25.07 Mutant-Allele Tumor Heterogeneity Scores Correlate With Neoadjuvant Therapy Response in Rectal Cancer

Posted on January 25, 2017

A. Greenbaum2, S. Ness4, T. Bocklage3, J. Lee1, A. Rajput2  1University Of New Mexico HSC,Epidemiology, Biostatistics And Preventative Medicine/Internal Medicine,Albuquerque, NM, USA 2University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 3University Of New Mexico HSC,Pathology,Albuquerque, NM, USA 4University Of New Mexico HSC,Internal Medicine,Albuquerque, NM, USA

Introduction:   Neoadjuvant chemoradiation is the standard of care for locally advanced adenocarcinoma of the rectum.  It is currently unknown which patients will respond to therapy.  We aimed to determine if Mutant-Allele Tumor Heterogeneity (MATH) scores, a novel bioinformatics tool, can predict response to neoadjuvant treatment in locally advanced rectal tumors.

Methods:    We performed high read-depth (“deep”) sequencing of >400 cancer-relevant genes on a group of 13 patients with locally advanced rectal adenocarcinoma.  Normal and tumor DNA were extracted from formalin-fixed, paraffin-embedded tissues.   DNA samples were analyzed using the Ion Ampliseq Comprehensive Cancer Panel™ assay. Sequencing was performed on the Ion Proton Next-Generation Sequencing™ instrument.   Mutant allele frequencies were determined and a calculated MATH score was used to quantify tumor heterogeneity.  Response to chemo therapy was determine by primary resection pathology report.

Results:  A total of 13 patients with locally advanced rectal cancer (T3/4 or N1/2) were analyzed.  The boxplot in Figure 1 shows the range of calculated MATH scores by neoadjuvant therapy response category. Four patients were noted to have complete response, 7 had minimal/moderate and 2 demonstrated poor response. Tumor heterogeneity (as shown in MATH scores) was found to be significantly different amongst the 3 response groups (p=0.026), with higher MATH scores correlating with poorer response to treatment.

Conclusion:  The novel approach of applying the shape of a whole bioinformatics data set to analyze tumor heterogeneity may provide a useful biomarker for locally advanced rectal cancer.  MATH scores may allow a means of predicting response to neoadjuvant chemoradiation therapy.

 

25.05 Pomalidomide enhanced antitumor effects of gemcitabine and nab-Paclitaxel in pancreatic cancer cells

Posted on January 25, 2017

N. SAITO1, Y. Shirai1, T. Horiuchi1, H. Sugano1, R. Iwase1, K. Haruki1, Y. Fujiwara1, K. Furukawa1, H. Shiba1, T. Uwagawa1, T. Ohashi2, K. Yanaga1  1The Jike University School Of Medicine,Department Of Surgery,Minato-ku, TOKYO, Japan 2The Jikei University School of Medicine,Division Of Gene Therapy, Research Center For Medical Sciences,Minato-ku, TOKYO, Japan

Introduction:  NF-κB plays an important role in chemoresistance. Although gemcitabine and nab-paclitaxel therapy (GN) has been effective for pancreatic cancer, the therapeutic efficacy is attenuated by anticancer agents-induced activation of NF-κB. Meanwhile, pomalidomide is a novel immunomodulatory drug derived from thalidomide. Since thalidomide is a NF-κB inhibitor in digestive cancer, we hypothesized that pomalidomide also inhibits NF-κB activation, and enhances antitumor effects of GN for pancreatic cancer cell lines. 

Methods:  In vitro, we used human pancreatic cancer cell lines (MIA PaCa-2, PANC-1). We compared the antitumor effect of pomalidomide plus GN (GNP) with GN. Concentration of NF-κB, cell proliferation, cell cycle, and induction of apoptosis were evaluated by each assay. In vivo, we created xenograft orthotopic pancreatic cancer model (BALB/c with PANC-1). The animals were treated with oral polmalidomide five times a week and i.p. injection of GN once a week for 5 weeks. We evaluated sequential tumor volume by MRI and conclusive tumor weight and volume. We assessed the in vivo protein and apoptosis levels examined in vitro.

Results: 

Pomalidomide suppressed GN-induced NF-κB activation (MIA PaCa-2; GN : GNP = 9.57 ± 0.47 : 7.09 ± 0.20 ng/mg; p<0.01, PANC-1; GN : GNP = 69.58 ± 5.18 : 32.34 ± 13.61 ng/mg; p<0.05). Cell viability in GNP was significantly lower than that in GN (MIA PaCa-2; GN : GNP = 51.6 ± 9.0 : 24.1 ± 7.7 % ; p?0.01 , PANC-1; GN : GNP = 70.4 ± 7.5 : 41.7 ± 2.7 % ; p?0.01).

Moreover, in GNP, the levels of apoptotic protein (cleaved caspase 8, cleaved caspase 3, cleaved PARP) were higher than those in GN. Similar to nuclear NF-κB concentrations, phosphorylated IκBα was lower in GNP than that in GN. In addition, pomalidomide suppressed the expression levels of VEGF of pancreatic cancer in a dose-dependent manner. 

In vivo, the tumor weight (GN : GNP = 538 ± 36 : 385 ± 88 mg; p<0.05) and tumor volume (GN : GNP = 587 ± 51 : 313 ± 89 mm3; p=0.01) were significantly lower in GNP than those in the GN after five weeks of treatment. Moreover, immunohistochemical staining revealed down-regulation of VEGF and Ki-67 in GNP. 

Conclusion: Pomalidomide inhibited NF-κB activation and enhanced the antitumor effects of GN on pancreatic cancer cells.
 

25.04 Loss of miR-155 Upregulates WEE1 in Metastatic Melanoma

Posted on January 25, 2017

J. A. Campbell1, I. Huffnagle1, G. P. Robertson2, C. R. Pameijer1  1Penn State Hershey Medical Center,Department Of Surgery,Hershey, PA, USA 2Penn State Hershey Medical Center,Department Of Pharmacology,Hershey, PA, USA

Introduction:  WEE1 is a protein kinase in the BRAF pathway and is over-expressed in melanoma. It is involved in regulating cell cycle progression and is involved in tumor progression in several malignant tumors. In melanoma, experimentally induced down-regulation of WEE1 has been shown to inhibit cell growth in vitro and in vivo. Like many other proteins, WEE1 expression is regulated by microRNA. MicroRNAs are small, non-coding segments of RNA that are involved in post transcriptional regulation of gene expression and have been implicated in oncogenesis. Melanoma tumors have been shown to have very low levels of microRNA-155 (miR-155), although the downstream effect of this low expression is unknown. We evaluated a group of melanoma patient tumor samples for miR-155 expression, clinical outcome and possible mechanism of action of miR-155.

Methods:  RNA was extracted from melanoma patient tumor samples. miRNA microarray analysis was performed, with confirmation by qRT-PCR. Melanoma cell lines transfected with miR-155 were used for a mouse experimental metastasis model. Luciferase reporter assay was used to show interaction between miR-155 and the 3’UTR of WEE1. Western blotting was used to show the effect of miR-155 transfection on the protein levels of WEE1 in melanoma cells.

Results: In patient tumor samples miR-155 was lost in patients who develop metastatic disease. In the mouse model, transfection of cells from two metastatic melanoma cell lines with miR-155 mimic decreased the metastatic potential of melanoma cells, with a significant difference in the number of lung metastases between miR-155 and control mice (figure 1a). Transfection of melanoma cells with miR-155 reduced WEE1 protein levels (figure 1b) while inhibition of endogenous miR-155 up-regulated WEE1 expression.  Luciferase reporter assay demonstrated that miR-155 interacts with the WEE1 3’UTR and impaired gene expression in melanoma cells.

Conclusion: miR-155 is lost in patients who develop metastatic melanoma. Loss of miR-155 allows for upregulation of WEE1 in melanoma. Our in vivo studies showed significant decrease in metastasis with cells over expressing miR-155 suggesting that in melanoma miR-155 affects metastasis through WEE1 kinase.

25.03 Use of Circulating Microvesicles, Exosomes, as a Biomarker to Track Disease Burden in Melanoma

Posted on January 25, 2017

J. A. Cintolo-Gonzalez1, W. Michaud1, S. Cohen1, D. Plana1, D. J. Panka2, R. J. Sullivan3, G. M. Boland1  1Massachusetts General Hospital,Surgery,Boston, MA, USA 2Beth Israel Deaconess Medical Center,Hematology/Oncology,Boston, MA, USA 3Massachusetts General Hospital,Hematology/Oncology,Boston, MA, USA

Introduction:
Exosomes are extracellular microvesicles, which contain a variety of nucleic acids (DNA, messenger RNA (mRNA), microRNA (miRNA)) and proteins. Exosomes can be analyzed from archived serum/plasma samples making them ideal biomarkers to study tumor-based changes. We examined the feasibility and accuracy of exosomal mRNA analysis to assess disease burden in patients undergoing surgical resection for metastatic melanoma.

Methods:

Melanoma cell lines (A375, RPMI 7951, SK-MEL-30, and MeWo) were purchased directly from ATCC. Serial tumor and blood samples were collected from melanoma patients under IRB approved protocols.  Exosomes were isolated using combined filtration and ultracentrifugation. Exosomal RNA was isolated using the exoRNA serum/plasma kit (Qiagen Inc). 

Quantitative PCR (qPCR) was used to assess concordance of gene expression between paired exosomes and cell lines.  Paired exosomal and tumor mRNA from patient samples underwent whole transcriptome sequencing using Affymetrix Whole Transcriptome Pico Array.  Patient plasma-derived exosomes were analyzed via qPCR at pre-resection and post-resection time points.

Results:
Paired exosomes and their parental cell lines demonstrated concordance in gene expression as assessed by qPCR.  Specifically, in cell lines harboring a BRAF V600E mutation, BRAF V600E was detected by qPCR in cell lines and paired exosomes.   Likewise, whole transcriptome analysis of patient-derived exosomes and paired tumors demonstrated 80% concordance of gene expression. In patients undergoing resection of BRAF V600E mutant metastatic lesions, there was a significant decrease in exosomal BRAF V600E mRNA in patients rendered NED or with minimal residual disease but not in patients undergoing palliative resection with multiple residual sites of disease (Figure 1).  Of the patients undergoing definitive resection, patient 410, who showed ongoing, albeit decreased, exosomal BRAF V600E expression, recurred 3 months following resection.

Conclusion:
Exosomal mRNA reflects parental tumor mRNA expression and accurately predicts the presence of residual disease after resection in melanoma.  These findings support the use of exosomes as a biomarker for assessing tumor burden after surgery. Work is ongoing to assess the utility of exosomal RNA to assess minimal residual disease and risk of recurrence in high risk melanoma patients.

25.02 PI3K/mTOR Inhibition Suppresses Pancreatic Cancer by Reprogramming Cancer-Associated Fibroblasts

Posted on January 25, 2017

J. L. Williams2, I. A. Elliott1, A. H. Nguyen1, C. Matsumura1, R. Ghukasyan1, P. A. Toste1, J. R. Capri3, S. G. Patel1, L. Li1, N. Wu1, C. G. Radu3, T. R. Donahue1,3  1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2Harbor-UCLA Medical Center,Department Of Surgery,Torrance, CA, USA 3David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, with an overall survival of less than one year.  Contributing to this poor prognosis is PDAC’s characteristically dense stroma, which is comprised predominantly of cancer-associated fibroblasts (CAFs). CAFs promote tumor growth, metastasis, and treatment resistance because when activated by environmental factors such as cancer cell (CC) exposure, hypoxia, and cytotoxic chemotherapy, they secrete protumorigenic cytokines, growth factors, and extracellular matrix components.  However, elimination of CAFs also increases tumor growth and invasion.  Therefore, rather than ablation, reprogramming of CAFs to an inactive or quiescent state is a potential PDAC treatment strategy.  In this study, we aimed to determine the effect of PI3K/mTOR inhibition, a pathway known to be involved in fibroblast activation, on PDAC tumorigenicity.

Methods:  Immortalized CAFs were treated with NVP-BEZ235 (BEZ), a dual PI3K/mTOR inhibitor, and exposed to gemicitabine (GEM), hypoxia (1% O2), or PDAC CC conditioned media (CM) for 48 hours.  After treatment, markers of CAF activation were determined via reverse transcription polymerase chain reaction (RT-PCR), Western blot, and mass spectrometry proteomics analysis of CAF CM.  Additionally, PDAC CCs (Mia PaCa-2 and PANC-1) exposed to CM from treated CAFs were tested with in vitro viability, migration, and invasion assays. PDAC CCs were also co-injected with treated CAFs in vivo, and tumor size was assessed.  A 3D co-culture model was used to assess the effect of BEZ treatment on CCs grown together with CAFs.

Results: After treatment with BEZ, gene expression of known markers of CAF activation, including alpha-smooth muscle actin (αSMA) and type I collagen, were decreased, even after exposure to activating stimuli.  Similarly, αSMA protein levels were reduced in BEZ-treated CAFs. CM from BEZ-treated CAFs contained lower levels of extracellular matrix components, proinflammatory cytokines, and growth factors. CCs exposed to CM from BEZ-treated CAFs were less viable, migratory, and invasive in vitro. Pre-treatment of PDAC CAFs with BEZ restricted PDAC growth in our in vivo co-implantation model (Figure 1).  In CC-CAF 3D co-culture, combination treatment of BEZ with GEM was more effective at suppressing CC growth than GEM treatment alone.

Conclusion: Inhibition of PI3K/mTOR decreases CAF activation, resulting in reduced PDAC CC tumorigenesis in vitro and in vivo. Additionally, combination treatment with BEZ, a dual PI3K/mTOR inhibitor, and GEM synergistically inhibits PDAC CC growth in the presence of CAFs. These data suggest that the addition of BEZ may improve the effectiveness of cytotoxic chemotherapy in PDAC.
 

25.01 Notch1—WISP-1 axis determines the regulatory role of MSC-derived fibroblasts in melanoma metastasis

Posted on January 25, 2017

P. P. Parikh1, H. Shao1, L. Cai1, M. Moller1, B. Issac2, R. Vazquez-Padron1, M. Owyong1, Z. Liu1,2  1University Of Miami,Surgery,Miami, FL, USA 2University Of Miami,Sylvester Comprehensive Cancer Center,Miami, FL, USA

Introduction:  Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis.

Methods:  MSC-DF were generated from bone marrow (BM)-derived MSC obtained from Notch1Flox/Flox and ROSAN1IC mice and transduced with Cre-GFP/Lentivirus or GFP/Lentivirus (control).  Various MSC-DF were co-cultured or co-grafted with human melanoma cells (labeled with Luc2) in vitro or in vivo. Melanoma growth and metastasis in vivo were examined by measuring tumor size and IVIS scanning. cDNA microarray was employed to identify downstream targets of Notch1 signaling.

Results: We demonstrated that the Notch1 pathway’s activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1-/- selectively promoted, while MSC-DFN1IC+/+ preferentially suppressed melanoma metastasis, but not growth, in mouse models. When co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1-/- supported, but MSC-DFN1IC+/+ inhibited melanoma cells to form spheroids. We also identified Wnt-induced secreted protein-1 (WISP-1) from MSC-DF as a key downstream secretory mediator of Notch1 signaling on melanoma metastasis. 

Conclusion: These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We demonstrate that the Notch1—WISP-1 axis functions as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis.

 

24.10 ECM and CEACAM Proteins Characterize Pancreatic Duct Exosomes in Patients with Pancreatic Cancer

Posted on January 25, 2017

J. Zheng1, J. Hernandez1, A. Doussot1, L. Bojmar2, B. Costa-Silva2, E. VanBeek2, C. Zambirinis1, M. Tesic Mark3, H. Molina3, M. Gonen1, T. Kingham1, P. Allen1, M. D’Angelica1, R. DeMatteo1, D. Lyden1,2, W. Jarnagin1  1Memorial Sloan-Kettering Cancer Center,New York, NY, USA 2Weill Cornell Medical College,New York, NY, USA 3Rockefeller University,New York, NY, USA

Introduction: Exosomes have emerged as important vehicles for intercellular communication between cancer cells and their microenvironment and may facilitate tumor progression. Exosomes in pancreatic duct fluid have not been previously studied. We hypothesize that exosomes can be detected in the pancreatic duct fluid and that exosomal proteins may be useful as diagnostic and/or prognostic biomarkers in patients with pancreatic ductal adenocarcinoma (PDAC).

Methods: Pancreatic duct fluid was collected from 22 patients at the time of partial pancreatectomy for PDAC (n=13), intraductal papillary mucinous neoplasm (IPMN) (n=4) and other benign pancreatic diseases (n=5). Serial ultra centrifugation was used to isolate exosomes. Exosomes were quantified using the DS500 nanoparticle characterization system (NanoSight), and confirmed by transmission electron microscopy (TEM). Exosomal proteins were identified and analyzed using liquid chromatography-mass spectrometry (LC-MS). Expression levels of exosomal proteins were compared between patients with PDAC and benign diseases using Wilcoxon rank-sum test. Survival analyses among patients with PDAC were performed using Kaplan-Meier with log rank test.

Results: Exosomes were successfully isolated from the pancreatic duct fluid of all patients. The mean concentration of exosomes isolated was 5.2 +/- 1 x 108 particles/mL, with a mean vesicle size of 186 +/- 4nm. The presence of exosomes was confirmed for each patient using TEM. Characteristic exosome surface markers, including tetraspanins CD9 and CD63, were identified. No differences in either size or concentration of EVs were detected when patients were stratified by diagnosis, but significant differences in protein composition were found. Laminin β3, Laminin C2, Tenascin C, and matrix metalloproteinases 1 and 7 were significantly overexpressed among patients with PDAC as compared to patients with IPMN and benign diseases (Table). Similarly, CEACAM1, CEACAM5, and CEACAM6 were significantly overexpressed among patients with PDAC (p=<0.001, 0.025, and 0.030, respectively). Among patients with PDAC, patients having expression of all three CEACAMs (n=6) had significantly worse overall and recurrence-free survival compared to patients lacking any of the CEACAMs (n=7) (p=0.01 and 0.005, respectively).

Conclusion: Exosomes can be isolated from pancreatic duct fluid. Exosomal ECM-associated proteins and CEACAMs appear to possess diagnostic and prognostic utility for patients with PDAC. Further evaluation of the mechanistic significance of these proteins in exosomes is warranted.

 

24.09 Tumor Homing Peptide Display on AAV2 Surface Enhances Viral Targeting of Pancreatic Cancer

Posted on January 25, 2017

S. Liu1, J. Lee3, J. Yu1, R. Sanchez1, M. Van Dam3, E. Rozengurt2, F. Brunicardi1  1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Division Of Digestive Disease,Los Angeles, CA, USA 3David Geffen School Of Medicine, University Of California At Los Angeles,Crump Institute For Molecular Imaging,Los Angeles, CA, USA

Background: Recombinant adeno-associated virus type 2 (AAV2) vectors transduce a wide variety of tissues in vivo and provide long-term gene expression with minimal immune responses and no pathological responses to the host, thus making AAV2 an attractive vector for gene delivery in vivo. However, its broad host range limits its usefulness in which transgene expression needs to be limited to a specific organ or cell type, such as cancer. Furthermore, low efficiency of wild type AAV2 (AAV2wt) transfection of pancreatic cancer (PDAC) cells further limits its effectiveness. In this study, we explored the possibility of directing rAAV2 transduction by incorporating a panel of tumor homing peptides to target human PDAC cells and tumors in mice.

Methods: 18 tumor homing peptides (TumorHoPe) were selected from literature and TumorHoPe database for AAV2 capsid display. Synthetic oligos were inserted into the rAAV2 capsid at R588. scAAV-eGFP and scAAV2-Gaussia Luciferase (GLuc) reporters were used. Real time-PCR was used to quantify the AAV2 titers. AAV infection of human PDAC cell lines (PANC-1, Mia PaCa2, Capan-2 and AsPC-1) and benign HPDE cells were performed, followed by peptide competition assays. GLuc secretory activity was determined by bioluminescence assay. In vivo imaging was performed in PANC-1 subQ xenograft using systemic delivery of AAV2TumorHoPe-GLuc imaging vector via tail vein.

Results: 18 TumorHoPe displayed AAV2  and one mutated AAV2 were created (AAV2TumorHoPe). The greatest viral infection was obtained in PANC-1 by AAV2RGD (CDCRGDCFC), Mia PaCa2 by AAV2RGR (CRGRRST), Capan-2 by AAV2RGD and AsPC-1 by AAV2LGL (RGDLGLS), with no infection of benign HPDE cells (Fig. Upper). There was no significant GFP expression in AAV2WT transfections of PDAC cells. To further identify whether the TumorHoPe display virus targeted PDAC cells and enhancing the infection efficiency, competition assays were performed on each cells infected by virus in the presence of excess exogenous TumorHoPe peptides; infection efficiency dropped to 42.5%, 34.3%, 25.6% and 36.9% in PANC-1, Mia PaCa2, Capan-2 and AsPC-1 cells, respectively. Systemic delivery of AAV2TumorHoPe-GLuc imaging vector in PANC-1 tumor model resulted in highly specific imaging of tumors without toxicity (Fig. Bottom A), whereas control AAV2 vector showed only non-specific transfection with none seen in the PANC1 tumor (Fig. Bottom B).

Conclusion: This study demonstrates that TumorHoPe display on AAV2 surface enhances viral targeting of PDAC cells and tumor, but not benign HPDE cells. The unique type of TumorHoPe on the AAV2 surface contributes to specific efficiency in PDAC cells. These preclinical data suggest that TumorHoPe AAV2 gene delivery could be used for targeted imaging of PDAC.

24.08 Cancer Stemness In Bone Marrow Micrometastases Of Human Breast Cancer

Posted on January 25, 2017

M. C. Kuo1, A. N. Kothari1, Z. Mi1  1Loyola University Chicago Stritch School Of Medicine,Surgery,Maywood, IL, USA

Introduction: Cancer cells metastasize to the bone marrow (BM) to create the pre-metastatic niche. Cancer stemness (or expression of stem cell characteristics) is regulated by the tumor microenvironment (TME) and associated with self renewal, clonal maintenance, and poor clinical outcomes. Osteopontin (OPN) induces mesenchymal stem cells (MSC) in the TME adopt a cancer-associated fibroblast (CAF) phenotype to potentiate cancer growth and metastasis. The mechanisms by which cancer cells and TME regulate stemness in the BM pre-metastatic niche are unknown.  

Methods: Human breast cancer cell lines, MB231-Luc (OPN+) and MCF7-Luc (OPN-), were used in an orthotopic murine xenograft model. 6-week-old female NOD scid mice were implanted with 2×106 tumor cells in the presence and absence of human MSC-GFP cells in the R4 position of the mammary fat pad. In selected instances,  MCF7-Luc transfected to express OPN (MCF7[lvOPN]) were co-implanted instead of MCF7-Luc. OPN aptamer (APT), which blocks and inactivates extracellular OPN, and/or inactive mutant APT (muAPT) were utilized. After 8 weeks, animals were sacrificed and femoral BM isolated. Stem cell markers, Sox2, Oct4 and Nanog, and CAF markers, SMA and vimentin, were measured by RT-PCR using human specific primers and normalized to Luc and GFP mRNA, respectively. Relative cell number was determined by titrating cell number to Ct value of GFP or Luc in vitro. Each treatment had 3-5 mice. Statistical analysis was performed using Student’s t-test; p-values < 0.05 were considered significant.

Results: 
In BM from MB231+MSC, expression of Sox2, Oct4 and Nanog was 4x greater than MB231 alone.(p<0.05) Administration of APT to block OPN decreased Sox2, Oct4 and Nanog to levels equivalent to MB231 alone and MB231+MuAPT. Total MB231 cell numbers were not different. In contrast, although CAF markers were not different among all treatments, CAF numbers in BM were 15-fold greater in MB231+MSC vs MB231 alone and MB231+MSC+APT.(p<0.05) In parallel gain-of-function studies, MCF7-Luc (which do not express OPN) co-implanted with MSC did not express increased stem cell markers in BM when compared to MCF-Luc alone. In contrast, using MCF7 expressing OPN, MCF7[lvOPN]+MSC had 5x greater Sox2, Oct4 and Nanog expression.(p<0.05) APT with MCF7[vOPN]+MSC ablated the increase in stem cell markers. Again, CAF markers were unchanged among treatments, but CAF numbers were 70x higher in MCF7[lvOPN] + MSC vs MCF7+MSC and MCF7[lvOPN]+MSC+APT.(p<0.05)

Conclusion:   

In this xenograft model of human breast cancer, our results indicate: 1) both breast cancer cells and accompanying CAF from the primary site metastasize to the BM to form the pre-metastatic niche, 2) tumor-derived OPN mediates CAF migration to the BM, and 3) cancer cells exhibit significantly increased stemness in the presence of CAF in the BM. We conclude that OPN-dependent migration of CAF is required for increased cancer cell stemness in the BM pre-metastatic niche.   

24.07 Nucleotide Depletion by Autophagy Inhibition Sensitizes Kras-driven PDAC to Replication Stress

Posted on January 25, 2017

I. A. Elliott1, J. L. Williams2, R. Ghukasyan1, C. C. Matsumura1, N. Wu1, L. Li1, W. Kim3, S. Poddar3, E. R. Abt3, A. M. Dann1, H. DeRubertis1, D. Braas4, T. M. Le3, C. G. Radu3, T. R. Donahue1,3  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2Harbor-UCLA Medical Center,Department of Surgery,Torrance, CA, USA 3University Of California – Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA 4University Of California – Los Angeles,Metabolomics Center,Los Angeles, CA, USA

Introduction:
Autophagy is a critical source of nucleotides, which are rate-limiting for Ras-driven cancer cell proliferation. Genetically disabling autophagy impairs energy and redox homeostasis by depletion of nucleotide pools, and sensitizes cancer cells to radiation. We hypothesized that the autophagy inhibitor chloroquine (CQ) would deplete deoxyribonucleotide triphosphate (dNTP) availability for incorporation into DNA, thus sensitizing cancer cells to replication stress and revealing a targetable liability in Ras-driven pancreatic ductal adenocarcinoma. 

Methods:
Human (MiaPaCa-2) and murine (KPC) PDAC cells were treated with CQ +/- the replication stress response (RSR) inhibitor VE822. S-phase cells were labeled by pulsing with 5’-ethynyl-2’-deoxyuridine (EdU) and cell cycle progression measured by flow cytometry. Newly synthesized dNTP incorporation into DNA was measured via [13C6]glucose labeling and our novel liquid chromatography mass spectrometry (LC-MS) platform. Global metabolomics analyses were performed by detection of relative amounts of metabolites using LC-MS. Western blots (WB) were done on cell lysates. Cell viability was measured by Cell-Titer-Glo assay. MiaPaCa-2 or KPC cells were injected s.q. in the flanks of NSG or C57BL/6 mice. Mice were treated 5x/week with CQ+/-VE822 (60mg/kg p.o.). Immunohistochemistry (IHC) was performed on explanted KPC tumors after 3 days of CQ+VE822.

Results:
We found that CQ caused S-phase arrest in MiaPaCa-2, and impaired incorporation of newly synthesized dNTPs into replicating DNA(Fig.1a), indicating nucleotide pool insufficiency. Global metabolomic profiling of MiaPaCa-2 revealed that this was due to depletion of purine and pyrimidine substrates for dN synthesis under CQ, and this was exacerbated by addition of the RSR inhibitor VE822(Fig.1b). We then tested the impact of this nucleotide depletion on cell fate; CQ+VE822 treatment led to synergistic induction of DNA damage (reflected by pH2A.X WB), and decreased viability of MiaPaCa-2 and KPC in vitro (Fig.1c). We also observed increased IHC staining for pH2A.X in KPC tumors and impaired growth of MiaPaCa-2 tumors after CQ+VE822 treatment in vivo. Finally, we confirmed the ability of CQ to induce oxidative stress as indicated by HO-1 levels on WB. Management of redox balance and DNA damage are critical to recovery from radiation; accordingly, we found that CQ+VE822 profoundly impaired survival after radiation of MiaPaCa-2 and KPC cells.

Conclusion:
Pharmacologic inhibition of autophagy by CQ impairs incorporation of dNTPs into DNA by depleting substrates for nucleotide biosynthesis. When combined with an RSR inhibitor, this leads to induction of DNA damage and synthetic lethality in PDAC cells in vitro and in vivo.

24.06 Functional Screens to Identify Gene Drivers of Pancreatic Cancer

Posted on January 25, 2017

N. Villafane1,2, Y. Tsang2, K. Scott2  1Baylor College Of Medicine,Michael E. DeBakey Department Of Surgery,Houston, TX, USA 2Baylor College Of Medicine,Department Of Molecular And Human Genetics,Houston, TX, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is not one of the most prevalent cancers but is by far one of the deadliest given its 5-year survival rate of <5%. The majority of PDAC tumors harbor activating mutations in the KRAS oncogene, a therapeutically undruggable and initiating “driver” event found in >90% of early-stage lesions. Following activation of KRAS, PDAC acquires other driver mutations that include inactivating events in tumor suppressor genes and numerous low frequency gene mutations, amplifications, and deletions likely required for KRAS-mediated tumor progression. Our objective is to determine which of these events are functional drivers or “driver effectors” required for KRAS biology, as targeting these events or their activated pathways offers hope of improving patient outcomes.

Methods:  We used a highly-vetted list of 250 amplified gene candidates (copy number > 5; GISTIC q-value < 0.075) that was previously defined at our lab by analyzing The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) PDAC datasets (derived from 218 tumors). We engineered pooled gene libraries consisting of DNA-barcoded open reading frame (ORF) clones encoding the amplified driver candidates. We used a lentiviral vector to deliver these ORFs to human pancreatic ductal epithelial (HPDE) cells engineered with a doxycycline-inducible KRASG12D allele in order to identify ORFs that drive tumor growth and metastasis in mice maintained on or off doxycycline diet to control for KRASG12D activation. 

Results: Our screens identified SEMA6A, a transmembrane protein member of the axon guidance pathway, as a high priority PDAC target. Our data indicate that SEMA6A expression is increased through KRASG12D– mediated down-regulation of miR145. This suggests that the SEMA6A/plexinA2/A4 signaling pathway may be hyper-activated in the presence of oncogenic KRAS, resulting in cell cycle progression and tumor growth. 

Conclusion: Screening of candidate driver pools in our laboratory has revealed potent drivers of PDAC, such as SEMA6A. High priority PDAC drivers resulting from these functional screens will lead to the discovery of detection biomarkers and potential targets for drug development efforts needed for patients suffering from this disease. 

 

24.03 Minnelide Decreases Tumor Burden in Murine Models of Malignant Melanoma

Posted on January 25, 2017

V. Sethi1, B. Giri1, B. Garg1, S. Modi1, S. Banerjee1, A. Saluja1, V. Dudeja1  1University Of Miami,Surgical Oncology, Department Of Surgery,Miami, FL, USA

Introduction:  Malignant melanoma is the most lethal of all skin cancers. Modern immune checkpoint inhibitors used to treat melanoma show response in < 50% of patients and are associated with various immune-related adverse events. Therefore, more effective and safer therapeutic strategies are urgently needed. Triptolide, a diterpenoid tri-epoxide and its water soluble pro-drug Minnelide, have been previously shown by our group to be highly effective in treating various solid tumors. Minnelide is currently undergoing phase I clinical trial for treating advanced GI malignancies. This study aims to evaluate the effect of Triptolide in vitro and of Minnelide in vivo against melanoma.

Methods:

In-vitro, B16-F10 cells, a highly metastatic murine melanoma cell line, were treated with various concentrations of Triptolide (0-75 nM). Cancer cell viability at 24 and 48 hours was measured using a formazan dye based colorimetric assay. Apoptosis at 24 and 48 hours was measured by quantifying Caspase 3 and 7 activities in a luminescence based assay. Effect of Triptolide on invasion and migration of melanoma cells across a chemotactic gradient was measured by using Transwells (having PET membrane with 8.0 μm  pore size) coated with and without Matrigel respectively.

 

In vivo, we treated mice with 0.21 mg/kg/day of Minnelide, a clinically relevant, well tolerated dose derived from phase I trial data. In the first model, 7.5 x104 B16-F10 cells were injected subcutaneously in mice and these were randomized into Minnelide and saline arms on day 3. Tumor volume was measured at endpoint (Day 19). In the second model, 1.5 x 105  cells were injected through an intrasplenic injection in a hepatic metastases model of melanoma. Randomization was done and treatment was started on day 5. Liver metastases were measured by quantifying liver volume and liver weight/body weight ratio at endpoint (Day 21).

Results: In vitro, Triptolide caused a significant decrease in viability and increase in apoptosis of B16-F10 cells in a dose dependent manner. Invasion and migration of melanoma cells was also significantly inhibited. In vivo, Minnelide treatment caused a significant decrease in volumes of subcutaneous tumors (mean tumor volume ± SEM was 814 ± 97.5mm3 in saline arm vs 467 ± 79.3mm3 in Minnelide arm; p < 0.05) and a drastic decrease in hepatic metastases burden as measured by liver volumes and liver weight/body weight ratio (mean liver volume ± SEM was 3.8 ± 0.4 ml in saline arm vs 2.2 ± 0.4 ml in Minnelide arm; p < 0.05)

Conclusion: Phase II clinical trial of Minnelide for treating GI cancers is starting soon. Minnelide has a potential to become an effective therapeutic option for treating advanced melanoma too.
 

24.04 High Depth Hybrid Capture Exome Sequencing Of Somatic Mutations For Personalized Cancer Therapy

Posted on January 25, 2017

N. Paez Arango1, L. Brusco2, K. R. Shaw2, K. Chen3, K. Eterovic4, V. Holla2, A. Johnson2, B. Litzenburger2, Y. B. Khotskaya2, N. Sanchez2, A. Bailey2, X. Zheng4, C. Horombe2, S. Kopetz5, C. Farhangfar6, M. Routbort7, R. Broaddus8, E. S. Bernstam9, J. Mendelsohn2, G. B. Mills2,4, F. Meric-Bernstam1,2,10  1University Of Texas MD Anderson Cancer Center,Department Of Surgical Oncology,Houston, TX, USA 2Univerisity Of Texas MD Anderson Cancer Center,Khalifa Institute For Personalized Cancer Therapy,Houston, TX, USA 3University Of Texas MD Anderson Cancer Center,Department Of Bioinformatics And Computational Biology,Houston, TX, USA 4University Of Texas MD Anderson Cancer Center,Department Of Systems Biology,Houston, TX, USA 5University Of Texas MD Anderson Cancer Center,Gastrointestinal Medical Oncology,Houston, TX, USA 6Carolinas Healthcare System,Levine Cancer Institute,Charlotte, NC, USA 7University Of Texas MD Anderson Cancer Center,Department Of Hematopathology,Houston, TX, USA 8University Of Texas MD Anderson Cancer Center,Department Of Pathology,Houston, TX, USA 9University Of Texas Health Science Center At Houston,School Of Biomedical Informatics,Houston, TX, USA 10University Of Texas MD Anderson Cancer Center,Department Of Investigational Cancer Therapeutics,Houston, TX, USA

Introduction: The increasing availability of molecular therapeutics targeting specific genomic alterations has led to an increased interest in the use of next-generation sequencing to help identify patients that could potentially benefit from targeted clinical trials and personalized cancer therapies.  Expanding the number of genes and incorporating copy number testing could potentially aid in identifying a higher number of genomic alterations in clinically actionable genes, and thus identify a greater number of patients that could likely benefit from targeted therapy. The purpose of this study was to look at patients who underwent routine genomic profiling under standard hotspot analysis and then using a deep targeted sequencing platform in the research setting, assess the added value to having a more comprehensive genomic test.

Methods: 1200 patients with advanced cancer underwent routine tumor profiling for characterization of common mutations on an 11, 46, or 50 gene hotspot sequencing platform in the Clinical Laboratory Improvement Amendments (CLIA) certified environment, as well as on 201 gene deep targeted sequencing platform in the research setting. 120 genes were considered actionable based on their potential to be targeted with available therapies.

Results:Five hundred and twenty eight patients (44%) had at least one mutation detected in a potentially actionable gene using standard hotspot genomic testing under a limited gene panel, with a total of 644 mutations detected. Under deep targeted sequencing in the research setting, we identified 686 patients (57%) that had at least one somatic mutation in a potentially actionable gene that was previously undetected in the standard testing, with a total of 2448 newly identified mutations. Additionally, we detected 654 (55%) patients with at least one potential copy number variation, with a total of 2784 potential copy number variations identified. A grand total of 5232 alterations in potentially actionable genes were found that would have been otherwise undetected (see table).

Conclusion:Expanded cancer gene sequencing for profiling somatic mutations identifies a greater number of alterations in potentially actionable genes that could have a clinical significance, allowing for the identification of an increased number of targets for personalized cancer therapies.

 

24.02 Targeting Neutrophil Extracellular Traps (NETs) prevents HCC in a mouse model of NASH

Posted on January 25, 2017

D. J. Van Der Windt1, V. Sud1, P. Varley1, J. Goswami1, H. Yazdani1, P. Loughran3, M. I. Minervini4, H. Huang1,2, R. L. Simmons1, A. Tsung1  1University Of Pittsburgh Medical Center,Surgery,Pittsburgh, PA, USA 2Huazhong University Of Science And Technology,Surgery,Wuhan, HUBEI, China 3Center For Biologic Imaging,Cell Biology,Pittsburgh, PA, USA 4University Of Pittsburgh Medical Center,Pathology,Pittsburgh, PA, USA

Introduction:  Non-alcoholic steatohepatitis (NASH) is a progressive, inflammatory form of non-alcoholic fatty liver disease. Its prevalence is rapidly increasing, resulting in more patients with NASH-related hepatocellular carcinoma (HCC). Surgical resection and liver transplantation are the only curative options for HCC. As a consequence, there is an increased number of patients with NASH undergoing liver resection, and those patients have higher risks of post-operative complications. The inflammatory pathogenesis of NASH progression to HCC remains incompletely understood. Neutrophils are increasingly recognized as cells that regulate the inflammatory environment and can affect the progression of inflammatory cancers. The formation of neutrophil extracellular traps (NETs) has been discovered as a novel neutrophil function, during which chromatin structures with pro-inflammatory proteins are exposed to the extracellular environment. Here we sought to evaluate the role of NETs in the inflammatory development of NASH and the progression to HCC.

Methods:  Wild type (WT) and peptidylarginine deiminase 4 knockout (PAD4 KO) mice (that are unable to form NETs) were exposed to a model of NASH-related HCC, and compared to healthy control (HC) mice. NASH was established by injection of streptozotocin to induce diabetes (200ug I.P., 1 dose within 5d from birth), and feeding of a high fat diet starting at age 3 weeks. WT and PAD4KO mice were sacrificed at various time points for analysis of liver inflammation and the formation of HCC. To test the efficacy of a clinically available treatment strategy for eliminating NETs, WT mice were treated with DNase1 (100U I.P. 3x/wk).

Results: WT male mice developed NASH as evidenced by progressive increase in NASH activity score (NAS), and developed multiple liver tumors by age 20 weeks. Neutrophil infiltration was seen as early as age 6 weeks as evidenced by flow cytometry (12.6±0.5% in WT vs. 4.0±0.2% of CD45+ liver non-parenchymal cells in HC, p<0.001). NETs were observed on liver immunofluorescence. At age 8 weeks, WT mice had elevated serum ALT and IL-6 levels, indicating liver injury and inflammation. ALT and IL-6 levels were reduced to baseline in PAD4 KO mice and by DNase1 treatment, respectively (ALT [U/L]: WT 166±35, PAD4 KO 102±4, DNase1 73±16, p<0.05. IL-6 [pg/mL]: WT 23±7.9, PAD4 KO 3.9±0.7, DNase1 5.8±1.2, p<0.05). Most importantly, PAD4KO mice developed significantly fewer tumors at age 20wks than WT mice (1.7±0.6 vs. 6.3±1.8, p<0.05).

Conclusion: Elimination of NETs reduces inflammation and prevents progression to HCC in murine NASH. Clinically, inhibition of NETs in patients with NASH may reduce their risk of developing HCC.

 

24.01 Acceleration of Pancreatic Cancer Development in KrasG12D Mice Deficient in Hormone Sensitive Lipase

Posted on January 25, 2017

M. Xu1, X. Jung1, A. Moro1, C. Chou1, H. Chang1, A. P. Stark1, A. Schmidt1, Y. Chen1, J. King1, O. J. Hines1, G. Eibl1  1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA

Introduction: Obesity has been associated with multiple types of cancer. Our previous work has shown that a high-fat, high-calorie diet (HFCD) leads to obesity in conditional KrasG12D (KC) mice with inflammation of the visceral adipose tissue (VAT) and acceleration of pancreatic intraepithelial neoplasia (PanIN) development. In this study, we investigated whether diet-independent inflammation in the visceral adipose tissue is sufficient to accelerate pancreatic cancer (PDAC) development using hormone-sensitive lipase (hsl) deficient mice. Hsl is one of the enzymes involved in the hydrolysis of stored triglycerides. Non-obese hsl deficient mice are characterized by a significant inflammation in visceral adipose tissue.  

Methods: KC mice were crossed into an hsl deficient background to generate KC;hsl+/+ and KC;hsl-/- mice. Mice were fed regular chow for six months and body weight recorded weekly. At sacrifice tissues were harvested for histological and immunohistochemical analysis.

Results: Over the study period of six months KC;hsl+/+ and KC;hsl-/- mice displayed similar weight gain. KC;hsl-/- males had an average weight of 29.5±4.5g at sacrifice, whereas KC;hsl+/+ males weighed 29.7±3.2g. KC;hsl-/- and KC;hsl+/+ females had an average weight of 25.2±1.8g and 24.3±1.8g, respectively. Twenty percent of the KC;hsl-/- mice developed invasive PDAC (n=20), whereas none of the KC;hsl+/+ mice developed PDAC (n=23; p<0.05). Interestingly, all KC;hsl-/- mice with PDAC were male. Histological and immune-histochemical analyses revealed that compared to KC;hsl+/+ mice KC;hsl-/- mice had significantly increased VAT inflammation as measured by the number of crown-like structures per high power field (hpf) (4.6±3.0 in KC;hsl-/- vs. 0.2±0.6 in KC;hsl+/+; p<0.01), enhanced activation of pancreatic stellate cells assessed by counting the cells per hpf positive for alpha-smooth muscle actin (44.5±9.0 in KC;hsl-/- vs. 15.8±5.2 in KC;hsl+/+; p<0.01) and more F4/80 positive macrophages per hpf in the pancreas (18.8±9.1 in KC;hsl-/- vs. 11.5±6.7 in KC;hsl+/+; p<0.01). PanIN lesions in KC;hsl-/- mice showed a higher rate of proliferation (per hpf) as measured by Ki67 staining (14.9±6.4 in KC;hsl-/- vs. 5.5±3.6 in KC;hsl+/+; p<0.01).

Conclusions: KC;hsl-/- mice had a significantly more robust VAT and pancreatic inflammation and accelerated PDAC development compared to KC;hsl+/+ mice. Our results suggest that VAT inflammation plays an important role in PDAC development. 

21.12 Prognostic impact of pancreastatin following chemoembolization for neuroendocrine tumors

Posted on January 25, 2017

D. S. Strosberg1, J. Onesti4, N. Saunders3, G. Davidson1, M. Shah5, M. Dillhoff1, C. Schmidt1, M. Bloomston2, L. A. Shirley1  1The Ohio State University Wexner Medical Center,Surgical Oncology,Columbus, OH, USA 221st Century Oncology,Fort Meyers, FL, USA 3Emory University School Of Medicine,Atlanta, GA, USA 4Mercy Health Grand Rapids,Grand Rapids, MI, USA 5The Ohio State University Wexner Medical Center,Medical Oncology,Columbus, OH, USA

Introduction: Transarterial chemoembolization (TACE) is a viable treatment option for patients with metastatic neuroendocrine tumors (NETs) to control tumor progression and palliate symptoms of hormone excess.  Pancreastatin, a split product of chromogranin, has been shown to correlate with survival in patients with NETs. The objective of this study was to investigate the prognostic impact of pancreastatin levels in patients with metastatic NETs treated with TACE.

Methods: Patients with metastatic NET treated with TACE at a single institution from 2000 to 2013 were analyzed. Clinical variables were analyzed with Chi-square, Fisher Exact, or independent T-test as appropriate.  Kaplan-Meier curves for overall survival (OS) were analyzed using log-rank testing for curve differences.

Results: 188 patients underwent TACE for metastatic NETs during the study period.  An initial pancreastatin level greater than 5000 pg/mL correlated with worse OS from time of first TACE (Median OS 58.5 months vs 22.1 months, p<0.001). A decrease in pancreastatin levels by 50% or more after TACE treatment correlated with improved OS (Median OS 53.8 months vs 29.9 months, p=0.032). Patients with carcinoid syndrome were more likely to have a subsequent increase in pancreastatin after initial drop post-TACE (percent of patient with increase 78.1% vs 55.2%, p=0.002). Patients who had an increase in pancreastatin levels after initial drop post-TACE were also more likely to have liver progression on axial imaging (70.7% vs 40.7%, p=0.005) as well as more likely to need repeat TACE (21.1% vs 6.7%, p=0.009).

Conclusion: For patients with liver metastases from NET, measurement of pancreastatin levels can be useful in several steps during potential TACE treatment.  Extreme high levels prior to TACE can predict poor outcomes, significant drops in pancreastatin after TACE correlate with improved survival, and a rise in levels after initial drop may predict progressive liver disease requiring repeat TACE.  As such, pancreastatin levels should be measured throughout the TACE treatment period.

 

21.04 Protease Tailored Flourogenic Substrates Outperform CEA in Identifying Mucinous Pancreatic Cysts

Posted on January 25, 2017

D. A. Dominguez1, S. Ivry3, S. Hatcher3, E. Gilbert2, S. Kumar2, W. Park6, M. Schmidt7, R. Brand4, A. O’Donoghue5, K. Kirkwood2, C. Craik3  1UCSF East Bay,Department Of General Surgery,Oakland, CA, USA 2UCSF,Department Of General Surgery,San Francisco, CA, USA 3UCSF,Department Of Pharmaceutical Chemistry,San Francisco, CA, USA 4University Of Pittsburgh,Department Of Gastroenterology,Pittsburgh, PA, USA 5UCSD,Department Of Pharmacy And Pharmaceutical Sciences,San Diego, CA, USA 6Stanford University,Department Of Gastroenterology,Palo Alto, CA, USA 7Indiana University,Department Of Surgery,Indianapolis, IN, USA

Introduction:  Risk stratification of pancreatic cystic lesions remains an area of great clinical uncertainty.  Measurement of cyst fluid CEA is used to predict which cysts are mucinous, and therefore may have malignant potential, but its accuracy limits its usefulness.  Due to this lack of definitive pre-operative characterization, some patients will die from undiagnosed cancers, while others undergo unnecessary pancreatic resections with significant morbidity.  Dysregulation of protease expression and activity has been previously reported in mucinous pancreatic cyst fluid.  We tested the hypothesis that differences in proteolytic activity between mucinous (MUC) and non-mucinous (NON-MUC) cysts could be used to improve our ability to identify pre-malignant pancreatic tumors.

Methods:  We first analyzed cyst fluid from a cohort of human pancreatic neoplasms (MUC, n=16; NON-MUC, n=7) using multiplex substrate-profiling by mass spectrometry (MSP-MS), which is an unbiased, comprehensive technology for analyzing patterns of proteolytic activity. We found that aspartyl protease activity was unique to MUC pancreatic cysts. Next, using shotgun proteomic analysis, we identified 2 proteases (Prot1, Prot2), that were selectively expressed in MUC cyst fluid.  Specific cleavage profiles were used to design a selective fluorescent substrate for each protease. Substrates were then used to assess the activity of each protease in a larger patient cohort (MUC, n=71; NON-MUC, n=39); ROC curves were generated for each substrate.  Performance was compared with CEA values (CLIA lab) using >= 192 ng/mL as a cut off for MUC cysts. 

Results:  Receiver operator characteristic (ROC) curves for Prot1 and Prot2 exhibited an area under the curve (AUC) of 0.98 and 0.82, respectively. Prot1 demonstrated a sensitivity of 93% and specificity of 100%.  Prot2 had a sensitivity of 70% and a specificity of 92%.  Testing required < 0.2 mL cyst fluid.  CEA had an AUC of 0.86, and a sensitivity and specificity of 65% and 94%, respectively.

Conclusion:  MSP-MS is a powerful technology to examine dysregulated proteolysis in complex biological fluids.  The resultant fluorogenic substrates outperformed CEA, the highest of which had a sensitivity of 93% and a specificity of 100%, for the detection of MUC pancreatic cysts.  Our fluorescent assay has the potential to be a rapid, inexpensive, and highly accurate predictor of MUC pancreatic cysts. 

 

21.03 DNA released from PAD4-mediated NETosis enhances tumor growth in murine pancreatic cancer

Posted on January 25, 2017

J. Miller-Ocuin1, W. R. Doerfler1, X. Liang1, B. Boone1, A. Singhi1, M. Lotze1, H. Zeh1  1University Of Pittsburgh,Surgical Oncology/Surgery/Medicine,Pittsburgh, PA, USA

Introduction: Activated neutrophils release intracellular material in a process known as neutrophil extracellular trap (NET) formation. NETs result from histone citrullination, chromatin decondensation, and ultimately DNA release from the cell. Peptidyl arginine deiminase 4 (PAD4) is an enzyme required for NET formation. Increased NET formation is associated with cancer progression in preclinical models of murine pancreatic cancer. We hypothesized mice deficient in PAD4 would demonstrate decreased tumorigenesis.

Methods: Luciferase-transfected Panc02 cells were injected into the pancreas of WT controls or PAD4-/- mice. Tumorigenesis continued for 5 weeks in untreated experiments. DNAse (5mg/kg IP) or vehicle was injected daily for 3 weeks in treated experiments. Mice were imaged weekly using an in vivo imaging system (IVIS) for tumor growth; area of interest (ROI) is represented as photon/sec/cm2/Sr. At conclusion of experiments, mice were sacrificed for tumor weights, immunohistochemistry (IHC) and serum DNA quantification. Bone marrow chimeras using Pdx-Cre KrasG12D (KC) transgenic mice, which develop spontaneous pancreatic cancer, were generated with lethal irradiation followed by reconstitution with PAD4-/- or WT bone marrow. 21 days after treatment with cerulein, which induces pancreatitis to accelerate tumor growth, animals were sacrificed and organs harvested. Hematoxylin and eosin stained tumor sections were evaluated by a pancreatic pathologist.

Results: PAD4-/-tumor bearing mice showed decreased NETs on IHC and decreased serum DNA (406ng/ml vs 858ng/ml, p=0.03), a surrogate NET marker. Tumorigenesis was significantly decreased in PAD4-/- vs. WT mice (921mg vs. 326mg, p=0.001; ROI: 2.4×107 vs. 1.6×106, p=0.05). DNAse treatment of WT mice lead to significantly decreased tumor growth vs. sham treated controls (336mg vs. 206mg, p=0.05; ROI: 9.9×105 vs. 3.8×105, p=0.05) while there was no significant change in tumor growth in PAD4-/- animals treated with DNAse (195mg vs. 217mg, p=0.29; ROI: 8.5×105 vs. 6.9×105, p=0.43). There was a trend toward decreased high-grade precursors and invasive cancers in PAD4-/- bone marrow recipients as compared to WT recipients (p=0.29) with significantly diminished NET formation by isolated bone marrow supernatant DNA levels (197ng/mL vs. 796ng/mL, p=0.001).

Conclusion: Murine pancreatic tumors in PAD4-/-mice show decreased tumorigenesis and decreased NET formation. DNA released during NET formation leads to increased tumor growth, which is suppressed by DNAse administration. Future studies will focus on the mechanism through which NET DNA promotes tumor growth.

 

21.02 Gut Microbiome Promotes Pancreatic Oncogenesis by Inducing Innate and Adaptive Immune Suppression

Posted on January 25, 2017

M. Hundeyin1, S. Pushalkar1, D. Daley1, G. Werba1, N. Mohan1, S. Lall1, B. Wadowski1, B. Aykut1, E. Kurz1, U. Soni1, E. Morales-Vicente1, D. Saxena1, G. Miller1  1New York University School Of Medicine,Surgery,New York, NY, USA

Introduction:
Pancreatic ductal adenocarcinoma (PDA) is the 3rd most lethal cancer in the United States and accounts for 85% of all pancreatic malignancies. The gut microbiome has emerged as an important regulator in the balance between health and disease, including oncogenesis. However, the microbiome has not been directly linked to pancreatic oncogenesis. We postulated that hosts with PDA harbor an altered pancreatic and gut microbiome and that dysbiosis influences PDA progression. 

Methods:
To determine whether endoluminal gut bacteria can access the pancreas, we administered fluorescently-labeled Enterococcus faecalis to WT mice via oral gavage. To determine whether bacteria promote the progression of pancreatic oncogenesis, we employed the slowly progressive p48Cre;LSL-KrasG12D (KC) mouse model of PDA and rederived KC mice in a germ-free environment. To identify possible perturbations in the gut microbiome associated with the progressive pancreatic oncogenesis, we evaluated the composition of the gut microbial community of KC mice compared with age-matched WT littermate controls by performing 16S gene sequencing. Lastly to determine the mechanism of immunosuppression; we injected F21242 pancreatic tumor cell line orthotopically into the pancreas of WT mice after bacterial ablation and vehicle, isolated the pancreatic leukocytes and analyzed them by flow cytometry. 

Results:
In the pre-morbid state the intestinal microbiome is similar in mice bearing pancreas-specific oncogenic mutations and in controls; however, as mice age, PDA-bearing hosts develop a unique gut microbiome including expansion of Actinobacteria and Deferribacteres. We found that gut bacteria access the pancreas and the cancerous pancreas harbors a distinct microbiome in mice. Further, genotypically identical PDA-bearing mice that exhibit divergent disease phenotypes harbor stage-specific microbiomes suggesting that microbial structure is associated with disease aggressiveness. Germ-free or ablative antibiotic treated mice were protected against PDA whereas transfer of gut bacteria from PDA-bearing mice, but not from control mice, reversed the tumor-protection. Bacterial ablation was associated with innate and adaptive immunogenic reprogramming of the PDA tumor microenvironment including a marked reduction in myeloid-derived suppressor cells and immune-suppressive macrophages, increased Th1 differentiation of CD4+ T cells, and expansion and activation of cytotoxic CD8+ T cells. In addition, we show that gut bacterial ablation and PD-1 blockade offer synergistic efficacy.

Conclusion:
These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has marked potential as a biomarker and therapeutic target in PDA.
 

21.01 Not all Readmissions are Created Equal – Index vs. Non-Index Readmissions After Major Cancer Surgery

Posted on January 25, 2017

S. Zafar1, A. A. Shah1, H. Channa2, L. L. Wilson4, N. Wasif3  1Howard University College Of Medicine,General Surgery,Washington, DC, USA 2Purdue University,Agricultural Economics,West Lafayette, IN, USA 3Mayo Clinic In Arizona,Surgical Oncology,Phoenix, AZ, USA 4Howard University College Of Medicine,Surgical Oncology,Washington, DC, USA

Introduction:
Hospital readmissions after major cancer surgery pose a major healthcare burden and are associated with increased costs and worse outcomes. Increasing regionalization of cancer surgery has the inadvertent potential to lead to fragmentation of care if readmissions occur at a different hospital from the index facility.  Using a national dataset we aim to quantify rates of readmission to non-index hospitals after major cancer surgery and to compare outcomes between index and non-index hospital readmissions.

Methods:
We used the National Readmissions Dataset (2013) as our data source. All adult patients undergoing a major cancer operation (defined as esophagectomies/gastrectomies, hepatico-biliary resections, pancreatectomies, colorectal resections, and cystectomies) within the first 9 months of the year were selected. Readmission characteristics including timing, cost, morbidity and mortality were analyzed. Discharge weights were used to calculate national estimates. Adjusting for clustering by facility, we used multivariate logistic regression to identify factors associated with non-index vs. index readmissions and also to identify differences in mortality, major complications and subsequent readmissions.  Generalized linear modeling was used to test for differences in length of stay (LOS) and hospital costs between the two groups.

Results:
A total of 57,362 patients with 86,362 hospital admissions were analyzed. Overall, the 90 day readmission rate was 23.1% and median time to readmission was 42 days (IQR 20-70 days). Weighted analysis revealed the total national cost for 90 day readmissions to be $682 million. Of the 17,020 readmissions, 22.0% were to a non-index hospital. Independent factors associated with 90 day readmission to a non-index hospital included younger age,  male gender, type of procedure, more comorbidities, Medicaid insurance, longer LOS, in-hospital complications, discharge to a nursing facility, and index surgery at a teaching hospital (p<0.05).  Following risk adjustment, patients readmitted to non-index hospitals had 32% higher odds of mortality (OR 1.32, 95% CI: 1.03-1.70) and 26% higher odds of having a major complication (OR 1.26, 95% CI: 1.10-1.43). Subsequent readmissions and hospital costs were not significantly different between the two groups.

Conclusion:
The 90 day readmission rate following major cancer surgery in the United States is 23.1%, of which a further 22% are to a non-index hospital. When compared to patients readmitted to the index hospital, readmission to a non-index hospital is associated with higher mortality and morbidity. Targeted interventions to reduce non-index readmissions may mitigate fragmentation of postoperative care and result in improved outcomes.
 

« Previous Page
Next Page »