 2013; 8:e55513 [DOI: 10.1371/journal.pone.0055513] [0127] 14. Sicard F, Gayral M, Lulka H, Buscail L, Cordelier P. Targeting miR-21 for the Therapy of Pancreatic Cancer. Mol Ther [Internet] 2013 [cited 2013 Mar. 18]; [DOI: 10.1038/mt.2013.35] Available from: http://www.nature.com/doifinder/10.1038/mt.2013.35 [0128] 15. Ryan DP1, Hong T S, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014 Sep. 11; 371(11):1039-49 [doi: 10.1056/NEJMra1404198].