Transferrin Receptor Targeting Nanomedicine Delivering Wild Type P53 Gene Sensitizes Pancreatic Cancer to Gemcitabine Therapy

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

no longer a contraindication to tumor resection. Here we analyze patients that required resection of the portal vein (PV) and/or SMV during pancreaticoduodenectomy (PD) at our institution. Methods: From 1993 through 2010 there were 1,928 PDs performed at our institution and recorded in a prospectively maintained database. We analyzed outcomes from PDs involving resection of the portal vein and/or SMV to identify prognostic risk factors for poor outcome. We examined whether techniques of PV/SMV resection and reconstruction had evolved over time. Results: One hundred and forty patients received PV and/or SMV resection and reconstruction during PD over the previous two decades with thirty and ninety day mortality rates of 0% and 3.9%. Reconstructions utilized primary repair or anastomosis most commonly (85%) and ‘‘complex reconstruction’’ with a graft or patch less frequently (15%). Pathohistological vascular invasion itself was not a prognostic variable for overall survival, however, complex vascular reconstruction did predict worse outcome. Complications after vascular resection (VR) were associated with worse long term survival. Recent trends in technique show that almost all VRs are now being reconstructed with primary anastomosis. Conclusions: In this large series, PV/SMV resection and reconstruction during pancreatic surgery was performed safely and efficaciously. Utilization of complex reconstruction was associated with worse outcomes. Evolution of the surgical technique at our institution with mobilization of perihepatic attachments and division of the splenic vein now permits almost all cases to be performed with primary anastomosis for optimal outcomes.

a marked survival advantage. Tumor differentiation and effective perioperative care correlate with an even more optimal prognosis. Current adjuvant therapies offer no detectable survival advantage in these particular patients.

TABLE 26.7. Resected Pancreatic Cancer with Favorable Prognosis: is Adjuvant Therapy Beneficial? C. M. Vollmer,1 R. Lewis,1 M. P. Callery,2 D. Fraker,1 J. Gates,1 J. A. Drebin1; 1 University of Pennsylvania - Department of Surgery, Philadelphia, PA; 2Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA Introduction: Despite the generally dismal prognosis of resected pancreatic ductal adenocarcinoma (PDAC), some patients achieve long-term survival (>5 yrs). We sought to characterize an optimal prognosis cohort and identify factors that determine their survival advantage. Specifically, we investigated whether adjuvant therapy (Chemo +/- Radiotherapy) provided any benefit for these particular patients. Methods: Databases containing PDAC resections performed at two institutions over a decade (2001-2011) were merged and studied. ‘‘Favorable’’ patients were initially defined a priori as R0/N0/M0 resections, and all others were deemed ‘‘Unfavorable’’. For further refinement of the Favorable group, univariate and multivariate survival analyses were conducted, applying demographic, pathologic, and perioperative variables. Next, Kaplan-Meier analysis compared these cohorts and assessed the impact of adjuvant therapy (primarily Gemcitabine-based Chemo/XRT) on each. Results: of 413 resections, 92 (22%) were R0/N0/M0. This Favorable group had median and actuarial 5-yr survivals of 43.8 months and 42% respectively (versus 19.1 months and 19% for the remaining 321 Unfavorable patients; p<0.0001), and they represented 44% of all actual 5-yr survivors. Within the Favorable group, multivariate predictors of diminished survival were poor tumor differentiation, percutaneous drain placement, and 30-day readmission. Using these, the Favorable group was further classified into Optimal (n¼48) and Less-Optimal (n¼44) prognosis cohorts (Table). The majority of Optimal patients survived five years, whereas Less-Optimal patients survived comparably to the Unfavorable group (Figure). While adjuvant therapy was associated with significantly improved survival when applied to Unfavorable patients (P<0.001), it afforded no survival benefit in either the Favorable group (p¼0.750), or the subgroups of Optimal (p¼0.659) or Less Optimal patients (p¼0.091). Conclusions: Over one-fifth of pancreatic resections for PDAC occur with a favorable margin- and node-negative pathology – a scenario which confers

Survival Analysis of 413 PDAC Patients Group Favorable (R0/N0/M0) Optimal (No: poor differentiation, percutaneous Less-Optimal (Any: poor differentiation, percutaneous drainage, or readmission) Unfavorable (Any R1 or N1 or M1)

Median 5-yr surv(m) surv

HR

P-value* <0.001

111.2

62%

0.33

24.0

22%

1

19.1

19%

1.2

0.358

*Comparison with Less-Optimal group; Cox Proportional Hazards Model. 26.8. Transferrin Receptor Targeting Nanomedicine Delivering Wild Type P53 Gene Sensitizes Pancreatic Cancer to Gemcitabine Therapy. E. R. Camp,1 C. Wang,1 P. Watson,1 E. Little,1 K. F. Pirollo,2 A. Rait,2 D. J. Cole,1 E. H. Chang,2 D. K. Watson1; 1Medical University of South Carolina, Charleston, SC; 2Georgetown Lombardi Cancer Center, Washington, DC Introduction: To overcome gene therapy barriers such as low transfection efficiency and non-specific delivery, liposomal nanoparticles targeted by a single chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild type human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Methods: Intra-splenic injection of 13106 Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastases. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled ODN (6FAM ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 mg of p53 IV) and gemcitabine (20 mg/kg IP) alone and in combination were administered biweekly compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wtp53 expression and transferrin were assessed by Western Blot analysis. Tumor burden was

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS quantified by liver weight. Results: Xenogen imaging demonstrated tumor specific uptake of TfRscFv-6FAM ODN. Exogenous human wtp53 protein was detected in the SGT-53 treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine, and the combination demonstrated improved median survival of 29, 30, and 37 days respectively (Figure). The combination treatment prolonged median survival when compared to single drug treatment and decreased tumor burden. Conclusions: SGT-53 represents a novel targeted liposomal-based nanoparticle capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. Further optimization is ongoing towards a Phase 1B/2 clinical trial.

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have completed chemotherapy and undergone successful surgical resection. One patient withdrew from the study on day 3. There were no dose limiting toxicities, no grade 4 or 5 events and no treatment delays with 8 patients receiving the maximum dose of HCQ. A single grade 3 event occurred in a patient with neutropenia, most likely secondary to GC treatment. 8 patients (67%) had a decrease in CA19-9 following treatment. 3 out of 7 patients (43%) with PET CT scans pre and post treatment demonstrated a partial radiologic response and 3 patients had stable disease. All patients underwent surgical resection as scheduled. Minor postoperative complications, grade 3 or less, affected 7 (58%) patients. One patient developed a grade 4 gastric leak after a modified Appleby procedure requiring percutaneous drainage, parenteral nutrition and monitoring in the intensive care unit. There was no perioperative mortality. Electron microscopy studies of PBMCs in two patients revealed decreases in autophagosomes with therapy. Conclusions: Neoadjuvant autophagy inhibition with HCQ in combination with GC is safe and tolerable at 1200 mg daily. Completion of the Phase II portion of the trial is warranted to correlate markers of autophagy with clinical outcomes and biologic activity.

26.10. MicroRNA-21 in Pancreatic Ductal Adenocarcinoma Tumor-Associated Fibroblasts Promotes Metastasis. B. E. Kadera, L. Li, P. Toste, N. Wu, D. W. Dawson, T. R. Donahue; University of California - Los Angeles School of Medicine, Los Angeles, CA

26.9. Phase I/II Trial of Autophagy Inhibition in Combination with Neoadjuvant Gemcitabine in High Risk Pancreatic Adenocarcinoma: Safety and Response to Treatment. B. A. Boone,1 A. Zureikat,1 N. Bahary,2 D. L. Bartlett,1 R. Amaravadi,3 M. T. Lotze,1 H. J. Zeh1; 1University of Pittsburgh - Department of Surgery, Pittsburgh, PA; 2 University of Pittsburgh - Department of Medicine - Division of Hematology/Oncology, Pittsburgh, PA; 3University of Pennsylvania - Department of Medicine - Division of Hematology/Oncology, Philadelphia, PA Introduction: Autophagy is a mechanism of cell survival in response to cellular stress in which organelles and proteins are recycled for energy production. Autophagy plays a critical role in resistance to therapy in pancreatic cancer and correlates adversely with prognosis. Hydroxychloroquine (HCQ), a commonly prescribed antimalarial drug, blocks acidification of the lysosome, inhibiting the last step in autophagy. Murine models demonstrate that CQ has significant anti-tumor activity and enhances cytotoxic effects of chemotherapy, suggesting a novel treatment strategy in patients with pancreatic cancer. Methods: A Phase I/II trial examining pre-operative gemcitabine (GC) in combination with oral HCQ in the treatment of high-risk pancreatic adenocarcinoma. Eligibility was limited to only those patients predicted to have limited survival advantage of surgical resection. Fixed dose GC (1500mg/m2) was administered every two weeks for two doses. HCQ (200mg/day-1200mg/day) was administered 48 hours prior to the first dose of GC and continued for 31 consecutive days until day of surgery. Toxicity was evaluated based on National Cancer Institute criteria. Response to treatment was assessed by decrease in CA19-9 levels and PET CT scans using the European Organization for Research and Treatment of Cancer criteria. Two weeks after the last dose of GC, patients underwent pancreatectomy. Perioperative mortality and morbidity were evaluated to 90 days postoperatively with complications graded according to the Clavien-Dindo classification. Serum and peripheral blood mononuclear cells (PBMCs) were collected pre-treatment, pre-surgery and postoperatively and evaluated for autophagosomes. Results: Twelve patients

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related mortality in the United States, and the incidence is rising. Current chemotherapeutic therapies have had little success; novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. While the tumor microenvironment has also been recognized to contribute to tumor progression, dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR21 expression in TAFs promotes TC invasion by regulating an epithelial-mesenchymal transition (EMT) program. Methods: In-situ hybridization for miR-21 was performed on the 153 patient UCLA Tissue Microarray. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. qRT-PCR for miR-21 was performed on early and late passages. Invasion assays were completed by transfecting TAFs with high baseline miR-21 expression, with miR-21 antisense inhibitor (Qiagen) or nontransformed primary lung fibroblasts with low baseline miR-21 expression, with miR-21 mimic (Qiagen) followed by coculture with GFP-MiaPaCa TCs in a modified Boyden chamber. qRT-PCR was then performed on FACS sorted GFP-TCs to assess for EMT markers. Results: miR-21 was upregulated in TAFs of 83% of tumors, and high miR-21 significantly correlated with decreased overall survival (P<0.001). Stromal miR-21 expression also significantly associated with advanced AJCC stage disease (P¼0.008) and lymph node invasion (P¼0.004), suggesting that it is driving TC spread. Coimmunofluorescence staining revealed that miR-21 colocalized with peritumoral fibroblasts expressing vimentin and a-smooth muscle actin. These TAFs are stably reprogrammed to express high levels of miR-21 as there was no decrease in expression over multiple passages. The mechanism whereby high miR-21 expression in TAFs promotes invasion of TCs is likely through EMT, as FSP-1 and ZEB1 were decreased in TCs at 48 hours when co-cultured with TAFs treated with a miR-21 inhibitor (P<0.05). Conclusions: This is the first study to demonstrate that miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion through EMT. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.