W1756

Expression of the Cancer Testis Antigens PLAC1 and MAGE3A in ColonTumors As Potential Targets for Specific Immunotheraphy for a Subset ofCRC PatientsM. C. Shantha Kumara H, Otavia L. Caballero, Su Tao, Aqeel Ahmed, Keith E. Hoffman,Vesna Cekic, Andrew J. Simpson, Carlos Cordon-Cardo, Richard L. Whelan

Introduction: Genes that encode Cancer testis (CT) antigens are normally expressed onlyin the human germline. High frequencies of expression in various tumors and restrictedexpression in normal tissues make the CT antigens attractive cancer vaccine targets. PLAC1,MAGE3A and GAGE are genes with high expression in placenta/or testis and different cancertypes with relatively restricted expression in normal tissues. The aim of this study was toevaluate PLAC1, MAGE3A and GAGE as vaccine targets in colorectal cancers. Method:Consenting CRC patients who underwent elective resection for whom tumor samples andpreoperative blood samples were available comprise the study population. Basic demographicand clinical as well as short term outcome data was prospectively collected. Tissues wereparaffin embedded and also stored at -80οC. Total purified RNA was isolated and cDNAsynthesized. Comparative quantitative PCR (QPCR) was performed using the SYBR Greenplatform. Comparative quantitative analysis was performed based on delta-delta Ct methodusing GAPDH as internal control. Expression levels in tumors were compared to expressionlevels in Testis (MAGE3 and GAGE ) or levels in Placenta (PLAC1) and samples found toexpress 0.1% or more of the testis or placenta levels were considered positive for theexpression of these genes. Results: 35 tumors and paired normal tissue samples were studied(83% colon, 17% rectal). Stage breakdown was as follows: stage 2, 18; stage 3, 16; andstage 4, 1 patient. Relative QPCR values of 35 malignant and paired normal samples forMAGEA3 and QPCR values of 31 pairs for PLAC1 and GAGE were available for analysis.Relative expression ratio of malignant to normal tissues over one(1) was higher for PLAC1(77%) and moderate for GAGE (35.5%) and MAGEA3 (31%). However, both expressionratio over one (1) and expression levels above 0.1% of the levels in testis or placenta werenoted for MAGE3A in 26%, PLAC1 (19%) but not for GAGE1. Conclusion: In a subset oftumors, the relative expression of MAGE3A and PLAC1 was considered above the expressionin paired normal colon tissues and had more than 0.1% of the levels in testis and placenta.Serological evaluation and immunohistochemistry studies are needed to further evaluate thetumor subsets presenting MAGEA3 and PLAC1 expression. These results suggest thatMAGEA3 and PLAC1 might be useful as vaccine targets for a subset of CRC patients andfurther studies are warranted.

W1757

Interstitial Cells of Cajal in AchalasiaReginald V. N. Lord, Yuri Y. Bobryshev, Michael Buckland, Steven R. DeMeester, Jeffrey A.Hagen, Murray C. Killingsworth, Tom R. DeMeester

Background: Interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinaltract. By connecting neuronal cells and smooth muscle cells, ICCs provide a continuousnetwork responsible for the spontaneous electrical and mechanical activity of smooth musclecells. In this study we tested the hypothesis that the absent peristalsis and impaired relaxationof the lower esophageal sphincter in achalasia are due to disappearance of ICCs in themuscular layer (muscularis externa or propria) of the esophagus. Methods: Representativedistal esophageal tissue specimens from nine patients who underwent esophagectomy forend stage achalasia were identified from the medical records. The diagnosis of achalasia wasconfirmed in all patients by manometry. Normal distal esophageal tissue specimens fromten patients who underwent esophagectomy for squamous cell carcinoma of the mid- orupper esophagus were used for controls. Formalin-fixed, paraffin-embedded tissues weresectioned and stained for CD117/c-kit using standard antigen retrieval immunohistochemicaltechniques. CD117-positive ICCs were distinguished from CD117-positive mast cells onthe basis of their stellate morphology and by the use of CD117/mast cell tryptase doubleimmunostainining. ICCs located in the circular and longitudinal layers of the muscularisexterna were counted in ten high power (x400) fields by two independent observers. Electronmicroscopy was performed to examine ICC ultrastructural features in achalasia. Results:ICCs were identified in the muscularis externa layer in all nine achalasia specimens and all10 normal control specimens. The median and (range) ICC numbers per high power fieldin the achalasia specimens were 13.85 (7.0 - 23.9) for the inner circular muscle layer and9.6 (7.8 - 11.5) for the outer longitudinal muscle layer, and 16.05 (6.5 - 26.7) and 10.9(5.2 - 18.5) for the normal esophagus inner and outer muscluaris externa layers. The ICCdensity was not significantly different in achalasia compared to normal control specimens inany layer. Electron microscopy revealed the presence of cells with typical ICC ultrastructuralappearance in achalasia tissue specimens. Conclusion: This study did not find a significantdifference in the density of ICCs in achalasia compared to normal esophagus specimens.These results suggest that the motility defects that characterize achalasia result from insuffi-cient neuronal input and perhaps impaired ICC activity rather than insufficient numbersof ICCs.

W1758

Laparoscopic Fundoplication: New Insights Into Neural Anatomy At theEsophagogastric JunctionHeinz Wykypiel, Erich Brenner, Helga Fritsch, Florian Struller, Thomas Muehlbacher,Judith Stempel, Gabriel Djedovic, Oliver Renz

Background: In laparoscopic fundoplication the mobilisation of the distal esophagus andthe proximal stomach are mandatory in order to obtain enough intraabdominal esophageallength and to enable a posterior fundoplication. In surgical literature and in most anatomicillustrations there are no nerves described between diaphragm and stomach. Moreover themechanism of the so-called postfundoplication complaints is not fully understood yet. Weintraoperatively observed small nerve branches penetrating the left crus of the diaphragmlateral of the hiatus, presumably going into the stomach. Their course (from lateral to medial)suggested that they could be branches of the splanchnic nerves or phrenic nerves rather

A-935 SSAT Abstracts

than branches of the vagus. It was the aim of the study to find these nerves in cadaversand to describe their origin and their target organ. Material and Methods: 53 cadavers (23male 30 female, age range: 35-103 years) were dissected with special attention to the courseof the nerves penetrating the left crus of the diaphragm. The whole course of the nerveswas documented with drawings and photos. Results: In 17 out of 53 cadavers, one or twosplanchnic nerves were found. Seen from below, they penetrated the left crus of the diaphragmin mean at the 4 o'clock (range: 2-5) position, in mean at a distance of 2cm (range: 0-5)from the hiatus, all of them going into the stomach (cardia or fundus). Ten out of themhad an additional branch going into the coeliac ganglion. In 14 cadavers, a branch of thephrenic nerve was found, penetrating the left crus at a 4 o'clock position, in mean at adistance of 7,3cm (range: 3-10). These phrenic nerves mostly went into the stomach (fundus),but in two cases, additional branches going to the coeliac ganglion were found. Conclusion:Branches of the splanchnic nerves and of the phrenic nerve, penetrating the left crus of thediaphragm at the 3-5 o'clock position are regularily found. Most of them go to the stomach,and some also have branches to the coeliac ganglion. The role of these nerves is not clearyet, but they all are cut regularily during a laparoscopic (posterior) fundoplication. Whetherthe dissection of these nerves during fundoplication contributes to postfundoplication symp-toms such as postprandial bloating has to be cleared in further studies.

W1759

Timing-Dependent Protection of Hypertonic Saline Solution Administration inExperimental Liver Ischemia/Reperfusion InjuryEstela R. Figueira, Telesforo Bacchella, Ana Maria M. Coelho, Sandra N. Sampietre, NilzaA. Molan, Regina Leitao, Marcel C. Machado

During liver ischemia, the drop in mitochondrial energy causes cellular damage, which isaggravated after reperfusion. This injury can trigger a systemic inflammation, also producingremote organ damage. Several substances have been employed to reduce this inflammatoryresponse during liver transplantation, liver resections and hypovolemic shock. Aim: Toevaluate the effects of hypertonic saline solution and the best timing of administration toprevent organ injury during experimental liver ischemia/reperfusion. Methods: Animalsunderwent one hour of warm liver ischemia followed by reperfusion. One hundred-twentyrats were allocated into six groups (n=20). S: sham; C: control animals submitted to parcialliver ischemia; ISSpi: rats received NaCl 0.9% 3.4mL/kg, 15 min before ischemia; HTSpi:rats received NaCl 7.5% 0.4mL/kg, before ischemia; ISSpr: rats received NaCl 0.9% 15 minbefore reperfusion; HTSpr: rats received NaCl 7.5% before reperfusion. Four hours afterreperfusion blood was collected for AST, ALT, IL-6 and IL-10 analyses. Evans blue dye wasintravenously administrated to eight animals from each group. Rats were killed for liverhistology, phosphorylation of liver mitochondria, edema of liver tissue and pulmonaryvascular permeability analyzes. Results: HTSpr group presented elevation of AST (1984±207UI/L) and ALT (1539±208 UI/L) significantly lower than C (AST 3141±303 UI/L, ALT2903±347 UI/L) and ISSpr (AST 3169±218, ALT 2791±245 UI/L) groups. The comparisonsof HTSpi group to ISSpi or to C groups were not statistically significant. Additionally HTSprgroup showed AST and ALT levels significantly lower than HTSpi group (AST 2691±257UI/mL, ALT 2484±328 UI/mL). No significant differences in IL-6 and IL-10 levels wereobserved. A significant reduction on mitochondrial dysfunction was observed in HTSprgroup compared to ISSpr and C groups (p<0.05). Also ADP/O ratio was significantly reducedin HTSpi group (1.63±0.032) compared to HTSpr (1.83±0.039). Liver tissue edema wassignificantly lower in HTSpr group (69.8±0.4%) compared to ISSpr (72±1,1%) and C(73±1,4%) groups. Pulmonary vascular permeability was significantly lower in HTSpr group(148±17 mg EBD/g tissue) compared to ISSpr (282±35 mg EBD/g tissue) and C (280±52mg EBD/g tissue) groups. No significant differences in myeloperoxidase activity wereobserved. The liver injury histological score was significantly lower inHTSpr group (8.1±1.32)compared to HTSpi group (11.4±0.93). Conclusions: HTS ameliorated liver and lung injuriesin experimental liver ischemia/reperfusion. Infusion of HTS in pre-reperfusion period iscrucial to accomplish the best results. FAPESP 05/042267

W1760

Human Tissue Slice Model for Evaluating Conditionally Replicative OncolyticAdenovirus Specificity to Pancreatic CancerAmanda K. Arrington, Julia Davydova, Eric J. Brown, Abhinav Humar, Eric H. Jensen,Selwyn M. Vickers, Masato Yamamoto

Pancreatic adenocarcinoma is the fourth leading cause of cancer-related deaths in the US.Conditionally replicative oncolytic adenoviruses (CRAds) have been developed and testedin clinical trial, but fell short of clinical expectation. We have developed new CRAds forpancreatic cancer with RGD modification to facilitate efficient transduction in CAR negativepancreatic cancer cells and Cox2 promoter-based replication control, which eliminatestoxicity in normal organs including liver and normal pancreas. A preclinical challenge thatoncolytic viruses face is the absence of suitable animal model which permits In Vivo viralreplication for toxicology. Given that human adenoviral shows replication tropism to naturalhost, human cells, the optimal model to use is human tissue. In this study, we used tissueresected from patients undergoing operative procedures to access our adenoviral vectors.METHODS: After obtaining IRB approval, we collected normal liver, normal pancreas,and pancreatic cancer tissue from specimens resected from patients undergoing plannedresections. The tissue was immediately sliced into 200 micron slices using the KrumdieckTissue Slicer and incubated overnight. They were then infected with Ad RGD wild type orRGDCox2CRAdF vectors. On day 3, samples were collected and DNA was extracted. E4copy number and B-actin was determined for each sample by PCR. Statistical analysis wasperformed by Student's T-test. RESULTS: After compensation with B-actin, the Ad RGD Cox2vector showed high replication in pancreatic adenocarcinoma tissues while its replication innormal liver and normal pancreas was minimal. In contrast, the replication of RGDWt washigh regardless of the origin of the tissues. When the replication of these two vectorswere compared in each type tissue, the percentage of compensated viral copy numberRGDCox2CRAdF in pancreatic adenocarcinoma tissue was significantly higher than thosein normal pancreas and normal liver (Table). CONCLUSION: RGDCox2CRAdF vectorsshow high specificity for pancreatic adenocarcinoma tissue compared to normal pancreas

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