40.20 Murine radical mastectomy model for adjuvant chemotherapeutic drug development

Posted on January 25, 2017

E. Katsuta1, E. J. Peterson2, S. J. Katner2, N. P. Farrell2, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Virginia Commonwealth University,Department Of Chemistry,Richmond, VA, USA

Introduction:  Despite massive expenditures in drug development for breast cancer, few agents pass clinical trials. Adjuvant chemotherapy is a systemic treatment for a patient after surgical removal of the primary tumor to reduce recurrence and prolong survival. However, vast majority of the preclinical studies for adjuvant therapy evaluate its effect on primary tumors. Lately our group and others have shown that the genetic profiles of metastatic lung tumors are significantly different from that of their primary mammary tumors. Therefore we hypothesized that metastatic/recurring tumors respond to drugs differently from primary tumors, and adjuvant chemotherapy needed to be evaluated by metastatic tumors. To date, there are few reports of murine mastectomy models used for preclinical study.

Methods:  Murine mammary adenocarcinoma 4T1-luc2 cells were inoculated into #2 right mammary fat pad under direct vision. Three groups; two underwent mastectomies; midline incision method and radical Halsted mastectomy, 8 days after inoculation and one with no operation, were treated with novel platinum drug, Triplatin NC, or Vehicle. Tumor burden were quantified using bioluminescence imaging.

Results: First, two methods of mastectomies were compared. There was no significant difference in the weight of resected tumors between these two techniques (p=0.751), however, the bioluminescence in midline incision model was significantly higher than Halsted mastectomy at the first day after operation that suggest remnant disease (p=0.003). By 30 days after operation, all (100%) the animals that underwent midline incision method developed chest tumor, whereas only 1 out of 7 cases (14%) did after Halsted mastectomy (p=0.005). No mice developed respiratory failure with tight wound closure of wide skin defect. We then evaluated the effect of new platinum drug, Triplatin NC, on orthotopic mammary pad implantation model that metastasize to lung, and our Halsted mastectomy model. Total tumor burden of orthotopic model showed higher bioluminescence in treatment group than control group, which implicated failure of the treatment. However, ex vivo bioluminescence of the lung demonstrated that treatment group had significantly less lung metastases than non-treatment group (p=0.025), which suggest that this drug possess efficacy on metastatic lesions, but not the primary tumor. We tested this drug after Halsted mastectomy and found that lung metastases were significantly less in treatment group than non-treatment group where this time we were able to monitor the metastatic growth in live animals by bioluminescence (p=0.038).

Conclusion: We have established a murine radical mastectomy model for evaluation of adjuvant chemotherapy. We found that some agents, such as Triplatin NC, have effect on metastatic tumor, but not on the primary tumor.

40.19 Baicalein and Metformin Synergistically Inhibit Breast Cancer Cell Proliferation

Posted on January 25, 2017

Z. Bostanci2, E. S. Han1, J. Yan2, Q. Xing2, W. Wen2, J. H. Yim2  1City Of Hope National Medical Center,Department Of Surgery, Division Of Gynecologic Oncology,Duarte, CA, USA 2City Of Hope National Medical Center,Department Of Surgery, Division Of Surgical Oncology,Duarte, CA, USA

Introduction:  Baicalein is a flavone with anti-cancer properties found naturally in thyme that upregulates DNA-Damage-Inducible-Transcript 4 (DDIT4) which in turn inhibits the mTOR pathway by acting on the TSC1/2 complex as we have previously demonstrated. Metformin activates AMP-activated protein kinase (AMPK) which also acts on the TSC1/2 complex, resulting in similar anti-cancer properties found with mTOR inhibition. This is the purported mechanism for metformin anti-cancer activity for which there are numerous clinical trials in multiple cancers, including breast cancer, to assess its clinical utility.  Because baicalein and metformin appear to target the TSC1/2 complex by different mechanisms we hypothesized that baicelein and metformin will have synergistic anti-proliferative effects which may allow for enhanced anti-cancer effects in patients.

Methods:  MDA468 (ER-/PR-/Her2-), MDA231(ER-/PR-/Her2-), SKBR3 (ER-/PR-, Her2+) cell lines were treated with vehicle or baicalein only (2.5 µM -40 µM), metformin only (0.625 mM -10 mM) and combinations of baicalein and metformin. Cell proliferation was measured with MTT assay and Chou Talalay Plots were performed depicting synergism at a combination index (CI) below 1. Western Blot was used to assess protein expression of DDIT4, pAMPK and pS6 as direct and downstream targets of baicalein and metformin on the mTOR pathway. 

Results: Cell proliferation decreased in all cell lines when baicalein and metformin were used separately and in combination in a dose dependent manner.  The combination of baicalein and metformin had a synergistic (CI<1) anti-proliferative effect on MDA231 and SKBR3 cells at 72 hours at every dose combination. In MDA468 cells, synergistic effect (CI<1) was present at higher doses at 72 hours. Western blotting in separate experiments with MDA468 cells showed that there was an increase in DDIT4 by baicalein and an increase in pAMPK by metformin, for which the mTOR end target pS6 was markedly more suppressed by the combination than either alone.  

Conclusion: Baicalein is present in the herb thyme and is an active component of the Traditional Asian Medicine (TAM) extract Huang Qin and has been tolerated well. Metformin is an inexpensive medication that has been commonly used to treat diabetes and is being studied in numerous clinical trials as an anti-cancer agent. Our study shows that targeting both AMPK and DDIT4 results in synergistic suppression of breast cancer cell growth and enhanced mTOR inhibition. The combination of baicalein and metformin or other compounds that activate AMPK may offer a therapeutic alternative or an adjunct to current anti-cancer agents in aggressive breast cancers. 

 

40.18 Organotypic Tumor Tissue Slice Cultures: Ex-Vivo Methods for Individualized Cancer Treatment Screening

Posted on January 25, 2017

T. Ivanics1, J. R. Bergquist1, R. P. Graham2, G. Liu1, M. C. Hernandez1, L. Yang1, M. J. Truty1  1Mayo Clinic,Department Of Surgery,Rochester, MN, USA 2Mayo Clinic,Department Of Laboratory Medicine And Pathology,Rochester, MN, USA

Introduction:
Patient-derived xenograft (PDX) models are increasingly being used to study cancer and have been shown to be strong predictors of response to therapy because they accurately recapitulate tumor phenotype and biology. We describe recent efforts to develop and optimize an ex-vivo assay utilizing thin slices of PDX tumors to facilitate rapid high throughput screening of multiple agents prior to formal in-vivo PDX testing.

Methods:
PDX tumors were embedded in 2% agarose and sliced into 100-400μm slices using a vibrating microtome. These tissue slices were then cultured ex-vivo for 5 days utilizing 2 different culture

Methods: Organotypic Teflon inserts (0.4μm pore) and Gelfoam. Media was DMEM with 10%Fetal-Bovine-Serum and 1% Antibiotic and was changed daily. 

Results:
Tissue viability was maximized at 5 days using the 200μm slice thickness although 300μm also showed good viability. The culture method with maximum viability was the Organotypic Teflon  insert (Table). 100μm slices were difficult to handle without offering significant benefit in tissue viability. 400μm slices showed extensive ischemic necrosis. 

Conclusion:

Utilization of a vibrating microtome makes thin slices of tumor easy to create. This data provides encouraging results showing viability of tumor slices in ex-vivo culture for up to 5 days. 200µm slice thickness maintained on organotypic Teflon inserts provides optimal conditions for maintenance of tissue viability. 400μm slices are too thick likely due to greater oxygen diffusion distance. This novel method allows for pre-screening of therapeutic agents prior to formal in-vivo testing and can ultimately be used to individualize treatment approach for cancer patients.

 

40.17 CD73 expression is associated with cancer stem cell genes and poor prognosis of pancreatic carcinoma

Posted on January 25, 2017

E. Katsuta1, S. N. Hochwald2, K. Takabe1  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Gastrointestinal Surgery, Department Of Surgical Oncology,Buffalo, NY, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is known as one of the most aggressive cancers with five year survival less than 10%. This is a reflection of the fact that PDAC is refractory to current treatment and novel targets for therapy are in urgent need. Recently, cancer stem cells and epithelial–mesenchymal transition (EMT) have been proposed to be one of the mechanisms of drug resistance that leads to poor survival. CD73 (5'-nucleotidase) degrades AMP into adenosine that results in the generation of an immunosuppressed and pro-angiogenic tumor microenvironment that promotes the onset and progression of cancer. CD73 is also known as one of the essential mesenchymal stem cell markers. Given these roles, we hypothesized that CD73 expression in PDAC is associated with survival.

Methods:  Clinical and RNA-seq data were all obtained from the Cancer Genome Atlas (TCGA). Of the 170 PDAC cases with tumor RNA-seq, 169 cases were usable for overall (OS) and 130 cases were usable for disease-free survival (DFS) analysis, respectively. Tumors were classified as either high or low expression of CD73 by the mean value. Sex, age T and N factor, histological grade, tumor location and CD73 expression were analyzed to identify patterns of tumor recurrence and factors that predict survival. Cox proportional hazard regression model was used for multivariate analysis.

Results: High expression of CD73 in the tumor was significantly associated with larger tumor size (4.2cm vs 3.6 cm; p=0.021). Interestingly, CD73 highly expressed cases demonstrated significantly worse survival in both OS (median 15.8 months vs 21.4 months; p=0.004) and DFS (median 9.6 months vs 17.3 months; p<0.001). On univariate analysis, residual tumor (R1, 2; p=0.008) and CD73 high expression (p<0.001) had a significant association with worse DFS. Multivariate analysis demonstrated that the independent factors negatively impacting DFS were residual tumor (p=0.045) and CD73 high expression (p=0.011). Lymph node metastasis (p=0.017), residual tumor (p=0.011) and CD73 high expression (p= 0.004) had a significant association with worse OS on univariate analysis. Multivariate analysis demonstrated that residual tumor (p=0.025) and CD73 high expression (p=0.007) were also related with poor OS. Correlation analysis revealed that CD73 expression was correlated with cell cycle related genes such as E2F7 (R = 0.65) and cancer stem cell related genes such as MET (R = 0.63). Only weak correlation was seen with epithelial–mesenchymal transition (EMT) related genes such as SNAI2 (R = 0.42) and TMEM132A (R = 0.42). No correlation was seen between CD73 and immune system nor angiogenesis related genes.

Conclusion: Tumors with high expression of CD73 have larger tumors and worse overall and disease free survival in PDAC. CD73 expression levels correlated with cell cycle related genes and cancer stem cell related genes, but not with immune system or angiogenesis factors. 

40.16 Expression of Phosphorylated Sphingosine Kinase 1 Is Associated with Invasiveness of Gastric Cancer

Posted on January 25, 2017

T. Hanyu1, H. Ichikawa1, T. Ishikawa1, S. Kosugi2, M. Nakajima1, J. Sakata1, T. Kobayashi1, H. Kameyama1, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Uonuma Institute Of Community Medicine Niigata University Medical And Dental Hospital,Department Of Digestive And General Surgery,Minami-Uonuma, NIIGATA, Japan

Introduction:

Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has been implicated as a key regulatory molecule in cancer through its ability to promote cell proliferation, migration, angiogenesis and lymphangiogenesis. Previous studies suggested that S1P produced by sphingosine kinase 1 (SphK1), which is activated by phosphorylation, plays important roles in progression of disease and metastasis. However, the associations between phospho-SphK1 (pSphK1) and clinical parameters in human gastric cancer have not been well investigated to date.

Methods:

We created pSphK1 expression profiles by immunohistochemical analysis for 136 patients, who underwent surgery for gastric cancer at Niigata University Medical and Dental Hospital between January 2007 and December 2009. pSphK1 expression and clinicopathological factors were compared by univariate and multivariate analyses.

Results:

The univariate analysis revealed that pSphK1 expression was significantly correlated with depth of tumor invasion (P = 0.022), lymph node metastasis (P = 0.040), distant metastasis (P = 0.042), histological differentiation (P = 0.048), and lymphatic invasion (P = 0.004). The multivariate analysis revealed that diffuse type (Odds ratio 2.169, 95% confidence interval 1.014–4.630, P = 0.046) and the presence of lymphatic invasion (Odds ratio 3.401, 95% confidence interval 1.451–8.000, P = 0.005) were independently associated with pSphK1 expression in patients with gastric cancer.

Conclusion:

Our results provide the first evidence that pSphK1 expression is associated with invasiveness of human gastric cancer, suggesting importance of S1P in patients with gastric cancer.

40.15 Effects of Intralipid on Serum Partitioning of Cancer-targeting Alkylphosphocholine Analogs

Posted on January 25, 2017

J. A. Lubin1, R. R. Zhang1,2,3, J. P. Weichert2,4, J. S. Kuo1,3,4,5  1University Of Wisconsin,Department Of Neurological Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Radiology,Madison, WI, USA 3University Of Wisconsin,Department Of Cellular And Molecular Biology,Madison, WI, USA 4University Of Wisconsin,Carbone Cancer Center,Madison, WI, USA 5University Of Wisconsin,Department Of Human Oncology,Madison, WI, USA

Introduction: Alkylphosphocholine (APC) analogs are lipophilic small molecules that selectively target a broad spectrum of human cancers. Radioactive iodine-labeled (CLR1404) or fluorescent APC analogs (CLR1501, CLR1502) are progressing to clinical trials for cancer imaging and therapeutic applications. Pharmacologic strategies to shift the serum partitioning of APC agents from the protein-bound layer (predominantly albumin) to the plasma lipoprotein layer may optimize pharmacokinetics, thus improving timing of APC administration with imaging or surgery. Intralipid, an FDA approved essential fat formulation composed primarily of triglycerides that yields free fatty acids following lipolysis, may decrease the amount of albumin-bound APC analogs through competitive binding of fatty acids. This study assesses whether Intralipid increases the lipoprotein concentration in human plasma to modify plasma:albumin partitioning of APC analogs.

 

Methods:  Fresh unfrozen human plasma samples were incubated with APC analogs (125I-CLR1404, CLR1501, CLR1502) and Intralipid and compared to control samples that contained plasma and APC analogs without Intralipid. To measure the drug partitioning in each layer following ultracentrifugation, assays for the percent activity or fluorescence corresponding to labeled APC were performed.

 

Results: Addition of Intralipid increased APC partitioning to the plasma lipoprotein layer from 0.64% to 3.78%, 7.33% to 14.40%, and 6.15% to 11.26% for 125I-CLR1404, CLR1501, and CLR1502 respectively.  Concomitantly, the albumin-binding percentage decreased from 98.07% to 94.48%, 85.93% to 79.32%, and 90.34% to 84.64% with the use of Intralipid (p<0.05).

Conclusion: These data suggest that the APC partitioning to the lipoprotein fraction increases at the expense of partitioning to the albumin-bound fraction. Because lipoprotein-bound drugs have increased bioavailability following high-fat diets, the use of pharmacologic interventions such as Intralipid administration may reduce time required between administration and cancer imaging and/or surgery applications by promoting tumor cell APC uptake. Further optimization of APC pharmacokinetics and pharmacodynamics profiling is in progress.

40.14 FAK Inhibition Decreases Cell Survival and Proliferation in PDX Models of Metastatic Wilms Tumor

Posted on January 25, 2017

J. M. Aye1, S. Mruthyunjayappa2, L. S. Stafman2, E. F. Garner2, J. E. Stewart2, E. Mroczek-Musulman3, K. J. Yoon4, S. L. Cramer1, E. A. Beierle2  1University Of Alabama at Birmingham,Department Of Pediatrics, Division Of Hematology Oncology,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Department Of Pathology,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Department Of Pharmacology And Toxicology,Birmingham, Alabama, USA

Introduction: Wilms tumor is the most common pediatric renal tumor.  The 4-year-relapse-free survival (RFS) rate for patients with non-metastatic, favorable histology Wilms tumor is greater than 90%; however, the 4-year-RFS rate for those with metastatic Wilms tumor is only 70%.  A concentrated research effort is needed for these patients. Limited cell lines are available for the study of metastatic Wilms tumor and long-term passaged cell lines do not always accurately represent the human condition.  We have developed patient-derived xenograft (PDX) models of metastatic Wilms tumor for in vitro and in vivo biology studies and these models were found to recapitulate human tumors.  Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that controls a variety of cellular pathways involved in tumorigenesis.  Inhibition of FAK has been found to decrease cell proliferation and survival in a number of renal tumors, including Wilms tumor.  To date, the role of FAK in metastatic Wilms tumor has not been investigated.

Methods: Cells from two PDXs of metastatic Wilms, COA 16, a lung metastasis, and COA 42, a liver metastasis, were utilized.  Immunohistochemical staining was used to detect FAK protein expression and phosphorylation in the PDXs.  Cells from COA 42 were treated with small molecule FAK inhibitors, PF-573228 and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), for 24 hours at increasing concentrations. Cell viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively. Results were compared with student’s t-test and p≤0.05 was considered significant.      

Results: Immunohistochemical staining confirmed FAK expression and phosphorylation in both COA 16 and COA 42.  Inhibition of FAK with PF-573228 (10 μM) decreased cell viability by 56% and proliferation by 79% in the COA 42 cells compared to untreated control cells.  Y15-induced FAK inhibition similarly resulted in decreased cell survival and proliferation.  Treatment of the COA 42 cells with Y15 (10 μM) diminished cell viability by 58% and proliferation by 33% compared to untreated control cells.

Conclusion: FAK protein is expressed and phosphorylated in human metastatic lung and liver Wilms tumor PDXs.  FAK inhibition with two small molecules led to decreased tumor cell viability and proliferation.  These findings suggest that further exploration of FAK as a target for metastatic Wilms tumor should be undertaken.   
 

40.13 Novel Chimeric Parapoxvirus HOV-189 as an Oncolytic Immunotherapy in Triple-Negative Breast Cancer

Posted on January 25, 2017

A. H. Choi1, M. P. O’Leary1, J. Lu1, S. Kim1, N. G. Chen1,2, Y. Fong1,2  1City Of Hope National Medical Center,Department Of Surgery,Duarte, CA, USA 2Beckman Research Institute,Center For Gene Therapy, Department Of Hematology And Hematopoetic Cell Transplantation,Duarte, CA, USA

Introduction:  Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a higher recurrence rate and poor prognosis. Oncolytic viruses possess a natural selectively for replication and destruction of cancer cells, making oncolytic immunotherapy an attractive treatment modality for a disease such as TNBC that lacks effective targeted therapy. Here we describe a novel chimeric parapoxvirus that efficiently kills TNBC in vitro and in vivo.

Methods:  A novel chimeric parapoxvirus (HOV-189) was generated in our lab and selected as a candidate virus through high-throughput screening. Cytotoxicity of HOV-189 was assayed in vitro in the TNBC cell line MDA-MB-468. Viral replication was examined through standard plaque assay. Orthotopic TNBC xenografts were generated by MDA-MB-468 implantation into the second and fourth mammary fat pads of athymic nude mice. Xenografts were treated with a single intratumoral injection of HOV-189 at 1×103 plaque-forming units (PFU), 1×104 PFU, or 1×105 PFU per tumor. Treatment response was assessed by measuring tumor size every 3 days.

Results: HOV-189 demonstrated dose-dependent cytotoxicity at low multiplicity of infection (MOI), with >90% cell death 6 days after treatment. Interestingly, the virus did not replicate efficiently in this cell line at low MOI. In vivo, statistically significant reductions in tumor size were observed two weeks after intratumoral injection of HOV-189 at doses as low as 1×103 PFU compared to saline-injected control tumors (P<0.01). This reduction in tumor size was sustained six weeks post-treatment without significant signs of viral toxicity.

Conclusion: HOV-189 demonstrated efficient cytotoxicity in vitro and potent anti-tumor effect in vivo at doses as low as 103 PFU. Taken together with its relatively poor in vitro replication, the anti-tumor effect observed in vivo may be secondary to HOV-189’s ability to prime the innate immune system against tumor rather than through direct oncolysis.

 

40.12 A Mechanistic Case for Paclitaxel-Eluting Polymer Films in the Treatment of Sarcoma

Posted on January 25, 2017

C. S. Digesu1, K. J. Hachey1, R. Liu1, C. P. Raut1, M. W. Grinstaff2, Y. L. Colson1  1Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 2Boston University,Department Of Biomedical Engineering,Boston, MA, USA

Introduction: Soft tissue sarcoma is primarily treated with surgical resection, yet despite macroscopically complete resection, locoregional recurrence (LRR) remains high. Traditional radiation and systemic chemotherapy have yielded relatively little benefit. However, our group previously demonstrated that local delivery of paclitaxel (Pax) via drug-eluting polymer films drastically reduces LRR and improves survival in murine models. We investigate the mechanisms of sustained delivery of high concentration Pax leading to these improved outcomes in murine models of human sarcoma.

Methods: To simulate in vivo drug levels obtained via systemic therapy or Pax-eluting polymer films, CS-1 cells derived from human chondrosarcoma, were treated with 10nM or 1000nM Pax via 4hr pulse dose (and subsequent culture in media alone for 26 hours) or 30hr sustained dose. Cells were harvested and analyzed for cell cycle, incorporation of pulse BrdU, and expression of the p53/p21 pathway. To confirm results, Nu/J mice were inoculated with CS-1. Tumors were sliced at 250um intervals using a vibratome and ex vivo tissue slices were cultured at 37o in the above Pax treatment groups. Immunohistochemistry (IHC) was carried out for expression of caspase-3, p21, and incorporation of BrdU.

Results: High concentration 1000nM Pax resulted in suspension of cells in G2/M of the cell cycle with both the 4hr pulse and sustained 30hr treatment; however, only cells treated with 1000nM Pax for 30hrs demonstrated upregulation of p21. Upstream p53 was constitutively expressed. A significant proportion of p21+ cells co-localized to cells in G2/M or a post-mitotic (4N) state (6.4% in control vs. 32.4% 1000nM for 30hrs, Fig 1). Incorporation of BrdU was similar between controls and 10nM Pax, whereas <10% of cells incorporated BrdU with 1000nM 30hr treatment. IHC on cultured ex vivo tissue slices revealed similar rates of caspase-3 and p21 expression among controls, 10nM, and 1000nM Pax at 4hrs; however there was an increase in p21 expression with 1000nM Pax at 30hrs (<10% for controls vs. 25%). A majority of p21+ cells were post-mitotic, multinucleated cells.

Conclusion: Paclitaxel-eluting polymer films locally deliver high-concentration doses of Pax not clinically feasible through traditional systemic chemotherapy. Our work demonstrates that sustained duration of drug exposure and concentration of drug are both required for the increased efficacy of these Pax-eluting polymer films likely via multifactorial mechanisms including upregulation of caspases as well as p21. The difference is striking and suggestive of a new mechanism of cell death secondary to local Pax delivery that could potentially lead to overall prolonged in vivo survival in sarcoma.

40.11 Activin Type ? Receptor as an Affordable Surrogate to Identify Microsatellite Instability

Posted on January 25, 2017

K. Yuza1, H. Ichikawa1, T. Hanyu1, T. Ishikawa1, Y. Shimada1, J. Sakata1, H. Kameyama1, T. Kobayashi1, H. Izutsu2, K. Kodama2, K. Takabe3, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Life Innovation Research Institute, Denka Innovation Center, Denka Co., Ltd.,Diagnostics Research Department,Machida City, TOKYO, Japan 3Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NEWYORK, USA

Introduction: There has been increasing evidence indicating importance of microsatellite instability (MSI) and mutations in the DNA mismatch repair pathway in association with the efficacy of 5-FU and immune checkpoint inhibitors in patients with gastrointestinal cancers. MSI has been shown to correlate with high somatic mutational rates in cancer as determined by whole exome sequencing. Some gene mutations, such as BRAF mutations, are known to be associated with MSI-high status (MSI-H). The activin type II receptors (ACVR2), which modulate signals for the transforming growth factor beta superfamily of ligands, are frequently mutated in MSI-H colorectal cancer. However, the incidence of ACVR2 mutations in gastric cancer patients has not well investigated yet. The aim of this study is to reveal the incidence of MSI-H and ACVR2A mutations, and to examine the association between the MSI-H and ACVR2A mutations in gastric cancer patients.

Methods: DNA samples extracted from 124 archived FFPE tumor tissues of Japanese patients with gastric cancer (stage I-IV), who were operated at Niigata University Medical and Dental Hospital or Niigata Cancer Center Hospital, were analyzed with the next generation sequencing-based comprehensive genomic test platform (CancerPlex). Overall mutational burden, mutations in DNA mismatch repair genes and MSI status (MSI-H or microsatellite stable; MSS) were evaluated.

Results: All 124 gastric cancer patients were successfully analyzed by comprehensive genomic test platform. 13 out of 124 patients (10.4%) showed MSI-H status and 111 patients showed MSS status. The median of mutation burden in patients with MSI-H is 35 (range: 19-102), while that in patients with MSS is 14 (range: 4-42) (P < 0.001). Mutations in mismatch repair genes were found in 6 of 13 (46.2.8%) MSI-H patients (1 in MLH1, 2 in MSH6, 2 in PMS2, and 1 in both MSH6 and PMS2). Interestingly, 10 of 13 MSI-H patients (76.9%), which is 8.0% of all gastric cancer examined, showed ACVR2A mutation, and no patients with MSS status (0%) showed ACVR2A mutation (P < 0.001).

Conclusion: 13 (10.4%) gastric cancer patients showed MSI-H status, and all of 10 that showed ACVR2A mutation has MSI-H. Our results implicate an intriguing possibility that ACVR2A mutations may be an affordable surrogate to identify MSI-H.

 

40.10 What Formalin-fixed Paraffin-embedded Samples Are Adequate For Deep Next Generation Sequencing

Posted on January 25, 2017

M. Ikarashi1, M. Endo1, J. Tsuchida1, K. Tatsuda1, C. Toshikawa1, M. Hasegawa1, M. Nakajima1, H. Ichikawa1, Y. Shimada1, C. Kanbayashi2, K. Kaneko2, N. Sato2, H. Izutsu3, K. Kodama3, K. Takabe4, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Niigata Cancer Center Hospital,Department Of Breast Oncology,Niiata City, NIIGATA, Japan 3Denka Co., Ltd.,Diagnostics Research Department, Life Innovation Research Institute, Denka Innovation Center,Machida City, TOKYO, Japan 4Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA

Introduction: Precision medicine utilizing next generation sequencing (NGS) is expected to bring paradigm shift from pathological microscopic-based to genetic signature-based diagnosis. Precision medicine is only possible in oncology practice if deeper sequencing of clinically targetable genes in fragmented DNA, such as DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples can be performed with NGS. Therefore, it is very important for clinicians like us to know the condition of FFPE samples to be usable for precision medicine. Further, it is important to clarify how much DNA can be harvested from minimal amount of sample, since often times only biopsy samples are available, such as those who will receive neoadjuvant chemotherapies. The aim of this study is to examine the quality and quantity of breast cancer tissue samples obtained by resected specimen and biopsy for deep sequencing with NGS.

Methods: 48 FFPE breast cancer tissue samples (28 resected specimens and 20 biopsy samples) of Japanese patients with breast cancer, who were operated at Niigata University Medical and Dental Hospital or Niigata Cancer Center Hospital, were used. DNA was extracted from 6 unstained sections with 20 μm thickness from FFPE for each case. The cases with enough DNA quantity were examined for DNA quality by Q-ratios, in which 41 bp and 129 bp targets are amplified and compared. The Q-ratio (129 bp/41 bp) > 0.1 was determined as high-quality DNA for NGS analysis.

Results:The median quantity of DNA was 1,197 (range: 7.2 – 12,848) ng from FFPE in 48 samples. The median quantity of DNA from resected specimens (n = 28) was 1,764 (range 86.1 – 12,848) ng, while that from biopsy samples (n = 20) was 620 (range 7.2 – 2,632) ng (P < 0.01). Enough quantity of DNA for deep targeted NGS (> 150 ng) was obtained from FFPE in 40 out of 48 (83.3%) samples examined. DNA quantity was enough in 26 out of 28 (92.9%) samples obtained from resected specimens, while that was enough in 14 out of 20 (70.0%) samples obtained from biopsy (P = 0.053). For the quality of DNA, high-quality DNA for NGS (Q-ratio > 0.1) was obtained from 37 out of 40 samples with enough quantity of DNA. 24 out of 26 (92.3%) resected specimens and 13 out of 14 (92.8%) biopsy samples showed high-quality. All the 3 DNA samples obtained from FFPE older than 6 years showed low-quality, while all the DNA samples obtained from more recent FFPE showed high-quality.

Conclusion:Our results indicate that enough quantity of DNA can be obtained from 6 unstained FFPE sections in most of the cases, although the quantity of DNA is smaller from biopsy samples than resected specimens. Samples older than 6 years may not be able to extract adequate quality DNA.

 

40.09 A Novel Strategy to Sensitize Neuroblastoma to Anti-Cancer Agents by Inhibiting DNA Ligase III

Posted on January 25, 2017

S. Chukkapalli1, N. Neamati2, T. Thomas1, V. Castle3, E. Newman1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Medicinal Chemistry,Ann Arbor, MI, USA 3University Of Michigan,Pediatrics,Ann Arbor, MI, USA

Introduction:  Neuroblastoma (NB) is the most common extracranial solid tumor in children, arising from neural crest stem cells. Despite multimodality therapies, the 5-year survival rate of patients with high-risk tumors is less than 50%. Previously, we found that tumorigenic and high-risk NB cells are defective in DNA double-strand break (DSB) repair, with elevated expression of alternate NHEJ (alt-NHEJ) pathway components, compared to classical NHEJ. We have shown that inhibiting DNA Ligase III (Lig3) and Ligase I (Lig1) lead to DSB accumulation and cell death. We hypothesize that inhibiting alt-NHEJ components by a novel alt-NHEJ ligase inhibitor (CH43) may sensitize NB cells to standard chemotherapeutic drugs.

Methods:  To test efficacy of a novel Lig3 inhibitor (CH43) on NB cells, a panel of stromal (SH-EP1), and tumorigenic (IMR32, SH-SY5Y) NB cell lines were cultured and treated with CH43 for 24, 48 and 72 hours.  At end-point, cell viability analysis was performed with CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) and IC50 values were calculated. In order to measure the DNA damage response due to Lig3 inhibition, CH43 treated cells were immunostained with γH2AX (a marker for DSB) followed by quantification of γH2AX foci in the nuclei of treated cells. Protein lysates were analyzed by immunoblot to assess the extent of Lig3 inhibition in CH43 treated cells. To evaluate for synergistic activity between CH43 and doxorubicin, NB cells were treated with CH43 for 24 hours, followed by doxorubicin treatment for 48 hours. Cell viability was assessed by the MTS assay, and combination index values were calculated with Compusyn Software.

Results: Each NB cell type was sensitive to CH43 in a dose-dependent manner, with IC50 values ranging from 2uM to 20uM. Immunoblotting analysis of protein lysates collected from CH43 treated cells confirmed inhibition of Lig3 and Lig1 in tumorigenic NB cells. We also noted increased expression of DNA Ligase IV, a classical NHEJ pathway component in these cells. Quantification of γH2AX foci in the nucleus of CH43 treated cells showed that inhibition of Lig3 with CH43 induced DSBs in NB cells. NB cells pretreated with CH43 were more sensitive to Doxorubicin compared to cells without pretreatment; suggesting CH43 and doxorubicin exhibit strong synergism against tumorigenic NB cell lines. 

Conclusion: Collectively, these findings strongly suggest that sensitizing tumorigenic NB cells to anti-cancer drugs by inhibiting alt-NHEJ components is an effective therapeutic approach to treat high-risk NB.
 

40.08 Rhodiola Crenulata Plant Extracts Alter Pyruvate Metabolism in NB-1691 Neuroblastoma Cells

Posted on January 25, 2017

K. E. Wong1, M. C. Mora1, N. Sultana2, K. Moriarty3, R. Arenas1, N. Yadava2, S. Schneider2, M. Tirabassi3  1Baystate Medical Center,Department Of Surgery,Springfield, MA, USA 2Pioneer Valley Life Sciences Institute,Springfield, MA, USA 3Baystate Childrens Hospital,Springfield, MA, USA

Introduction:  Rhodiola crenulata (RC) extracts are derived from the roots of adaptogenic Tibetan plants. The extracts are known to have anti-neoplastic properties in a variety of cancers. RC exerts a striking cytotoxic effect on MYCN amplified NB-1691 neuroblastoma cells. However, the mechanism of RC cytotoxicity is not yet known. Because rapidly proliferating cancer cells, such as neuroblastoma, are more dependent on glycolysis, we evaluated if RC cytotoxicity is mediated by alterations in cellular metabolism.

Methods:

NB-1691 cells were treated with 200ug/mL RC or ethanol vehicle control (VC) +/- 1mM pyruvate (PYR) in cell culture. Viability was evaluated using trypan blue exclusion with a cell viability counter following 24 hours of treatment. Pyruvate kinase (PK) activity, lactate dehydrogenase activity (LDH), NAD quantification and pyruvate dehydrogenase activity (PDH) assays were performed per protocol. NB-1691 cells were treated with either RC 200μg/mL or VC for two hours, metabolic assays were performed and differences in cells treated with VC and RC were evaluated.

Results:
Upon supplementation of media with PYR, the viability of NB-1691 was rescued from the cytotoxic effects of RC. RC treatment resulted in a 69% reduction in viability without PYR (p<0.001), while the addition of PYR with RC resulted in only 16.6% reduction in viability (p=0.0361) compared to VC (figure 1A). PK activity was significantly increased upon treatment with RC compared to VC (p=0.0081, figure 1B). LDH activity was significantly reduced upon treatment with RC compared to VC treated cells (p=0.0196, figure 1C). Total NAD (NAD++NADH) levels were reduced upon treatment with RC compared to VC (p<0.001). The total amount of NAD was observed to increase upon addition of PYR resulting in no significant difference between treatment groups in the presence of PYR (figure 1D). A significant increase in NADH generated by PDH was also observed upon treatment with RC compared to VC (p=0.03, figure 1E).

Conclusion:
RC extracts and the fractions contained within it hold promise as a potential chemotherapeutic option for neuroblastoma treatment given the cytotoxic effects it exerts upon NB-1691 cells in vitro. RC mediates neuroblastoma cytotoxicity through reprogramming optimal cellular metabolic functioning involving pyruvate metabolism. Reduced NAD levels observed upon RC treatment can halt glycolysis and limit intracellular pyruvate production by glycolysis made evident by the rescue of viability upon  pyruvate supplementation. The changes in PK, PDH and LDH activities may be compensatory responses to pyruvate limitation following RC treatment. Together, these findings identify a potential mechanism of RC cytotoxicity upon treatment of NB-1691 cells in vitro.
 

40.07 Development of a Novel Murine Model for Intrahepatic Cholangiocarcinoma

Posted on January 25, 2017

R. K. Marcus1,2,4, W. Foo3, A. Maitra1, S. Gupta1  1University Of Texas MD Anderson Cancer Center,Sheikh Ahmed Bin Zayed Al Nahyan Center For Pancreatic Cancer Research,Houston, TX, USA 2University Of Texas MD Anderson Cancer Center,Department Of Surgical Oncology,Houston, TX, USA 3University Of Texas MD Anderson Cancer Center,Department Of Pathology & Laboratory Medicine,Houston, TX, USA 4Medical College Of Wisconsin,Department Of Surgery,Milwaukee, WI, USA

Introduction:  Cholangiocarcinoma (CCA) is a poorly understood cancer of the biliary epithelium that represents the second most common type of liver cancer. Surgical resection is currently the only treatment associated with prolonged disease free survival. Unfortunately, most patients with CCA present with unresectable disease and have poor responses to standard chemotherapy, resulting in a median survival of less than one year. While elucidating the molecular pathogenesis of CCA may enable identification of pathways that form the basis for targeted therapies and improve early detection efforts, inquiry into this disease has been hampered by a paucity of genetically faithful animal models. The Cancer Genome Atlas analysis and other recent studies have revealed discrete epigenetic perturbations amongst CCAs originating from different anatomic sites. Intrahepatic cholangiocarcinoma (ICC) arises from the intrahepatic bile ducts and its incidence is currently rising in the United States. A subset of ICCs is characterized by loss-of-function mutations in genes encoding for chromatin regulatory factors such as BRCA associated protein 1 (BAP1). This gene encodes a nuclear deubiquitinase with a role in chromatin remodeling. BAP1 is mutated in 3-33% of ICCs. Loss of this protein may be associated with global epigenomic and transcriptomic alterations that contribute to tumor progression and metastasis dissemination.  

Methods:  A genetically engineered mouse model that incorporates a deletion of BAP1 with an activating mutation of Kras was developed. An Albumin-Cre promoter was used to induce hepatoblast-specific mutations in these genes.

Results: Conditionally deleted BAP1 mice develop focal biliary precursor lesions and frank ICC within a background of hepatocellular carcinoma (HCC). True mixed ICC-HCC lesions also develop. Tissue-specific activation of Kras alone results in the development of HCC with low penetrance and long latency. By combining Kras activation with heterozygous deletion of BAP1, the presence of biliary disease is induced and disease latency shortened. Homozygous deletion further shortens disease latency.

Conclusion: As hepatoblasts are bipotential, differentiating into both cholangiocytes and hepatocytes, the ICC phenotype of our model may be enhanced by biliary tree-specific deletion of BAP1 using adenoviral Cre enzyme (Ad-Cre) to achieve combinatorial specificity. A novel surgery enabling retrograde biliary tree administration of Ad-Cre was developed. To establish a proof-of-principle, Ad-Cre injection into ROSAmT/mG mice to confirm appropriate localization of Cre recombinase expression within cholangiocytes is ongoing. We will build on this by performing Ad-Cre injections in BAP1fl/fl;K-rasLSL-G12D/+ mice to activate Kras and knock out BAP1. Ideally, this model will develop ICC in a timely manner that recapitulates features of human ICC, and, as such, enables interrogation of the oncogenic pathways involved in development of this disease.

40.06 Lung-Selective Drug Delivery as a Novel Treatment Strategy for Colorectal Cancer Lung Metastasis

Posted on January 25, 2017

P. Rychahou1,2, Y. Bae4, Y. Zaytseva1, E. Y. Lee1,3, H. L. Weiss1, B. Evers1,2  1University Of Kentucky,Markey Cancer Center,Lexington, KY, USA 2University Of Kentucky,Department Of Surgery,Lexington, KY, USA 3University Of Kentucky,Pathology And Laboratory Medicine,Lexington, KY, USA 4University Of Kentucky,Department Of Pharmaceutical Sciences,Lexington, KY, USA

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is important for CRC progression and metastasis; SN-38 is an active metabolite of irinotecan with 1000 times more activity than irinotecan itself. The purpose of our study was to: (i) develop an effective nanocarrier for lung-selective delivery of pan-PI3K inhibitor PX-866 and SN-38 as a targeted therapy for CRC lung metastasis, and (ii) evaluate the effect of lung selective delivery of PX-866 and SN-38 loaded nanoparticles on CRC metastasis.

Methods: (1) HT29 LungM3 cell line was derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection for lung metastasis in immunodeficient mice. (2) Polymeric nanoparticles were constructed and loaded with fluorescent dye (Alexa 547), pan-PI3K inhibitor PX-866 or SN-38. (4) HT29 LungM3 cell line was injected intravenously to establish lung metastasis. Lung metastasis was treated with PX-866 and SN-38 loaded nanoparticles.

Results: (1) Lung selective accumulation of Alexa-546 fluorescently-labeled nanoparticles was confirmed by confocal imaging of frozen tissue sections from lung, liver and spleen. (2) Selective PI3K inhibition in lung tissue was confirmed by western blot of protein extracts after intravenous administration of PX-866-loaded nanoparticles. (3) Treatment with PX-866, an irreversible pan-PI3K inhibitor currently being evaluated in clinical trials, demonstrated a marked suppression of lung metastasis growth. Combination treatment with PX-866 and SN-38 loaded nanoparticles (four intravenous doses) completely eliminated lung metastases.

Conclusion: We demonstrate selective and efficient delivery of drug-loaded nanocarriers to lungs, suggesting that lung selective drug delivery is a viable treatment strategy for CRC lung metastasis.

 

40.04 Detection of Tumor-Specific Mutations in Plasma DNA: A Potential Esophageal Adenocarcinoma Biomarker

Posted on January 25, 2017

M. Egyud1, J. Jackson1, E. Yamada1, A. Stahlberg1,2, P. Krzyzanowski3, V. Litle1, L. Stein3, T. Godfrey1  1Boston University School Of Medicine,Department Of Surgery,Boston, MA, USA 2University Of Gothenburg – Sahlgrenska Cancer Center,Department Of Pathology,Gothenburg, , Sweden 3Ontario Institute For Cancer Research,Toronto, ON, Canada

Introduction: Recent studies have shown that tumor-specific DNA from multiple types of tumors can be detected circulating in plasma and this has raised the possibility of “liquid biopsies” using mutated tumor DNA as a potential diagnostic and prognostic biomarker. Detection of mutations with allele frequencies below 0.1% remains challenging however given that circulating cell-free DNA is highly degraded and in low abundance.  Detection of multiple different mutations in the same sample presents an additional challenge particularly when the mutation panel may change from patient to patient. We have developed a novel approach, called SimSen-Seq, to introduce molecular barcodes into sequencing libraries with DNA inputs as low as 5ng. Barcodes enable differentiation of true mutants from background noise introduced by Taq polymerase errors and permits detection of variant alleles with frequencies below 0.1%. The barcodes are protected from mis-priming using a hairpin structure which permits a high degree of multiplexing and flexibility for detection of multiple mutations from one plasma sample.  We are using this technology to test the utility of liquid biopsy as a biomarker for esophageal adenocarcinoma (EAC) diagnosis and disease monitoring.

Methods: Blood samples were obtained at a single time point from patients with various stages of EAC and longitudinal blood samples were also collected from patients undergoing neoadjuvant therapy followed by surgery. Tumor samples were obtained from biopsy or resection specimens. Tumor DNA was sequenced using a targeted EAC panel to identify mutations in each tumor. SiMSen-Seq assays were designed to identify these mutations in plasma, and hairpin barcodes were attached. Sequencing libraries were generated from circulating DNA, sequenced and analyzed using the barcodes to reduce background noise.

Results: Mutations were identified in tumor samples from 32 patients. To date, 24 patients have had plasma analyzed; 4 stage I, 8 stage II, 9 stage III, and 3 stage IV. Of these 24, the same mutations have been identified in 11 plasma DNA samples (0% stage I, 50% stage II, 67% stage III, 100% stage IV. All mutant alleles were present at <1% frequency and five patients demonstrated multiple mutant alleles in plasma DNA.

Conclusion: SimSen-Seq shows promise as a novel ultra-sensitive, highly multiplexed sequencing method for identifying rare circulating mutations. Possible applications include prognostication in early stage patients and rapid monitoring of therapeutic response and recurrence. Further work is to evaluate this is ongoing.

 

40.03 MYCN Amplification Negatively Regulates MYCC and the long non-coding RNA SPRY4-IT1 in Neuroblastoma

Posted on January 25, 2017

J. Mazar1, A. Rosado1, J. Shelley2, J. Marchica2, T. Westmoreland1  1Nemours Children Hospital,Surgery,Orlando, FL, USA 2Sanford Burnham Prebys Medical Discovery Institute,Orlando, FL, USA

Introduction: MYCN amplification is among the most significant biomarkers associated with the diagnosis and poor prognosis of human childhood neuroblastoma.  Over the last 20 years, there has been only limited improvement in the overall survival rate of children with this gene amplification.  As a result, we are investigating novel molecular events surrounding neuroblastoma, with an emphasis on long non-coding RNAs (lncRNAs). We recently identified the lncRNA SPRY4-IT1, a regulator of tumor growth in various human cancers, as differentially expressed in MYCN-amplified versus MYCC-expressing neuroblastomas.  It is our hypothesis that MYCN amplification controls the regulation of SPRY4-IT1 expression through the regulation of MYCC. 

Methods: In order to test this hypothesis, we prepared total cellular RNA, isolated from the human MYCN-amplified neuroblastoma cell lines CHLA-122, IMR-32, SK-N-Be(1), and SMS-KAN, as well as the non-amplified cell lines SK-N-AS, LAN-6, SK-N-SH.  Knockdown of MYCN was performed by transfection of MYCN-specific siRNA into the IMR-32, SK-N-Be(1), and SMS-KAN cell lines.  Exogenous expression of MYCC was performed by transfection of a MYCC expression plasmid (pcDNA6/MYCC) into the IMR-32 and SMS-KAN cell lines.  Total cellular RNA was isolated 48 hours post-transfection.  The expression of MYCN, MYCC, and the lncRNA SPRY4-IT1 were measured using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). GAPDH was measured as an internal load control.

Results:The expression of MYCN was confirmed as highly expressed in all the MYCN-amplified cell lines as compared to the MYCN non-amplified cells.  Interestingly, MYCC expression inversely correlated with MYCN expression, showing dramatically higher expression in the MYCN non-amplified cells.  This expression pattern also positively correlated with that of the lncRNA SPRY4-IT1.  Knockdown of MYCN in all three MYCN-amplified cell lines resulted in dramatic increases in both MYCC and SPRY4-IT1. Surprisingly, exogenous expression of MYCC in the MYCN-amplified cells had no effect on MYCN expression, but highly up-regulated SPRY4-IT1.  

Conclusion:The inverse correlation of MYCN and MYCC suggests competitive inhibition.  Since the loss of MYCN leads to significant increases in both MYCC and SPRY4-IT1, and exogenous expression of MYCC had no effect on MYCN, but dramatically increased SPRY4-IT1 expression, we can conclude that MYCN is an upstream negative regulator of MYCC, and MYCC is an upstream positive regulator of SPRY4-IT1.  This offers the promising possibility of SPRY4-IT1 as a prognostic biomarker of MYCN down-regulation. Together, these data identify a promising network of regulation in MYCN-amplified cells, allowing for the possibility of circumventing MYCN activity in these difficult to treat childhood cancers.  

 

40.02 Immune-modulator FTY720 potentiate Doxorubicin in obesity-related breast cancer

Posted on January 25, 2017

K. Takabe1, E. Katsuta1, M. Nagahashi2  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, , Japan

Introduction:  Obesity is one of the most prevalent health issues in the US. It has been shown that obesity-related breast cancer is more aggressive with poor prognosis, which is partly explained by the low-grade inflammation caused by obesity. Recently we have published that sphingosine-1-phosphate (S1P), a signaling lipid mediator, link inflammation and cancer in colitis-associated colon cancer. FTY720, a functional antagonist of S1P signaling, is an immune-modulator that dramatically reduces peripheral lymphocyte counts and other inflammatory cells. We hypothesized that addition of FTY720 thus suppress the effect of obesity-mediated inflammation should enhance anti-cancer effect of doxorubicin, which is one of the most commonly used anti-cancer drug for breast cancer as part of the standard of care.

Methods:  Female B6.cg-Lepob (ob/ob) mice fed with high fat diet for 2 weeks prior to implantation of 1×106 murine mammary adenocarcinoma E0771 cells into #2 right fat pads as previously described (Katsuta et al JSR 2016), were used as an obesity model, and litter mate control mice fed with normal diet were used as a control. Both ob/ob and control mice were randomized into 4 groups in each group; vehicle, Doxorubicin, FTY720 and Combination of Doxorubicin and FTY720. Doxorubicin was administrated 5 mg/kg on Day 0 and 3 i.p. FTY720 was administered 1 mg/kg daily p.o. during the entire course. Tumor growths were measured daily by caliper measurements. Tumor weights were measured on 21 days after cell inoculation.

Results: The body weight of obesity model was significantly heavier than control mice at the time of cancer cell inoculation (44.1 g vs 19.4 g; p < 0.001). In non-treatment group, tumor weight in obesity group was significantly heavier than control mice (1232 mg vs 966 mg; p = 0.049), which is consistent with the dogma that obesity worsen cancer progression. As expected, tumor weight in non-treatment group was heavier than any treatment group, and that in combination treatment of doxorubicin and FTY720 was lightest in both of obesity group and control group. Interestingly, tumor reduction rate in obesity group compared with non-treatment group is significantly greater than control group (Doxorubicin: 83% vs 19%, p=0.001; FTY720: 80% vs 46%, p=0.027, Doxorubicin + FTY720: 93% vs 64%, p=0.011). Over 15% weight loss were seen in obesity doxorubicin group and obesity combination treatment group.

Conclusion: Immune-modulator FTY720 enhanced the efficacy of doxorubicin particularly in obese mice, which implicate a novel approach to treat obesity-associated breast cancer.

40.01 The Use of Fluorescent-labeled Cetuximab for Image-guided Soft Tissue Sarcoma Surgery

Posted on January 25, 2017

N. K. Behnke1, A. C. Prince2, J. M. Warram1  1The University Of Alabama At Birmingham,Department Of Surgery,Birmingham, AL, USA 2University Of Alabama,School Of Medicine,Birmingham, Alabama, USA

BACKGROUND: Soft tissue sarcomas (STS) are a heterogeneous group of solid malignancies whose treatment includes margin-negative resection. Large tumor size and anatomic constraints make margin assessment challenging. Fluorescence-guided surgical resection can help delineate intraoperative margins; preclinical studies demonstrate improved oncologic outcomes in other malignancies using cancer-specific imaging probes.  Recent literature describes cathepsin activated probes selective for STS, but no studies using disease-specific chemotherapeutic agents conjugated to imaging probes.  This novel strategy has potential to decrease unnecessary healthy tissue resection and improve oncologic outcomes by reducing margin-positive resections.

 

Epidermal growth factor receptor (EGFR) is overexpressed in multiple subtypes of STS and is a potential target for fluorescence-guided surgery.  Recent studies show fluorescently labeled cetuximab, an FDA-approved, anti-EGFR antibody, to be safe and useful for margin assessment in head and neck cancer. Our aim was to evaluate its potential for STS, examining tumor-targeting specificity of two drug-probe conjugates, and comparing them to the described cathepsin-activated probes.  We hypothesize the drug-probe conjugates would successfully target STS, with superior tumor specificity.  We also aimed to determine the smallest tumor detectable by our imaging probe conjugate.

Methods: Athymic nude mice with subcutaneous HT1080 fibrosarcoma tumors were injected with one of five probes:  IRDye800CW fluorescent probe conjugated to either cetuximab (anti-EGFR) or DC101 (anti-VEGFR2), IRDye800CW conjugated to an isotype control (IgG), or a cathepsin-activated probe (IntegriSense 750 and Prosense 750). Fluorescence imaging was performed daily with open- and closed-field systems. Tumor-to-background ratios (TBR), signal washout times and normalized signal averages were evaluated.  On day 9, smallest resectable game evaluation was performed to assess sensitivity for detecting residual post-resection tumor

Results: At day 9 post-injection, the TBR of the cetuximab-IRDye800CW group (11.1) was significantly greater than Integrisense750 (6.88, p=0.005), the IgG-IRDye800CW control (4.44, p=0.00005), Prosense750 (2.35, p=0.00009), and DC101-IRDye800CW (1.87, p=0.00003). During in vivo imaging, cetuximab-IRDye800CW outperformed all other agents by several folds of contrast enhancement. The smallest resectable game evaluation demonstrated 1mm3 fragment detection using the cetuximab-IRDye800CW probe.

Conclusions: This study demonstrates superiority of cetuximab-IRDye800CW for disease-specific imaging in a subcutaneous animal model of STS.  The novel strategy of coupling improved margin-negative surgical resection with established chemotherapy has considerable translational significance and is an avenue for exploration with other drugs used to treat STS. 

 

25.10 UAB30, a novel retinoid, decreases tumor burden in a model of disseminated medulloblastoma

Posted on January 25, 2017

E. F. Garner1, G. K. Friedman3, V. R. Atigadda2, L. Nan3, B. P. Moore3, T. Etminan3, L. L. Stafman1, J. E. Stewart1, D. D. Muccio2, C. J. Grubbs4, E. A. Beierle1  1University Of Alabama at Birmingham,Division Of Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Department Of Chemistry,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Division Of Pediatric Hematology And Oncology,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA

Introduction:  Medulloblastoma (MB) is the most common malignant brain tumor in children.  There are four molecular subtypes of MB: WNT, SHH, Group 3 and Group 4, with Group 3 tumors having the most dismal prognosis. Our laboratory has previously shown that the novel retinoid, 9-cis-UAB30 (UAB30), inhibited MB proliferation in vitro and prolonged animal survival in a localized human patient-derived xenograft (PDX) MB mouse model. We hypothesized that UAB30 would also be effective in limiting the metastatic spread in a disseminated MB mouse model.

Methods:  We used a mouse PDX model with bioluminescent imaging to follow the spread of disease. The group 3 MB PDX cell line D341 labeled with GLuc-GFP was used for the in vivo study.  5 x 105 cells were stereotactically injected into the right lateral ventricle of 5-week-old athymic nude mice. The mice were randomized into three groups to receive control chow (n=8), 13-cis-retinoic acid (RA)-treated chow (n=8), or UAB30-treated chow (n=8). Treatment was initiated on the day of injection. Bioluminescence imaging was performed on days 7, 11, 14, 18, and 20 post-tumor injection using the IVIS Lumina III (Perkin Elmer). The animals were euthanized on day 20 when they met IACUC parameters. Tumor burden was quantified using total flux (photons/sec) in both the brain and spine. Student’s t-test was used to compare the mean bioluminescence between groups with p ≤ 0.05 considered significant. 

Results: Control mice had significantly greater tumor burden (9.87 x 107 photons/sec) in the brain at day 20 when compared to RA-treated (3.61 x 107 photons/sec; p = 0.02) and UAB30-treated (3.84 x 107 photons/sec; p = 0.03) mice (Fig. 1A and 1B). The UAB30-treated mice had significantly less spinal metastasis compared to the control mice at day 18 and day 20 (p = 0.02 and p= 0.04, respectively) (Fig. 1A and 1C). The RA-treated mice initially had less spinal metastasis than control mice at day 18 (p =0.05), but this difference was not significant at day 20 (Figure 1 C). 

Conclusion: In this PDX model of disseminated MB, treatment with UAB30 significantly delayed progression of tumor burden in the brain and spine compared to untreated mice.  These data suggest that this novel retinoid should be further explored as a potential therapeutic for MB. 

 

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