219 RAS Mutation Status Impacts Survival in Patients Undergoing Repeat Hepatectomy for Colorectal Liver Metastases

218 Inhibition of Pancreatic Cancer Cell and Stroma Growth With a Downstream Hedgehog Antagonist Kim C. Honselmann, Moritz F. Pross, Ulrich F. Wellner, Hryhoriy Lapshyn, Ekaterina Petrova, Tobias Keck, Dirk Bausch Introduction/Background Pancreatic cancer is still a dismal disease today. Advances on effective chemotherapeutic agents have not led to significant clinical changes in survival yet. This is mostly due to a highly desmoplastic stroma, which seems to have different functions for the tumor, one being to promote tumor growth and chemo resistance. Paracrine Hedgehog (Hh) signaling is one key regulator of the tumor stroma, but not the cancer cells. This is why upstream Hh inhibition has not proven to be an effective chemotherapeutic agent against pancreatic cancer. But there is growing evidence for a non-canonical Hh-pathway in the tumor cells themselves. Therefore the aim of this study was to find out, if direct inhibition of the transcriptional factor GLI, a downstream Hh effector, harms pancreatic tumor cells and the tumor stroma, especially in comparison to inhibition of the upstream factor SHH. Methods Five pancreatic cancer cell lines were treated with the synthetic GLI inhibitor GANT-61 and assessed for viability in vitro. In the following in vivo experiments, we employed Panc1 or HPAF2 cells into a subcutaneous mouse model with athymic female nu/nu mice and treated them either with PBS, GANT-61 (40 mg/kg body weight) or 5E1 Antibody (300ug per mouse/per day) three times a week for 21 days. Tumor size was measured twice a week and tumors were harvested on day 21 for Immunohistochemistry and RNA-isolation. RT-PCR was done to evaluate the effect of GANT-61 and a hedgehog antibody 5E1 on the Hedgehog pathway genes in the stroma (mouse) and the tumor cells (human). Results Tumor cell viability in all cell lines was reduced by up to 94.1% under treatment with GANT-61 in a concentration dependent manner. Tumor size was reduced in mice with Panc1 tumors treated with Gant 61 (69%) and 5E1 (48%) for 21 days (Fig 1). Tumors in mice with HPAF2 tumors were reduced by 64% (GANT-61) and by 85% (5E1) (Fig 1). Growth inhibition was most likely due to Hh pathway gene expression seen in both the tumor and stroma cells in GANT-61 treated mice. 5E1 demonstrated complete inhibition of the Hh pathway in the stroma but not in the tumor cells (Fig.2) Discussion/ Conclusion The Gli-antagonist GANT-61 was able to inhibit tumor growth by up to 69% in vivo. This effect was due to tumor cell and stroma Gli inhibition. Gant 61 could possibly be a new effective therapeutic agent in the fight against pancreatic cancer as it targets both the tumor stroma and the cancer cells. Proteomic analyses in order to find out the Gli effectors are underway. 220

SSAT Abstracts

Intratumoral CD3+ and Nkp46+ Cells Protect Against Tumor Progression in Resected Colorectal Liver Metastases Treated With Neoadjuvant Chemotherapy Matteo Maria Cimino, Matteo Donadon, Kelly Hudspeth, Luca Di Tommaso, Max Preti, Massimo Roncalli, Domenico Mavilio, Guido Torzilli Background. Chemotheraphy (CHT) response is one of the most important prognostic factor in the oncosurgical approach to colorectal liver metastases CRLM. Several tumoral and nontumoral factors, such as the innate immune system (IIS) may play an important role in chemosensitivity. The aim of this study was to investigate the role of the IIS in a setting of patient affected by CRLM treated with neoadjuvant CHT and liver resection considering pathological features and oncological long-term outcome. Methods: In 121 patients undergoing liver resection for CRLM. between 2005 and 2013 preoperatively treated with standard CHT with or without biological agents we tested immunoreactivity to CD3+ and NKp46+ cells inside the tumor, at the border between the tumor and the normal liver, and inside the normal liver with computer-assisted image analyses. The relationship between IIS, chemotherapy response rate, and long-term survival were investigated. Results: At univariate analysis T1/T2- and N0-status of the primary tumor, metachronous CLM, the radiological response, and the higher density of intratumoral CD3+ (>1%) and of NKp46+ (mean>1) were found to be significantly associated with prolonged survival, but only intratumoral CD3+ (>1%) and NKp46+ (mean>1) were significant on multivariate analysis (P=0.005 and P=0.004 respectively). On logistic regression analysis the metachronous CLM (OR=2.781; P=0.002), the use of irinotecan and cetuximab (OR= 3.891; P=0.001) and the radiological response (OR=3.219; P=0.001) were found to be associated with increasing density of intratumoral CD3+ and NKp46+ cells. No significant associations were found with CLM number or size, CEA, or number of CHT courses. Combining the intratumoral CD3+ and NKp46+ cells density we defined four stages of survival (P=0.003): patients presenting intratumoral CD3+ (>1%) and NKp46+ (mean>1) had 100% overall survival at 5 years. Conclusions: Intratumoral immunoreactivity to CD3+ and NKp46+ cells seems to play an important role in CHT response rate. Increased intratumoral density of IIS cells seems to improve overall Survival. Some chemotherapy regimens may be more effective to enhance intratumoral CD3+ an NKp46+ migration. Further external validations are required to confirm our promising findings. Survival by intratumoral CD3+ and NK cells density.

219 RAS Mutation Status Impacts Survival in Patients Undergoing Repeat Hepatectomy for Colorectal Liver Metastases Jason Denbo, Guillaume Passot, Yun Shin Chun, Suguru Yamashita, Scott Kopetz, Dipen Maru, Kristoffer W. Brudvik, Steven Wei, Claudius Conrad, Thomas Aloia, Jean-Nicolas Vauthey Background: Repeat hepatectomy for recurrent colorectal liver metastases (CLM) is safe and can provide long-term survival. In patients who undergo hepatectomy for CLM, RAS mutation status has been shown to impact recurrence and survival. Objective: To evaluate the impact of RAS mutation status in patients undergoing repeat hepatectomy for CLM. Methods: Identified all patients who underwent hepatectomy with known RAS mutation status for CLM between January 2005 and November 2014. Then, we compared the outcomes of patients who underwent repeat curative-intent hepatectomy with respect to their RAS mutation status, and performed multivariate analysis for predictors of recurrence free survival (RFS) and overall survival (OS). Results: RAS mutation status was known for 922 patients who underwent hepatectomy. Ninety-nine of these patients underwent repeat hepatectomy, and 33 (33%) harbored a RAS mutation. The RAS wild type (WT) and mutant groups had similar clinicopathologic characteristics. Recurrence-free survival (RFS) was not different for RAS WT and mutant groups, 8.5 months and 7 months, respectively (p=0.683). Median overall survival (OS) was 62.5 months for the RAS WT group, compared to 39.3 months for the mutant group, (p=0.028). On multivariate regression analysis, only tumor size >3cm [hazard ratio (HR)=2.0, p=0.009] was associated with worse RFS, while RAS mutation (HR= 2.0, p=0.046) and tumor size >3cm (HR=2.5, p=0.018) predicted worse OS, and major hepatectomy (HR=0.36, p=0.032) improved OS. Conclusion: RAS mutations and large tumor size were associated with worse OS following repeat hepatectomy, thus consideration should be given to RAS mutation status preoperatively.

SSAT Abstracts

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