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In the last decades, HOX proteins have been extensively studied due to their pivotal role in transcriptional events. HOX proteins execute their activity by exploiting a cooperative binding to PBX proteins and DNA. Therefore, an increase or decrease in HOX activity has been associated with both solid and haematological cancer diseases. Thus, inhibiting HOX-PBX interaction represents a potential strategy to prevent these malignancies, as demonstrated by the patented peptide HTL001 that is being studied in clinical trials. In this work, a computational study is described to identify novel potential peptides designed by employing a database of non-natural amino acids. For this purpose, residue scanning of the HOX minimal active sequence was performed to select the mutations to be further processed. According to these results, the peptides were point-mutated and used for Molecular Dynamics (MD) simulations in complex with PBX1 protein and DNA to evaluate complex binding stability. MM-GBSA calculations of the resulting MD trajectories were exploited to guide the selection of the most promising mutations that were exploited to generate twelve combinatorial peptides. Finally, the latter peptides in complex with PBX1 protein and DNA were exploited to run MD simulations and the ΔGbinding average values of the complexes were calculated. Thus, the analysis of the results highlighted eleven combinatorial peptides that will be considered for further assays.

The anti-inflammatory properties of products that contain high levels of humic acid, such as peat, sapropeles, and mumie, are well described. The aim of this study was to establish the clinical efficacy of brown coal-derived potassium humate, as in vivo animal models had suggested similar efficacy of humate to prednisolone. In this double-blind, placebo-controlled study, atopic volunteers were recruited and randomized to placebo or 1.8 g potassium humate/day (three divided dosages) when they presented with hay fever. The efficacy parameters used were skin-prick tests for wheal and flare reactions, nasal smears for inflammatory cell accumulation, and cytokine levels. Patients also filled out daily symptom score cards to determine efficacy against symptoms of hay fever. Potassium humate resulted in a significant decrease in wheal and flare reactions as well as nasal eosinophil counts. These findings were supported by similar changes (not significant) in cytokine levels. Changes in symptom scores did not reach significance. This proof of concept study clearly demonstrated the potential of potassium humate in the treatment of inflammatoryconditions such as hay fever. Drug Dev Res 71:358-363, 2010. C!J 2010Wiley-Liss, Inc.

This research was financially supported by Unique Health Trust and a grant from the South African National Research Foundation (NRF).

To date cancer vaccines have yet to show efficacy in a phase III trial. However, the clinical benefit seen with monoclonal antibody mediated therapies (e.g., Herceptin) has provided proof of principle that immune responses directed against tumour-associated antigens could have therapeutic potential. The failure of past cancer vaccine trials is likely due to several factors including the inappropriate choice of tumour antigen, use of an unoptimised antigen delivery system or vaccination schedule or selection of the wrong patient group. Any one of these variables could potentially result in the induction of an immune response of insufficient magnitude to deliver clinical benefit. Live recombinant viral vaccines have been used in the development of cancer immunotherapy approaches for the past 10 years. Though such vectors are self-adjuvanted and offer the ability to express multiple tumour-associated antigens (TAAs) along with an array of immune co-factors, arguably, they have yet to demonstrate convincing efficacy in pivotal clinical trials. However, in recent years, more coordinated studies have revealed mechanisms to optimise current vectors and have lead to the development of new advantageous vector systems. In this review, we highlight that live recombinant viral vectors provide a versatile and effective antigen delivery system and describe the optimal properties of an effective viral vector. Additionally, we discuss the advantages and disadvantages of the panel of recombinant viral systems currently available to cancer vaccinologists and how they can work in synergy in heterologous prime boost protocols and with other treatment modalities.

Tumour cells are able to evade the immune system by using several 'escape mechanisms'. Downregulation of molecules involved in the processing and presentation of self-antigens has been reported. However, these adaptations have not been compared in metastases in different anatomical locations but derived from a single patient. We investigated three melanoma cell lines--MJT1 from the parietal lobe of the brain, MJT3 from the cerebellum and MJT5 from the left side of the neck--established from biopsies excised from a 45 year old female patient. Although human leukocyte antigen (HLA) class I was detected in all three cell lines by flow cytometry using an anti-HLA monomorphic antibody, further serological analysis demonstrated HLA B38 loss in all three cell lines, HLA B7 downregulation in MJT5 (skin metastases) and B7 loss in MJT3 and MJT1 (brain metastases) compared with the HLA type of the patient's normal autologous lymphocytes. Interferon-gamma (IFNgamma) treatment increased the expression of HLA class I and transporters associated with antigen processing 1 (TAP1) in all three cell lines. De novo HLA class II molecule expression was observed after IFNgamma treatment in MJT3 and MJT5. Western blot and reverse transcription-polymerase chain reaction results revealed heterogeneity of melanoma-associated antigen (MAA) expression in the cell lines: MJT3 cells expressed higher levels of MAAs than the other two cell lines. In conclusion, this study has demonstrated that three metastatic lesions from a single patient can have differential expression of molecules involved in antigen processing (TAP1) and presentation (HLA I and II), but that expression of these molecules is modulated by IFNgamma to a similar degree in all cell lines. In contrast, the downregulation of expression of specific MAAs between the three cell lines was unaffected by the addition of IFNgamma.

Soft tissue sarcomas are rare tumors derived from mesenchymal cells. The histologic diversity of sarcomas is vast, and new discoveries in molecular alterations are enhancing our understanding of soft tissue sarcomas. The majority of sarcomas develop in the extremities and the retroperitoneum. A detailed history and physical exam along with high-quality cross-sectional imaging are important first steps in the workup of a soft tissue sarcoma. Management of sarcomas relies upon a multidisciplinary approach with multiple treatment options including surgery, radiation, and cytotoxic chemotherapy.

Although studies have suggested that exposure to cigarette smoke (CS) may be associated with the development of atopy, the mechanisms underlying this are not clearly understood. It has been suggested that CS impairs the barrier function of the airway epithelium, leading to increased access of allergens such as those of the house dust mite (HDM) Dermatophagoides pteronyssinus (Der p) to antigen-presenting cells, with subsequent allergic sensitization. In order to test this hypothesis, we established primary explant cultures of human bronchial epithelial cells (HBEC) in cell culture inserts, and exposed these for 20 min, 1 h, 3 h, and 6 h to CS or air in the absence or presence of 300 ng/ml Der p, and then further incubated the cultures over a period of 24 h. The HBEC cultures were assessed for changes in permeability as measured by changes in: (1) electrical resistance (ER); and (2) passage of 14C-labeled bovine serum albumin (14C-BSA) and Der p allergens across the HBEC cultures. We also assessed the effects of protease inhibitors and the antioxidant glutathione (GSH) in this experimental system. Damage to HBEC cultures was assessed by the release of [51Cr]sodium chromate from prelabeled cells, and by release of lactate dehydrogenase (LDH). Twenty minutes of exposure to CS as compared with exposure to air did not significantly alter either the ER or passage of 14C-BSA across the HBEC cultures. In contrast, incubation with Der p led to a significant increase in the permeability of HBEC cultures, an effect that was enhanced by exposure to CS but was abrogated by the specific protease inhibitors and GSH. Passage of Der p was also increased by exposure to CS. Exposure of HBEC cultures to CS led to a significant release of 51Cr and LDH from these cells as compared with cells exposed to air. This effect was augmented further when HBEC cultures were incubated with Der p. Exposure of HBEC cultures for 1 h, 3 h, and 6 h to CS led to a markedly significant dose- and time-dependent increase in the permeability of these cells. These results suggest that exposure to CS significantly enhances Der p-induced decreases in electrical resistance and the increased passage across HBEC cultures of 14C-BSA and of the Der p allergen itself.

Live attenuated bacterial vectors, such asSalmonella typhimurium, have shown promise as delivery vehicles for DNA. We have examined two new strains ofS. typhimuriumand their impact on dendritic cell maturation (CD12-sifA/aroCmutant and WT05-ssaV/aroC, both in TML background). Strain WT05 matured dendritic cells in a more efficient way; caused higher release of cytokines TNF-α, IL-12, IL-1β; and was efficient for gene transfer. These findings suggest that the genetic background of the attenuation can influence the pattern of inflammatory immune response toSalmonellainfection.

e20063 Background: Among melanoma patients, prior reports show that groups with lower socioeconomic status (SES) may be diagnosed later. We sought to develop a fuller understanding of the character...

Sentinel lymph nodes provide an excellent opportunity to study early immune responses to cancer. However, harvesting live cells has not previously been possible, because it conflicts with the need to preserve tissue for histological interpretation. This study used scrape cytology on 26 sentinel and 8 non-sentinel nodes, harvested from 17 stage I/II melanoma patients undergoing sentinel node biopsy. Numbers of viable cells harvested before and after cryopreservation were measured and the effect on subsequent histology assessed. The mean number of cells harvested from 26 sentinel nodes was 7.06×106 (range 0.1–32.2), with a mean viability of 99.5% (range 87–100, lower 95% CI 98.5%). Furthermore, counts and viabilities were well maintained after cryopreservation. Flow cytometry confirmed CD3+, CD20+ and lineage-1−/HLA-DR+ subpopulations, consistent with T-lymphocytes, B-lymphocytes and dendritic cells, respectively. Importantly, there was no discernible change in histological detail and the proportion of positive sentinel nodes remained unchanged. This technique will allow more functional and quantitative approaches to sentinel lymph node research.

175 Background: Circulating tumor cells (CTCs) are cells shed into circulation from tumors. They are increasingly recognized to be important biomarkers of disease burden in patients with solid tumors. Studies in pancreatic ductal adenocarcinoma (PDAC) have been limited due in part to low sensitivity of existing assays with extremely low numbers detected (0-15 per 7.5-15ml of blood). The development of newer microfluidic platforms has resulted in the ability to detect substantially greater numbers of CTCs. Methods: 15 patients with PDAC were enrolled in an IRB-approved study. Blood was collected in EDTA tubes and processed within 3 hours. CTCs were selected from 2-3ml of whole blood using monoclonal antibodies against epithelial cell adhesion molecule (EpCAM) and enumerated using a novel microfluidic platform. CTCs were confirmed by DAPI, CK8/18/19 and CD45 staining. Results: Patients with metastatic disease (n=12) had a mean of 29.7, median of 22 (3.5-107) CTCs per 1ml of blood. Patients with localized disease (n=3) had a mean of 3.8, median of 2.9 (2.3-6.2) CTCs per 1ml of blood. 0.5-1 CTCs per 1ml were detected in normal controls (n=2). The number of CTCs was significantly different between localized, metastatic and normal patients (p=0.01). 1 patient, initially thought to have localized disease by standard imaging but found to be metastatic at time of operation, had a mean of 45.9 CTCs per 1ml of blood compared to a mean of 3.8 CTCs per 1ml in patients who underwent a curative resection (p=0.009). Conclusions: Studies of CTCs in PDAC have been very limited. Our ability to detect large numbers of CTCs with good dynamic range suggests that further investigation into CTCs as a prognostic marker in PDAC is warranted. This is the first study that we are aware of to find CTCs in patients with localized disease. The presence of CTCs in patients with localized PDAC is surprising and may be associated with findings of unexpected metastatic disease at surgery. Further follow-up will be needed to determine if the presence of CTCs in these patients is a harbinger of shorter progression-free survival and overall survival after curative operations.

444 Background: It is well known that KRAS mutations limit the efficacy of anti-EGFR therapy in patients with metastatic colorectal cancer (mCRC). However the role of KRAS mutations in patients who undergo a curative liver resection for mCRC is less clear. The purpose of our study was to evaluate the relationship between KRAS mutation status and survival in this patient population. Methods: We examined an IRB approved tissue repository and retrospective database of 129 patients from 1998-2010 who underwent curative liver resection for mCRC. Tumors were sequenced for KRAS codons 12, 13, and 61 mutations using pyrosequencing. Overall survival (OS) and disease-free survival (DFS) were analyzed using the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards regression method. Results: The median follow-up for our cohort was 20.4mo (0.4-112). Mean age was 61.4±12.3. Prior to surgical resection 55 (43%) patients received chemotherapy. 35 (27%) tumors were KRAS mutant (mt), 83 (64%) were wild-type (wt), and 11 (9%) were not characterized. Median OS for KRAS wt patients was 40.3mo vs. 27.1mo for KRAS mt patients (p=0.046). Median DFS for KRAS wt was 13.6mo vs. 7.7mo for KRAS mt patients (p=0.037). 8 patients received cetuximab post–operatively. Cetuximab status was unknown in 50 patients. When we excluded those treated with cetuximab, the median OS was 40mo for KRAS wt vs. 25mo for KRAS mt patients (p=0.007). There were no differences in OS or DFS in patients who received cetuximab (p=0.7). In a multivariable model with pre-operative chemotherapy (p=0.2), extent of resection (p=0.053), and cetuximab therapy (p=0.7), the presence of KRAS mutation was independently associated with poor prognosis (HR=2.7 [1.3-5.5]). Conclusions: In patients undergoing curative liver resection for mCRC, KRAS mutation status is independently predictive of a worse outcome regardless of cetuximab therapy. KRAS status may be associated with more aggressive tumor biology. Our data supports the critical need to define KRAS mutation status and to develop therapies against KRAS and its downstream effectors.