1034 Spectral Biomarkers of Pancreatic Cancer

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Role of Sex Hormones and Sex Chromosomes in Mechanically-Induced Visceral Hyperalgesia in Mice Muriel H. Larauche, Mandy Biraud, Ryan Mackie, Arthur Arnold, Yvette Tache

Prevalent and Incident Lesions Identified With Pancreatic Cancer Screening in High Risk Individuals Sean T. McCarthy, Amy E. Hosmer, James Scheiman, Victoria M. Raymond, Richard S. Kwon, Elena M. Stoffel

BACKGROUND: Characterized by recurrent abdominal pain and altered bowel habits, irritable bowel syndrome (IBS) is more common in women. The mechanisms underlying this sex difference in prevalence remain unclear. The sex-biased proximate factors causing sex differences in phenotype include direct effects of gonadal hormones (organizational or activational) and of genes represented unequally in the genome because of their X- or Ylinkage. To address the role of sex hormones vs. sex chromosomes in the modulation of visceral sensitivity in rodents, we used the "four-core genotypes" (FCG) mice (XX and XY mice with ovaries, and XX and XY mice with testes). AIM: To determine the influence of sex hormones and sex chromosomes on the visceral hyperalgesia induced by repeated noxious colorectal distension (CRD) when monitored non invasively. METHODS: Intact and gonadectomized (GDX) FCG male (XY(Sry+) and XX(Sry+), mice with testes) and female (XX and XY, mice with ovaries) mice (4-6 months old; n=6-8/group) were used. Mice were subjected to 4 sets of isobaric phasic distensions (each set: 3 CRDs at 55 mmHg, 10-s duration, 5-min intervals). Visceromotor response (VMR) was recorded using manometry. The 1st CRD set served as a baseline response. Results were expressed in AUC/min. Data were analyzed using 2-way ANOVA and Bonferroni post-hoc test. RESULTS: Visceral hypersensitivity developed in response to repeated noxious CRD in intact XX mice at the 3rd set of CRD, in XY(Sry+) at the 4th set, and in XX(Sry+) mice in the 2nd, 3rd and 4th sets but not in XY mice. The VMR between groups of males (XY( Sry+) and XX(Sry+)) and females (XY and XX) was similar. When pooled together, gonadal males exhibited visceral hyperalgesia at the 4th set of CRD (p<0.01) and gonadal females at the 3rd set of CRD (p<0.05), with males presenting higher VMR than females to all sets of CRD reaching significance at the 4th set (p<0.01). Gonadectomy reduced the baseline VMR to the 1st set of CRD in all groups compared to intact mice. In addition, GDX gonadal males and gonadal females all presented a strong visceral hyperalgesia at the last two sets of CRD, including the 2nd set only for XYfemale mice. No difference in VMR was detected between groups of GDX males and females. When pooled together, GDX males and females exhibited visceral hyperalgesia at the 2nd, 3rd and 4th set of CRD (p<0.01), and their VMR were comparable. Males GDX exhibited lower VMR at all sets of CRD compared to intact males, unlike GDX females which except for the 1st of CRD, had a similar VMR to CRD than intact females. CONCLUSIONS: These data support a major role of sex hormones, but not sex chromosomes, in the modulation of visceral hypersensitivity in response to repeated noxious colorectal distensions under non stress conditions. Supported by NIH DK-57238 (YT), 1K01DK088937 (ML), 1R01NS043196 (AA)

Background: Expert opinion advocates screening for pancreatic cancer (PC) in individuals with inherited predisposition, in hopes that early detection will reduce mortality; however outcome data are lacking. Our aim was to describe our center's experience screening a cohort of high-risk individuals (HRIs) for pancreatic cancer. Methods: Subjects with a history suggestive of genetic risk for PC were identified through queries of the Cancer Genetics Registry at the University of Michigan. HRIs met ‡1 of the following criteria: 1) familial pancreatic cancer (defined as 3 or more relatives, or 2 first degree relatives (FDRs), with PC), 2) germline CDKN2A mutation associated with familial atypical multiple mole melanoma syndrome (FAMMM), 3) germline STK11 mutation associated with Peutz-Jeghers syndrome, 4) germline mutation of PRSS1 or SPINK associated with hereditary pancreatitis, 5) germline BRCA1 or BRCA2 mutation with PC in a FDR, 6) DNA mismatch repair (MMR) mutation with Lynch syndrome with PC in a FDR. Chart reviews were performed to collect demographics, findings of screening tests and clinical outcomes. Results: A total of 93 HRIs were identified, with the largest proportion fulfilling criteria for familial pancreatic cancer (n=34, 37%). Of the 93 individuals, 43 (46%) were counseled to undergo screening and 28 (30%) underwent at least one screening examination (Table 1). Results of screening tests were available in 23 patients. Initial screening test included EUS (n=8) and MRI/MRCP (n=15). Abnormal findings were noted on initial examinations in 11/23 patients; 47% and 50% of MRI and EUS exams, respectively. Cysts were identified in 4 initial MRI exams, but no initial EUS examinations (Table 2). Subjects underwent an average of 3 screening exams (range 1-9) with average follow up of 30.3 months (range 0-327). Six patients developed incident abnormalities on subsequent screening examinations, including new cysts (n=4), progressive chronic pancreatitis changes (n=2), and a solid mass (n=1). Two individuals ultimately underwent surgery. The mass (2 cm pancreatic adenocarcinoma in the head of pancreas) was diagnosed in a CDKN2A mutation carrier 14 months after a previous MRI which had noted only a tail cyst. Another patient had a strong family history of PC and underwent a Whipple resection of a focally dilated pancreatic duct (final pathology showed benign fibrotic tissue). Conclusions: Less than one-third of patients meeting consensus criteria undergo screening for PC. Nearly half of HRIs screened had abnormalities noted on initial examinations, with cysts and chronic pancreatitis changes being the most common. Overall, 17/23 (74%) HRIs had at least one abnormal screening test. Emergence of PC within one year of a screening MRI highlights the need for studies to identify the most sensitive and specific screening modality.

1032 Development of a Novel Preparation Based on a Transgenic CGRPa Reporter Mouse to Directly Correlate Functional Properties of Colorectal Primary Afferent Neurons With Their Neurochemical Phenotype Timothy J. Hibberd, Melinda A. Kyloh, Simon J. Brookes, David Wattchow, Nicholas J. Spencer Several types of spinal afferent neurons have been characterised in the mouse colorectum. These include low threshold muscular and muscular mucosal afferents, high threshold serosal (or vascular) afferents, and a population of distension-insensitive afferents. In the DRG, several neurochemical classes of colorectal afferents have been defined, including a large population expressing CGRP. Most previous electrophysiological recordings from colorectal afferents have been made without identifying their neurochemical phenotype. This study aimed to develop a preparation, using a novel transgenic mouse, in which functional properties of primary afferents could be directly correlated with their neurochemistry. To do this, we generated a transgenic knock-in mouse expressing the fluorescent reporter, mCherry, under the CGRPa promoter. Decentralised preparations of L6 and S1 DRGs, with intact connections to the colorectum were set up in vitro. The distal 4cm of colorectum was cannulated to apply graded intraluminal distensions, whilst recording intraluminal pressure. Sharp intracellular electrophysiological recordings were made from L6 and S1 DRG nerve cell bodies using micropipettes filled with 5% 5, 6-carboxyfluorescein. Carboxyfluoresceinfilled neurons were analysed for colocalization with mCherry fluorescence immediately after recording. Colorectal afferents were identified by antidromic action potentials evoked by an electrical stimulus applied to the mesenteric arcade, close to the gut wall. Twenty-eight colorectal afferent neurons were identified by stimulation of the mesentery (n=11, 23 in L6). All had C-fibre conduction velocities ( £1.0 m/s). Most colorectal afferents discharged action potentials to intra-somal depolarization (8/11 tested, rheobase 0.26 ± 0.09 nA), showed inwardly rectifying IH-like currents during hyperpolarizing constant current pulses (500ms, 8/9 cell tested) and had passive membrane properties typical of small to medium sized DRG neurons (Em -53 ± 1.4mV, Rinput 66 ± 20 mV). Twenty neurons were insensitive to pressure distensions up to 60cm H2O. Focal electrical stimuli confirmed that they had axons in the gut wall (4/4 cells tested). Five of six distension-insensitive neurons filled with carboxyfluorescein colocalized with mCherry fluorescence. Eight neurons were sensitive to colorectal distension (up to 60cm H2O, n=5). Seven of 8 neurons had low thresholds (10cm H2O), and fired up to 42Hz at 40cm H2O (instantaneous frequency 25 ± 12Hz, amplitude: 59.2 ± 8.8mV, half-peak duration: 1.4 ± 0.3ms). All low-threshold, distension-sensitive nerve cell bodies tested colocalized with mCherry (3/3 cells), while a single high-threshold afferent lacked mCherry labelling. In conclusion, we have developed a preparation that can rapidly determine functional, electrophysiological and neurochemical phenotypes of colorectal afferent neurons.

1034 Spectral Biomarkers of Pancreatic Cancer Bohus Bunganic, Michal Tatarkovicˇ, Martin Laclav, Stepan Suchanek, Lucie Stovickova, Lucie Kocourkova, Vladimir Setnicka, Miroslav Zavoral The search and definition of biomarkers of pancreatic cancer (PC) remains a subject of great interest. The specificity and sensitivity of the current tested biomarkers are below the required values. In order to make a diagnosis of early pancreatic cancer, establishing a new biomarker is essential. During cancer diseases, many changes may occur within the 3D structure of proteins, peptides and other biomolecules. Therefore, we tested a new approach in PC diagnosis based on a specific molecular signature of blood plasma components using chiroptical and vibrational spectroscopy. Chiroptical methods such as electronic circular dichroism (ECD) and Raman optical activity (ROA) are inherently sensitive to these 3D structures and were supplemented by conventional infrared (IR) absorption and Raman spectroscopies. We collected blood plasma samples from 23 healthy control persons and 34 pancreatic cancer patients (PCs). The ECD spectra of the PCs showed generally not only a lower intensity profile in the UV spectral region than the healthy controls, but also the spectral patterns were slightly changed. There were as well distinct differences between the Raman and ROA spectra of PCs and controls. The Raman and ROA spectra of controls showed several bands in the amide I and extended amide III regions. Their positions and spectral band-shapes are characteristic of a mainly alpha-helical peptide/protein conformation with a low content of beta-sheet structures. The more intense negative band in the amide I region of the ROA spectra of PCs cohered with changes in intensities of bands belonging to unordered and

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beta-sheet structures. Other observed changes corresponded to aliphatic and saccharide/ glycoprotein moieties. The spectra obtained from all four spectral methods were consequently processed by linear discriminant analysis (LDA) showing clear separation of controls and PCs (Figure 1). The quality of the established statistical model was confirmed by leave-oneout cross-validation (Table 1), where sensitivity and specificity reached 91% and 87%, respectively. The results obtained in this pilot study show a high potential of the combination of chiroptical and vibrational spectroscopy as a promising tool in the identification of new spectral biomarkers for PC diagnosis. The LDA confusion matrix for the cross-validation results

^Mean +/- Standard deviation *Median (Interquartile range) Figure 1. Graphical results of LDA of blood plasma samples analyzed by a combination of ECD, Raman, ROA and IR absorption spectroscopy: control persons (blue) and pancreatic cancer patients (red)

1035 Risk for Developing Adverse Outcomes in Suspected Intraductal Papillary Mucinous Neoplasms Without High Risk or Worrisome Features Saurabh S. Mukewar, Anupama Aryal-Khanal, Naoki Takahashi, Santhi Swaroop Vege, Mark Topazian, Michael J. Levy, Suresh T. Chari Background: International Consensus Guidelines for intraductal papillary mucinous neoplasm (IPMN) at Fukuoka provide indications for surgical resection in pancreatic cystic lesions (PCL) suspected to be IPMN. However, data concerning the outcome of conservative follow-up of PCLs without high-risk or worrisome features (Fukuoka negative) are limited. In such lesions we estimated the 3 and 5-year risk of adverse cyst outcomes defined by new development of high risk or worrisome features, pancreatic surgery and/or pancreatic cancer (PC). Methods: From Mayo Clinic Rochester's electronic databases we identified 766 randomly selected patients who had PCLs diagnosed on CT or MRI. We excluded patients with i) inflammatory PCL (n= 104) ii) definite or suspected non-IPMN PCL (n=29), iii) Fukuoka positive (with high risk or worrisome features) PCLs (n=106), main duct IPMN (n=4) and mixed IPMN (n=14) on initial imaging, iv) <1 year follow up, and v) pancreatic surgery or pancreas cancer <1 year from cyst diagnosis (n=153). We re-reviewed all cross-sectional imaging and abstracted clinical and follow-up data on Fukuoka negative PCLs suspected to be branched duct IPMN (n=287). We estimated adverse cyst outcomes in this cohort and in subgroups categorized by maximum diameter of cyst size (<1cm, 1-<2 cm, 2-<3 cm). Results: Of the 287 patients with suspected IPMN 39% were males; their mean age was 65.9 +/- 12.6 years at baseline and size distribution was as follows: 121 had cyst <1cm, 133 had cyst 1 to <2 cm and 33 had cyst 2 to 3 cm in size. They had a median follow up of 4.8 (1 to 14.2) years. Probability of developing worrisome or high-risk features (Fukuoka positive), pancreatic surgery for PCL or PC were 8.2%, 1.4% and 1.4% for cysts <1cm; 2.9%, 0% and 0% for cysts 1-<2 cm; 42.9%, 4.8% and 0% for cysts 2-<3 cm at 5 years. Conclusions: On conservative follow up PCLs without high risk or worrisome features (Fukuoka negative) suspected to be IPMN have a extremely low risk of developing pancreatic cancer or undergoing cyst related surgery. Cyst characteristics of included cases

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1037 Serum Human Telomerase Reverse Transcriptase Messenger RNA As a Novel Marker for Pancreatic Ductal Adenocarcinoma Kazuya Matsumoto, Norimasa Miura, Yohei Takeda, Takumi Onoyama, Soichiro Kawata, Kenichi Harada, Yoshikazu Murawaki Background Endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNA) used as a diagnostic modality for a pancreatic mass has an accuracy of 85-91%. However, the institutions that are capable of performing EUS-FNA are limited. The conventional biomarkers for pancreatic ductal adenocarcinoma (PDAC) have an accuracy of at most around 70%, and are especially difficult to use to distinguish between a PDAC and a pancreatic inflammatory lesion or a benign stricture of the main pancreatic duct (MPD), and between intraductal papillary mucinous carcinoma (IPMC) and intra-ductal papillary mucinous neoplasm (IPMN). Therefore, a novel surrogate marker is needed for clinical use. Using a very small amount of mRNA from the human serum, we have established a method to quantify human telomerase reverse transcriptase mRNA (hTERT mRNA) expression. Objective: We examined the usefulness of the serum hTERT mRNA as a biomarker for PDAC. Methods: Eighty-three cases (45 pancreatic cancers, 38 non-pancreatic cancers) were treated in our hospital from December 2011 to October 2012. After removing the cellular component from the serum by centrifugal separation, we did RNA extraction and purification with a DNase treatment, finally hTERT mRNA levels were measured by using a KAPA SYBR FAST Universal OneStep qRT-PCR Kit with SYBR Green I (KAPA BIOSYSTEMS, Boston, USA). Results: The serum hTERT mRNA was significantly higher for PDAC (5285.6±15320.0 copy/serum 10µl) than for pancreatic inflammatory lesions or benign strictures of the MPD (1050.9±807.5 copy/serum 10µl, P< 0.05). Furthermore, the average serum hTERT mRNA of IPMC was significantly higher (2863.6±1291.8 copy/serum 10µl) than that of IPMN (846.5±926.1 copy/serum 10µl, P< 0.05). Using a cut-off value of 924 (copy/serum 10µl), calculated based on a receiver-operating characteristic curve analysis, the pancreatic malignancies diagnostic ability of hTERT mRNA had a sensitivity of 97.8%, a specificity of 76.3%, a positivepredictive value (PPV) of 83.0%, a negative-predictive value (NPV) of 96.7% and an accuracy of 88.0%. Compared with conventional tumor markers, the sensitivity, NPV and the accuracy of hTERTmRNA for pancreatic malignancies were significantly improved. Regarding the T factor in the pancreatic malignancies, the sensitivity of T1/ T2/ T3/ T4 was 100%/ 100%

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