- Email: [email protected]
Enhanced Growth of Syngeneic Pancreatic Cancer in CXCR2 Deficient Mice
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS fold change in the LRCC compared to the NLRCC. Canonical pathway analysis of the upregulated subsets of genes noted increased activity of the Wnt-b-catenin and Hedgehog pathway. Gene ontology analysis revealed increased expression of negative regulators of apoptosis in the LRCC population. Conclusions: We show that 3 pancreatic cancer cell lines contain LRCC. Based on the fact that LRCC are hypothesized to be putative stem cells, and we have demonstrated that pancreatic LRCC have a gene signature consistent with stem cells, we propose that pancreatic LRCC may represent a novel class of pancreatic cancer stem cells. Additionally, the unique gene signature of LRCC may provide novel targets for therapy specifically directed towards pancreatic cancer stem cells.
40.7. Clinical Significance of Serum COL6A3 in Pancreatic Ductal Adenocarcinoma. J. Wang, G. Chipitsyna, M. Lazar, T. Hyslop, M. Chu, D. Relles, C. J. Yeo, H. A. Arafat; Thomas Jefferson University, Philadelphia, PA Introduction: Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils and constitutes a major component of the prominent desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that a subunit of COL6, COL6A3, is expressed in high levels in PDA tissue and that the COL6A3 gene undergoes tumor-specific alternative splicing. the aim of this study is to investigate the diagnostic value and clinical significance of circulating COL6A3 in PDA. Methods: Serum samples were obtained from patients that underwent pancreatic resection at Thomas Jefferson University Hospital between 2006 and 2009. COL6A3 levels in the sera from patients with pathologically confirmed PDA (n¼44), intraductal papillary mucinous neoplasms (IPMN) (n¼22), cystadenomas (n¼15), chronic pancreatitis (n¼10) and neuroendocrine tumors (NET) (n¼7) were analyzed by ELISA. Serum CA19-9 values were obtained from the electronic medical records. the prediction levels for malignancy were determined by the area under the receiver operating characteristic curve (AUC). Immunohistochemical staining for COL6A3 was performed on 12 tissue samples where both serum and paraffinized tissue samples from the same patients were available. Results: Circulating COL6A3 levels were significantly (p¼0.0035) elevated in PDA patients when compared to all benign lesions. Compared to IPMN alone, COL6A3 levels were significantly higher in PDA (p¼0.014). There were no significant differences between the levels in PDA and NET (p¼ 0.59). Interestingly, high COL6A3 serum levels correlated with lymph node invasion (p¼0.006) and with tumor size (p¼0.045). We assessed the level of association of log(col6a3) and log(ca19-9) with cancer status using a logistic regression model. With each increasing unit of log COL6A3, a patient is 12.9 times more likely to have cancer, with 95% CI (1.9, 86.8), p¼0.009. the odds for each increasing unit of log CA19-9 are 2.4 times, with 95% CI (1.6, 3.7), p<0.001. the overall area under the receiver-operator curve (ROC) is 0.90. Immunohistochemical analysis revealed a strong COL6A3 staining in PDA and NET and its localization to the perineoplastic stroma and the malignant cells. Conclusions: Our data show for the first time the potential clinical significance of circulating COL6A3 levels in the diagnosis of pancreatic malignancy. Correlation of the high COL6A3 levels with tumor size and lymph node invasion suggests a role for COL6A3 in PDA progression and metastatic potential. 40.8. Notch1 Upregulates PDX-1 and Acts Synergistically in the Development of Islet Cell Neoplasia. S. Liu,1 G. Zhou,1 K. M. Shahi,1 J. Nemunaitis,2 D. Dawson,3 W. E. Fisher,1 F. Brunicardi1,3; 1Baylor College of Medicine, Houston, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3David Geffen School of Medicine at UCLA, Los Angeles, CA Introduction: Notch and PDX-1 are embryonic pancreatic transcription factors and pancreatic oncogenes, however a relationship between
309
the two has not yet been demonstrated. the purpose of this study was to determine whether Notch1 upregulates PDX-1 in the development of islet neoplasia. Methods: HEK293 cells, stably transfected with human PDX-1 (HEK293-hPDX-1), and mouse insulinoma b TC-6 cells, which express endogenous mPDX-1, were (co)transfected with human and mouse notch1 intracellular domain (hNICD, mNICD), respectively, verified by Western blot. Proliferation of both cells lines was determined by MTS assay. Glucose-stimulated insulin secretion (GSIS) of b TC-6-mNICD cells was determined by ELISA assay. Beta cell-specific overexpressing NICD (bNICD+/+) mice were generated by breeding LSL-ROSANICD and rat insulin promoter-CRE mice; genotype was verified using PCR. Serial glucose and insulin levels were measured and IPGTT was performed in 6 mo old mice. IHC for insulin, PDX-1 and cell cycle proteins was performed on islets of bNICD+/+ mice, appropriate control litters and human pancreatic neuroendocrine tumor specimens (hPNT; n¼34). Islet cell apoptosis was determined via TUNEL assay. Results: 88% and 100% of hPNT strongly expressed Notch1 and PDX-1, respectively (figure). HEK293-PDX-1-hNICD and b TC-6-mNICD cells had increased PDX-1 expression and proliferation (both p<0.05 vs controls). b TC-6-mNICD cells had 2 fold increase in GSIS (p<0.05 vs controls). bNICD+/+ mice developed islet neoplasia: 1. glucose levels of 87mg/L vs 122mg/L and insulin levels of 3.5ng/L vs 1.3ng/L vs controls (p<0.05 for both); 2. IPGTT revealed significantly decreased glucose levels at each time point, 3. islets demonstrated enlargement of islets size, increased PDX-1 and insulin expression, disruption of cell cycle proteins and reduced islet cell apoptosis vs controls (Figure). Conclusions: Notch1 and PDX-1 were over-expressed in hPNT, suggesting a synergistic relationship in human tumors. Cell lines transfected with NICD demonstrated islet neoplasia with increased PDX-1, proliferation and GSIS. bNICD+/+ transgenic mice developed islet neoplasia associated with increased PDX-1, hyperinsulinemia and hypoglycemia. These data support the hypothesis that Notch1 up-regulates PDX-1 and synergistically contributes to the development of islet neoplasia.
FIG. 1. Expression of Notch1 is associated with overexpression of PDX-1 in human pancreatic neuroendocrine tumor specimens (hPNT) and islets of bNlcDþ/þtransgenic mice. 40.9. Enhanced Growth of Syngeneic Pancreatic Cancer in CXCR2 Deficient Mice. G. W. Donald,1 M. Assifi,2 A. Moro,1 M. Chen,1 G. Eibl,1 H. A. Reber,1 D. Dawson,3 V. G. Li,4 J. HInes O.1; 1Department of Surgery, UCLA Center for Excellence in Pancreatic Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Department of Surgery, Jefferson Medical College, Philadelphia, PA; 3Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA; 4Department of Biostatistics, UCLA School of Public Health, Los Angeles, CA Introduction: Evidence in our laboratory has identified the CXC chemokine group as potent mediators of disordered growth and angiogenesis in pancreatic cancer. Clinically, the overexpression of these ligands predicts survival of pancreatic cancer patients following resection. These observations appear to be mediated through the CXCR2 receptor. We hypothesized that pancreatic cancer growth is dependent
310
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
upon CXCR2 expression. Utilizing a CXCR2 knockout strategy, we investigated this hypothesis in a syngeneic, preclinical model of pancreatic cancer. Methods: CXC ligand expression during pancreatic carcinogenesis was evaluated by immunohistochemistry utilizing the C57BL/6 PDX1-Cre; LSL-KRASG12D murine model of pancreatic cancer. Pancreatic cancer cells (mKRAS) derived from these transgenic animals were cultured and chemokine expression determined by ELISA. C57BL/6 CXCR2 +/- mice were bred and genotyping confirmed from newborn mice tail samples. Mice between the ages of 4 and 8 weeks were injected subcutaneously with 2x106mKRAS cells. the tumors were allowed to grow for four weeks after which time the mice were sacrificed and the tumors harvested. the tumor samples were measured and analyzed following H&E staining. Descriptive statistics such as mean and standard error (SE) were summarized for all variables. P values were calculated with two-tailed t-test. Results: PDX1-Cre; LSL-KRASG12Dmice were sacrificed at 4, 7, and 10 months. Successful excision-recombination eventswere confirmed by the presence of a single LoxP site in thepancreas. Immunohistochemistry for the CXCL5[LIX] revealed that LIX was absent from normal ductal epithelium, but progressively expressed in mPanIN lesions. Analysis of cell supernatant from cultured mKRAS cells confirmed CXCL5 expression in vitro (6,7726101 pg/ml). CXCR2+/- breeding produced only one CXCR2 -/- pup and no tumor growth was found in this animal.Tumors were larger in the CXCR2+/- compared to CXCR2+/+ animals (104.9629.9 vs 35.17644.75 mm3, p<0.07). Gross histological appearance showed no difference in central tumor necrosis, mitotic activity, or in-growth of blood vessels. Conclusions: Initial data indicate that the transgenic mouse model of pancreatic cancer, PDX1-Cre; LSL-KRASG12D, express CXCL5 as has been observed in human pancreatic cancer. Cancer cells derived from this model establish a more aggressive phenotype when grown in a CXCR2+/- background since these tumors were more than twice the size of those in wildtype mice. These findings may be the result of altered immunity and diminished surveillance known to exist in CXCR2 transgenic animals. Given these findings the CXCL:CXCR2 biologic axis may have disparate activity during pancreatic cancer initiation and promotion. 40.10. Axl Receptor Tyrosine Kinase is Essential to Pancreatic Tumor Progression and Metastases. A. R. Kirane,1 M. T. Dellinger,1 K. T. Ostapoff,1 J. E. Toombs,1 R. E. Schwarz,1 J. B. Lorens,2 R. A. Brekken1; 1Department of Surgery, Division of Surgical Oncology, Hamon Center for Cancer Therapeutics, UT Southwestern Medical Center, Dallas, TX; 2Deparent of Biomedicine Univeristy of Bergen, Bergen, Hordaland Introduction: Expression of the receptor tyrosine kinase Axl in pancreatic cancer is associated with increased metastasis and reduced survival. Axl participates in angiogenesis, proliferation, invasion and transformation, but the precise function of Axl in pancreatic cancer is ill-defined. in this study, we investigate the therapeutic benefit of inhibiting Axl or its ligand, Gas6. Methods: Mia PaCa2 cells stably expressing a shRNA targeting Axl were generated and validated (1132 cells). 1132 cells were injected orthotopically into SCID mice and growth was compared to control cells. Primary tumor size and metastatic burden were measured at sacrifice and Axl expression was evaluated by qPCR and IHC. Warfarin, an inhibitor of Gas6, was used to evaluate the efficacy of targeting the Axl ligand. Three cell lines, Panc-1 and AsPC-1 (Axl+) and Axl non-expressing Capan-1 were evaluated for in vitro response to warfarin by MTS, migration, and anchorage independent growth assays. These lines were then implanted orthotopically into SCID mice. in addition, Pan02 murine cancer cells were implanted into C57BL/J6 mice. Subtherapeutic warfarin was initiated on post-implantation day 7. Inhibition of Axl signaling was determined in tumor lysates by Western blot analysis. Results: 58 days post implantation, mice bearing 1132 tumors had no discernable tumor burden while control mice were significantly moribund. By day 72 half of the 1132 animals had developed tumors approaching control size. IHC and qPCR demonstrated that 1132 tumors that grew showed
high levels of Axl expression while, tumors that failed to progress showed no Axl expression. Metastatic incidence was significantly lower for all 1132 tumor bearing animals. in vitro, warfarin treatment sensitized cells to gemcitabine by MTS assay proportional to Axl expression. Furthermore, Panc-1 cells demonstrated dramatic inhibition of migration and growth in soft agar with warfarin treatment, AsPC-1 had a moderate response and Capan-1 cells were unaffected. in vivo, Panc-1 and AsPC-1 tumors were of smaller average weight in warfarin treated animals, but this finding was not significant. However, metastatic incidence was decreased profoundly with almost no metastases found in warfarin treated animals (p<0.05). Warfarin had no affect on Capan-1 tumors. Additionally, Pan02 tumors were significantly smaller (p<0.05) and, notably, non-metastatic in warfarin treated animals. Analysis of Axl signaling in Panc-1 tumor lysates demonstrated that inhibition of Gas6 with warfarin resulted in decreased phosphorylation of Akt and Erk as well as increased Parp cleavage. Conclusions: We conclude that Axl is important in the progression of pancreatic cancer. Inhibition of Axl signaling with subtherapeutic warfarin resulted in a reduction of metastases for Axl expressing tumors and sensitized cells to gemcitabine in vitro. This finding warrants further evaluation of Axl as a novel target and strategy to augment chemotherapy for pancreatic cancer.
ONCOLOGY 7: TUMOR BIOLOGY 41.1. Epithelial Sarcomas: Experience From a Single Institution. A. A. Guzzetta,1 E. A. Montgomery,1 P. P. Emmanouil,1 T. Fu,1,2 N. Ahuja1; 1Johns Hopkins University, Baltimore, MD; 2Daping Hospital, Third Military Medical University, Chongqing, Yuzhong District Introduction: Epithelioid sarcomas are rare soft tissue sarcomas of unknown histiogenesis. the clinical behavior and response to treatment are poorly described in the literature due to the rarity of this disease. Methods: The centralized cancer registry and the pathology archives of this institution were queried and 22 patients were diagnosed with epithelioid sarcomas from 1980 to present. Two patients had inadequate information for analysis and were excluded. the charts of all patients were reviewed and histology was confirmed by re-review by a pathologist with expertise in sarcomas. This study was performed with IRB approval. Results: The 20 patients diagnosed with epithelioid sarcoma were examined. Mean age at diagnosis was 31 years and 75% of patients were male. 95% of patients were treated with surgery and 10% of patients were found to have metastasis to regional lymph nodes. of patients treated with surgery at this institution, 73% underwent R0 resection, 18% underwent R1 resection and 9% underwent R2 resection. Overall survival and recurrence free survival trended with margin status but was not significant (p>0.05). 5-year survival was 86% and median overall survival was 56.4 months. 60% of patients received adjuvant therapy. 55% of patients recurred at a mean of 35 months, regardless of chemotherapy or radiation (p>0.05). 35% of all patients recurred two or more times and the maximum number of recurrences was 5 though the number of recurrences did not affect survival (p>0.05). Patients with at least one recurrence demonstrated a nonsignificant improvement in survival (figure 1). Patients with 2 or more recurrences benefitted from continued resection with a median overall survival of 58 months. Location of recurrence, including lung metastasis was not associated with worse prognosis (p>0.05). Conclusions: Our study reveals that patients with epithelioid sarcomas can have long-term survival following surgical resection. Neither radiation nor chemotherapy affect outcome or recurrence. Importantly, patients with multiple recurrences continued to benefit from aggressive surgical treatment. Unlike prior reports, patients with lung metastasis did not have worse outcomes in our study. Epitheloid sarcomas are unique as they metastasize to regional nodal basins and all patients undergoing wide local excision should undergo simultaneous exploration of local lymph nodes.
40.7. Clinical Significance of Serum COL6A3 in Pancreatic Ductal Adenocarcinoma. J. Wang, G. Chipitsyna, M. Lazar, T. Hyslop, M. Chu, D. Relles, C. J. Yeo, H. A. Arafat; Thomas Jefferson University, Philadelphia, PA Introduction: Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils and constitutes a major component of the prominent desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that a subunit of COL6, COL6A3, is expressed in high levels in PDA tissue and that the COL6A3 gene undergoes tumor-specific alternative splicing. the aim of this study is to investigate the diagnostic value and clinical significance of circulating COL6A3 in PDA. Methods: Serum samples were obtained from patients that underwent pancreatic resection at Thomas Jefferson University Hospital between 2006 and 2009. COL6A3 levels in the sera from patients with pathologically confirmed PDA (n¼44), intraductal papillary mucinous neoplasms (IPMN) (n¼22), cystadenomas (n¼15), chronic pancreatitis (n¼10) and neuroendocrine tumors (NET) (n¼7) were analyzed by ELISA. Serum CA19-9 values were obtained from the electronic medical records. the prediction levels for malignancy were determined by the area under the receiver operating characteristic curve (AUC). Immunohistochemical staining for COL6A3 was performed on 12 tissue samples where both serum and paraffinized tissue samples from the same patients were available. Results: Circulating COL6A3 levels were significantly (p¼0.0035) elevated in PDA patients when compared to all benign lesions. Compared to IPMN alone, COL6A3 levels were significantly higher in PDA (p¼0.014). There were no significant differences between the levels in PDA and NET (p¼ 0.59). Interestingly, high COL6A3 serum levels correlated with lymph node invasion (p¼0.006) and with tumor size (p¼0.045). We assessed the level of association of log(col6a3) and log(ca19-9) with cancer status using a logistic regression model. With each increasing unit of log COL6A3, a patient is 12.9 times more likely to have cancer, with 95% CI (1.9, 86.8), p¼0.009. the odds for each increasing unit of log CA19-9 are 2.4 times, with 95% CI (1.6, 3.7), p<0.001. the overall area under the receiver-operator curve (ROC) is 0.90. Immunohistochemical analysis revealed a strong COL6A3 staining in PDA and NET and its localization to the perineoplastic stroma and the malignant cells. Conclusions: Our data show for the first time the potential clinical significance of circulating COL6A3 levels in the diagnosis of pancreatic malignancy. Correlation of the high COL6A3 levels with tumor size and lymph node invasion suggests a role for COL6A3 in PDA progression and metastatic potential. 40.8. Notch1 Upregulates PDX-1 and Acts Synergistically in the Development of Islet Cell Neoplasia. S. Liu,1 G. Zhou,1 K. M. Shahi,1 J. Nemunaitis,2 D. Dawson,3 W. E. Fisher,1 F. Brunicardi1,3; 1Baylor College of Medicine, Houston, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3David Geffen School of Medicine at UCLA, Los Angeles, CA Introduction: Notch and PDX-1 are embryonic pancreatic transcription factors and pancreatic oncogenes, however a relationship between
309
the two has not yet been demonstrated. the purpose of this study was to determine whether Notch1 upregulates PDX-1 in the development of islet neoplasia. Methods: HEK293 cells, stably transfected with human PDX-1 (HEK293-hPDX-1), and mouse insulinoma b TC-6 cells, which express endogenous mPDX-1, were (co)transfected with human and mouse notch1 intracellular domain (hNICD, mNICD), respectively, verified by Western blot. Proliferation of both cells lines was determined by MTS assay. Glucose-stimulated insulin secretion (GSIS) of b TC-6-mNICD cells was determined by ELISA assay. Beta cell-specific overexpressing NICD (bNICD+/+) mice were generated by breeding LSL-ROSANICD and rat insulin promoter-CRE mice; genotype was verified using PCR. Serial glucose and insulin levels were measured and IPGTT was performed in 6 mo old mice. IHC for insulin, PDX-1 and cell cycle proteins was performed on islets of bNICD+/+ mice, appropriate control litters and human pancreatic neuroendocrine tumor specimens (hPNT; n¼34). Islet cell apoptosis was determined via TUNEL assay. Results: 88% and 100% of hPNT strongly expressed Notch1 and PDX-1, respectively (figure). HEK293-PDX-1-hNICD and b TC-6-mNICD cells had increased PDX-1 expression and proliferation (both p<0.05 vs controls). b TC-6-mNICD cells had 2 fold increase in GSIS (p<0.05 vs controls). bNICD+/+ mice developed islet neoplasia: 1. glucose levels of 87mg/L vs 122mg/L and insulin levels of 3.5ng/L vs 1.3ng/L vs controls (p<0.05 for both); 2. IPGTT revealed significantly decreased glucose levels at each time point, 3. islets demonstrated enlargement of islets size, increased PDX-1 and insulin expression, disruption of cell cycle proteins and reduced islet cell apoptosis vs controls (Figure). Conclusions: Notch1 and PDX-1 were over-expressed in hPNT, suggesting a synergistic relationship in human tumors. Cell lines transfected with NICD demonstrated islet neoplasia with increased PDX-1, proliferation and GSIS. bNICD+/+ transgenic mice developed islet neoplasia associated with increased PDX-1, hyperinsulinemia and hypoglycemia. These data support the hypothesis that Notch1 up-regulates PDX-1 and synergistically contributes to the development of islet neoplasia.
FIG. 1. Expression of Notch1 is associated with overexpression of PDX-1 in human pancreatic neuroendocrine tumor specimens (hPNT) and islets of bNlcDþ/þtransgenic mice. 40.9. Enhanced Growth of Syngeneic Pancreatic Cancer in CXCR2 Deficient Mice. G. W. Donald,1 M. Assifi,2 A. Moro,1 M. Chen,1 G. Eibl,1 H. A. Reber,1 D. Dawson,3 V. G. Li,4 J. HInes O.1; 1Department of Surgery, UCLA Center for Excellence in Pancreatic Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Department of Surgery, Jefferson Medical College, Philadelphia, PA; 3Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA; 4Department of Biostatistics, UCLA School of Public Health, Los Angeles, CA Introduction: Evidence in our laboratory has identified the CXC chemokine group as potent mediators of disordered growth and angiogenesis in pancreatic cancer. Clinically, the overexpression of these ligands predicts survival of pancreatic cancer patients following resection. These observations appear to be mediated through the CXCR2 receptor. We hypothesized that pancreatic cancer growth is dependent
310
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
upon CXCR2 expression. Utilizing a CXCR2 knockout strategy, we investigated this hypothesis in a syngeneic, preclinical model of pancreatic cancer. Methods: CXC ligand expression during pancreatic carcinogenesis was evaluated by immunohistochemistry utilizing the C57BL/6 PDX1-Cre; LSL-KRASG12D murine model of pancreatic cancer. Pancreatic cancer cells (mKRAS) derived from these transgenic animals were cultured and chemokine expression determined by ELISA. C57BL/6 CXCR2 +/- mice were bred and genotyping confirmed from newborn mice tail samples. Mice between the ages of 4 and 8 weeks were injected subcutaneously with 2x106mKRAS cells. the tumors were allowed to grow for four weeks after which time the mice were sacrificed and the tumors harvested. the tumor samples were measured and analyzed following H&E staining. Descriptive statistics such as mean and standard error (SE) were summarized for all variables. P values were calculated with two-tailed t-test. Results: PDX1-Cre; LSL-KRASG12Dmice were sacrificed at 4, 7, and 10 months. Successful excision-recombination eventswere confirmed by the presence of a single LoxP site in thepancreas. Immunohistochemistry for the CXCL5[LIX] revealed that LIX was absent from normal ductal epithelium, but progressively expressed in mPanIN lesions. Analysis of cell supernatant from cultured mKRAS cells confirmed CXCL5 expression in vitro (6,7726101 pg/ml). CXCR2+/- breeding produced only one CXCR2 -/- pup and no tumor growth was found in this animal.Tumors were larger in the CXCR2+/- compared to CXCR2+/+ animals (104.9629.9 vs 35.17644.75 mm3, p<0.07). Gross histological appearance showed no difference in central tumor necrosis, mitotic activity, or in-growth of blood vessels. Conclusions: Initial data indicate that the transgenic mouse model of pancreatic cancer, PDX1-Cre; LSL-KRASG12D, express CXCL5 as has been observed in human pancreatic cancer. Cancer cells derived from this model establish a more aggressive phenotype when grown in a CXCR2+/- background since these tumors were more than twice the size of those in wildtype mice. These findings may be the result of altered immunity and diminished surveillance known to exist in CXCR2 transgenic animals. Given these findings the CXCL:CXCR2 biologic axis may have disparate activity during pancreatic cancer initiation and promotion. 40.10. Axl Receptor Tyrosine Kinase is Essential to Pancreatic Tumor Progression and Metastases. A. R. Kirane,1 M. T. Dellinger,1 K. T. Ostapoff,1 J. E. Toombs,1 R. E. Schwarz,1 J. B. Lorens,2 R. A. Brekken1; 1Department of Surgery, Division of Surgical Oncology, Hamon Center for Cancer Therapeutics, UT Southwestern Medical Center, Dallas, TX; 2Deparent of Biomedicine Univeristy of Bergen, Bergen, Hordaland Introduction: Expression of the receptor tyrosine kinase Axl in pancreatic cancer is associated with increased metastasis and reduced survival. Axl participates in angiogenesis, proliferation, invasion and transformation, but the precise function of Axl in pancreatic cancer is ill-defined. in this study, we investigate the therapeutic benefit of inhibiting Axl or its ligand, Gas6. Methods: Mia PaCa2 cells stably expressing a shRNA targeting Axl were generated and validated (1132 cells). 1132 cells were injected orthotopically into SCID mice and growth was compared to control cells. Primary tumor size and metastatic burden were measured at sacrifice and Axl expression was evaluated by qPCR and IHC. Warfarin, an inhibitor of Gas6, was used to evaluate the efficacy of targeting the Axl ligand. Three cell lines, Panc-1 and AsPC-1 (Axl+) and Axl non-expressing Capan-1 were evaluated for in vitro response to warfarin by MTS, migration, and anchorage independent growth assays. These lines were then implanted orthotopically into SCID mice. in addition, Pan02 murine cancer cells were implanted into C57BL/J6 mice. Subtherapeutic warfarin was initiated on post-implantation day 7. Inhibition of Axl signaling was determined in tumor lysates by Western blot analysis. Results: 58 days post implantation, mice bearing 1132 tumors had no discernable tumor burden while control mice were significantly moribund. By day 72 half of the 1132 animals had developed tumors approaching control size. IHC and qPCR demonstrated that 1132 tumors that grew showed
high levels of Axl expression while, tumors that failed to progress showed no Axl expression. Metastatic incidence was significantly lower for all 1132 tumor bearing animals. in vitro, warfarin treatment sensitized cells to gemcitabine by MTS assay proportional to Axl expression. Furthermore, Panc-1 cells demonstrated dramatic inhibition of migration and growth in soft agar with warfarin treatment, AsPC-1 had a moderate response and Capan-1 cells were unaffected. in vivo, Panc-1 and AsPC-1 tumors were of smaller average weight in warfarin treated animals, but this finding was not significant. However, metastatic incidence was decreased profoundly with almost no metastases found in warfarin treated animals (p<0.05). Warfarin had no affect on Capan-1 tumors. Additionally, Pan02 tumors were significantly smaller (p<0.05) and, notably, non-metastatic in warfarin treated animals. Analysis of Axl signaling in Panc-1 tumor lysates demonstrated that inhibition of Gas6 with warfarin resulted in decreased phosphorylation of Akt and Erk as well as increased Parp cleavage. Conclusions: We conclude that Axl is important in the progression of pancreatic cancer. Inhibition of Axl signaling with subtherapeutic warfarin resulted in a reduction of metastases for Axl expressing tumors and sensitized cells to gemcitabine in vitro. This finding warrants further evaluation of Axl as a novel target and strategy to augment chemotherapy for pancreatic cancer.
ONCOLOGY 7: TUMOR BIOLOGY 41.1. Epithelial Sarcomas: Experience From a Single Institution. A. A. Guzzetta,1 E. A. Montgomery,1 P. P. Emmanouil,1 T. Fu,1,2 N. Ahuja1; 1Johns Hopkins University, Baltimore, MD; 2Daping Hospital, Third Military Medical University, Chongqing, Yuzhong District Introduction: Epithelioid sarcomas are rare soft tissue sarcomas of unknown histiogenesis. the clinical behavior and response to treatment are poorly described in the literature due to the rarity of this disease. Methods: The centralized cancer registry and the pathology archives of this institution were queried and 22 patients were diagnosed with epithelioid sarcomas from 1980 to present. Two patients had inadequate information for analysis and were excluded. the charts of all patients were reviewed and histology was confirmed by re-review by a pathologist with expertise in sarcomas. This study was performed with IRB approval. Results: The 20 patients diagnosed with epithelioid sarcoma were examined. Mean age at diagnosis was 31 years and 75% of patients were male. 95% of patients were treated with surgery and 10% of patients were found to have metastasis to regional lymph nodes. of patients treated with surgery at this institution, 73% underwent R0 resection, 18% underwent R1 resection and 9% underwent R2 resection. Overall survival and recurrence free survival trended with margin status but was not significant (p>0.05). 5-year survival was 86% and median overall survival was 56.4 months. 60% of patients received adjuvant therapy. 55% of patients recurred at a mean of 35 months, regardless of chemotherapy or radiation (p>0.05). 35% of all patients recurred two or more times and the maximum number of recurrences was 5 though the number of recurrences did not affect survival (p>0.05). Patients with at least one recurrence demonstrated a nonsignificant improvement in survival (figure 1). Patients with 2 or more recurrences benefitted from continued resection with a median overall survival of 58 months. Location of recurrence, including lung metastasis was not associated with worse prognosis (p>0.05). Conclusions: Our study reveals that patients with epithelioid sarcomas can have long-term survival following surgical resection. Neither radiation nor chemotherapy affect outcome or recurrence. Importantly, patients with multiple recurrences continued to benefit from aggressive surgical treatment. Unlike prior reports, patients with lung metastasis did not have worse outcomes in our study. Epitheloid sarcomas are unique as they metastasize to regional nodal basins and all patients undergoing wide local excision should undergo simultaneous exploration of local lymph nodes.