40.04 Diagnosis of Pancreatic Adenocarcinoma via Protein Signatures from Fine Needle Aspirations

Posted on January 18, 2018

M. H. Gerber1, D. Delitto1, B. DiVita1, S. Han1, R. M. Thomas1, J. Trevino1, S. J. Hughes1  1University Of Florida,Department Of Surgery,Gainesville, FL, USA

Introduction:  Cytological analysis of fine needle aspiration (FNA) of pancreatic lesions fails to confirm pancreatic ductal adenocarcinoma (PDAC) in up to one third of patients. We discovered that a 4-analyte protein signature from tumor lysates distinguishes PDAC from other lesions including chronic pancreatitis, thus raising the notion protein signatures could prove superior to FNA cytological analysis in the diagnosis of PDAC.  Here, we aim to translate this observation to the FNA biopsy platform.

Methods:  At time of operation, a “virtual” FNA biopsy of various pancreatic pathologies was obtained using a 19-gauge needle with six passes through the intestinal wall into the region of interest. Biopsy samples were placed in various quantities of lysis buffer and protein concentrations of each sample determined. IL-6 concentrations (one of the informative analytes in the diagnostic protein signature) were determined for each FNA biopsy sample, with and without spiking of “contaminant” plasma.

Results: Samples were collected from 27 consecutive patients undergoing pancreatectomy (PDAC n = 18; normal n = 3; pancreatitis n = 6). Protein concentrations from FNA biopsy samples diluted in 150 ul of lysis buffer ranged from 1.8 –  260.8 mg/ml with a median of 17.1 mg/ml (Interquartile range (IQR): 11.5 – 32.6 mg/ml). IL-6 levels in the FNA samples ranged from 185 – 2941 pg/ml with a median of 486 pg/ml (IQR: 247 – 740 pg/ml). Plasma concentrations of IL-6 from the same patients ranged from 59 – 312 pg/ml with a median of 111 pg/ml (IQR:  65 – 203 pg/ml).  When patient FNA samples were compared to plasma samples, FNA samples had IL-6 concentrations 3.1 to 14.9 times higher than the matched patient plasma concentration.

Conclusion: Pancreatic FNA biopsy produces adequate quantities of protein for multiple replications on a variety of protein assay platforms. Normalization of cytokine concentrations to total protein may be subject to blood/plasma contamination inherent to the FNA procedure; thus alternative normalization to a resident, soluble protein may be necessary.  Soluble protein analysis of pancreatic FNA biopsy samples remains a realistic supplement to cytology in the diagnosis of PDAC.
 

40.05 Sphingosine Kinase Type 1 and Type 2 Works Differently in Pancreatic Cancer

Posted on January 18, 2018

M. Nakajima1, K. Yuza1, J. Tsuchida1, Y. Hirose1, K. Miura1, H. Ichikawa1, Y. Shimada1, T. Kobayashi1, J. Sakata1, H. Kameyama1, M. Abe2, K. Sakimura2, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata, NIIGATA, Japan

Introduction: Pancreatic cancer is one of the most lethal diseases known, and it is important to develop new therapeutic agents. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival, migration, angiogenesis and lymphangiogenesis, which are all factors involved in cancer progression. S1P, which functions intra- and extracellularly, is generated inside the cell by two sphingosine kinases (SphK1 and SphK2). We have reported that SphK1 plays an important role in S1P secretion (J Biol Chem 2010) and cancer progression (Cancer Res 2012, J Surg Res 2016), and that SphK2 has a unique role in regulating cellular functions in the liver (Hepatology 2015). Little is known, however, about the role of SphK1 and SphK2 in pancreatic cancer progression. The aim of this study is to investigate the role of SphK1 and SphK2 in pancreatic cancer progression using SphK-knockout (KO) cells generated by CRISPR/Cas9 technology.

Methods: We generated Pan02 murine pancreatic cancer cell lines with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. To investigate the role of SphK1 or SphK2 in cellular proliferation, we assessed cell growth by a spectrophotometric technique using the water-soluble tetrazolium salt, WST-8. Cell migration was measured by an in vitro scratch assay. In the animal experiments, we assessed the prognosis of C57BL/6 mice injected with the SphK1 KO or SphK2 KO Pan02 cells described above intraperitoneally.

Results: SphK2 KO Pan02 cells were significantly less proliferative than WT cells. Unexpectedly, SphK1 KO cells were significantly more proliferative than WT cells. The in vitro scratch assay indicated that SphK2 KO cells were less migratory than WT cells, and that SphK1 KO cells had greater migratory ability than WT cells. Furthermore, the animal experience showed that mice injected with SphK1 KO cells had shorter prognosis than those injected with SphK1 WT cells, while mice injected with SphK2 KO cells had longer prognosis than those injected with SphK2 WT cells. These results indicate that SphK2, rather than SphK1, may have important roles in proliferation and migratory behavior in pancreatic cancer cell lines and pancreatic cancer progression. On the other hand, SphK1 KO cells treated with gemcitabine had more survival rate than WT cells and SphK2 KO cells treated with gemcitabine had less survival rate than WT cells. These results indicate that SphK1 may have important roles in resistance against chemotherapy.

Conclusion: Our findings indicate that S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cell. Targeting both SphK1 and SphK2 signaling pathways may be a potential strategy for pancreatic cancer treatment.

 

40.03 IL-23 Plays an Important Role in Short-Term Survival after Pancreatic Adenocarcinoma Resection

Posted on January 18, 2018

B. A. Krasnick1, S. M. Husain2, Y. Bi1, P. V. Dickson2,3, J. Deneve2,3, D. Shibata2,3, R. Fields1, W. G. Hawkins1, E. S. Glazer2,3  1Washington University,Surgery,St. Louis, MO, USA 2University Of Tennessee Health Science Center,Surgery,Memphis, TN, USA 3University Of Tennessee West Cancer Center,Surgery,Memphis, TN, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) carries the highest case fatality rate of any cancer and will likely become the second leading cause of death by 2020.  TGF-ß has a paradoxical relationship with survival in PDAC patients where it is a tumor suppressor in early stage PDAC and a tumor promotor in late stage PDAC. While TGF-ß is known to drive inflammation in the tumor microenvironment (TME), the role of interleukins in the pancreatic TME is not well understood.  We hypothesized that IL23, a pro-inflammatory interleukin associated with suppression of cytotoxic T cells, is associated with survival in PDAC.

Methods:  24 long-term survivors (>30 months) and 24 short-term survivors (<12 months) with resected PDAC were identified. Tumor sections were taken from formalin fixed, paraffin embedded blocks and protein expression of IL-23 and TGF-ß were independently investigated with immunohistochemistry utilizing quantitative analysis with CellProfiler image analysis software. Immunohistochemistry expression of IL23 or TGF-ß was determined to be high (highest quartile), low (lowest quartile), or median (within the interquartile range) based on 5 representative images of each tumor section. Comparisons with clinical outcomes were investigated with Student’s t-test, ANOVA, or multivariate regression.

Results: There was no significant difference in the average age (66 ± 12 years), gender (44% male), or clinical stage between the two groups. Patients with low TGF-ß protein expression were more likely to be in the short-term survival group (OR=2.2, P=0.018). Tumors from short-term survivors were significantly more likely to have low IL23 expression (OR=0.48, P=0.019).  In long-term survivors, neither TGF-ß nor IL-23 protein expression was associated with survival (P>0.05). There was no difference in IL23 expression in low or median TGF-ß expressing tumors (P>0.5), however, in high TGF-ß expressing tumors, long-term survivors were associated with 20% higher IL23 protein expression (P=0.008). Multivariate analysis demonstrated that long-term survival was linearly associated with increasing IL23 expression (OR=3, P=0.001) but not TGF-ß expression (P=0.07). Overall, we also found a statistical association between IL23 expression and TGF-ß expression (P<0.001) and that long-term survival was associated with a higher ratio of IL23 / TGF-ß expression (P=0.04).

Conclusion: We found that IL23 tumor expression is associated with survival after PDAC resection in short-term survivors and statistically related to TGF-ß expression for all patients.  While other groups have shown that TGF-ß is associated with survival in PDAC, we demonstrated that IL23 may be a critical component to understanding the relationship between TGF-ß expression and survival in patients with survival less than 12 months.

 

40.01 Modeling of Early Pancreatic Neoplasia Transcriptional Regulation in Mice

Posted on January 18, 2018

J. K. Thompson1, H. Crawford3,4, M. Pasca Di Magliano1,2, F. Bednar1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Cell And Developmental Biology,Ann Arbor, MI, USA 3University Of Michigan,Molecular And Integrative Physiology,Ann Arbor, MI, USA 4University Of Michigan,Internal Medicine,Ann Arbor, MI, USA

Introduction: KRAS is the primary oncogenic driver in pancreatic ductal adenocarcinoma (PDA). Pancreatic acinar cells are most susceptible to transformation by Kras in mouse models of PDA. The earliest stage of transformation consists of conversion of the acinar cells to a duct-like progenitor phenotype in a process called acinar-ductal metaplasia (ADM). Networks of developmental transcription factors (TFs) are involved in fate specification and maintenance of acinar cells. We hypothesize that oncogenic Kras alters these networks to establish the neoplastic cell state within the pancreas. In this work, we aimed to validate a genetically engineered mouse model that will allow us to analyze changes in gene regulatory networks driven by oncogenic Kras in early pancreatic neoplasia.

Methods: Mice with a pancreatic acinar cell-specific, tamoxifen inducible Cre recombinase (Ela-CreER) were bred with strains containing the oncogenic Kras G12D allele and the fluorescent protein tdTomato in the Rosa26 locus. To activate the oncogenic Kras and the tdTomato lineage tracer, Ela-CreER; Kras G12D/+; R26 tdTomato mice and littermates lacking oncogenic Kras, were treated with daily gavages of tamoxifen (4mg/day) for five consecutive days. Mice were sacked one week after the initial tamoxifen gavage. We analyzed the expression of tdTomato by fluorescence microscopy and fluorescence-activated cell sorting (FACS). Total cell RNA was isolated from the sorted cells using the RNeasy Micro Kit (QIAGEN) and complementary DNA (cDNA) was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). We utilized a panel of 26 pancreatic developmental TFs and three acinar and duct markers (amylase, elastase, keratin 19 – CK19) with specific quantitative PCR (qPCR) TaqMan probes to characterize the isolated cells. Differences in TF expression between the wildtype and oncogenic Kras-containing mice were analyzed with a Kruskal-Wallis rank test. Statistical significance was set at p<0.05.

Results: Tamoxifen gavage induced high level of tdTomato expression in Ela-CreER mice. Fluorescence microscopy confirmed that the tdTomato+ cells were also amylase positive. RT-qPCR analysis after FACS sorting confirmed high amylase, high elastase, and low CK19 expression in the tdTomato+ cells. We also consistently found measurable levels of 23/26 (88%) of pancreatic developmental TFs in the isolated acinar cells. Preliminary analysis did not reveal significant differences in TF levels in oncogenic Kras-expressing pancreata versus wildtype controls.

Conclusion: We established a genetically engineered mouse model of early pancreatic neoplasia, which allows for specific isolation of acinar cells and their progeny. Early analysis of the model suggests that one-week activation of oncogenic Kras does not yet lead to significant developmental TF expression changes in the adult pancreas.

 

40.02 HO-1 Polymorphism and Acute Necrotic Pancreatitis Through V-cam and E-selectin Expression

Posted on January 18, 2018

A. K. Gulla1,2, A. Gulbinas3, G. Barauskas3, Z. Dambrauskas3  1Georgetown University Medical Center,Department Of Surgery,Washington, DC, USA 2Vilnius University Hospital, Santaros Clinics,Department Of Surgery,Vilnius, SANTARISKIU 2, Lithuania 3Lithuanian University Of Health Sciences, Kaunas Clinics,Department Of Surgery,Kaunas, , Lithuania

Introduction:  Acute pancreatitis is a severe and frequently a life-threatening disease, which can lead to pancreatic necrosis, acute lung injury, SIRS and MODS. The inducible enzyme heme oxygenase-1 (HO-1) is an anti-oxidative, anti-inflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains (GT)n dinucleotide repeats and is highly polymorphic in the population. In this study, we hypothesized that the number of GT repeats in HO-1 promoter can influence the occurrence of acute necrotic pancreatitis through v-cam and e-selectin expression due to its protective function. Patients with acute pancreatitis are more likely to have long repeats than controls.

Methods: Acute pancreatitis patients (n=135) and age- and sex-matched healthy controls (n=33) were studied. Peripheral blood samples from pancreatitis patients were collected on admission. Genomic DNA was extracted from the blood samples of patient and control groups. The HO-1 promoter region with the GT repeats was PCR amplified with fluorescent tagged primers and levels of cytokines were measured.

The PCR products were analyzed by ABI 3130 genetic analyzer and the exact size of the PCR products was determined by GeneMapper software.  A short allele was defined as containing 27 GT repeats or fewer, whereas a long allele was more than 27 repeats.

Results: The subjects were categorized into 3 groups based on the genotype

Results: one short and one long alleles (S/L), two short alleles (S/S) and two long alleles (L/L). The presence of S/L was similar between the patient group (41.2%) and the controls (39.4%). Interestingly, 46.6% of patients were carriers of two long repeats (L/L) while 11.1% v-cam and 11.1 % e-selectins levels (p<0.05) vs 24.2% of control subjects, whereas 12.2% of patients were carriers of two short repeats vs 36.4% of control population. 

Conclusion: Our data demonstrate a strong bias toward longer alleles and higher levels of v-cam and e-selectins among patients with acute pancreatitis. Thus, polymorphism of the GT repeats in the HO-1 promoter region may be a risk factor for developing acute pancreatitis. Further studies are now underway to analyze the pancreatic levels of HO-1 protein in acute pancreatitis patients and controls and to determine whether the presence of the short alleles facilitate HO-1 upregulation and consequently promote its protective anti-inflammatory function in acute pancreatitis.

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