27.03 Circulating Tumors Cells as a Preoperative Biomarker of Metastatic Disease in Pancreatic Cancer

Posted on December 22, 2014

J. Ankeny1, C. Court1, H. Tseng2, J. S. Tomlinson1  1University Of California – Los Angeles,Surgical Oncology,Los Angeles, CA, USA 2University Of California – Los Angeles,Molecular & Medical Pharmacology,Los Angeles, CA, USA

Introduction: The majority of pancreatic ductal adenocarcinoma (PDAC) patients who undergo pancreatic resection ultimately succumb to metastatic disease. This fact strongly points to our inability to accurately stage patients at the time of disease presentation. We are in serious need of a biomarker which predicts systemic disease in order to better select our patient’s for surgery versus systemic therapy. Our goal was to investigate the presence of circulating tumor cells (CTCs) in early stage PDAC as a biomarker of metastatic disease.

Methods: We obtained a venous blood (VB) in the preoperative setting from 21 consecutive PDAC patients deemed to be AJCC Stage I or II based on preoperative imaging. Four milliliters of VB was evaluated for the presence and number of CTCs. Capture and enumeration was carried out with a novel microfluidic NanoVelcro technology enhanced by anti-EpCAM enrichment. CTCs were defined by immunocytochemical staining (CK+ or CEA +, CD45-, DAPI+). CTC number was correlated with postoperative AJCC stage and the presence or absence of metastatic disease discovered at surgery.

Results:Of the 21 PDAC patients taken to the operating room, 7 were found to have visible macroscopic metastatic disease in the peritoneal cavity and thus the planned resection was aborted. CTC number correlated with stage and distinguished patients with local disease versus metastatic disease. Mean CTC counts for stage I, 2A, 2B and 4 were 0, 0.67, 0.78, 13.1 respectively.  AUROC utilizing a CTC cutoff of > 3 CTCs/4ml VB was 0.93 (95% CI (0.772-1.086) p-value = 002).

Conclusion:In this small prospective study, the presence of 3 or more CTCs in 4ml VB demonstrate a strong ability to predict macroscopic metastatic disease undetectable by preoperative cross sectional imaging. CTCs are a promising biomarker which may allow for strong preoperative prediction of metastatic disease in the PDAC patient and guide first line treatment decisions. Longer follow-up with the addition of outcomes data is needed to firmly establish CTCs as a predictive biomarker in PDAC.

 

27.05 ERK MAP Kinase Interacts with and Up-regulates Pancreatic Duodenal Homeobox-1

Posted on December 22, 2014

G. Zhou1, E. Rozengurt2, J. Sinnett-Smith2, S. Liu1, J. Yu1, J. Wu1, R. Sanchez1, F. C. Brunicardi1  1University Of California – Los Angeles,General Surgery/Surgery/David Geffen School Of Medicine,Los Angeles, CA, USA 2University Of California – Los Angeles,Digestive Diseases/Medicine/David Geffen School Of Medicine,Los Angeles, CA, USA

Introduction: Pancreatic and duodenal homeobox-1 (PDX-1) is a key pancreatic transcription factor known to be involved in tumorigenesis and markedly overexpressed in pancreatic ductal adenocarcinoma (PDAC). However, little is known about the related oncogenic signaling pathways causing the overexpression. The activation of the extracellular signal-regulated kinases (ERK) pathway driven by the K-RAS mutation plays a critical role in promoting survival, invasion and migration of PDAC cells. The purpose of this study is to determine the role of ERK in regulation of PDX-1 expression in PDAC.

..

Methods: Antibody array screen was performed in GFP-PDX-1 stable HEK293 cells using a membrane filter arrayed with 400 antibodies, which was preblocked in a buffer containing 5% nonfat milk. After overnight incubation at 4°C, the presence of the antibody-antigen-PDX-1 complex was detected by horseradish peroxidase-conjugated anti-GFP antibody, followed by chemiluminescence. The ERK-PDX-1 interaction was confirmed in PDAC cells by performing immunoprecipitation/Western blotting using anti-ERK and anti-PDX-1 antibodies. Protein expression levels were determined by performing Western blotting using appropriate antibodies. Lipofectamine 2000 was used for transient transfection of PDX-1, ERK1, c-Jun N-terminal kinase 1 (JNK1) and Ubiquitin into HEK293 cells. PDAC cells were treated with various concentrations of epidermal growth factor (EGF) in the presence of 2% serum. Serine 268 residues of human PDX-1 were mutated into alanine by performing site-directed mutagenesis as instructed by the manufacture’s manual. The mutations were confirmed by standard PCR sequencing. Ubiquitination of PDX-1 was monitored by immunoprecipitating PDX-1, followed by followed by Western blotting using an antibody against ubiquitin.

Results:1) ERK1 and ERK2 were identified as PDX-1-interacting proteins in an antibody array screen; 2) The ERK-PDX-1 interaction was confirmed by immunoprecipitation/Western blotting in PDAC cells in response to EGF; 3) PDX-1 expression was specifically up-regulated by ERK1, but not by JNK1; 4) EGF enhanced PDX-1 expression in PDAC cells; 5) ERK inhibitor blocked EGF-stimulated PDX-1 expression in PDAC cells; and 6) Phosphorylation of proline-directed Ser 268 suppressed PDX-1 ubiquitination and stabilized PDX-1, thus, Ser 268 is a potential ERK phosphorylation site within PDX-1, whose phosphorylation contributes to the stabilization of PDX-1.

Conclusion:These data show that 1) ERK MAP kinase is a positive regulator of PDX-1; and 2) ERK up-regulates PDX-1 expression via a mechanism involving promoting phosphorylation of Ser 268, leading to decreased ubiquitination and enhanced stabilization of PDX-1. Thus, these data suggest that ERK MAP kinase plays an important role in PDX-1 overexpression in PDAC.

 

23.01 Immune-mediation Alters The Cytokine Profile Of Hormone-dependent And Hormone-independent Breast Cancer Cells In A 3d In Vitro Culture System

Posted on December 22, 2014

T. N. Augustine1, R. Duarte2, G. P. Candy2  1School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa 2School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Immune infiltration is a fundamental determinant of tumour progression and response to therapy, with interactions between immune, and tumour cells mediated by cytokines. In advanced breast cancer, a dominance of regulatory T (TREG) lymphocytes and the scarcity natural killer (NK) cells may reflect the primacy of adaptive immunity in the induction of tumour tolerance. We thus developed 3-dimensional (3D) models of the tumour microenvironment to determine induced cytokine profiles under immune-mediation.

Three-dimensional culture models were established by co-culturing CD4+CD25+ TREG lymphocytes and NK cells with hormone-dependent MCF-7 or hormone-independent MDA-MB-231 cell lines in growth factor reduced-Matrigel. Cytokine production was measured using a multiplex cytokine assay. Multivariate analyses were used to determine significant differences in cytokine production, and to explore associations between cytokines.

The results show that cytokine secretions in a 3D simulated breast tumour microenvironment are associated with the hormone-dependency of tumours. The effects of NK cell-mediation induced a significant increase in CCL2, CCL4 and CXCL8 secretion in the MCF-7 culture model. In the MDA-MB-231 culture model NK cells alone induced a significant increase in IL-12 and CCL2 inferring the dominance of TREG cell-mediation of cytokine secretion. Cluster analysis and principal components analysis indicate that IL-6 plays a significant role in the induction of a chemokine cascade in both culture models, with IL-1β implicated in the induction of proinflammatory environment in the MDA-MB-231 culture model.

The cytokine data suggests that hormone-dependent MCF-7 cells, as a weakly metastatic cell line, are capable of subverting cytokine secretion of NK cells and TREG lymphocytes to enhance their invasive potential. In contrast, hormone-independent MDA-MB-231 cells, a more aggressive phenotype, subvert TREG cell-mediated cytokine secretion for the maintenance of a proinflammatory microenvironment for tumour progression. Further, the data suggests a role for IL-6 and IL-1β in promoting such immune evasion and tissue invasion.

23.02 NIR Photoimmunotherapy of Pancreatic Cancer Significantly Decreases Recurrence after Resection

Posted on December 22, 2014

A. A. Maawy1, Y. Hiroshima4, Y. Zhang3, M. Garcia-Guzman5, L. Makings5, R. Heim5, G. A. Luiken2, R. M. Hoffman1,3, M. Bouvet1  1University Of California – San Diego,Surgery,San Diego, CA, USA 2OncoFluor, Inc.,San Diego, CA, USA 3AntiCancer, Inc.,San Diego, CA, USA 4Yokohama City University,Surgery,Yokohama City, , Japan 5Aspyrian Therapeutics,San Diego, CA, USA

Introduction:   Photoimmunotherapy (PIT) is a selective treatment modality in the treatment of cancer. Tumor selectivity and targeting is based on the use of a monoclonal antibody  specific to cancer epitopes conjugated to a near infra red (NIR)phthalocyanine dye (IR700). While surgical resection of pancreatic cancer offers the only real chance at a cure, recurrence rates and overall mortality is still high. PIT, if efficacious, could serve as a useful adjunct in the surgical treatment of pancreatic cancer that would help eliminate invisible microscopic disease on the tumor bed.  In this study, we chose to use anti-carcinoembryonic antigen (CEA) as the monoclonal antibody because it highly expressed in pancreatic cancer.

Methods:   Athymic nude mice were orthotopically implanted with the GFP expressing human pancreatic cancer cell line BxPC3. After engraftment, the mice were divided into two groups: Bright light surgery (BLS) + anti-CEA-IR700 + 690nm laser (PIT) and BLS only.  The anti-CEA-IR700 conjugate (100 μg) was administered to the treatment group via tail vein injection 24 hours prior to therapy. After 24 hours, tumors were surgically exposed and treated with the designated phototherapy intraoperatively at an intensity of 150 mW/cm2 for 30 minutes and serially imaged non-invasively for 8 weeks using the OV-100 small animal imager.

Results:  Over the course of 8 weeks there was a significant difference in tumor size between the PIT-BLS (2.14 mm2, 95% CI [6.34, -2.06] and BLS group (115.2 mm2, 95% CI [141.6, 88.8]) with p<0.001. There was also a significant difference in tumor weight between the PIT-BLS (6.65 mg, 95% CI [19.65, -6.35] and BLS (1100 mg, 95% CI [1406, 794] at 8 weeks with p<0.001. In the PIT-BLS group, there was no tumor detectable in 86% of the mice vs a 100% recurrence rate in the BLS group (p=0.04).

Conclusion:  PIT causes significant tumor cell death with high specificity in the treatment of pancreatic cancer as a surgical adjuvant in orthotopic nude mouse models. Animals treated with BLS-PIT appear to be cured from an aggressive metastatic pancreatic cancer.  PIT holds promise in the treatment of this highly lethal cancer and may serve as a useful adjunct to surgery in the eradication of otherwise undetectable microscopic disease.  The results of the present report suggest that BLS-PIT be evaluated clinically in the future.

 

23.03 Defining Immunological Aspects of Regional Chemotherapy Using Immunocompetent Murine Melanoma Model

Posted on December 22, 2014

M. Tsutsui1, Z. Sun1, P. Speicher1, P. Dolber1,2,3, J. Dannull1, S. Nair1, D. Tyler1,2  1Duke University Medical Center,Surgery,Durham, NC, USA 2Durham VA Medical Center,Durham, NC, USA 3Duke University Medical Center,Pathology,Durham, NC, USA

Introduction:

The effectiveness of regional therapeutics may be related to the degree of local and systemic anti-tumor immune response generated. We utilized an immunocompetent mouse model of advanced extremity melanoma to define the immune aspects of regional chemotherapy infusion.

Methods:

B16F10.9OVA melanoma cells were inoculated subcutaneously in one or both hindlimbs of C57BL/6 mice. After tumor diameter reached 5 mm, isolated limb infusion (ILI) was performed using melphalan or vehicle in one hindlimb with or without anti-CTLA-4.  Mice experiencing a complete response were rechallenged with a second tumor inoculation.

Results:

Tumor doubling and quadrupling time were significantly longer using melphalan ILI with systemic anti-CTLA-4 than without (P = 0.005, Hazard Ratio: HR 1.96 and P = 0.02, HR 1.72, respectively); no anti-CTLA-4 effect was observed on tumors that received vehicle ILI. Of the mice rechallenged with a second tumor inoculation, inhibition of tumor growth at the second inoculation site was only observed in mice treated with melphalan ILI and systemic anti-CTLA-4. In animals carrying tumors on both hindlimbs, tumor doubling and quadrupling times on the non-ILI-treated side were significantly longer in animals whose contralateral hindlimb was treated with melphalan ILI and systemic anti-CTLA-4 than with melphalan ILI alone (P = 0.011, HR 5.05 and P = 0.004, HR 9.1, respectively). Systemic tumor specific immune responses were detectable in the regional draining lymph nodes only in animals treated with both melphalan ILI and systemic anti-CTLA-4.

Conclusion:

Optimizing immunologic aspects of regional melanoma therapy may significantly complement systemic immunotherapy strategies.

23.04 Melanoma-Associated Fibroblasts Are a Promising Therapeutic Target

Posted on December 22, 2014

Z. Liu1, H. Shao1, M. G. Moller1, O. C. Velazquez1  1University Of Miami,Surgery/Sylvester Comprehensive Cancer Center,Miami, FL, USA

Introduction:  Cancer-associated fibroblasts (CAF) play critical roles in promoting primary tumor development, growth and progression by stimulating tumor cell proliferation, survival, migration, angiogenesis as well as providing a niche supporting the metastatic colonization of disseminated tumor cells in distant organs. Moreover, CAF appear to be relevant to the development of drug resistance and tumor recurrence. Hence, CAF are a promising therapeutic target. We have previously demonstrated that activation of the Notch1 signaling pathway confers normal human dermal fibroblasts a suppressive phenotype to melanoma growth. Here, we further investigated whether manipulation of Notch signaling in melanoma-associated fibroblasts (MAF) isolated from human melanoma patients alter their regulatory phenotype by which affects melanoma growth. 

Methods:  MAF were isolated from human primary and metastatic melanoma lesions and characterized. Notch pathway activity in MAF versus normal human dermal fibroblasts was examined using Notch pathway RT2-PCRArray and immunoblotting. Enforced activation of Notch pathway was achieved by transducing MAF with lentiviral vector encoding active form of Notch1 (NIC). The effect of MAF engineered to carry high Notch activity on melanoma growth was tested by in vitro co-culture and in vivo co-engrafting animal model. 

Results: Isolated MAF are a-smooth muscle actin (a-SMA) and fibroblast activation protein (FAP) positive. MAF exhibited a relatively lower Notch activity compared to normal human dermal fibroblasts. Enforced activation of Notch1 pathway downgraded cellular activities of MAF. MAF engineered to carry high Notch activity significantly inhibited melanoma cell growth in vitro and retarded xenografted human melanoma cell growth on mouse skin. 

Conclusion: 

Notch signaling pathway appears to be a ‘molecular switch’ in determining the function of tumor stromal fibroblasts. Notch signaling activity is lower in MAF. Increasing Notch signaling activity in MAF confers them a tumor-suppressing phenotype towards melanoma growth. Our study demonstrated that Notch signaling functions as a critical molecular determinant in governing the tumor-regulating role of tumor stromal fibroblasts and provided a novel approach to target tumor microenvironment by manipulation of Notch signaling in stromal fibroblasts.

 

 

23.05 Tumor Stroma and Melanoma Progression Depends on Host Endothelial Cell Derived SDF-1

Posted on December 22, 2014

Z. N. Maan1, M. S. Hu1, A. J. Whittam1, L. H. Fischer1, D. Duscher1, G. G. Walmsley1, G. W. Krampitz1, J. Barrera1, D. Atashroo1, M. Rodrigues1, A. Whitmore1, M. Findlay1, P. H. Lorenz1, M. T. Longaker1, G. C. Gurtner1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction: The surrounding vascularized stroma of malignant cells provides a microenvironment critical for tumor development and subsequent progression of cancer. Understanding the signaling mechanisms governing the microenvironment and its interplay with tumor cells has the potential to inform more efficient tools for cancer management. Stromal-derived factor-1 (SDF-1) has been implicated in regulating stem cell microenvironments and has also been shown to influence cancer biology, though its specific role remains unclear. Utilizing newly developed murine models, we investigated the role of host endothelial derived SDF-1 in mediating endothelial-fibroblast interactions during tumor stroma formation and cancer progression.

Methods: Murine B16 melanoma cells were seeded onto a pullulan-collagen hydrogel, which were subcutaneously implanted in SDF-1 endothelial (eKO) and fibroblast knockout (fKO), CXCR4 fibroblast knockout (frKO), and floxed control mice. The mice were photographed and weighed and had their tumor size measured at regular intervals. After 28 days, tumors were harvested, weighed and processed for histology. The effects of SDF-1 on fibroblast proliferation, migration, survival and angiogenic profile were assessed in vitro.

Results: eKO mice demonstrated significantly reduced tumor burden compared to fKO, frKO, and control mice, in terms of height (*p < 0.05), weight (*p < 0.05) and volume (*p < 0.05). SDF-1 increased fibroblast proliferation (*p<0.001), migration (*p<0.01), and survival (* p < 0.05) in vitro. Co-culture demonstrated that decreased endothelial production of SDF-1 significantly reduced fibroblast expression of VEGF (*p < 0.05) and FGF-2 (*p < 0.05) in vitro.

Conclusion: Endothelial cell SDF-1 (eSDF-1) plays a pivotal role in cancer biology, regulating the expression of cytokines responsible for neovascularization and modulating fibroblast behavior and survival capacity, thereby modulating tumor stroma formation and tumor progression.

 

23.06 Neutrophil Extracellular Traps Promote Tumor Progression after Liver Ischemia Reperfusion

Posted on December 22, 2014

S. Tohme1, H. Huang1, A. Al-Khafaji1, A. Tsung1  1University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA

Introduction: Previous studies have shown that after liver ischemia-reperfusion (I/R), the growth of hepatic metastatic tumors increase; however, the mechanisms remain unclear. We have previously shown that during I/R neutrophils release Neutrophil Extracellular Traps (NETs), extracellular fibers composed of DNA and neutrophil proteins, which heighten the inflammatory response and subsequent liver injury. Some emerging data suggests that NETs may play a role in tumor progression, but the role of NETs in I/R-mediated acceleration of tumor growth is unknown. We hypothesize that NETs formed in response to liver I/R promote tumor growth and progression.

Methods: In-vitro, neutrophils were harvested from mice bone marrow. Neutrophils were treated with PMA, a well-known stimulator of NET formation, for 4hrs. Media was collected and co-cultured with mc38 cancer cells. MTT assays, Western blots, invasion and migration assays were used for analysis. In-vivo, colorectal liver metastases were induced in C57BL/6 mice by spleen injection of mc38 cells. Animals were then subjected to partial liver I/R vs. sham surgery followed by splenectomy with or without daily DNase I injections, a known inhibitor of NETs. The livers were harvested 3 weeks later for analysis.

Results: In vitro, there was a significant increase in proliferation of mc38 cells co-cultured with stimulated neutrophil media compared to untreated mc38 cells by MTT assay. This surge in proliferation was significantly decreased when DNaseI was added. Similarly, there a was significant increase in the invasion and migration of mc38 cells treated with stimulated media compared to control or addition of DNaseI. By using western blot analysis, the addition of neutrophil media resulted in the activation and phosphorylation of STAT3, a protumorigenic transcription factor, and p38, p65 and JNK, mitogen activator protein kinases implicated in tumor growth and development under stressful conditions. The activation of these proteins was significantly decreased with the addition of DNaseI. In vivo, Mice in the I/R group had grossly significantly more tumor growth and higher liver/body weight when compared to mice in the sham group. Addition of DNaseI to mice receiving I/R resulted in a significant decrease in tumor growth. Histologically, the tumors from the I/R + DNaseI group has less proliferation by Ki67 staining compared to the I/R group. There was a similar decrease in the activation of the MAP kinases from the tumor tissue obtained from the I/R + DNase1 mice compared to the I/R group. There was no difference in tumor growth between mice in the sham groups with or without DNaseI.

Conclusions: Liver I/R is a strong stimulus for metastatic tumor growth. NETs formed during I/R contributes to tumor growth by activating protumorigenic signaling pathways. DNaseI may represent a novel therapy for targeting NET-mediated tumor growth during liver I/R.  

23.07 Rhodiola Crenulata Inhibits Wnt/β-Catenin Signaling in the Treatment of Glioblastoma Multiforme

Posted on December 22, 2014

M. C. Mora1,2, K. E. Wong1,2, M. V. Tirabassi3, R. B. Arenas1,2, S. Schneider2  1Baystate Medical Center,Surgery,Springfield, MA, USA 2Pioneer Valley Life Science Institute,Springfield, MA, USA 3Baystate Children’s Hospital,Surgery,Springfield, MA, USA

Introduction:
Extracts from Rhodiola crenulata, a Tibetan plant, have anti-neoplastic effects on a variety of cancers. The purpose of this study is to determine if Rhodiola crenulata extract exhibits anti-neoplastic properties on Glioblastoma Multiforme (GBM) in-vitro.

Methods:
Human U-87MG GBM cell line was pretreated with 200ug/ml of RC or vehicle control for 24, 48, 72, and 96 hours.  Cell proliferation was then measured using a MTS calorimetric assay. Clonogenicity assay was used to further evaluate cell proliferation in which 100 cells were treated with vehicle control, 100ug/ml of RC, radiation, or radiation and RC together and were observed for colony growth.  Neurosphere formation was evaluated by phase contrast microscopy comparing treatment with 100ug/ml RC to vehicle control.  For the remainder of experiments cells were pretreated with 200ug/ml RC for 72 hours. qRT-PCR was performed to evaluate the expression of proliferation and differentiation genes. Expressions of β-catenin and Glial fibrillary acidic protein (GFAP), a protein marker of differentiation, were measured with immunocytochemistry (ICC). To assess transcriptional activity of the Wnt/β-Catenin axis, a luciferase-β-Catenin-reporter assay was conducted. After transfection, cells were treated with 200μg/ml of RC or vehicle control and a dual luciferase reporter assay was performed to quantify luciferase activity up to 72 hours following RC treatment.

Results:
MTS assay revealed a 65% decrease in proliferation with RC therapy starting at 48 hours (p=0.01) and a 70% reduction observed at 96 hours (p=0.0006).  Colony formation was reduced by more than half in cells treated with RC (p=0.03) and colonies were further reduced if treated with radiation and RC together (p=0.009). Neurosphere formation was eliminated in all wells after treatment with RC (Figure 1A-B).  C-myc and cyclin-D1, genes of proliferation associated with the Wnt/ β-catenin pathway, had a 60-fold and 55-fold reduction in expression respectively (p<0.05) after RC treatment. ID-1, oct-4, and snail, genes of differentiation were noted to have a 95-fold, 50-fold, and 40-fold reduction in expression respectively, (p<0.05) after treatment with RC.  ICC revealed that RC induced GFAP expression (Figure1C-D) and decreased nuclear expression of β-catenin. Luciferase assay performed on luciferase-β-Catenin-reporter transfected cells revealed decreased Wnt promoter activity (p=0.0041) following treatment with RC.

Conclusion:
Rhodiola crenulata extract effectively suppresses proliferation, stimulates differentiation, and eliminates tumorsphere formation of GBM cells in-vitro. The effects observed are likely secondary to the inhibition of the Wnt/β-catenin signaling pathway. 
 

23.08 Patient-Derived Pancreatic Cancer Xenografts Reflect Patient Tumor Biology and Predict Outcome

Posted on December 22, 2014

R. Marayati1, C. J. Tignanelli2, J. Yeh1,2,3  1Lineberger Comprehensive Cancer Center,Chapel Hill, NC, USA 2University Of North Carolina At Chapel Hill,Department Of Surgery,Chapel Hill, NC, USA 3University Of North Carolina At Chapel Hill,Department Of Pharmacology,Chapel Hill, NC, USA

Introduction: Pancreatic cancer is a lethal malignancy with an extremely poor prognosis and lack of effective therapy. Patient-derived xenograft (PDX) models have been used as a preclinical platform to investigate the underlying biology of pancreatic cancer and to evaluate new biomarkers and anti-cancer therapies. We sought to assess whether PDX models reliably reflect the tumor biology and course of disease in patients with pancreatic cancer.

Methods: Surgically resected pancreatic ductal adenocarcinomas were obtained from 56 de-identified patients after IRB approval. Tumors were engrafted either orthotopically or subcutaneously into immunocompromised mice and passaged over time. PDX tumors were considered successful if they reached a volume of at least 200 mm3 and were passaged at least twice. The time to 200 mm3 was defined by the number of weeks it took for a tumor to reach a volume of 200 mm3 in the initial passage. At each passage, pancreatic ductal adenocarcinoma histology was confirmed by hematoxylin and eosin staining and KRAS mutation status was determined by pyrosequencing. Overall survival (OS) and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method and compared using the log-rank test. Pearson’s chi-square and Fisher's exact tests were used to compare categorical variables (IBM SPSS Statistics v20).

Results: Out of 56 engrafted patient tumors, 37 (66%) met our criteria for success. The median follow-up of our patient cohort was 34 months. Patients with successfully engrafted tumors had a significantly shorter median OS (12 months vs. 21 months, p=0.039). Successful engraftment did not correlate with known pathological variables such as differentiation, lymph node involvement, stage of disease, or tumor margin status. Furthermore, successful engraftment was independently predictive of OS in a multivariate Cox regression model that included lymph node involvement, stage, and tumor margin status, with a hazard ratio of 0.497 (95% CI [0.248, 0.997], p=0.049). Of the 37 successfully engrafted tumors, 79% had KRAS mutations. Patients whose tumors took longer than 20 weeks to reach a volume of 200 mm3 had a significantly longer median RFS (18 months) than those patients whose tumors took less than 20 weeks (9 months, p=0.013). In a multivariate Cox regression model that included KRAS mutation status, lymph node involvement, stage, and tumor margin status, time to 200 mm3 was the single independent predictor of RFS with a hazard ratio of 3.429 (95% CI [1.075, 10.936], p=0.037).

Conclusions: Our results show that PDX models accurately recapitulate the growth pattern and inherent tumor biology of patients with pancreatic cancer, in that tumors that grow faster are associated with earlier recurrences. In addition, patients with successfully engrafted tumors have a significantly poorer overall survival, suggesting that these tumors represent a more aggressive subset for which novel therapies are needed.

23.09 PI3K and Pan-ErbB Inhibition Overcomes ErbB Cross-Talk in Pancreatic Ductal Adenocarcinoma

Posted on December 22, 2014

C. J. Tignanelli1, J. Stratford2,3, R. A. Moffitt2, J. Yeh1,2,3  1University Of North Carolina At Chapel Hill,Department Of Surgery,Chapel Hill, NC, USA 2University Of North Carolina At Chapel Hill,Lineberger Comprehensive Cancer Center,Chapel Hill, NC, USA 3University Of North Carolina At Chapel Hill,Department Of Pharmacology,Chapel Hill, NC, USA

Introduction:
 

     Resistance to single kinase inhibition is one of the main challenges in the treatment of cancer patients. We have previously shown that treatment with BKM120 (a pan-class 1 PI3K inhibitor, currently in Phase I/II clinical trials) resulted in tumor growth inhibition (p = 0.017) but not regression in a pancreatic ductal adenocarcinoma (PDAC) patient derived xenograft (PDX) mouse model, suggesting that tumors may be adapting to PI3K inhibition. When evaluating possible mechanisms of resistance we identified ErbB1, ErbB2 and ErbB3 activation in response to BKM120 treatment in both cell lines and PDX tumors.  The ErbB family is a well-established therapeutic target in multiple cancers. Intense cross-talk is known to occur between ErbB isoforms perhaps contributing to the limited effectiveness of single ErbB inhibition seen in the clinic in the case of erlotinib in PDAC. We hypothesized that pan-ErbB inhibition would be required in combination with BKM120 for optimal tumor response.

Methods:

      PDAC cell lines were treated with 475 nM of BKM120 and either 25 nM of siErbB1, siErbB2, siErbB3, or 40nM dacomitinib (a pan-ErbB inhibitor currently in Phase III clinical trials). Target inhibition was confirmed by immunoblotting.  Cellular viability was measured using a cellular growth assay after 72 hours of treatment.

Results:
 

     We found that the combination of BKM120 and dacomitinib inhibited proliferation in 10 of 10 PDAC cell lines (p < 0.01) and was more effective than BKM120 alone. Furthermore, BKM120 and dacomitinib showed impressive synergy across all cell lines with a mean combination index of 0.24 (0.00245 – 0.49). We next evaluated whether inhibition of any single ErbB family member would be sufficient for synergy with BKM120. We observed significantly greater growth inhibition after treatment with BKM120 and dacomitinib (68%) in the HPAC cell line compared with BKM120 + siErbB1 (48%, p < 0.001), BKM120 + siErbB2 (51%, p < 0.001), or BKM120 + siErbB3 (55%, p = 0.002), suggesting that single ErbB inhibition is not as effective as pan-ErbB inhibition.

Conclusion:

     Our results suggest that inhibition of any single ErbB will not be sufficient to overcome the adaptive response of tumors to PI3K inhibition. Instead, combined treatment with a pan-ErbB and PI3K inhibitor will be necessary.  Combination studies in PDX models are ongoing. Pan-ErbB and PI3K inhibition in PDAC may be more effective than either single agent alone and should be considered in clinical trials.

23.10 Identification of Glioblastoma Stem-Like Cell-Binding Human Antibodies by Yeast Biopanning

Posted on December 22, 2014

J. S. Kuo1, M. Zorniak1, E. V. Shusta2, J. S. Kuo1  1University Of Wisconsin,Neurological Surgery,Madison, WI, USA 2University Of Wisconsin,Chemical And Biological Engineering,Madison, WI, USA

Introduction:  Glioblastoma multiforme (GBM) is a poorly treated human brain cancer with few tools available for detection and isolation of their therapeutic-resistant, glioblastoma cancer stem-like cell (GSC) populations. A non-immune, human single-chain antibody (scFv) yeast display library was mined for human GSC-specific antibodies via biopanning.

Methods:  Standard molecular biology techniques were employed, along with novel yeast biopanning strategy involving positive and negative selections (Wang et al, Nature Methods. 2007 Feb;4(2):143-5) followed by screening identified clones against patient-derived GSC and control normal neural stem cell lines. Briefly, GSC-binding scFv candidates were isolated after combining nine rounds of enrichment via positive screening with several rounds of negative screening against normal human astrocytes, neural stem cells, and serum-cultured GBM tumor. 

Results: Clonal scFv assessment by restriction enzyme fingerprinting revealed 62 unique scFv clones. Each yeast-displayed scFv clone was characterized for qualitative binding selectivity against 12 distinct human lines of normal, GSC, and patient-matched GBM cells. Clone scFv-9.7, in particular, demonstrated substantial binding specificity for five GSC lines representing three different classes of tumor invasiveness and various neural progenitor lineages. GSC-specificity was further verified using secreted and purified scFv-9.7, which detected highly infiltrative GSCs from tumor xenografts via flow cytometry, and successfully targeted and fluorescently visualized tumor xenografts in vivo when conjugated with a near-infrared dye. 

Conclusion: In summary, rapid screening via yeast antibody library biopanning identified human GSC-specific antibodies for potential development into immunotargeted diagnostics and therapeutics in brain cancer. 

 

24.01 Demonstration of the Effects of Portal Vein Ligation on Glucose Metabolism Using In Vivo Multi-modal PET/MRI Measurements in Healthy Rat Liver

Posted on December 22, 2014

A. Fülöp1, A. Budai1, D. Korsós1, V. Hegedűs1, L. Harsányi1, I. Horváth2, N. Kovács3, D. Máthé3, K. Szigeti2, A. Szijártó1  11st Department of Surgery, Semmelweis University, Budapest, Hungary 2Department of Biophysics and Radiation Biology; Semmelweis University; Budapest; Hungary 3CROmed Translational Research Centers; Budapest; Hungary

Introduction:
Portal vein ligation (PVL) results in ipsilateral atrophy and hypertrophy of contralateral liver segments. It is unknown how PVL affects metabolic patterns of hepatic tissues. The aim of this study is to evaluate the effect of PVL on glucose metabolism, using multi-modal PET/MRI imaging in healthy rat liver. 

Methods:
Male Wistar rats (n=30) underwent PVL. 2-deoxy-2-(18F)fluoro-D-glucose (FDG) PET/MRI imaging and morphological/histological examination were performed before; 1-, 2-, 3-, 7-days after PVL. Dynamic PET data were collected and the standardized uptake values (SUV) for ligated and non-ligated liver lobes were calculated in relation to cardiac left ventricle (SUVVOI/SUVCLV) and mean liver SUV (SUVVOI/SUVLiver). 

Result:
PVL induced atrophy of ligated lobes, while non-ligated liver tissue showed compensatory hypertrophy. Dynamic PET scan revealed altered FDG kinetics in both ligated and non-ligated liver lobes. SUVVOI/SUVCLV significantly increased in both groups of lobes, with a maximal value at 2nd postoperative day and returned near to the baseline 7 days after the ligation. After PVL, ligated liver lobes showed significantly higher tracer uptake compared to the non-ligated lobes (significantly higher SUVVOI/SUVLiver values were observed at postoperative day 1, 2 and 3). The homogenous tracer biodistribution observed before PVL reappeared by 7th postoperative day. 

Conclusion:
Our study demonstrated an altered glucose metabolism in both ligated and non-ligated liver lobes. The observed alterations in FDG uptake dynamics should be taken into account during the assessment of PET data until the PVL induced atrophic and regenerative processes are completed. 

24.02 Photodynamic Therapy of Human Lung Cancer Xenografts in Mice

Posted on December 22, 2014

C. Nwogu1, P. Pera1, K. Attwood1, W. Bshara1, R. Pandey1  1Roswell Park Cancer Institute,Buffalo, NY, USA

Introduction: Photodynamic therapy may be effective for treatment of peripheral tumors in patients unable to tolerate surgery. We hypothesized that a novel photosensitizer, PS1, would be more effective than the standard agent, Porfimer sodium (Photofrin® or PFII), in treating human lung cancer xenografts in mice.

Methods: Patient-derived NSCLC xenografts were established subcutaneously in 20 SCID mice. There were two treatment and two control groups. Two groups of 5 mice were injected with PS1 or PFII. 24 hours later, the subcutaneous tumors in these mice were treated with laser light at a wavelength of 630nm for PFII and 665nm for PS1. 4 mice were treated with laser light with no photosensitizer and 6 mice received no treatment at all. The mice were observed for 60 days. Bonferroni adjusted methodology was used to compare the tumor growth rates between treatment groups. The tumor growth endpoint, time-to-1000mm3, was evaluated using standard Kaplan-Meier methods and compared between groups using the log-rank test. All analyses were conducted in SAS v9.3 (Cary, NC). One representative tumor in each group was cut and stained with H&E and Caspase3 to evaluate necrosis and apoptosis.

Results: Tumor re-growth pattern in the mice is illustrated in figure 1. The median time-to-1000mm3 was 12, 12, 26 and 52 days for the control, light only, PF II and PS1 groups (p<0.001). H&E analysis revealed <1%, 0%, 67% and 80% necrosis, respectively from representative samples in the same four groups. Caspase3 positivity in these groups was 2%, <1%, 17% and 39% respectively.

Conclusion:

The mice treated with PS1 exhibited a longer time for tumor regrowth, showed more tumor necrosis and apoptosis compared to the other treatment groups. Thus, the novel photosensitizer, PS1, was demonstrated to be more effective than Porfimer sodium in treating human lung cancer xenografts in a preclinical pilot study and deserves further study.

 

Figure 1: Effect of photodynamic therapy on human NSCLC implanted in SCID mice

 

24.03 Phase I Clinical Trial of Nitroglycerin added to 5-FU & IR for Treatment of Operable Rectal Cancer

Posted on December 22, 2014

S. Huerta1,2, D. H. Wang2, J. Dowell2, W. J. Hittson2, J. R. Torrosi2, H. Illum2  1University Of Texas Soutwhestern,Surgery,Dallas, TX, USA 2North Texas VA Health Care System,Surgery/Hematology Oncology/Radiation Oncolgy,Dallas, TX, USA

Introduction:
Neoajuvant chemoradiation (CRT) is currently the standard of care for patients with rectal cancer. The ability of neoadjuvant CRT to reduce tumor load in rectal cancer is heterogeneous and unpredictable.  The nitric oxide (NO) donor DETANONOate reduced tumor load in vitro and in vivo models of rectal cancer subjected to ionizing radiation (IR).  Low doses of NO from nitroglycerine (NTG) radio-sensitized breast cancer cells and prostate cancer xenografts.  Transcutaneous NTG patches in combination to chemotherapy were safe and effective in patients with lung cancer in clinical trials.  Our hypothesis is that NO from NGT can radiosensitize tumors more effectively when added to conventional treatment in patients with rectal cancer.  

Methods:
We designed an open label, non-randomized, multi-cohort, dose escalation, Phase I Clincal Trial with primary endpoint to evaluate the safety, tolerability, feasibility and maximum tolerated dose (MTD) of topical nitroglycerin in addition to 5-flourouracil and radiation therapy for neo-adjuvant treatment of loco-regionally advanced operable rectal cancer. A secondary endpoint was rate of pathological complete response (pCR) and tumor response assessed by tumor regression (TGR).  Patients were assigned to 4 sequential cohorts (3 each) of escalating dose levels of commercially available nitro glycerin patches (0.2; 0.4; 0.6 and 0.8mg/hour). All patients received radiation therapy [45-50 Gy] in 25-28 fractions to the pelvis along with continuous infusion 5-FU [225mg/m2 / day] for the duration of the radiation therapy. The radiation therapy was planned and delivered as per institutional standard of care.

Results:
From 12/2010 to 8/2014, 15 patients were enrolled in the trial as a dose-escalation protocol. They were all male age (59.9±SD 8.5; range 42-70 years-old). The observed toxicities during the study protocol were mild to moderate and manageable. Four patients developed asymptomatic grade 3 lymphopenia during CRT that resolved promptly upon completion. This was not considered a serious side-effect and was not used for decision making in regards to therapy adjustments or dose-escalation.  Two other patients developed grade 3 toxicity (diarrhea and mucositis).  These were well managed and were not considered to be related to the NTG patches.  Seven patients developed headaches [HA] (5/15 grade I, 1/15 grade II, and 1/15 grade III).  One patient in the 0.2 mg dose developed a grade III HA requiring and additional group at this dose.   No further grade III HAs were observed following dose escalation.  HAs were managed with oral pain medication.  Two patients experience pCR (0.2 mg and 0.4 mg), six had a TGR of 1 and three of 2.  All of the patients in the 0.6 mg group were down staged from stage III to stage II.  Patients in the 8 mg cohort await surgical intervention.

Conclusion:
This data demonstrate that NTG patches can be safely utilized in conjunction with neoadjuvant CRT in patients with rectal cancer.

 

24.04 Targeting Colorectal Cancer Metastasis with Folate-Conjugated Ultrastable RNA Nanoparticles

Posted on December 22, 2014

P. Rychahou1,2, F. Haque4,5, Y. Shu4,5, Y. Zaytseva1, H. L. Weiss1, E. Y. Lee1,2,3, W. Mustain2, J. Valentino2, P. Guo1,4,5, B. M. 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,Nanobiotechnology Center,Lexington, KY, USA 5University Of Kentucky,Department Of Pharmaceutical Sciences,Lexington, KY, USA

Introduction: The majority of deaths from all cancers, including colorectal cancer (CRC), is the result of systemic metastasis, usually to the liver and lung. If advances are to be made in the survival of patients with stage IV CRC, more selective, highly innovative, and better targeted therapies are required. The purpose of this study was to: (i) test primary and metastatic CRCs for expression of folate receptor α (FRα), which could be used to target nanoparticles, and (ii) construct multifunctional and chemically stable RNA nanoparticles, that take advantage of receptor targeting, to specifically target CRC liver and lung metastases.

Methods: (1) To determine FRα expression, we obtained primary CRCs (n=12), CRC liver metastases (n=22) and CRC lung metastases (n=10). All samples were tested for FRα expression by immunohistochemistry (IHC) and blindly analyzed by a pathologist. (2) Thermodynamically stable 3-way junction (3WJ) RNA nanoparticles were constructed and confirmed by atomic force microscopy (AFM) and gel electrophoresis; folate and the fluorescent dye Alexa647 were conjugated to the nanoparticles to serve as a ligand for the binding to CRC cells and to provide a fluorescent marker, respectively. Nanoparticle binding to KM20 and HT29 human CRC cells was evaluated in vitro with confocal microcopy. (3) The conjugated RNA nanoparticles were next evaluated in murine (athymic nude mice) models of human CRC liver and lung metastasis.

Results: (1) FRα expression was detected in 78% of primary CRCs, 91% of CRC liver metastases, and 80% of CRC lung metastases. (2) In vitro evaluation demonstrated that folate conjugated RNA nanoparticles are selectively taken up by CRC cells via receptor-mediated endocytosis. (3) In vivo evaluation showed, for the first time, that folate-conjugated RNA nanoparticles remained intact after systemic injection and strongly bound to CRC liver and lung metastases. Conversely, no accumulation of nanoparticles was detected in normal liver or lung parenchyma. 

Conclusion: RNA nanoparticles specifically targeted metastatic liver and lung CRC cells, demonstrating the therapeutic potential of RNA-3WJ nanoparticles as a selective delivery system for the treatment of CRC metastasis. Our enthusiasm for current project is driven not only by its inherent scientific importance, but also by its translational potential, clinical impact, and the possibility to provide a more effective and less toxic delivery system targeting CRC metastases specifically in the liver and lung.

 

24.05 Nanoparticle-targeting of breast cancer stem cells improves efficacy and durability of chemotherapy.

Posted on December 22, 2014

P. T. White1, C. Subramanian1, P. T. Grogan1,2, S. Cai3, M. L. Forrest3, M. S. Cohen1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Kansas,Department Of Pharmacology, Toxicology And Therapeutics,Kansas City, KS, USA 3University Of Kansas,Department Of Pharmaceutical Sciences,Lawrence, KS, USA

Introduction: As the second leading cause of cancer-related death in US women, breast cancer recurrence, metastatic spread, and drug resistance remain a significant challenge despite current therapeutic advances. If only a small subpopulation of tumor cells called progenitor or stem cells survive treatment, they have the ability to transform and recreate a tumor or metastasize. To date no therapeutic specifically targets this cancer stem cell (CSC) population mainly due to the heterogeneity of its surface markers, although CD44 positivity is a commonality among most CSCs. We have developed a CD44-targeted hyaluronic acid nanoparticle drug-delivery platform and hypothesize that this targeting when conjugated to standard chemotherapy drugs will selectively target breast CSCs leading to an improved, more durable therapeutic response.

Methods: A panel of validated human breast cancer cell lines (SUM159,MDA-MB-468LN,T47D,SK-BR-3) were evaluated for CD44 expression levels by flow cytometry(FC). Cy5-labeled HA-drug conjugates (cisplatin, doxorubicin, and docetaxel) were evaluated by FC and fluorescent microscopy for cellular uptake and CD44 targeting. Breast CSCs were identified in the cell lines by CD44,ALDH1,CD133 marker positivity. Drug efficacy, toxicity and survival was performed using our prior published MDA-468LN triple neg model in female Nu/Nu mice via breast fat pad injection(10^6 cells). Animals were treated with either 50% or 75% MTD cis, dox, docetaxel(ACT) combination therapy weekly x 3wks vs. same doses conjugated to 35kDa HA nanoparticles vs. control then followed for survival, relapse, and tumor CD44 expression by Western Blot (WB).

Results: Receptor-mediated active drug uptake was CD44 dependent with SUM159 and MDA468LN having 80-90% uptake and >90% expression compared to 1-5% expression and uptake with CD44-low SK-BR-3 and T47D cells (p<0.001). Uptake is time dependent with a peak at 19hrs. Drug uptake was abrogated by 70-80% via CD44 saturation with excess free HA or CD44 antibody vs. unsaturated(p<0.01). 100% of HA-ACT therapy mice(75%MTD) had a complete clinical response during treatment without observed toxicity vs. 40% std ACT group(p<0.01). 85% of the HA-ACT mice(75% MTD) had a complete durable pathologic response 8 weeks post end of treatment vs. 0% survival in the std ACT and control arms by end of study(p<0.001). Tumor lysates of the HA-triple therapy mice had almost complete inhibition of CD44 expression by WB even 8 weeks after completion of treatment compared to no inhibition with the std. ACT and control groups.

Conclusions: HA-conjugation of chemotherapeutics is a novel approach to selectively target CD44 expressing breast tumor cells which includes the vast majority of breast CSCs. This in combination with improved tumoral drug delivery lead to significant improvement in survival and durability of response compared to standard combination therapy supporting further translation toward clinical applications.

20.06 Primary Gastrointestinal Tract Melanoma: Epidemiology And Outcomes For 1,044 Patients

Posted on December 22, 2014

V. Chakravorty1,2, K. Mahendraraj1, R. S. Chamberlain1,2,3  1Saint Barnabas Medical Center,Department Of Surgery,Livingston, NJ, USA 2St. George’s University School Of Medicine,St. George’s, St. George’s, Grenada 3New Jersey Medical School,Department Of Surgery,Newark, NJ, USA

Introduction: Primary melanoma of the gastrointestinal tract (GIM) are exceedingly rare entities, with an overall incidence of ~0.47 cases per million. While outcomes for cutaneous melanomas have been extensively examined, clinical information related to GIM is scant and derived from small case series. This study examined a large cohort of GIM patients to determine demographic, clinical, and pathologic factors affecting outcomes.

Methods: Demographic and clinical data on 1,044 patients with GIM was abstracted from the SEER database (1973-2010) and analyzed using the Chi square test, t-test, and multivariate analysis. Kaplan-Meier analysis was used to compare long-term survival between groups.

Results: 1,044 cases of GIM were identified, involving the oropharynx in 307 patients (29.4%), esophagus in 48 (4.6%), stomach in 26 (2.5%), small bowel in 38 (3.6%), large bowel in 19 (1.8%), rectum in 250 (23.9%), and anal canal in 356 (34.1%). Overall, there were 569 women (54.5%) and 475 men (45.5%; female-to-male ratio 1.2:1, p<0.001) with an overall mean age of 68+15 years. GIM was most common in Caucasians (73.7%, p<0.001) often presenting with localized (40.1%, p<0.001), poorly differentiated (68.1%, p=0.01) tumors measuring 2-4cm (34.5%, p<0.001), with uncommon lymphatic spread (37.9%, p<0.001). Mean survival for GIM was 3.8+0.3 years with longest survival seen among oropharyngeal GIMs (5.4+0.6 years, p<0.005) and shortest in gastric GIMs (0.7+0.2 years, p<0.005). Overall and cancer-specific mortality for GIM were 80.3% and 72.6%, respectively, (p=0.01). Surgical resection was the most common GIM therapy (64.8%, p<0.001), followed by surgery and radiotherapy (15.8%, p<0.001).  Survival was greatest with combination surgical and radiotherapy was used (4.4+0.8 years, p<0.001) compared to surgical resection only (4+0.3 years, p<0.001). Multivariate analysis identified regional (OR 1.9, CI 1.1-3.1) or distant disease (OR 4.5, CI 2.3-8.8), lymph node positivity (OR 4.5, CI 1.7-9.8), and age >60 (OR 6.2, CI 2.4-15) as independently associated with increased mortality for GIM (p<0.005).  Specific site of disease was not associated with increased mortality.

Conclusion: GIM is a rare malignancy that occurs in all parts of the GI tract with the highest incidence in the oropharyngeal and anorectal regions. GIMs are more common in Caucasian women in the seventh decade of life.  The majority of GIMs are localized, often poorly differentiated, with gastric GIM having the worst prognosis. Advanced age, stage and lymph node positivity are associated with increased mortality. Surgical resection with or without radiotherapy confers a significant survival advantage, and is standard therapy for all GIM patients with resectable disease.

20.07 Impact of Age and Gender on Immune Response and Clinical Outcome After Cancer Vaccination

Posted on December 22, 2014

A. G. Ramirez1, N. A. Wages2, M. E. Smolkin2, C. L. Slingluff1  1University Of Virginia,Surgery,Charlottesville, VA, USA 2University Of Virginia,Public Health Sciences,Charlottesville, VA, USA

Introduction:

Cancer vaccines have promise as monotherapy or in combination immunotherapy regimens.  The impact of patient age and gender on immune response and clinical outcome after cancer vaccinations are not known. Decline in immunogenicity with increasing age have led to uncertainty over inclusion of elderly patients in cancer vaccine trials. Gender differences in response to injury and to vaccination for infectious diseases suggest an independent effect of gender on immune response. We hypothesized younger age and female gender may be predictive of higher rates of immune response to a multipeptide cancer vaccine. 

Methods:

Patients with resected stage IIB-IV melanoma were enrolled in three clinical trials: Mel43, Mel44, and Mel48, received 6 vaccinations with 12 Class I MHC restricted peptides from melanocytic differentiation antigens and cancer testis antigens. T cell responses were detected by direct IFN-y ELIspot assay.  Clinical data, including age and gender, were collected. Cumulative incidence (CI) analyses of immune response measureable by Week 7 were used to compare patients based on age and gender, overall and within study arms. Kaplan-Meier estimates and log-rank tests were used to compare overall survival (OS) and disease-free survival (DFS). Chi-squared tests were performed to compare the percentage of immune responders by age and gender.

Results:

T cell responses were evaluated in 327 patients. Immune responses were detected in 49.5% (111/224) males and 47.6% (49/103) females. There was no difference in immune response by gender (p=0.74 by CI analysis; p=0.73 by Chi-squared test). Younger females responded earlier; however, the difference equilibrated over time. Menopausal status did not appear to be associated with difference in immune response (p=0.63). Interestingly, males experienced improved DFS (p=0.04) and a trend towards better OS (p=0.08). Female patients had a median DFS of 19.9 months versus 53.3 months for males. Immune responses were detected in 53% (130/249) of patients less than 64 yo but in only 38.5% (30/78) of older patients (p=0.02 by CI over time; p=0.03 by Chi-squared test). Sub-analysis of individual study arms showed comparable differences (p=0.02). No difference was detected in OS or DFS by age group (p=0.6 and 0.9, respectively).

Conclusion:

Our study reveals a decline in rates of immune response to peptide vaccines with increasing age; however, a significant percentage of elderly patients do develop immune responses to vaccination. Male patients had improved clinical outcomes, but gender differences were not associated with immune response.  Thus, in clinical trials with immunologic endpoints, age should be considered in comparing outcomes between study arms. Older patients can be immunized and should not be excluded on the basis of age.  Discordance between these clinical variables for immune response and clinical outcomes suggests complex interactions that may deserve further study with other immune therapies.

21.04 Staging Studies are of Limited Utility for Newly Diagnosed Clinical Stage I-II Breast Cancer

Posted on December 22, 2014

A. Linkugel1, J. Margenthaler1, A. Cyr1  1Washington University,General Surgery/College Of Medicine,St. Louis, MO, USA

Introduction:   For patients diagnosed with clinical Stage I-II breast cancer, treatment guidelines recommend against the routine use of radiologic staging studies in the absence of signs or symptoms suggestive of distant metastasis. However, these tests continue to be used for many early-stage breast cancer patients. This study aims to determine the utilization and yield of these studies at a National Comprehensive Cancer Network (NCCN) member institution.

Methods:   Female patients presenting with AJCC 7th Edition clinical stage I-II invasive breast cancer between 1998 and 2012 at Siteman Cancer Center, an NCCN member institution, were identified in a prospectively maintained institutional surgical database. Patients treated with neoadjuvant chemotherapy were excluded. Charts were reviewed to verify clinical stage and to document staging studies performed within six months of diagnosis.  Staging studies of interest included computed tomography (CT) of the chest, abdomen, and/or pelvis, bone scan, and positron emission tomography (PET).  Results of staging studies and additional diagnostic studies or procedures were recorded.  Descriptive statistics were used for the analysis.

Results:  A total of 3291 patients were included in the analysis (2044 were stage I and 1247 were stage II). Of these, 882 (27%) received CT of the chest, abdomen, and/or pelvis; bone scan; or PET within 6 months of diagnosis. A total of 691/882 (78%) received chest CT, 705/882 (80%) abdominal/pelvic CT, 704/882 (80%) bone scan, and 70/882 (8%) PET. Of these 882 patients, 312 were stage I (15% of the stage I cohort) and 570 were stage II (46% of the stage II cohort). Of the 882 patients imaged, 194 (22%) required additional imaging (x-ray, CT, bone scan, sonogram, or PET) and/or biopsies to follow-up abnormalities seen on the staging studies. However, only 11 of those 194 (6%) were confirmed to have metastatic disease (1.2% of the 882 imaged patients, 0.33% of the total study cohort). Of these 11 patients, one was clinically stage I at presentation, and 10 were stage II. Metastatic sites identified included lung (n=3), bone (n=4), liver (n=1), and a combination of sites (n=3). Numbers of patients determined to have metastatic disease were too small for comparative analysis.

Conclusions:  The identification of distant metastasis among clinical Stage I-II patients in this study was rare (0.33% of the total cohort). Even among patients judged appropriate for staging studies (CT, bone scan, and/or PET), only 1.2% were diagnosed with metastatic disease. These findings suggest that even at an NCCN member institution, staging studies are overused and lead to additional procedures in over 20% of patients.

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