Tu1927

Heat Shock Protein 27 Expression Is Inversely Correlated With IntraepithelialNeoplasia and Positively Correlated With Poor Differentiation of GastricCancerYoshiaki Nagata, Toshiharu Sakurai, Masaki Takayama, Tomoyuki Nagai, MasanoriKawasaki, Yutaka Asakuma, Satoru Hagiwara, Naoshi Nishida, Shigenaga Matsui, HiroshiKashida, Masatoshi Kudo

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflamma-tion and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaper-one, inhibit reactive oxygen species (ROS) accumulation. In previous reports have beensuggested that HSP27 plays an important role in inflammation and carcinogenesis. Weinvestigated HSP27 expression in gastric neoplasia and background gastric mucosa to assessits involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerasechain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosaeof 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients withoutgastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastriccancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis.HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P=0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased celldeath and ROS accumulation that link inflammation to cancer. Poorly differentiated tumorsmost frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated withan epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells,HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscleactin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastricmucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastriccancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Tu1928

Tissue Expression of Heat Shock Protein 27, 70 and 90 Might Be Utilized As aDiagnostic and Prognostic Biomarker in Patients With GastricAdenocarcinomaHamid R. Sima, Mahdi Zardadi, Kamran Ghaffarzadehgan, Ali Taghizadeh-Kermani,Mostafa Parizadeh, Mostafa Abrishami, Monavar Afzalaghaee, Majid Ghayour-Mobarhan

Introduction: Gastric cancer is the second leading cause of cancer-related death worldwideand the most common gastrointestinal cancer in Iran. Heat Shock Proteins (HSP) play animportant role in some inflammatory and neoplastic disorders. We sought to assess thetissue expression of HSP 27, 70 and 90 in tumoral and non-tumoral tissues in patients withgastric adenocarcinoma and their association with histological grade and tumor stage as wellas patients' survival. Methods: Eighteen patients with gastric adenocarcinoma who hadundergone gastrectomy were enrolled. Marginal non-tumoral tissues were considered as thecontrol group. Demographic and epidemiological characteristics were reviewed. Histologicaltype and grade and tumor stage (TNM) were determined by single pathologist. Tissueexpression of Heat Shock Proteins 27, 70 and 90 in tumoral and non-tumoral tissues wereassessed by IHC. Patients were followed and survival data were collected. Data were analyzedusing SPSS software version 16. Results: Fifty-eight patients (%72.5) were male with meanage of 64 years old. HSP 27, 70 and 90 were significantly expressed more in tumoral ratherthan non-tumoral tissues (P..001).Patients with gastric body tumor were found to havehigher HSP 27 expression (P=.049). Younger patient expressed more HSP 70 (P =.040).HSP 90 overexpression was seen more in patient with well differentiated or intestinal subtypeof tumor (P=.017 and P=.009, respectively). HSP 27 overexpression was associated withpoor prognosis (P =.018), however HSP 70 overexpression was associated with betterprognosis (P =.016). Conclusion: Tissue expression of Heat Shock Protein 27, 70 and 90mightbe utilized as a diagnostic and prognostic biomarker in patients with gastric adenocarcinoma.

Tu1929

Detection of Locally Methylated Septin 9 of Colorectal Cancer and Adenomain Peripheral Blood Is Related to Circulating Free DNA AmountBéla Molnár, Kinga Tóth, Ferenc Sipos, Katalin Leiszter, Alexandra Kalmár, Árpád V.Patai, Andrea Scholler, Gabor Valcz, Barnabás Wichmann, Zsolt Tulassay

Background: Methylated Septin 9 (SEPT9) is a sensitive and specific biomarker for colorectalcancer (CRC) and adenoma on tissue level. In CRC it was proven to be a highly sensitiveperipheral blood marker. However, its detection in peripheral blood for adenoma screeningis rarely studied. Aims: 1) Compare SEPT9 methylation from matched plasma and tissuesamples from healthy, adenoma and CRC. 2) Evaluation of Septin 9 in relation to circulatingfree DNA (cfDNA) amount 3) SEPT9 detection from higher amount of plasma samples inadenoma cases. Material and Methods: Plasma samples were collected from patients withno evidence of disease (NED), adenomas and CRC (n = 40 per class). Matching biopsytissues were available from 33 NED, 26 adenoma and 34 CRC patients. Plasma sampleswere processed using the Epi proColon kit (Epigenomics AG, Berlin, Germany). Total DNAfrom tissue was prepared using High Pure PCR Template Preparation Kit followed by EpiproColon bisulfite treatment. Quantitative determination of total DNA and SEPT9 wasperformed using the Epi proColon RT-PCR assay. In 6 adenoma specimens SEPT9 wasevaluated from higher amount (from 7 to 10.5 ml) than the suggested plasma volume (3.5ml). Results: SEPT9 PMR (percent methylation reference) values larger than 1%were detectedin 30.3% (10/33) NED, 100% (26/26) adenoma and 100% (34/34) CRC biopsy tissues. Inlaser microdissected epithelial cells, Septin 9 significantly underexpressed in CRC comparedto healthy controls (p,0,001), but not in stroma samples. In plasma from the same patients,SEPT9 PMR values larger than 0.01%. It was detected in 5% (2/40) NED, 25% (10/40)adenoma and 87.5% (35/40) CRC. cfDNA amount was similar in normal and adenomacases, but significantly increased in CRC (p,0.01). SEPT9 methylation was detected in 50%(3/6) from higher plasma amount of adenoma samples. Conclusions: Healthy and cancerplasma and tissue samples showed strong correlation in SEPT9 methylation, however it was

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not maintained for adenoma samples due to lower cfDNA amount. Sensitivity for adenomasamples could be increased with the evaluation of higher amount plasma.

Tu1930

Next Generation Sequencing Analysis of Cfdna in Patients With ColorectalCancer Compared to IBD, Adenoma and Healthy ControlsAndrea Scholler, Sandor Spisak, Árpád V. Patai, Alexandra Kalmár, Barnabás Wichmann,Barbara Bartak, Zsofia Nagy, Béla Molnár, Zsolt Tulassay

Introduction: During the progression of colorectal cancer and inflammaton the circulatingcell free DNA (cfDNA) level increases significantly in the plasma from 10 - 20 ng/ml to 100- 200 ng/ml or more. DNA methylation is a well known epigenetic regulation of genefunctions and its alteration can be observed during cancer development. Aims: Our aimwas to detect changes in cfDNA isolated from peripheral blood of healthy, IBD, colorectaladenoma and cancer patients using recent new generations sequencing techniques in relationto methylation pattern changes. Materials and methods: We investigated 50-50 patients withadenoma, IBD, colorectal cancer and healthy control patients. CfDNA was isolated from theplasma with QIAamp Circulating Nucleic Acid Kit (Qiagen).From each sample 50-100 ngextracted DNA was assembled in each groups and pooled DNA was applicated for furthersequencial analysis (SOLiD sequencing). After matching native plasma samples to the refer-ence genome, coverage and pile up diagrams were defined. We sought for positions at leasttwentyfive times covered and also showing differencies between healthy and cancer samples.Results: Region between 92075 and 92115 derived from SEPT9 gene has shown 6200 timeshigher number of reads in CRC samples in 50-50% from reverse and forward direction toensure that is not artifact. This position is exactly matched with region detected by EpiProCo-lon Kit (Epigenomics), a method based on the enrichment of methylated fragments. It isalready proven that methylated SEPT9 (mSEPT9) is present 94,7% in CRC patients. Thecoverage of mSEPT9 was 245 times higher in adenoma and only 8 times higher in healthypatients plasma sample. SFRP1, SFRP2, MAL and PRIMA1 genes proceeds the same resultwhich means that enrichment of methylated regions were significantly higher than in ade-noma, IBD, and healthy samples. Conclusion: General hypomethylation and local hypermeth-ylation is characteristic in tumours that is reflected in cfDNA content. The detection of thesespecific epigenetic changes from peripheral blood can be important step to develop screeningand diagnostic assays.

Tu1931

Magnetic Resonance Imaging of Hand/Wrist to Evaluate Growth in Coeliacand Non-Coeliac ChildrenMaria Bavastrelli, Monica Montuori, Mariarita Maimone, Lucilla Caivano, Daniela Merletti,Raffaella Nenna, Francesco M. Perla, Ernesto Tomei, Margherita Bonamico

Objectives: The aim of the present study is to evaluate the growth of children, through theassessment of bone age with Magnetic Resonance Imaging (MRI) of hand/wrist in coeliacand non-coeliac children. We also studied the bonemarrow intensity in all children. Methods:We enrolled 91 children afferent to the Coeliac and Malabsorption Diseases Unit of theDepartment of Pediatrics and infantile Neuropsychiatry, Sapienza University of Rome. 45patients (26F, 19M) average age 7,3 years, were affected by coeliac disease. The diagnosiswas based on the ESPGHAN criteria of 1990. These patients were all following a gluten-free diet since at least one year, with excellent compliance, proved by the normal level ofanti-transglutaminase antibodies. 46 children (22F,24M) median aged 6,9 years, were notaffected by coeliac disease. The growth of all patients was evaluated through a clinicalcriteria: the percentile charts that represent the distribution of height according to age andsex. All children underwent MRI of hand/wrist. A single sequence T13DSE thin layer (1,3mm)in an open magnet (0,2 T) was performed after parents' informed consent, approved by theEthics Committee of our University. We assessed skeletal age through the comparison ofthe MRI of hand/wrist of 180 healthy children matched by sex and age, grouped in a"Greulich and Pyle" like atlas, created by the Department of radiology of Sapienza UniversityRoma. Results: In 85/91 (93,4%), the bone age assessed through MRI corresponded to thepercentiles. Only 6 children (6,6%) 4 with CD and 2 without, showed correlation betweenskeletal and anagraphic age, whereas there was no correspondence with the percentile charts(minus 2 SD). Finally an increased bone marrow density in 16/45 (35,5%) CD patients wasevidenced, in comparison to the non -coeliac patients group, where no modifications werefound. Conclusion: We believe that this innovative method of bone age assessment throughthe use of MRI in alternative to the X-rays, could represent a new and safer marker ofgrowth. It is therefore possible to follow growth through MRI taken at shorter time periods.The meaning of the bone marrow intensity alteration is until today unknown and neverresearched before in pediatric populations. Such alteration was found on a consistent groupof children (35,5%). All were affected by CD; we emphasize the importance of future studieson the correlation between MRI growth's assessment, bone marrow intensity and coeliacdisease on a larger group of children.

Tu1932

Endoscopic Features of Food Protein-Induced Proctocolitis in Neonates andInfantsYoshiko Nakayama, Yousuke Shima, Mai Kusakari, Nao Hidaka, Sawako Kato, AkiraHoriuchi, Kenichi Koike

Background and aims: Food protein-induced proctocolitis (FPIPC) is a non-IgE mediatedfood hypersensitivity disorder. It presents with overt rectal bleeding in otherwise healthyinfants and is characterized by an eosinophilic infiltration. The aim of this study was todetermine the endoscopic features of FPIPC in neonates and infants. Methods: Twenty-onepatients, who presented with persistent or recurrent rectal bleeding, were evaluated bycolonoscopy under sedation at the endoscopic units. Biopsies were taken from the everylandmark. The diagnosis of FPIPC was based on the eosinophil infiltration by histology(.20/hpf) and the repose to elimination of regular milk or maternal diet. Results: Of the

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