James M. Lindberg

A thirteen-gene expression signature predicts survival of patients with pancreatic cancer and identifies new genes of interest.

Background Currently, prognostication for pancreatic ductal adenocarcinoma (PDAC) is based upon a coarse clinical staging system. Thus, more accurate prognostic tests are needed for PDAC patients to aid treatment decisions. Methods and Findings Affymetrix gene expression profiling was carried out on 15 human PDAC tumors and from the data we identified a 13-gene expression signature (risk score) that correlated with patient survival. The gene expression risk score was then independently validated using published gene expression data and survival data for an additional 101 patients with pancreatic cancer. Patients with high-risk scores had significantly higher risk of death compared to patients with low-risk scores (HR 2.27, p = 0.002). When the 13-gene score was combined with lymph node status the risk-score further discriminated the length of patient survival time (p<0.001). Patients with a high-risk score had poor survival independent of nodal status; however, nodal status increased predictability for survival in patients with a low-risk gene signature score (low-risk N1 vs. low-risk N0: HR = 2.0, p = 0.002). While AJCC stage correlated with patient survival (p = 0.03), the 13-gene score was superior at predicting survival. Of the 13 genes comprising the predictive model, four have been shown to be important in PDAC, six are unreported in PDAC but important in other cancers, and three are unreported in any cancer. Conclusions We identified a 13-gene expression signature that predicts survival of PDAC patients and could prove useful for making treatment decisions. This risk score should be evaluated prospectively in clinical trials for prognostication and for predicting response to chemotherapy. Investigation of new genes identified in our model may lead to novel therapeutic targets.

Abstract C19: The role of resident liver macrophages in suppressing the progression of hepatic micrometastases from pancreatic ductal adenocarcinoma

Background: The majority of patients with localized pancreatic ductal adenocarcinoma (PDAC) die from metastatic disease, typically of the liver, despite a margin-negative (R0) resection. Therefore, these patients likely harbor occult metastatic disease in the liver at the time of surgery. In this study, we evaluated the role of resident liver macrophages in suppressing the progression of hepatic micrometastases using a murine model of micrometastatic PDAC with patient-derived xenografts (PDXs). Methods and Results: Low-passage, patient-derived KRAS-mutant tumor cells expressing firefly luciferase were injected into the spleens of athymic nude mice resulting in liver metastases, followed by splenectomy to remove the primary tumor. Hepatic tumor burden and metastatic growth kinetics were evaluated by bioluminescent imaging on post-injection days 1, 2, 3, 7, and weekly thereafter. Each of the PDX tumors exhibited a decline in tumor burden over the initial days after injection followed by a period of quiescence with a reproducible, PDX-specific time to proliferative outgrowth ranging from 15 days to greater than 200 days. To assess the role of apoptosis in initial tumor cell clearance and suppression of outgrowth, mouse liver preparations were analyzed for expression of cleaved caspase-3 and cleaved PARP in tumor cells using flow cytometry. There was low expression of both apoptosis markers, suggesting that apoptosis is not a major cell-clearance mechanism. Athymic nude mice lack cell-mediated immunity but have functioning innate immunity. Because there is a large population of resident macrophages in the liver, we hypothesized that macrophages play an important role in the clearance and suppression of hepatic PDAC metastases. To test this, hepatic metastasis outgrowth was assessed following macrophage ablation with liposomal clodronate treatment of mice 48 hours prior to tumor cell injection. Following macrophage ablation, there was a trend toward less robust initial tumor cell clearance and a significantly decreased time to proliferative outgrowth compared with control (13 days vs 26 days, p =0.039). HE PDX 366: 40.4% vs 76.3% clearance at seven days, p =0.024). Average relative hepatic bioluminescence at 21 days was significantly increased for PDX 608 in the NSG mice (14.7 vs 0.355, p =0.014) and there was a trend toward increase for PDX 366 (3.07 vs 0.025, p =0.065). Conclusions: In a preclinical model of hepatic micrometastatic PDAC, resident liver macrophages are implicated in the initial clearance and the suppression of proliferation of tumor cells. Further investigation of the interaction of resident hepatic macrophages and micrometastatic PDAC cells may lead to novel strategies for therapy. Citation Format: Alex D. Michaels, Timothy E. Newhook, James M. Lindberg, Sara J. Adair, Sarbajeet Nagdas, Matthew G. Mullen, Edward B. Stelow, J. Thomas Parsons, Todd W. Bauer. The role of resident liver macrophages in suppressing the progression of hepatic micrometastases from pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C19.

Genomic profiling of intrahepatic cholangiocarcinoma: Refining prognostic determinants and identifying therapeutic targets.

210 Background: The molecular alterations that drive tumorigenesis in intrahepatic cholangiocarcinoma (ICC) remain poorly defined. We sought to define the incidence and prognostic significance of mutations associated with ICC among patients undergoing surgical resection. Methods: 138 patients who underwent resection at 6 centers in the United States and Europe were included in the cohort. Mutational profiling was performed using nucleic acids that were extracted from resected ICC tumor specimens; mutations were identified using a multiplexed mutational profiling platform. The frequency of mutations was ascertained and the impact on outcome determined. Results: Most patients had a solitary tumor (82%) and median tumor size was 6.0cm. Most patients had R0 resection (89%); 19% patients had N1 disease, while 15% had microscopic vascular invasion. A minority received adjuvant therapy (30%). The majority (55%) of patients had no genetic mutation identified. Among the 62 (45%) patients with a genetic mutation, o...

Mo1612 Morbidity and Mortality of Hepatectomy for Benign Liver Tumors

S A T A b st ra ct s factors in patients undergoing hepatectomy with NCNELM and establish appropriate patients selection [Methods]One hundred and sixty one patients who underwent liver resection for NCNELM between January 1990 and December 2009 at a single institution were reviewed retrospectively. Patient demographics, primary disease, tumor characteristics, treatment, and postoperative course were analyzed. [Results]Patient age ranged from 17-81 years, with a median age of 58. There were 64 women (40%) and 97 men (60%). Liver metastases were synchronous in 43 of 161 (27%). Hepatectomy was performed for liver metastases from gastric carcinoma (n=59; 36.6%), genitourinary tumors (n=32; 19.9%), and soft tissue primary tumors (n=28; 17.4%). There are four patients from breast cancer and two patients from melanoma. Overall 5-year and 10-year survival rates were 36% and 27%, respectively. The average follow-up period was 47.4 months (2-260 months). Five-year survival in patients with liver metastases from gastric carcinoma, genitourinary tumors, soft tissue tumors, and others were 25%, 55%, 30%, and 29%, respectively. Genitourinary tumors had a best survival with a significant difference. (p=0.05) Five-year survival for synchronous andmetachronous liver metastases was 36% and 34%, respectively. There was no significant difference between both of them. There were 19 patients who underwent repeat hepatectomies in this study. Most of the patient (n=8) were liver metastases from soft tissue tumors of gastrointestinal tract. There is a patient who underwent 4 times hepatectomies for liver metastasis from gastro-intestinal stromal tumor of stomach (alive at 118 months from the primary tumor surgery). [Conclusions]NCNELM could be resected safely and with accepted survival outcomes in the selected patients. Hepatectomy for genitourinary tumors metastases might be beneficial among the patients with NCNELM. It is important to consider biological behavior of the primary disease and to confirm neither rapid tumor growth nor increase in tumor numbers within a short time interval in appropriate patient selection.

Abstract 808: Combination therapy with a MEK inhibitor plus T-type calcium channel inhibitor is highly effective in patient-derived pancreatic ductal adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and novel therapeutic strategies are needed. Mutations in the KRAS oncogene are important drivers of PDAC progression; however, monotherapies targeting the RAS pathway have shown only modest efficacy. The RAS pathway is an activator of calcium (Ca2+) signaling, which has been implicated in PDAC progression. We hypothesized that MEK inhibition combined with the T-type calcium channel inhibitor mibefradil would result in enhanced growth inhibition of PDAC tumors. Methods and Results: Gene expression profiling of patient-derived PDAC tumors (relative to normal pancreas) and tumors chronically treated with the MEK inhibitor trametinib in vivo revealed an upregulation of Ca2+-signaling related genes including members of the calpain pathway and the calmodulin pathway, both implicated in PDAC progression. To assess the in vivo efficacy of combined T-type calcium channel inhibition and MEK inhibition, mice were engrafted orthotopically with patient-derived KRAS-mutant (Tumor 366) and KRAS-wild type (Tumor 738) PDAC tumors and treated with control, the MEK inhibitor trametinib, the T-type calcium channel inhibitor mibefradil, or combination. Combination therapy with trametinib plus mibefradil was highly effective with greater inhibition of PDAC growth than either therapy alone in the KRAS-mutant Tumor 366, however mibefradil did not augment the level of growth inhibition achieved by trametinib alone in the KRAS-wild type Tumor 738. To evaluate combination trametinib plus mibefradil therapy in patient-derived PDAC tumors following chronic treatment with trametinib, mice were engrafted orthotopically with patient-derived PDAC tumors, allowed to grow to 300-500mm3, and treated with trametinib for 4-6 weeks. Following initial treatment, tumors were harvested, re-implanted, and again exposed to therapy over 4-6 weeks. The cycles of treatment and reimplantation were continued until tumor growth was not significantly reduced by trametinib treatment. Therapy with trametinib plus mibefradil nearly completely inhibited growth in KRAS-mutant PDAC tumors (Tumors 608 and 366) previously resistant to chronic trametinib therapy. Conclusions: Combination therapy with the MEK inhibitor trametinib plus the T-type calcium channel inhibitor mibefradil results in significant growth inhibition of KRAS-mutant patient-derived PDAC tumors. Moreover, this combination therapy results in near complete growth inhibition of PDAC tumors that have acquired resistance to trametinib. The combination therapy of mibefradil plus trametinib should be further evaluated in clinical trials for patients with PDAC. Citation Format: Timothy Eric Newhook, James M. Lindberg, Sara J. Adair, Edik Blais, Jason Papin, Lloyd Gray, J. Thomas Parsons, Todd W. Bauer. Combination therapy with a MEK inhibitor plus T-type calcium channel inhibitor is highly effective in patient-derived pancreatic ductal adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 808. doi:10.1158/1538-7445.AM2014-808

Abstract 4040: The MEK inhibitor trametinib delays tumor outgrowth and prolongs survival in a patient-derived mouse model of occult hepatic metastatic pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and the majority of patients succumb to metastatic disease. Even for those with localized PDAC, most will die from metastatic disease despite margin-negative resection and adjuvant therapy. Therefore, these patients must harbor occult metastatic PDAC at presentation. We have developed a PDAC model of occult liver metastases using patient-derived xenografts (PDXs) to study the growth of PDAC within the metastatic microenvironment of the liver and evaluated the role of KRAS-MEK-ERK signaling on tumor progression. Methods and Results: Extensively characterized low passage, patient-derived KRAS-mutant (Tumors 608, 366, and 654) and wild-type (Tumors 738 and 215) PDAC cells expressing luciferase were injected into the spleens of athymic, nude mice and allowed to circulate for 10 minutes, after which a splenectomy was performed. To evaluate metastatic cell growth kinetics in the liver, tumor burden was monitored by sequential bioluminescent imaging. Each of the PDX tumors exhibited a characteristic and reproducible time to proliferative outgrowth ranging from 20 days (Tumor 608) to greater than 100 days (Tumor 654). To evaluate the role of KRAS signaling in maintaining dormant cell survival and proliferative outgrowth, tumor 608 cells were injected and mice were treated with the MEK inhibitor trametinib (0.3 mg/kg, daily) or control beginning 48 hours post-injection. Trametinib significantly reduced metastatic tumor burden, delayed time to proliferative outgrowth, and greatly prolonged survival as compared to control (med. survival: 114 vs. 43 days, p<0.001). In contrast, in an orthotopic model with 250-500 mm3 tumors trametinib led to limited inhibition in tumor growth for Tumor 608. To characterize these PDAC cells, we isolated Tumor 608 cells from the liver 48 and 72 hours, 10 and 28 days after splenic injection using magnetic column separation with human EpCAM (CD326)-targeted magnetically labeled microbeads. Flow cytometric analyses of retrieved cells revealed that decreased cellular markers of proliferation and increased PARP cleavage correlated with decreased tumor burden observed at these timepoints in mice treated with trametinib. Conclusions: Using a model of occult liver metastatic PDAC, patient-derived tumors exhibited characteristic, albeit different growth kinetics in the liver microenvironment. Further, inhibition of KRAS-MEK-ERK signaling with the MEK inhibitor trametinib decreased metastatic cellular proliferation, increased apoptosis, prolonged metastatic tumor outgrowth, and significantly increased survival. Further investigation into factors promoting PDAC cell survival within the hepatic microenvironment will lead to development of rational therapeutic strategies for patients with occult metastatic PDAC. Citation Format: Timothy Eric Newhook, James M. Lindberg, Sara J. Adair, J. Thomas Parsons, Todd W. Bauer. The MEK inhibitor trametinib delays tumor outgrowth and prolongs survival in a patient-derived mouse model of occult hepatic metastatic pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4040. doi:10.1158/1538-7445.AM2014-4040

Preoperative bowel preparation for pancreaticoduodenectomy: Is it necessary?

279 Background: Preoperative bowel preparation (PBP) before pancreaticoduodenectomy (PD) is commonly performed; however, the effect of PBP on intraoperative fluid requirements and acute renal failu...

Targeting occult metastatic disease: A hematogenously derived xenograft model of human pancreatic tumor growth in the murine liver.

198 Background: Most pancreatic cancer patients will die following surgery due to recurrent metastatic disease. Thus, better systemic therapies are needed to treat occult metastases to improve survival. We have developed a model of occult liver metastasis from pancreatic cancer in order to evaluate novel treatment strategies. Methods: Pancreatic cancer cells (MAD 09-366, 08-608, MPanc96) transduced with green fluorescent protein (GFP) and luciferase were injected into the spleens of athymic, nude mice to generate hepatic metastases. Ninety-six hours after injection, tumor-bearing mice were treated with MEK1/2 inhibitor (trametinib, 0.3mg/kg, daily), gemcitabine (100mg/kg, twice weekly), or vehicle control. Sequential bioluminescence imaging, flow cytometry, and histologic evaluation were used to assess hepatic tumor growth and behavior. Results: All injected cell lines generated hepatic metastases. Different cell lines exhibited different growth kinetics. MPanc96 injected mice demonstrated a 64% decrease ...

Abstract 935: Inhibition of pancreatic cancer growth via MEK-targeted therapy is significantly enhanced by concomitant inhibition of EGFR and Her2.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: MAPK and EGFR family signaling promote tumor proliferation in pancreatic ductal adenocarcinoma (PDAC). Previously we have shown that lapatinib (EGFR/Her2 inhibitor) significantly improved the ability of trametinib (MEK1/2 inhibitor) to inhibit PDAC tumor growth. Here, we investigated whether combined panitumumab (EGFR inhibitor), trastuzumab (Her2 inhibitor) and trametinib treatment would more effectively inhibit tumor growth than trametinib monotherapy in a patient-derived (PD), orthotopic, xenograft model of PDAC. Methods: In vitro proliferation assays were performed with PD-PDAC cell line MAD09-366 exposed to trametinib, panitumumab, trastuzumab, and combination therapies. Western blot analysis was performed on treated cell lysates. Athymic, nude mice were orthotopically implanted with 3 different PD-PDAC xenografts (MAD09-366, 08-608, and 08-738). Established murine tumors were treated with control, trametinib (0.3mg/kg,PO, qDay), panitumumab (500ug, IP, BIW), trastuzumab (200ug, IP, BIW) or combination for 2-4 weeks. Interval volumetric MRI was used to measure tumor growth. Phospho-RTK and MAPK arrays were used to assess tumor molecular response. Results: In vitro studies demonstrated improved growth inhibition of MAD09-366 (Kras mut.) cells exposed to combination therapy with all 3 inhibitors relative to control or each inhibitor alone. Western blot analysis revealed that EGF stimulation increased Ras pathway signaling in this Kras mutant cell line. Under conditions of EGF stimulation, the greatest inhibition of Ras pathway signaling was seen in cells exposed to all 3 inhibitors. In vivo studies in all PD-PDAC xenografts revealed that triple inhibitor therapy significantly decreased the rate of tumor growth relative to control, trametinib alone, panitumumab alone, or panitumumab plus trastuzumab. In two of three PD-PDACs assessed, triple therapy was superior to trametinib plus panitumumab. The greatest response was seen in MAD08-738 (Kras wt) triple-therapy-treated mice whose tumor size decreased by 9.3%. Phospho-array analysis demonstrated increased activity of Akt and its downstream effectors in trametinib treated tumors. Relative Akt phosphorylation returned to levels seen in control treated tumors with the addition of panitumumab and trastuzumab under triple therapy. Conclusions: Combination therapy with trametinib, panitumumab, and trastuzumab produced the most complete in vitro Ras signaling blockade and most effective in vivo growth inhibition. Phospho-array data suggests that a compensatory increase in pro-survival PI3K/Akt signaling in response to Ras pathway blockade alone is mitigated by the concomitant inhibition of EGFR/Her2 signaling. This combination treatment strategy should be considered for a future clinical trial in pancreatic cancer patients. Citation Format: James M. Lindberg, Timothy E. Newhook, Sara J. Adair, Clifford A. Cutchins, Alison Kim, J. Thomas Parsons, Todd W. Bauer. Inhibition of pancreatic cancer growth via MEK-targeted therapy is significantly enhanced by concomitant inhibition of EGFR and Her2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 935. doi:10.1158/1538-7445.AM2013-935

Acquired resistance to combination therapy with lapatinib and MEK 1/2 inhibitor GSK1120212 in an in vivo murine model of pancreatic cancer.

208 Background: Mutations of the oncogene KRAS and activation of cell-surface receptor tyrosine kinases are important and preserved mechanisms of tumorgenicity in pancreatic cancer. Dual inhibition of the downstream KRAS effector MEK 1/2 and tyrosine kinases EGFR and Her2 results in effective inhibition of patient-derived tumor growth in a murine orthotopic transplantation model. Because combinatorial therapies are moving rapidly into clinical trials, we sought to develop a model of acquired tumor resistance to this combination therapy. METHODS Patient-derived pancreatic tumor xenografts MAD 09-366 (KRAS mut), MAD 08-608 (KRAS mut) and MAD 08-738 (KRAS wt) were implanted orthotopically in nude mice and treated with combination lapatinib (EGFR/Her2 inhibitor) and GSK1120212 (MEK1/2 inhibitor) and tumor volume was measured by MRI. Following 4-6wks of treatment, tumors were reimplanted in second and third generation mice and retreated. Tumors were evaluated by phospho-RTK and phospho-MAPKinase array. RESULTS Acquired resistance developed in all three tumor xenografts. Treated tumors demonstrated a relative volume decrease in the original (F0) generation. All second re-implantation (F2) tumors, demonstrated relative tumor volume increases despite treatment. A comparison of pre-treatment mean tumor volumes showed a significant decrease in tumor size from the F0 to F2 generations suggesting selection for slower growing tumors. Array data demonstrated increased activation of FGFR1, VEGFR1/3, GSK-3β, p38, and Akt in resistant tumors as compared to their pre-treatment controls. These may represent mechanisms of tumor resistance and warrant further investigation. CONCLUSIONS Repeated tumor exposure in vivo to combination treatment with GSK1120212 and lapatinib was used to develop a preclinical, orthotopic murine model of acquired drug resistance in patient-derived pancreatic cancers. This model provides the opportunity to define the mechanism of resistance in an appropriate tumor microenvironment and to develop alternative strategies for treating tumors resistant to this and other emerging therapies.