21.06 Increased Malignancy Rates in Surgical Patients with Incidentally-Discovered Thyroid Nodules

Posted on December 22, 2014

A. R. Marcadis1, M. Rodriguez1, S. Liu1, B. Wang1, J. I. Lew1  1University Of Miami Miller School Of Medicine,Division Of Endocrine Surgery,Miami, FL, USA

Introduction: With the advent of better imaging technology and its widespread utilization in the clinic setting, incidental thyroid nodules are often discovered while evaluating patients for unrelated disease. If the risk of underlying thyroid malignancy in such incidental nodules is common, further evaluation is required. However, if thyroid cancers are exceedingly rare in such incidentally discovered thyroid nodules, further costly evaluations can be avoided. The purpose of this study is to compare the rate of malignancy in incidentally-discovered thyroid nodules (IDTN) by imaging to nonincidentally-discovered thyroid nodules (NDTN) in surgical patients.

Methods: A retrospective review of prospectively collected data of 1370 patients who underwent thyroidectomy at a single institution was performed. Before surgical resection, all patients underwent surgeon-performed ultrasound (SUS) and fine-needle aspiration (FNA). Patients who had IDTN by imaging studies for unrelated reasons (n=536) and patients who had NDTN (n=834) were further stratified according to age, gender, FNA results, SUS characteristics and final pathology. Rates of malignancy in IDTN and NDTN were calculated and statistical significance was determined by  two-tailed Z-test.

Results: Of 1370 patients, 536 presented with IDTN by imaging and 306 (57%) were found to have malignancy on final pathology. Of patients with IDTN found to have malignancy, 92% (n=283) had papillary thyroid cancer, 5% (n=14) medullary thyroid cancer, and 3% (n=9) follicular thyroid cancer. Of the patients with NDTN, 401 (48%) were found to have malignancy following surgery with 44% (n=366) papillary thyroid cancer (PTC), 2.2% (n=18) medullary thyroid cancer, 2% (n=14) follicular thyroid cancer, and <1% anaplastic thyroid cancer (n=3). The rate of PTC on final pathology for IDTN was statistically higher than the rate of PTC found in NDTN (p<0.05). Patients with IDTN had statistically higher rates of microcalcifications (34%), hypoechogenicity (60%), and irregular borders (35%) by SUS compared to patients with NDTN (p<0.05). There was also a significantly higher rate of patients with IDTN who had all three SUS features together (14%) compared to patients with NDTN (10%) (p<0.05). There was no significant difference in percentage of malignant FNA results between patients with IDTN and NDTN.

Conclusion: For IDTN, malignancy rates may be higher than expected in surgical patients. Furthermore, certain characteristics of SUS may help predict malignancy in IDTN. The high rate of malignancy suggests that total thyroidectomy by an experienced surgeon should be strongly considered when managing such patients with IDTN.

 

22.04 Deficiency of the Immunostimulatory Cytokine IL-21 Promotes Intestinal Neoplasia

Posted on December 22, 2014

M. M. Shapiro1,2, B. Nandi1,2, G. Gonzalez1, R. Prabhala1,2,3, Q. Huang1,4, N. C. Munshi1,2,3, N. Y. Frank1,2,4, J. S. Gold1,2,3  4Brigham And Women’s Hospital,Boston, MA, USA 1VA Boston Healthcare System,West Roxbury, MA, USA 2Harvard School Of Medicine,Brookline, MA, USA 3Dana Farber Cancer Insititute,Boston, MA, USA

Introduction: Interest in the use of the cytokine IL-21 as an immunotherapeutic agent grew out of the initial success with interferon gamma and IL-2 in the treatment of melanoma. IL-21 functions by increasing proliferation and survival of B and T cells. It also increases granzyme B-mediated cytotoxicity of NK and T cells. Due to these properties, IL-21 has been investigated in the treatment of both renal cell carcinoma and melanoma. The role of IL-21 in spontaneous intestinal carcinogenesis has not been fully explored.

Methods: Mice with a targeted knockout of IL-21 (KO) were bred with APCMIN/+ (MIN) mice. MIN mice spontaneously develop numerous intestinal adenomas. Wild-type C57/Bl6 mice (WT) were used as a control. Mouse small intestines were harvested at 15 weeks. Polyps were measured and counted under a dissecting microscope. Mouse ileum was also either preserved for paraffin embedding and immunohistochemical staining or snap frozen for cDNA preparation and q-RT-PCR.

Results: Polyp-bearing ileum from MIN mice had a five-fold increase in IL-21 expression by q-RT-PCR as compared to the non-polyp bearing ileum of WT mice (p=0.03). MIN mice lacking IL-21 had increased intestinal polyp number and tumor load as compared to MIN mice with functional IL-21 (55 vs. 40 polyps, p=0.007; Figure panel A; tumor load 88 vs. 68, p=0.02). The differences in polyp number and tumor load were significant in the jejunum and ileum but not in the duodenum (duodenum 5.4 vs. 4.1 polyps, p=0.3 and tumor load 14 vs. 7.8, p=0.5; jejunum 21 vs. 14, p=0.02 and 31 vs. 22, p=0.04; ileum 29 vs. 22, p=0.01 and 44 vs. 36, p=0.03). KO-MIN mice had fewer CD3+ cells (T cells) and B220+ cells (B cells) in the polyp-bearing ileum than MIN mice (50 vs. 78 cells/0.07mm2, p<0.001 for T cells; 13 vs. 18, p<0.001 for B cells). Similarly, the number of granzyme B+ cells was much lower in polyp-bearing ileum of KO-MIN mice when compared to that of MIN mice (20 vs. 41 cells/0.07 mm2, p<0.001; Figure panel B).

Conclusion: Adenoma development is associated with upregulation of IL-21 in the intestine in a mouse model of spontaneous intestinal neoplasia. Deficiency of IL-21 leads to accelerated tumor development in this model. Loss of IL-21 is also associated with a decrease in B and T cell populations in the polyp-prone intestine as well as a concomitant decrease in granzyme B-releasing cells. These data support the hypothesis that IL-21 is involved in mediating spontaneous anti-tumor immunity controlling adenoma development. The use of IL-21 for the treatment of colorectal cancer warrants further investigation.

22.06 Histone Deacetylase Inhibitors Induce a Pro-inflammatory Phenotype in Pancreatic Cancer Fibroblasts

Posted on December 22, 2014

A. H. Nguyen1, S. Patel1, M. Vogelauer2, P. A. Toste1, N. Wu1, J. Williams3, L. Li1, D. W. Dawson4, S. Kurdistani2, T. R. Donahue1  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Department Of Biological Chemistry,Los Angeles, CA, USA 3Harbor-UCLA Medical Center,Department Of Surgery,Torrance, CA, USA 4University Of California – Los Angeles,Department Of Pathology And Laboratory Medicine,Los Angeles, CA, USA

Introduction:
Histone deacetylase inhibitors (HDACi) are currently being investigated in early phase clinical trials for patients with pancreatic ductal adenocarcinoma (PDAC). Although there have been measurable responses in patients with hematologic malignancies treated with HDACi, similar results have not been demonstrated for solid organ tumors. We hypothesize that tumor associated fibroblasts (TAFs), the predominant cell type in the PDAC stoma, may contribute HDACi therapy resistance.

Methods:
Primary TAFs were isolated from human PDAC tumor samples. MTT assay was used to determine cell viability. Western blot was performed of whole cell lysate and acid extracted histone-enriched samples. Gene expression was determined by qRT-PCR. A modified Boyden chamber was utilized to assess tumor cell (TC) invasion. ChIP-seq was performed for HDAC2 and was overlaid with publically available H3K4me1 and H3K27ac ChIP data from the NIH Roadmap Epigonics Mapping Consortium website.

Results:
As has been described, PDAC TC (PANC-1, MIA PaCa-2) viability was significantly decreased (p<0.01) by all tested class I and II HDACi's (vorinostat, entinostat, panobinostat). However, slowly dividing primary TAFs showed neither growth arrest nor an upregulation of CDKN1A, despite an appropriate increase in global levels of acetylated histones H3 and H4. HDACi treatment of TAFs induced a tumor supportive secretory milieu, as conditioned media from treated cells increased TC invasion (p<0.001) and viability (p=0.05). HDACi treatment on primary TAFs increased secretion of a series of pro-inflammatory proteins (p=0.05) including CXCL1, IL-6, IL-8, and SPP1, by upregulating NFκB target genes (p<0.01). By western blot, we identified markers suggesting the cytosolic activation of NFκB, STAT3, and p38 MAPK pathways, yet, inhibition of STAT3 and p38 MAPK failed to completely abrogate the HDACi-induced inflammatory response and NFκB inhibition proved to be lethal to HDACi-treated fibroblasts. To elucidate how HDAC inhibition may directly affect gene regulation, pro-inflammatory gene enhancers were identified from published H3K4me1 ChIP-seq on normal fibroblasts. We overlaid our HDAC2 ChIP-seq from naïve primary PDAC TAFs to these poised enhancer regions. Our ChIP-seq shows HDAC2 binds these enhancer regions, and importantly, in cells that normally express the active enhancer mark H3K27ac. The promoter regions for these genes both show motif and ChIP-seq data supporting expression from STAT3 and NFκB mediated factors, suggesting HDAC inhibition provides a permissive chromatin landscape for the expression of inflammatory genes in primary PDAC TAFs.

Conclusion:
HDACi's are effective against PDAC TCs in culture, but induce a tumor supportive phenotype in primary PDAC TAFs, which may explain their disappointing results in solid organ tumors. These studies are beginning to uncover the mechanism of this detrimental response in PDAC TAFs

22.07 Primary Gastrointestinal Tract Melanoma: Epidemiology And Outcomes For 1,044 Patients

Posted on December 22, 2014

V. Chakravorty1,2, K. Mahendraraj1, R. S. Chamberlain1,2,3  1Saint Barnabas Medical Center,Department Of Surgery,Livingston, NJ, USA 2St. George’s University School Of Medicine,St. George’s, St. George’s, Grenada 3New Jersey Medical School,Department Of Surgery,Newark, NJ, USA

Introduction: Primary melanoma of the gastrointestinal tract (GIM) are exceedingly rare entities, with an overall incidence of ~0.47 cases per million. While outcomes for cutaneous melanomas have been extensively examined, clinical information related to GIM is scant and derived from small case series. This study examined a large cohort of GIM patients to determine demographic, clinical, and pathologic factors affecting outcomes.

Methods: Demographic and clinical data on 1,044 patients with GIM was abstracted from the SEER database (1973-2010) and analyzed using the Chi square test, t-test, and multivariate analysis. Kaplan-Meier analysis was used to compare long-term survival between groups.

Results: 1,044 cases of GIM were identified, involving the oropharynx in 307 patients (29.4%), esophagus in 48 (4.6%), stomach in 26 (2.5%), small bowel in 38 (3.6%), large bowel in 19 (1.8%), rectum in 250 (23.9%), and anal canal in 356 (34.1%). Overall, there were 569 women (54.5%) and 475 men (45.5%; female-to-male ratio 1.2:1, p<0.001) with an overall mean age of 68+15 years. GIM was most common in Caucasians (73.7%, p<0.001) often presenting with localized (40.1%, p<0.001), poorly differentiated (68.1%, p=0.01) tumors measuring 2-4cm (34.5%, p<0.001), with uncommon lymphatic spread (37.9%, p<0.001). Mean survival for GIM was 3.8+0.3 years with longest survival seen among oropharyngeal GIMs (5.4+0.6 years, p<0.005) and shortest in gastric GIMs (0.7+0.2 years, p<0.005). Overall and cancer-specific mortality for GIM were 80.3% and 72.6%, respectively, (p=0.01). Surgical resection was the most common GIM therapy (64.8%, p<0.001), followed by surgery and radiotherapy (15.8%, p<0.001).  Survival was greatest with combination surgical and radiotherapy was used (4.4+0.8 years, p<0.001) compared to surgical resection only (4+0.3 years, p<0.001). Multivariate analysis identified regional (OR 1.9, CI 1.1-3.1) or distant disease (OR 4.5, CI 2.3-8.8), lymph node positivity (OR 4.5, CI 1.7-9.8), and age >60 (OR 6.2, CI 2.4-15) as independently associated with increased mortality for GIM (p<0.005).  Specific site of disease was not associated with increased mortality.

Conclusion: GIM is a rare malignancy that occurs in all parts of the GI tract with the highest incidence in the oropharyngeal and anorectal regions. GIMs are more common in Caucasian women in the seventh decade of life.  The majority of GIMs are localized, often poorly differentiated, with gastric GIM having the worst prognosis. Advanced age, stage and lymph node positivity are associated with increased mortality. Surgical resection with or without radiotherapy confers a significant survival advantage, and is standard therapy for all GIM patients with resectable disease

2.02 Hypoxia Inducible Factor-1α Enhances Hepatic Metastasis in Murine Pancreatic Adenocarcinoma

Posted on December 22, 2014

J. A. Yi1, E. E. Moore1,2, A. Banerjee1, K. El-Kasmi1, C. C. Barnett1,2  1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA

Introduction:  Pathological studies have demonstrated that hypoxia inducible factor-1α (HIF1α) is a marker of advanced malignancy in pancreatic cancer patients.  HIF1α is a transcription factor that regulates multiple cellular processes including oxygen transport, angiogenesis, metabolism, cell adhesion, and inflammation. We hypothesize that HIF1α activity is important for the development of hepatic metastases in pancreatic adenocarcinoma.

 

Methods:  The Pan02 murine pancreas adenocarcinoma cell line was modified using short-hairpin RNA targeting HIF1α via lentiviral transduction to create Pan02-SH+ cells lacking HIF1α activity. Knockdown of HIF1α activity by >80% was confirmed using polymerase chain reaction prior to injection of the tumor cells.  C57/Bl6 mice underwent splenic injection of 200,000 Pan02 or Pan02-SH+ cells followed by hemisplenectomy to create intraabdominal metastatic tumor dissemination. After 4 weeks, mice were euthanized for necropsy performed by blinded observers.  Tumor volume was calculated as (height x width2).  Statistical analysis was performed using Student’s t-test and Fisher’s exact test, with significance determined to be α<0.05.

 

Results:  Abrogating HIF1α activity of the tumor cells resulted in a decreased number of hepatic metastases (p=0.0397) and decreased hepatic tumor volume (Pan02 mean volume=1275 mm3 ± 236.08, Pan02-SH+ mean volume=16.4 mm3 ± 13.98; p=0.0007). There was no significant difference in non-hepatic intraabdominal metastases by count (p=0.1347) or by tumor volume (Pan02 mean volume=1193.8 mm3 ± 264.97, Pan02-SH+ mean volume=358.6 mm3 ± 278.55; p=0.0616) with HIF1α shRNA knockdown. 

 

Conclusion:  HIF1α activity is important for the development of hepatic metastases in pancreatic adenocarcinoma.  This was reflected by both reduced number of malignant lesions and tumor volume in the liver.  Non-hepatic metastases were not significantly decreased by reduction of HIF1α activity.  HIF1α may represent an important target in hepatic directed therapy for pancreatic adenocarcinoma.

2.03 A Novel Orthotopic Mouse Model of Pancreatic Cancer with Immunocompetent Milieu and Robust Stroma

Posted on December 22, 2014

K. Majumder1, S. Modi1, N. Arora1, R. Chugh1, A. Nomura1, S. Banerjee1, R. Dawra1, A. Saluja1, V. Dudeja1  1University Of Minnesota,Surgery,Minneapolis, MN, USA

Introduction:  The development of novel therapeutics for pancreatic cancer has been hindered by a lack of relevant preclinical models. The introduction of the KPC (KrasLSL.G12D/+; p53R172H/+; PdxCretg/+) mouse model has shed some light on tumorigeneis and progression in pancreatic cancer and has been a step in the right direction. An accurate tumor model, besides recapitulating the tumorigenic properties, has to recapitulate the immune and stromal microenvironment, the components lacking in commonly used subcutaneous or orthotopic models in immunodeficient mice (SCID, Athymic nude). While these components are present in the KPC model, this genetic model is fraught with inconsistency making this unsuitable for study of various component of tumorigenesis as well as the study of novel therapies. For example, the time to invasive disease in this model varies from 47-355 days of life. The amount of pre-invasive disease and invasive adenocarcinoma varies between litter mates and up to 20% of animals never develop tumor. Furthermore, the entire pancreas in this model has Kras-p53 mutation as opposed to the sporadic mutations generally seen in human pancreatic cancer. We therefore propose a novel orthotopic tumor model with tumors from KPC mice implanted in C57Black6 mice, which recapitulates the tumor microenvironment and circumvents the drawbacks of the above mentioned models.

Methods:  6 month KPC mice with palpable pancreatic tumors were sacrificed and the tumors were cut into ~3mm3 pieces using core biopsy punch. Laparotomy was performed using a midline incision on female C57BL/6 mice and the tumor piece was sewn into a pocket of pancreas using a figure of 8 stitch of 7-0 prolene incorporating the superior and inferior border of the pancreas. Mice were sacrificed at two time points, 4-weeks and 8 weeks after implantation. Tumor volume and weight was measured. Stromal component and immune infiltration were studied by immunohistochemistry.

Results: Tumor take rate was ~90%. Tumor volume was very similar in all mice suggesting similar growth rate (tumor volume. 4 weeks: 331 ± 122 mm3, 8 weeks: 433 ± 77 mm3). Mortality at 8 weeks was ~20%. Immunohistochemistry confirmed presence of pancreatic adenocarcinoma at both time points. Staining for stromal components (collagen and αSMA) showed intense desmoplastic stromal reaction at both 4 and 8 weeks after implantation. CD45 staining demonstrated intense infiltration of tumor and surrounding pancreas with leukocytes.

Conclusion: The proposed model of pancreatic cancer offers all the advantage of state of the art genetic model along with the predictability of a conventional orthotopic model. This model mimics the stromal reaction and immune infiltration observed in human pancreatic ductal adenocarcinoma as well as circumvents the issue of variability seen in previous mouse models. This clinically relevant model can be a valuable tool to evaluate novel therapeutics in pancreatic cancer.

 

2.04 The Tumor Supportive Phenotype of Chemotherapy Conditioned PDAC Fibroblasts Depends on NfkB

Posted on December 22, 2014

S. G. Patel1, L. Li1, A. Nguyen1, P. Toste1, N. Wu1, C. Choi1, J. Smogorzewski1, T. Donahue1  1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA

Introduction:
We have previously shown that exposure of pancreatic cancer (PDAC) tumor associated fibroblasts (TAFs) to cytotoxic chemotherapy unleashes a tumor supportive and pro-inflammatory phenotype, increasing tumor cell (TC) growth and invasion in vitro and in vivo. We hypothesize that Nfkb is the predominant transcription factor driving this TAF response to DNA damage, and therefore genetic knockout of the activator subunit, p65 in TAFs will attenuate the induction of these genes and tumor cell viability.

Methods:
Immortalized PDAC TAFs (Logsden Lab) were homozygously deleted for p65 using CRISPR:Cas9.  RT-PCR and MTT assay were done using standard protocols.  Conditioned media was collected for 24 hours from the TAFs preconditioned with 48 hours of 100 nM gemcitabine or basal media. PANC-1 TCs were cultured in this media for two days and MTT assay used to assess cell viability and proliferation.  CHIP-Seq data and promoter motif enrichment was derived from NIH Epigenome roadmap public data and processed using GREAT. 

Results:
Gemcitabine treatment of PDAC TAFs induced a pro-inflammatory gene expression program including statistically significant (p < 0.05) upregulation of a number of cytokines: IL-6 (9.2 fold), IL-8 (17.8 fold), IL-1a (62.1 fold), and Spp1 (2.7 fold) [Fig 1A].  The genes are part of a DNA damage response collectively known as the senescence associated secretory phenotype (SASP).  Each of these genes have Nfkb binding sites identified from Chip-Seq for p65 or from binding site motif identification.  The expression of these gemcitabine-induced SASP genes followed the cytosolic to nuclear translocation of the activating subunit of the Nfkb complex: p65 [Fig 1B].  Homozygous knockout of p65 in TAFs (TAF p65 -/-) resulted in loss of upregulation of multiple SASP mediators in gemcitabine treated cells as compared to wild-type TAFs with intact p65 (p<0.05).  Additionally, two genes in our panel expressed at low levels under basal conditions in WT TAFs (IL8, IL-6) were silenced in the p65 KO TAFs.  Conditioned media collected from naïve and 48 hour gemcitabine treated WT TAFs or non-target control (RelA NC) increased the proliferation of PDAC TCs (Panc-1, p<0.05); however, conditioned media from gemcitabine treated p65 KO (-/-) TAFs did not permit cancer cell growth in low serum conditions [Fig 1C].

Conclusion:
Gemcitabine treatment of PDAC TCs shows a cytotoxic effect in vitro, but is complicated by the emergence of resistance in vivo.  TAFs influence on TCs is typically not appreciated in the in vitro setting.  We show that culture media conditioned by TAFs is supportive of TC growth, in vitro, and inflammatory cytokines expressed subsequent to induction of DNA damage in TAFs are silenced in the absence of p65.   We conclude that in the presence of gemcitabine, Nfkb signaling in stromal cells can promote cancer cell growth.  
 

2.05 E-Cadherin Expression in Obesity-Associated, Kras-Initiated Pancreatic Ductal Adenocarcinoma

Posted on December 22, 2014

A. P. Stark1, W. Sheppard1, X. Jung1, K. Hertzer1, A. Moro1, H. Chang1, M. Xu1, O. J. Hines1, G. Eibl1  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA

Introduction: Epithelial-mesenchymal transition (EMT) is increasingly considered to be a necessary first step in the development of invasive disease. Accordingly, loss of the epithelial marker E-cadherin is associated with decreased survival in human pancreatic ductal adenocarcinoma (PDAC). Alarmingly, EMT has been demonstrated prior to tumor formation in a mouse model of PDAC—and is accelerated by inflammation. Obesity is a known pro-inflammatory state and an established risk factor for PDAC. We have previously demonstrated accelerated pancreatic tumorigenesis as a result of a high fat, high calorie diet; however, the direct effect of diet-induced obesity on EMT and E-cadherin expression is unknown.

Methods: Conditional KrasG12D (KC) mice were fed a control diet (CD; 3726 kcal/kg, 12% fat) or a high fat, high calorie diet (HFCD; 4536 kcal/kg, 40% fat). Mice were weighed weekly. To demonstrate the spectrum of pancreatic epithelial neoplasia (PanIN 1-3), cohorts were sacrificed at 3 and 9 months of age. Pancreata were preserved in formalin and embedded in paraffin. Immunohistochemistry (IHC) was performed using antibody against E-cadherin. Immunoreactivity was characterized by staining intensity, location (cell membrane, cytoplasm, nucleus), and proportion of positive cells. 

Results: For the 3-month cohort, implementation of the diet led to significantly increased weight gain among HFCD mice (n=7) vs. CD mice (n=7), 15.8gm vs. 5.6gm (p<.001). In the 9-month cohort, HFCD mice (n=7) also had higher average weight gain compared to CD mice (n=5), 19.8gm vs. 12.9gm (p = .007). Compared to CD-fed animals, mice fed the HFCD for 3 and 9 months showed more robust inflammatory features in the pancreas as demonstrated by a loss of acinar tissue and formation of desmoplasia. Mice fed the HFCD for 9 months had more severe pathology than mice fed the HFCD for 3 months. No invasive cancer was detected. Overall, E-cadherin expression was ubiquitous, strong, and membranous regardless of diet in both 3- and 9- month cohorts. Staining intensity was intermittently decreased in higher-grade PanIN lesions, but no differences between groups were noticed (See Figure 1).

Conclusions: Despite evidence of more advanced disease, KC mice fed a HFCD did not show evidence of grossly altered E-cadherin expression. If early EMT is part of the mechanism underlying diet-induced acceleration of pancreatic tumorigenesis, it may occur despite E-cadherin expression.

2.06 Increased Stromal Integrin-Linked Kinase Expression in IPMN is Associated with Worse Survival

Posted on December 22, 2014

L. A. Shirley1, B. Swanson2, W. Frankel2, T. Bekaii-Saab3, M. Bloomston1, C. Chen4  1Ohio State University Wexner Medical Center,Surgical Oncology,Columbus, OH, USA 2Ohio State University Wexner Medical Center,Pathology,Columbus, OH, USA 3Ohio State University Wexner Medical Center,Medical Oncology,Columbus, OH, USA 4Ohio State University College Of Pharmacy,Medicinal Chemistry,Columbus, OH, USA

Introduction: Intraductal papillary mucinous neoplasms (IPMNs) are pre-malignant lesions of the pancreas that are typically found incidentally.  The presence of an IPMN presents a clinical dilemma in that it is difficult to predict which patients will go on to succumb to pancreatic cancer.  Integrin-linked kinase (ILK) is a serine-threonine kinase known to be overexpressed in pancreatic cancer, correlating with worse survival.  We examined resected pancreata with IPMN to see if similar correlations exist.

Methods: Intraductal papillary mucinous neoplasms (IPMNs) are pre-malignant lesions of the pancreas that are typically found incidentally.  The presence of an IPMN presents a clinical dilemma in that it is difficult to predict which patients will go on to succumb to pancreatic cancer.  Integrin-linked kinase (ILK) is a serine-threonine kinase known to be overexpressed in pancreatic cancer, correlating with worse survival.  We examined resected pancreata with IPMN to see if similar correlations exist.

Results: In our cohort, 10 samples (35.7%) had an invasive component.  Mean IPMN ILK score was 0.56, while mean stromal ILK score was 1.96.  If an invasive component was present, the mean ILK score in this region was 0.6.  There was no correlation between IPMN (P=0.46) or invasive component (P=0.55) ILK scores and patient survival.   However, high ILK expression (score of 3) in the stroma surrounding the IPMN was associated with a significantly worse overall survival (median survival not reached vs. 33.5 months, 5-year survival 69.8% vs. 22.2%, P=0.016) (Figure 1).  This correlation remained significant when stratified by the presence of an invasive component (non-invasive median survival not reached vs. 39.6 months, 5-year survival 75.2% vs. 50%, invasive median survival 62.6 vs. 19.9 months, 5-year survival 60% vs. 0%, P=0.029).

Conclusion: Stromal ILK over-expression in IPMNs was associated with worse survival, independent of the presence of an invasive component.  Stromal ILK expression could be used in the future as a marker to stratify patients with IPMNs that need more aggressive therapy.  Additionally, targeting ILK overexpression may provide a novel therapeutic strategy. 

 

2.07 Poor PDA Patient Prognosis Predicts Xenograft Tumor Engraftment Likelihood

Posted on December 22, 2014

H. L. Beal1, J. Shea1, M. Firpo1, S. Mulvihill1,2, C. Scaife1,2  1University Of Utah,Department Of Surgery Research,Salt Lake City, UTAH, USA 2Huntsman Cancer Institute,Salt Lake City, UTAH, USA

Introduction:

Pancreatic adenocarcinoma (PDA) is an exceptionally lethal disease. The development of new efficacious therapies could be improved through the development of animal models that more faithfully replicate disease biology.  One such approach is to utilize patient-derived primary mouse xenograft tumors.  Here we describe the results of our xenograft tumor banking program from 2011-2013 and compare the xenograft phenotype to the patients, as well as patient prognosis, and compare characteristics of the xenograft tumor with the patient tumor. We tested the hypothesis that a more aggressive tumor phenotype will grow more frequently in mice.

Methods:

With IRB and IACUC approval, tumor samples from suspected PDA cases were obtained within an hour of resection, placed in media, cut into approximately 1mm3 pieces, mixed with Matrigel, and implanted subcutaneously into SCID hairless female mice. Upon reaching 2cm in diameter, the tumor was processed for histology and re-implanted in a new mouse.  Tumors for histology were fixed in formalin, embedded in paraffin and then stained with hematoxylin and eosin or immunohistochemically stained with epithelial markers (E-cadherin and  β-catenin).  Each patient’s chart was reviewed for patient demographics, diagnosis, recurrence, and overall and disease-free survival.

Results:
In the three year study period, 54 patients with periampullary adenocarcinoma were enrolled.  Twenty-two patients were female (age 68 +/- 7 years) and 32 were male (age 63 +/- 9 years).  We implanted 47 PDA tumors, 4 cholangiocarcinoma tumors, and 3 ampullary tumors. The overall xenograft take rate was 44%.  Take rates for PDA (13/39; 9 under observation), cholangiocarcinoma (4/4), and ampullary (3/3) tumors were 33%, 100%, and 100%, respectively. Once established, 84% of tumors successfully grew in a second mouse.

Histologically, mouse PDA xenograft tumors were remarkably similar to the original patient tumor with all samples demonstrating extensive desmoplastic tissue surrounding tumor cells.  The histological characteristics of the tumor appear to be stable over several passages. The xenograft tumors cells were positive for both E-cadherin and β -catenin. Mean survival in patients with succefully engrafted PDA tumors was 10 +/- 6 months, while patients whose tumor did not engraft had mean survival of 21 +/- 10 months (p=0.005).

Conclusion:

We have successfully established 13 patient derived PDA, 3 ampullary and 4 cholangiocarcimonas. The PDA xenograft tumors maintain histological similarities with the patient tumor and preliminary survival analysis suggests that more aggressive tumors are more likely to establish in the mouse model. The tumors established in our xenograft banking program, are an important resource that can be utilized to evaluate new therapeutics treatments. 

 

2.08 Generation of Patient-Derived Xenografts from Small Volume Biopsy Samples

Posted on December 22, 2014

D. Roife1,2, Y. Kang1, R. Zhang1, L. Wang3, B. Fang3, M. Katz1, J. Gershenwald1, C. Dinney4, J. Fleming1  1University Of Texas MD Anderson Cancer Center,Surgical Oncology,Houston, TX, USA 2University Of Texas Health Science Center At Houston,General Surgery,Houston, TX, USA 3University Of Texas MD Anderson Cancer Center,Thoracic And Cardiovascular Surgery,Houston, TX, USA 4University Of Texas MD Anderson Cancer Center,Urology,Houston, TX, USA 5University Of Texas MD Anderson Cancer Center,Melanoma,Houston, TX, USA

Introduction:  Patient-derived xenografts have become a powerful tool for cancer research in recent decades. One of the most significant barriers to growing xenografts from patient tumors is the need for surgical resection of tumor tissue. Thus far, patient derived xenografts have been grown from tumor tissue obtained after surgical resection of the primary tumor, and occasionally from metastatic tumors, if surgery was medically warranted. However, many cancer patients never undergo surgery for a variety of reasons, and therefore they are never given the opportunity of having their tumor tissue grown for research purposes via xenografts. We hypothesized that xenograft tumors could be grown from smaller volumes of patient tissue, such as those obtained during diagnostic biopsies. 

Methods:  Surgical specimens were obtained after resection of primary or metastatic lesions of the following cancers: pancreatic adenocarcinoma, lung adenocarcinoma, bladder (transitional cell) carcinoma, and melanoma. At least 10 cases of each cancer were included in this study. These cases did not exclude any patient in regards to neoadjuvant treatment or clinical characteristics. To mimic clinical biopsies, small fragments of the surgical specimens were biopsied with a 22g needle and the needle contents were injected subcutaneously in immunocompromised mice. The tumor fragment from which the biopsy was taken was also implanted subcutaneously in the contralateral side of the same mouse as a control.

Results: Success rates of the traditional method of xenograft implantation ranged from 30-70%, similar to what is reported in the literature. Success rates of the experimental biopsy technique ranged from 10%-40%. Since the experimental biopsy technique demonstrated successful results, the study culminated with the first patient derived xenograft grown from a percutaneous, outpatient biopsy. A patient with metastatic pancreatic adenocarcinoma underwent percutaneous ultrasound-guided core needle biopsy of a liver lesion. We divided this core biopsy and implanted the fragments into three immunocompromised mice, all of which grew into large tumors in under two months. 

Conclusion: The lower success rate of the experimental biopsy technique was to be expected due to the smaller volume of tumor tissue implanted. However, it should be noted that these biopsy derived tumors reached comparable sizes as traditionally implanted tumor fragments in the same time frame, despite implanting significantly fewer cells. Also, metastatic tumor sites had much higher successful engraftment rates than samples taken from the primary tumor site. In conclusion, we have shown that it is possible to successfully grow patient-derived xenografts with small volumes of tumor samples that are obtainable during diagnostic biopsies, which potentially can open the xenograft technique to a wider scope of cancer patients.

 

2.10 Novel actionable genomic analysis of individual PDAC specimens in real time

Posted on December 22, 2014

J. Yu1, G. Zhou1, S. Liu1, J. Wu1, R. Sanchez1, D. Dawson1, W. Fisher2, F. C. Brunicardi1  1University Of California – Los Angeles,Surgery Department,Los Angeles, CA, USA 2Baylor College Of Medicine,Houston, TX, USA

Introduction:  Recent molecular characterizations of pancreatic ductal adenocarcinoma (PDAC) have identified hundreds of genetic alterations and aberrant gene expression in key biological processes and signaling pathways. However, PDAC global database analyses are insufficient in identifying relevant PDAC biomarkers for individual patients. Our objective was to develop a list of PDAC signature genes that could serve as potential biomarkers for individuals with PDAC using a novel combination of real time genomic analysis and published PDAC databases.  

Methods:  PDAC and normal matching pancreas specimens (n=8) were procured and processed for genomic sequencing. RNA sequencing libraries were generated using Illumina TruSeq RNA v2.0 and sequenced on an Illumina HiSeq 2500 platform. Sequencing reads were aligned to the human genome hg19 using TopHat and Bowtie. Three microarray gene expression datasets from the GEO database were collected and processed with R and Bioconductor packages. PDAC signature genes were identified by comparing whole genome transcriptome profiles from gene expression microarrays on independent cohorts and microarray platforms. Weighted Gene Co-expression Network Analysis (WGCNA) was then conducted to identify hub genes that highly correlate with PDAC. Hub genes were further validated with RNAseq and QPCR in PDAC and normal tissue control specimens.

Results: 1007 differentially expressed genes were identified with a cutoff (log2 fold-change > 0.5, FDR < 0.01) by comparing whole transcriptome of PDAC with normal matching tissues from three independent cohorts of two microarray platforms. GO-term enrichment analysis revealed that these 1007 genes were significantly enriched in ECM-receptor interaction, focal adhesion, complement and coagulation cascades, and glycolysis and gluconeogenesis pathways. With higher stringency (log2 fold-change > 2 and FDR < 0.01), 83 PDAC signature genes that distinguish PDAC from the normal pancreas were identified. To identify modules of highly co-regulated genes, co-expression gene networks using the transcriptome data of the entire 1007 gene list were constructed, revealing five modules and 12 hub genes with high connectivity and significant correlation with PDAC. Three hub genes, MBOAT2, LAMC2, and TSPAN1, were validated using QPCR of individual PDAC and normal pancreas specimens.

Conclusion: This novel actionable genomic analysis utilizes a comparison of RNAseq in real time to microarray on a large number of PDAC samples and WGCNA using a multifactorial genomic analysis and reveals a list of 83 PDAC signature genes, including MBOAT2, LAMC2 and TSPAN1. The data suggest that this actionable genomic analysis platform will help increase the understanding of signaling pathways and the identification of potential therapeutic targets in real-time for each patient with PDAC.

 

2.11 Hypoxia Inducible Factor-1α Increases Pro-Tumorigenic Macrophage Activation in Pancreatic Cancer

Posted on December 22, 2014

J. A. Yi1, K. El Kasmi1, E. E. Moore1,2, C. C. Barnett1,2  1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA

Introduction:

Tumor-associated macrophages (TAMs) mediate tumor progression.  Macrophage polarization is categorized as M1 (classical) or M2 (alternative) activation pathways.  M1 cells are pro-inflammatory and involved in innate immunity, while M2 cells function in wound healing and cell proliferation.  The M2 phenotype is most commonly seen among TAMs, correlates with poor patient prognosis, and is considered pro-tumorigenic.  Hypoxia inducible factor-1α (HIF1α) is a transcription factor activated in hypoxia and inflammation that modulates numerous critical functions for tumor growth and metastasis.  Increased HIF1α expression in pancreatic adenocarcinoma is a marker of advanced malignancy.  We hypothesize that pancreatic cancer modulates local macrophages towards a pro-tumorigenic M2 phenotype via a HIF1α-dependent mechanism.

 

Methods:

A murine pancreas adenocarcinoma cell line (Pan02) was modified using short-hairpin RNA targeting HIF1α via lentiviral transduction to create Pan02 SH+ cells lacking HIF1α activity.  Pan02 and Pan02 SH+ cells were then incubated until 80% confluent prior to collection of supernatant.  RAW 264.7 cells, a murine macrophage line, were exposed to Pan02 or Pan02 SH+ supernatant in culture for 24 hours.  At 24 hours, the RAW 264.7 cells were harvested for mRNA extraction.  Real-time polymerase chain reaction was used to determine macrophage phenotype based on suppressor of cytokine signaling (SOCS) 1 or 3 expression, accepted markers of M1 and M2 phenotypes respectively.  Statistical analysis was performed using one-way analysis of variance with significance determined by α<0.05.

 

Results:

 

There was a modest increase in SOCS1 expression by RAW 264.7 cells exposed to either Pan02 or Pan02 SH+.  The relative expression of SOCS1 was not significantly different between the two treatment groups (mean fold change in expression of Pan02-exposed = 1.5399 ± 0.2151, Pan02 SH+-exposed = 10.3159 ± 5.1322, p=0.1384).  SOCS3 expression was significantly greater than SOCS1 in both Pan02 and Pan02 SH+ treatment groups (p=0.0015).  Among RAW 264.7 cells exposed to Pan02 SH+ supernatant, SOCS3 expression was significantly reduced as compared to the Pan02-exposed cells (mean fold change in expression of Pan02-exposed = 152.206 ± 25.795, Pan02 SH+-exposed = 66.088 ± 10.617, p=0.0368).

 

Conclusions:

 

There was an increase in M2 versus M1 differentiation upon exposure to both Pan02 and Pan02 SH+ cell supernatant. Importantly, exposure to Pan02 supernatant significantly increased M2 differentiation compared to Pan02 SH+ supernatant, implicating HIF1α in the modulation of a pro-tumorigenic M2 macrophage activation state. This may represent a therapeutic target for treatment of pancreatic cancer.

2.12 Triptolide Mediates Post-Translational Modification of Histones in Pancreatic Cancer

Posted on December 22, 2014

K. Majumder1, R. Chugh1, S. Modi1, N. Arora1, S. Banerjee1, R. Dawra1, A. Saluja1, V. Dudeja1  1University Of Minnesota,Surgery,Minneapolis, MN, USA

Introduction:  Triptolide, a diterpene triepoxide from the Chinese plant Tripterygium wilfordii, is markedly effective against pancreatic cancer cells both in vivo and in vitro. Minnelide, a water soluble analog of triptolide, is currently in phase I clinical trials. While our data suggests that downregulation of transcription factors HSF-1 (Heat shock factor -1), Sp-1 and NFκB contribute to the effect of triptolide on cancer cells, the mechanism of action of triptolide remains elusive. There is increased understanding of epigenetics in the pathogenesis of cancer and altered histone modifications can play a role in aberrant protein expression profile in cancer cells. In general post-translational modification of histones such as methylation represses gene transcription whereas acetylation activates gene transcription. For example binding of histone-3 trimethylated at lysine-9 residue (tri-methylated H3K9) leads to repression of transcription. In the current work we have evaluated whether epigenetic modulation plays a role in triptolide’s mechanism of action.

Methods:  Pancreatic cancer cells (MIA Paca2 and S2-VP10) were treated with triptolide (0-200nM). Protein and mRNA were extracted and analyzed by western blotting, qPCR and PCR array for histone methylation/acetylation and levels of chromatin modifying enzymes. CHIP qPCR was performed to analyze binding of tri-methylated histones to the promoter region of genes regulated by HSF-1 (e.g. HSP70), Sp-1 (FGFR-1) and NFκB (TNFα). 

Results: Dose dependent increase of tri-methyl histones (H3Lys27, H3Lys9 and H3Lys4) and a concurrent decrease of acetylated histones (Acetyl histone H3Lys9 and H3Lys18) were observed on western blotting suggesting that triptolide has widespread impact on histone post-translational modification. Western blotting and qPCR showed time and dose dependent decrease in levels of both the demethylases and methyltransferases with triptolide. CHIP qPCR showed increased binding of trimethylated H3K9 but not trimethylated H3K27 to promoter of FGFR-1, HSP70 and TNFα in triptolide treated Mia Paca2 cell lines as compared to untreated cells (% Fold change in occupancy of H3K9me3 on triptolide 200nM treatment when compared to no treatment: HSP70 promoter = 321%, FGFR-1 promoter = 262% and TNFα promoter= 208%).

Conclusion: Triptolide alters histone methylation and acetylation profile suggesting an epigenetic component to its action. Corresponding to these changes there is an increased binding of tri-methylated H3K9, to the response element of the transcription factors (HSF-1, Sp-1and NFκB) which are downregulated by triptolide. This is the first report suggesting that epigenetic modulation may be the mechanism of anti-tumor action of triptolide. Dissecting this mechanism further may lead to elucidation of novel therapeutic target in pancreatic cancer.

2.13 CDK4/6 Inhibitors are Potent Suppressors of Pancreatic Carcinoma Growth

Posted on December 22, 2014

N. A. Borja1, J. Franco2, E. Knudsen2,3, J. Mansour1,3, M. Choti1,3, A. Witkiewicz2,3  1UT Southwestern,Department Of Surgery,Dallas, TX, USA 2UT Southwestern,Department Of Pathology,Dallas, TX, USA 3Simmons Cancer Center,UT Southwestern,Dallas, TX, USA

Introduction: Pancreatic ductal adenocarcinoma (PDA) carries a dismal prognosis, even in patients who undergo successful surgical interventions.  Unlike other cancers, an approach tailored to its specific genetic features has not yet been developed. Loss of the CDKN2A tumor suppressor is an exceedingly frequent occurrence in PDA. The CDKN2A gene encodes the p16ink4a protein, which is a potent inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6).  Prior studies have shown that p16ink4a is dominant to KRAS and can serve to constrain oncogenic proliferation in many contexts.  Specific CDK4/6 pharmacological inhibitors have been developed and could represent a means to restore the physiological loss of CDKN2A and treat PDA.  Here we utilized multiple models to investigate the impact of CDK4/6 inhibition on PDA.

Methods: Three specific model systems were utilized to determine the therapeutic efficacy of CDK4/6 inhibition in the treatment of PDA.  First, a panel of established PDA cell lines was employed to delineate overall features of the response to CDK4/6 inhibition, mechanisms of resistance, and define novel combination treatments. Second, in order to recapitulate the complex microenvironment of PDA, we utilized a primary tumor explant model where slices from 13 clinically resected PDA specimens were cultured on semi-solid support. These explants maintained the histologic architecture, biomarker profile, and proliferative index of the primary surgical specimen. Third, a panel of patient-derived xenografts representative of resected disease were developed and utilized to examine the response to CDK4/6 inhibition in vivo.

Results: The established cell lines had variable response to PD-0332991 (CDK4/6 inhibitor) as a single agent; however, in combination with mTOR or MEK inhibitors provided substantial efficacy across all models studied. Surprisingly,  this diversity of response was not recapitulated in tumor explants. In this context, treatment with PD-0332991 led to profound suppression of proliferation in all models, with the exception of a single resistant case harboring a loss of the RB tumor suppressor.  Similarly, patient-derived xenografts exhibited profound inhibition of Ki-67 proliferation marker and growth suppression. These data indicate that CDK4/6 inhibition is effective in suppressing the growth of PDA under diverse physiological contexts.

Conclusion: Our data demonstrates that PDA growth is profoundly inhibited by selective targeting of the CDK4/6 in primary tumor explants and patient-derived xenografts. The variable response to PD-0332991 in established cell lines likely reflects a more aggressive phenotype, and potentially the altered biology of models propagated in long term culture.  Importantly, the data suggest that CDK4/6 inhibition could be particularly effective in the control of resectable disease given the potent suppression of tumorigenic growth in both explant and patient-derived xenograft models.
 

2.14 Nafamostat mesilate enhances antitumor effect of chemotherapy for pancreatic cancer.

Posted on December 22, 2014

T. Horiuchi1,2, H. Shiba1, Y. Shirai1,2, R. Iwase1,2, K. Haruki1, Y. Fujiwara1, K. Furukawa1, T. Uwagawa1, T. Ohashi2, K. Yanaga1  1The Jikei University School Of Medicine,Department Of Surgery,Tokyo, , Japan 2The Jikei University School Of Medicine,Department Of Gene Therapy, Research Center For Medical Science,Tokyo, , Japan

Introduction: ~Pancreatic cancer has a poor prognosis among visceral cancers, with an overall 5-year survival rate of around 5%. Currently, intravenous gemcitabine is a standard therapy for patient with advanced pancreatic cancer. Recently, gemcitabine plus nab-paclitaxel therapy was reported to be superior to gemcitabine alone in patients with advanced pancreatic cancer. However, the tumor resistance to chemotherapy is common and activation of nuclear factor-kappa B (NF-κB) is a key player in attenuation of anti-tumor effect of chemotherapy in pancreatic cancer. NF-κB is a transcription factor that plays an important role in the regulation of cell apoptosis, inflammation, and oncogenesis. Therefore, targeting downregulation of NF-κB activation is an important strategy to improve the efficacy of chemotherapy and outcome of patients with pancreatic cancer. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, inhibited NF-κB activation and induced antitumor effects for pancreatic cancer. We hypothesized that nafamostat mesilate downregulates the activity of NF-κB and improves therapeutic outcome of pancreatic cancer. The purpose of this study is to prove that addition of nafamostat mesilate to gemcitabine and nab-paclitaxel enhances their antitumor effect against pancreatic cancer.

Methods: ~We assessed NF-κB activity and cell viability of human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, ASPC-1) in the following five groups:  ① those treated with gemcitabine alone, ② gemcitabine and nab-paclitaxel,
③ nafamostat mesilate alone, ④ triple combination (gemcitabine, nab-paclitaxel and nafamostat mesilate), or ⑤ vehicle as control. In combination groups, the cells were treated with gemcitabine and nab-paclitaxel at the same time. In triple combination group, the cells were incubated with nafamostat mesilate at 3 hours before chemotherapy. Activation of NF-κB was evaluated by measuring nuclear localization of p65 protein.

Results:~Cell viability was assessed by the MTT assay. Cell viability in triple combination group was lower than that in gemcitabine plus nab-paclitaxel group (PANC 1: p<0.001, MIA PaCa-2: p=0.004, ASPC-1: p<0.001). The nuclear extracts were assessed using ELISA. NF-κB activity in gemcitabine plus nab-paclitaxel group was higher than that in control group, and NF-kB activity was significantly surpressed in nafamostat mesilate group and in tiple combination group compared with control group and gemcitabine plus nab-paclitaxel group, respectively (PANC-1: p<0.001, MIAPaCa-2: p<0.001, ASPC-1: p<0.001).

Conclusion:~Nafamostat mesilate significantly inhibited NF-κB activation and enhanced anti-tumor effect of gemcitabine plus nab-paclitaxel against human pancreatic cancer.

 

2.15 Role of Sphingosine Kinase 1 of the Host in the Pancreatic Cancer Peritoneal Carcinomatosis.

Posted on December 22, 2014

H. Aoki1, A. Raza1, P. Mukhopadhyay1, K. P. Terracina1, C. C. Barnett3, S. Spiegel2, K. Takabe1,2  1Virginia Commonwealth University,Surgery,Richmond, VA, USA 2Virginia Commonwealth University,Biochemistry And Molecular Biology,Richmond, VA, USA 3University Of Colorado Denver,Surgery,Aurora, CO, USA

Introduction:  Pancreatic cancer remains one of the deadliest cancer. Despite the recent improvement of survival of the small resectable tumors, prognosis of unresectable pancreatic cancer that collectively represent over 80% of individuals remains dismal with the 5-year survival of 5% and median survival is within ten months. Prognosis of patients with peritoneal carcinomatosis (PC), dissemination of cancer cells throughout the abdominal cavity, are particularly poor with median survival of only 6 weeks. This poor overall 5-year survival rate has not significantly changed over the past 5 decades, which reflects the fact that there is no effective treatment available for this condition. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinase 1 (SphK1), plays critical roles in many aspects of cancer progression, such as cell proliferation, migration, angiogenesis and lymphangiogenesis. We have published in Cancer Cell 2013 that S1P link inflammation and cancer in colitis-associated cancer development. Given the fact that inflammation is known to be essential for establishment and progression of PC, where cancer cells need to adhere to the peritoneum and form a nodule, we hypothesized that S1P generated by SphK1 in the host animal, including peritoneum, is necessary for progression of pancreatic cancer PC.

Methods:  Panc02 cells were obtained from ATCC. Panc02-luc cells were provided in collaboration with CCB. PC models were generated by injecting the cells intraperitonealy to C57Blk6 mice. For the orthotopic model, Panc02-luc cells were implanted in the tail of the pancreas mixed in matrigel. Survival was assessed by Kaplan-Meier method. Progression of Panc02-luc cell PC in either SphK1 wild type (WT) or knockout (KO) mice were assessed by the total tumor burden determined by bioluminescence on the indicated days.

Results: Panc02 cells developed significantly less PC by intraperitoneal injections (5 out of 11 animals with less amount of nodules). On the other hand, 96% of mice developed PC when Panc02-luc cells were injected (52 out of 54 mice with more than four-fold increased amount of nodules). The median survival of Panc02-luc PC model was 18.7 days, whereas orthotopic model was 40 days, despite the fact that 7 out of 10 orthotopically implanted animals eventually developed PC. This mimics human pancreatic cancer progression that PC has remarkably shorter survival. Panc02-luc PC in SphK1 WT mice continue to progress with significant increase in tumor burden quantified by bioluminescence, whereas PC in SphK1 KO mice did not progress.

Conclusion: Our result demonstrated that intraperitoneal injection of Panc02-luc cells in C57Blk6 mice will generate orthotopic pancreatic cancer peritoneal carcinomatosis model that mimic human disease, and SphK1 expression in the host animal, including peritoneum, is necessary for progression of pancreatic cancer PC.

 

2.16 Microbial Determinants of Clinical Outcomes in Patients Undergoing the Whipple Procedure

Posted on December 22, 2014

V. G. Aveson1, R. Brower-Sinning1, B. Firek1, B. Boone1, J. Steve1, M. Hogg1, A. Zureikat1, H. J. Zeh1, M. J. Morowitz1  1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA

Introduction:
Important recent studies have detailed surprising ways in which the human microbiome contributes to human health and disease. However, contributions of the microbiome to surgical outcomes have not been studied. Here, we conducted a pilot study to investigate bacterial populations present at multiple body sites in patients undergoing pancreatico-duodenectomy (PD) for pancreatic ductal adenocarcinoma (PDA). 

Methods:
We collected intraoperative swabs of pancreatic fluid, bile, and jejunal contents (PB&J) as well as postoperative fecal samples from 12 patients undergoing PD for PDA. After isolation of bacterial DNA, 16S ribosomal RNA gene sequences were sequenced on the Illumina MiSeq platform. Analysis of sequencing results with QIIME software allowed for taxonomic identification and statistical analysis of microbial communities. For each patient, we recorded preop clinical variables (biliary stent, neoadjuvant chemotherapy, antibiotics) and postop outcomes (anastomotic leak, abscess formation, wound infection, cancer recurrence, death).

Results:
Bacterial diversity was surprisingly high in PB&J samples (mean Shannon index 3.7, 2.7, and 2.7 respectively). We detected and characterized bacterial DNA within PB&J samples from 8 of 12 patients. The most abundant taxa within PB&J samples were gram-negative pathogens from the family Enterobacteriaceae (e.g. Citrobacter and Enterobacter), oral pathogens (e.g. Fusobacterium and Aggregatibacter), and intestinal commensals (e.g. Bacteroides). Correlations between PB&J samples varied. One patient had a marked abundance of Fusobacterium in each of the PB&J samples. Interestingly, a high number of DNA sequences from an Enterobacter-like species was found in the PB&J samples from a patient that developed a postop Enterobacter wound infection. Fecal samples from the 12 PDA patients were notable for a relative lack of bacterial diversity (mean Shannon diversity index 3.1), perhaps reflecting perioperative antibiotic therapy.  Additionally, several of the fecal samples contained unusually large populations of gram-negative pathogens (e.g. Citrobacter and Enterobacter). One fecal sample contained a highly unusual dominant population of Enterococcus (relative abundance >90%). Six patients developed a pancreatic anastomotic leak or postop abscess. Due to small sample size, we did not identify associations between the microbiome and either postop complications or preop exposures.

Conclusion:
Microbial diversity in biologic samples collected from PD patients has not previously been studied. Notable findings in this study were the high diversity within bile and pancreatic duct fluid and the presence of oral pathogens known to cause extraoral infections. We are currently enrolling additional study subjects. Future analyses may allow us to identify associations between intraop microbial profiles and postop outcomes. 

2.18 Characterization of a Novel Mutation in Fibrolamellar Hepatocellular Carcinoma

Posted on December 22, 2014

K. M. Riggle1, R. S. Yeung1, H. L. Kenerson1, K. J. Riehle2  1University Of Washington,Surgery,Seattle, WA, USA 2Seattle Children’s Hospital,General And Thoracic Surgery,Seattle, WA, USA

Introduction:  Hepatocellular carcinoma (HCC) is a heterogenous disease that commonly arises in a background of cirrhosis.  Fibrolamellar HCC (FL-HCC) is a subtype of HCC occurring in children and young adults in the absence of known liver disease.  Currently, there is no effective therapy for unresectable or metastatic FL-HCC.  Recent genomic analysis identified a recurrent mutation in FL-HCC involving a deletion on chromosome 19.  The mutation results in a common chimeric transcript containing the 5’-region of a heat shock protein (DNAJB1) fused to the catalytic subunit of protein kinase A (PRKACA).  We sought to characterize this chimeric protein and its effects on PKA activity in human FL-HCC. 

Methods:  We prepared tissue lysates from four snap-frozen FL-HCC samples along with paired, non-tumor liver tissues.  PRKACA expression was determined by immunoblot analysis.  PKA activity was determined via a radioactive kinase assay in the presence of cAMP, a known activator of PKA, with and without PKI, a specific inhibitor of PKA accounting for background activity.  RNA was extracted using TRIZOL reagent, and used to create cDNA for qRT-PCR analysis of the mutant transcript.     

Results:  We confirmed that all tumor samples expressed a 46-kDa fusion gene product in addition to the wild-type 41-kDa PKA protein.  The paired normal liver samples only expressed the wild-type protein.  Further, the mutant protein was not detected in ‘classic’ HCCs nor cancer-associated fibroblasts isolated from a case of FL-HCC.  Using qPCR we found that the FL-HCC tumors expressed the chimeric transcript at levels that were 10.59±4.2 fold higher than normal liver (p = 0.016).  Basal PKA activities from freshly lysed tumors and paired livers were not significantly different, but cAMP-stimulated PKA activity was significantly higher in FL-HCC tumors when compared to normal liver.  In a dose response experiment, the PKA activity in FL-HCC was 3.62, 5.52, and 6.41 pmol/min/mg (vs. normal liver with PKA activity of 2.60, 3.14, and 4.95 pmol/min/mg) at cAMP concentrations of 0.05, 0.5, and 5 uM respectively.

Conclusion:  Our data verify the unique expression of the DNAJB1-PRKACA fusion protein in all FL-HCC samples tested, but absent in the adjacent non-tumor liver and non-FL-HCCs.   Further, the lack of mutant protein expression in fibroblasts derived from FL-HCC highlights the primary effects of the mutation on transformed hepatocytes and not the stromal component.  The expression of the mutant transcript was significantly greater than that of the native PKA indicative of higher intrinsic promoter activity of DNAJB1 compared to that of PRKACA.  Importantly, PKA activity in the FL-HCCs remains cAMP-dependent but with increased sensitivity to cAMP without evidence of enhanced basal activity.  These findings suggest that the expression of DNAJB1-PRKACA in FL-HCC leads to over-activation of PKA under conditions of cAMP production, which may contribute to tumor development.

2.19 Incidental Gallbladder Pathology in Patients Undergoing Cholecystectomy

Posted on December 22, 2014

R. A. Rodriguez1, H. Overton2, K. Morris1, I. Nir1, M. Williamson3, A. Rajput1  1University Of New Mexico,Division Of Surgical Oncology, Department Of Surgery,Albuquerque, NM, USA 2University Of New Mexico,School Of Medicine,Albuquerque, NM, USA 3University Of New Mexico,Department Of Radiology,Albuquerque, NM, USA

Introduction: The highest incidence rates of gallbladder cancer (GBC) in the continental United States are in New Mexico and amongst the minority-majority Native American and Hispanic populations. This cancer is often fatal as most patients present with advanced stages of disease. Early diagnosis remains a challenge as specific signs and symptoms are absent. Early stages of disease, which have a more favorable outcome, are often found incidentally at time of cholecystectomy. The purpose of this study was to determine the incidence of gallbladder pathology in patients undergoing cholecystectomy and to correlate any pre-operative radiographic findings with the pathology.

Methods: Healthsystem database was queried for patients who underwent cholecystectomy between 1991 and 2013. A total of 6793 patients were identified. Data extracted included: demographics, previous ultrasound report, surgical and pathologic reports. Pathologic findings including gallbladder cancer, dysplasia, metaplasia and polyps were recorded. Radiographic reports were reviewed to determine if pre-operative finding predicted final pathologic diagnosis.

Results: There were 4993 (74%) females. Female patients were more likely to present at an older age (52.5) compared to males (45.4); patients who were found to have GBC were more likely to be older (73.4). Out of a total of 17 GBCs identified, 5 (29%) patients were known to have malignancy prior to surgery and 12 (71%) were discovered to have malignancy on pathologic analysis. Pre-operative radiographic findings correlated to pathological findings in only 3 (18%) cases. GB pathology stratified by ethnic group is shown in table 1. GB pathology was more likely to be found in Hispanics and Native Americans although the incidence of pathology was not statistically significant amongst ethnicities in our data set.

Conclusion: Although rare, gallbladder pathology is incidentally found at the time of cholecystectomy.  The diagnosis of cancer, dysplasia, metaplasia and polyps was more common in Hispanics and Native Americans as compared to non-Hispanic whites. Although ultrasonography continues to be a good diagnostic method for gallstones and cholecystitis, its utility in other gallbadder pathologies remains unproven. There is a need to develop new screening and diagnostic methods for patients with gallbladder cancer.

 

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