Su1891 Identification of Methylome Signatures in Cancer and Adenoma vs. Normal Colon Tissues by Next...
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AGA Abstracts (10.8-33.9)-fold in patients harboring remote dysplasia (p=0.002) compared to normal controls. Compared to UC patients without dysplasia, UC patients harboring remote dysplasia showed 8.2 (4.6-14.5)-fold upregulation of REG1α (p=0.001). UC patients harboring remote dysplasia had increased epithelial staining of REG1α compared to UC patients without dysplasia (p=0.04) and normal controls (p=0.04). Control individuals showed no colonic mucosal REG1α immunostaining. UC patients harboring remote dysplasia had a 7.6 ± 5.8 -fold increase in crypt staining compared to UC patients without dysplasia. In a tissue microarray of 29 UC-associated cancers, 25/29 cancers showed positive glandular epithelial staining. Of these, nine had focal weak positivity, two had diffuse weak positivity, eight had focal strong positivity, and six had diffuse strong positivity. Conclusions: REG1α transcript and protein levels were increased in UC patients harboring remote dysplasia compared to UC patients without dysplasia and normal controls. By immunohistochemistry, UC-associated colon cancers demonstrated increased glandular staining of REG1α. These findings suggest that REG1α may play an important role in the development of UC-associated cancer and could serve as a potential biomarker for early detection of neoplastic lesions in this high- risk population. Su1888 Prognostic Significance of Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Operable Esophageal and Gastric Cancer David S. Chan, Tom D. Reid, Guy Blackshaw, Tom Crosby, Wyn G. Lewis Introduction. Trastuzumab (Herceptin) allied to standard chemotherapy has been demon- strated to improve survival in patients with advanced metastatic gastric cancer, yet reports on the role of HER2 overexpression in patients with operable esophagogastric cancer have conflicted. The primary aim of this study was to determine the prognostic significance and relative HER2 receptor expression in patients with operable esophagogastric cancer, undergoing radical D2 lymphadenectomy. Methods. Eighty-five consecutive patients dia- gnosed with esophagogastric adenocarcinoma [18 esophageal (EC), 32 junctional (JC), 35 gastric (GC), and 25 neoadjuvant chemotherapy] undergoing R0 resection were studied retrospectively. Immunohistochemistry was used to determine patients' HER2 status at the endoscopic index biopsy and the final operative resection specimen. The primary outcome measure was survival. Results. Twenty (23.5%) patients had HER2 positive tumors, and positive HER2 status was commoner in JC (14/32, 44% vs. 2/18, 11% in EC and 4/35, 11% in GC; Chi2 = 11.66, p=0.003). Comparison of HER2 expression status between the index biopsy and final operative resection specimen revealed sensitivity, specificity, positive and negative predictive values were 56%, 93%, 63%, 91% respectively. The weighted Kappa statistic (Kw) was 0.504 95% CI 0.128 - 0.856, p<0.0001). Cumulative five year survival related to HER2 status 30% for the HER2 positive cohort compared with 43% for the HER2 negative cohort (p=0.221). With regard to tumor site 5 year survival in EC HER2 positive vs. negative cohorts was 100% and 36% (p=0.167) compared with 14% and 44% (p= 0.0726) in JC and 50% and 46% (p=0.942) in GC respectively. Conclusions. The overall proportion of HER2 positive tumours was significantly higher in patients with junctional adenocarcinomas when compared with EC and GC, and was associated with poorer long term survival. HER2 expression as defined by the index endoscopic biopsy had a high specificity and negative predictive value suggesting a larger study is needed to confirm these findings because of the implications for neoadjuvant and adjuvant chemotherapy. Su1889 Pre-Treatment Neutrophil to Lymphocyte Ratio as a Marker for Predicting Chemotherapeutic Response and Prognosis for Metastatic Advanced Gastric Cancer In Rae Cho, Jun Chul Park, Ji Young Yoon, Kyung Seok Cheoi, Hyuk Lee, Sung Kwan Shin, Sang Kil Lee, Yong Chan Lee Background and Aims Advanced gastric cancer (AGC) is one of the most common cause of cancer-related death in worldwide. However, only few studies investigated prognostic factors in metastatic AGC patients who are receiving palliative chemotherapy. Neutrophil to lympho- cyte ratio (NLR) in the peripheral blood has been shown to be a prognostic factor of various cancers in several studies. However, only limited information on the clinical and prognostic significance of NLR in patients with gastric cancer has been reported, especially in metastatic status. Therefore, we examined whether the NLR can be used as a marker for predicting chemotherapeutic response and prognosis for metastatic AGC who are receiving palliative chemotherapy. Method Total 269 patients diagnosed with metastatic AGC were enrolled between January 2006 and November 2009. NLR was calculated from lymphocyte and neutrophil counts on complete blood cell count taken before 1st chemotherapy. Patients were divided into two groups: higher than median N/L ratio and lower than median. Then, we compared two groups by chemotherapeutic response rate, progression-free survival (PFS) and overall survival (OS). Results The median follow-up period was 340 days (range 72 ~1796 days) and median N/L ratio was 3.06 (range 0.18~18.16). 139 patients (NLR ≥ 3.0) were placed in the high NLR group, and 129 patients (NLR < 3.0) were placed in the low NLR group. Low NLR group patients had a significantly higher chemotherapeutic response rate than high NLR group patients (89.9% versus 80.7%, p=0.034). Univariate analyses reveled that there is a significant relationship between the NLR and patients survival. (both PFS and OS) Patients in low NLR group had longer PFS and OS than high NLR group patients. (186 vs. 150 days; p= 0.001; 414 vs. 285 days; p=0.001, respectively). In multivariate analysis with Cox-regression model, NLR was shown a significant relationship with PFS (HR 1.466, 95% CI 1.137-1.889) and OS (HR 1.492, 95% CI 1.163-1.914). Conclusion These results suggest that the elevated pre-treatment NLR can predict poor chemotherapeutic response rate, progression-free survival and overall survival in metastatic advanced gastric cancer patients. S-528 AGA Abstracts Su1890 Colorectal Cancer Screening by Breath Analysis: A Specific Pattern of Volatile Organic Compounts (VOCs) Can Discriminate Between Patients and Healthy Controls Maria Di Lena, Francesca Porcelli, Livia Trizio, Simona Giuratrabocchetta, Elisabetta Travaglio, Maria Tutino, Gianluigi De Gennaro, Donato F. Altomare Introduction: early diagnosis of colorectal cancer by non-invasive and sensitive screening method is still a major goal in oncological research. Recent investigations have evidenced the presence of specific pattern of Volatile Organic Compounds (VOCs) in the breath of patients with lung and liver cancer and with mesotelioma, as a consequence of an increased oxidative stress and metabolic derangements of cancer cells. Aim of this preliminary prospect- ive comparative trial is to investigate whether colorectal cancer patients have a disease specific patter of VOCs in their breath compared with healthy population. Patients and Methods: exhaled breath was collected in an inert bag (Tedlar®) according to a validate sampling protocol using a dedicated device from non-smoker patients with histologically proved colorectal cancer and non-smoker healthy controls (subjects negative at colonoscopy per- formed for colorectal cancer screening). The exhaled breath was processed offline in a thermal desorber gas chromatography mass spectrometry (TDGSMS) in order to evaluate the spectrum of the VOCs which were identified using an international library of compounds. For each spectrum spike the area was calculated. A training phase was carried out to set up the predictive model which was tested prospectively in a blind fashion in the prospective trail. Statistics: VOCs produced by the bags and these with extreme variability within each group were screened out. The Support vector machine (SVM)methods validated by the “leave one out” method were used to identify the pattern of VOC which better discriminate the two groups. Results: the VOCs of 34 patients and 36 controls, well matched for sex, but not for age, were examined. 58 VOCs were detected, 53 of which were identified by the library. 23 VOCs (mainly long chain aldeids) were selected in the training phase to set up the prognostic specific pattern able to discriminate patients from controls. Using the SVM method 100% of the cases were correctly allotted and 86% of them were still correctly recognized in the internal validation test with the leave-one-out. The sensitivity and specificity of the breath test was 83% and 88% respectively and the area under the ROC curve 0.944. In the prospective blind trial phase further 10 colorectal cancer patients and 10 healthy controls were tested confirming an accuracy of about 80%. Discussion: analysis of VOCs in oncology is a new promising diagnostic method and, although our data needs further confirms, it seems to be a very powerful method for the screening of colorectal cancer. Su1891 Identification of Methylome Signatures in Cancer and Adenoma vs. Normal Colon Tissues by Next Generation Sequencing Hassan Ashktorab, Sudhir Varma, Hassan Brim Background: Colorectal adenoma (CRA) to cancer (CRC) progression involves epigenetic changes including DNA, miRNA, LINE, and SINE methylation. Here we performed whole genome Bisulfite Next-Generation Sequencing (WGBNGS) on a normal, a tubular adenoma and a tumor patient's tissues DNA to elucidate epigenetic drivers in normal to cancer progression. Patients and Methods: Genomic DNA was isolated from fresh frozen tissues from a patient with normal colon, from a tubular adenoma lesion (<0.5 cm) and from a carcinoma . A WGBNGS at >20X reads depth was performed on these DNA samples. Alignment, mapping and CpG methylation analyses were done. Pyrosequencing and immuno- histochemistry (IHC) were used to confirm selected methylation targets . Results: We identi- fied 94 unique CpG sites hypermethylated in 4 novel genes in adenoma compared to normal and 180 unique CpG sites in 7 genes in cancer. Ingenuity pathway analysis (IPA) showed that the methylated genes including a novel gene ATXN7L1 were involved in WNT pathway, ubiquitination process and cross-talk with B-catenin, in the adenoma. Three of the hyperme- thylated genes in cancer were; Loc100506436: A novel gene with 17 unique CpG sites (EGFR pathway, cRNA CpG site hypermethylated is part of MEK1/2), GPNMB with 7 unique CpG sites (membrane protein likely in complexes with integrins) and TNFAIP2 with 8 unique CpG sites (its role was noted in Head and neck cancer). IHC confirmed the lack of GPNMB expression in cancer tissues. We also identified several miRNA including miR-192 that, along with miR215, influence resistance to 5FU treatment. SINE and LINE hypermethyl- ation also identified in CRC vs. adenoma. Conclusion: Comparing methylomes of CRC, adenoma and normal revealed a subset of differentially methylated promoters and strikingly divergent methylation in LINE, SINE and miRNA subfamilies, with an apparent carcinogenic impact that is evident in the underlying methylom/genomic sequence. Thus, our study potentially will reveal DNA methylation features with therapeutic and prognostic value. Su1892 Sept9 DNA Methylation in Healthy, Adenoma and Colorectal Cancer Patients: A Comparison Between Tissue and Plasma Specimens Kinga Tóth, Orsolya Galamb, Ferenc Sipos, Katalin Leiszter, Gabor Valcz, Reinhold Wasserkort, Zsolt Tulassay, Béla Molnár Background: Colorectal cancer (CRC) is one of the most frequent causes of cancer-related death worldwide. Colonoscopy is the gold standard for early detection of CRC, but patient compliance in CRC screening programs is low. Blood-based tests are thought to increase patient compliance for cancer screening. Epi proColon, a product based on detection of SEPT9 DNA methylation (mSEPT9), has shown to be suitably sensitive and specific for the detection of CRC in blood. Aims: 1) Correlation of mSEPT9 in matched peripheral blood and biopsy tissue samples. 2) Evaluation of mSEPT9 for adenoma detection in blood. 3) Correlation between mSEPT9 and Septin9 protein expression in tissue. Material and Methods: Plasma samples were collected and analyzed from patients with no evidence of disease (NED), adenomas and CRC (n = 20 per class). Matching biopsy tissues were available from 13 NED, 10 adenoma and 16 CRC patients. Total DNA from tissue was prepared using QIAamp DNA extraction followed by custom bisulfite treatment. Plasma samples were processed using the Epi proColon kit (Epigenomics AG, Berlin, Germany). Quantitative
Transcript of Su1891 Identification of Methylome Signatures in Cancer and Adenoma vs. Normal Colon Tissues by Next...
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s(10.8-33.9)-fold in patients harboring remote dysplasia (p=0.002) compared to normalcontrols. Compared to UC patients without dysplasia, UC patients harboring remote dysplasiashowed 8.2 (4.6-14.5)-fold upregulation of REG1α (p=0.001). UC patients harboring remotedysplasia had increased epithelial staining of REG1α compared to UC patients withoutdysplasia (p=0.04) and normal controls (p=0.04). Control individuals showed no colonicmucosal REG1α immunostaining. UC patients harboring remote dysplasia had a 7.6 ± 5.8-fold increase in crypt staining compared to UC patients without dysplasia. In a tissuemicroarray of 29 UC-associated cancers, 25/29 cancers showed positive glandular epithelialstaining. Of these, nine had focal weak positivity, two had diffuse weak positivity, eight hadfocal strong positivity, and six had diffuse strong positivity. Conclusions: REG1α transcriptand protein levels were increased in UC patients harboring remote dysplasia compared toUCpatients without dysplasia and normal controls. By immunohistochemistry, UC-associatedcolon cancers demonstrated increased glandular staining of REG1α. These findings suggestthat REG1α may play an important role in the development of UC-associated cancer andcould serve as a potential biomarker for early detection of neoplastic lesions in this high-risk population.
Su1888
Prognostic Significance of Human Epidermal Growth Factor Receptor 2(HER2) Expression in Operable Esophageal and Gastric CancerDavid S. Chan, Tom D. Reid, Guy Blackshaw, Tom Crosby, Wyn G. Lewis
Introduction. Trastuzumab (Herceptin) allied to standard chemotherapy has been demon-strated to improve survival in patients with advanced metastatic gastric cancer, yet reportson the role of HER2 overexpression in patients with operable esophagogastric cancer haveconflicted. The primary aim of this study was to determine the prognostic significanceand relative HER2 receptor expression in patients with operable esophagogastric cancer,undergoing radical D2 lymphadenectomy. Methods. Eighty-five consecutive patients dia-gnosed with esophagogastric adenocarcinoma [18 esophageal (EC), 32 junctional (JC), 35gastric (GC), and 25 neoadjuvant chemotherapy] undergoing R0 resection were studiedretrospectively. Immunohistochemistry was used to determine patients' HER2 status at theendoscopic index biopsy and the final operative resection specimen. The primary outcomemeasure was survival. Results. Twenty (23.5%) patients had HER2 positive tumors, andpositive HER2 status was commoner in JC (14/32, 44% vs. 2/18, 11% in EC and 4/35, 11%in GC; Chi2 = 11.66, p=0.003). Comparison of HER2 expression status between the indexbiopsy and final operative resection specimen revealed sensitivity, specificity, positive andnegative predictive values were 56%, 93%, 63%, 91% respectively. The weighted Kappastatistic (Kw) was 0.504 95% CI 0.128 - 0.856, p<0.0001). Cumulative five year survivalrelated to HER2 status 30% for the HER2 positive cohort compared with 43% for the HER2negative cohort (p=0.221). With regard to tumor site 5 year survival in EC HER2 positivevs. negative cohorts was 100% and 36% (p=0.167) compared with 14% and 44% (p=0.0726) in JC and 50% and 46% (p=0.942) in GC respectively. Conclusions. The overallproportion of HER2 positive tumours was significantly higher in patients with junctionaladenocarcinomas when compared with EC and GC, and was associated with poorer longterm survival. HER2 expression as defined by the index endoscopic biopsy had a highspecificity and negative predictive value suggesting a larger study is needed to confirm thesefindings because of the implications for neoadjuvant and adjuvant chemotherapy.
Su1889
Pre-Treatment Neutrophil to Lymphocyte Ratio as a Marker for PredictingChemotherapeutic Response and Prognosis for Metastatic Advanced GastricCancerIn Rae Cho, Jun Chul Park, Ji Young Yoon, Kyung Seok Cheoi, Hyuk Lee, Sung KwanShin, Sang Kil Lee, Yong Chan Lee
Background and Aims Advanced gastric cancer (AGC) is one of the most common cause ofcancer-related death in worldwide. However, only few studies investigated prognostic factorsin metastatic AGC patients who are receiving palliative chemotherapy. Neutrophil to lympho-cyte ratio (NLR) in the peripheral blood has been shown to be a prognostic factor of variouscancers in several studies. However, only limited information on the clinical and prognosticsignificance of NLR in patients with gastric cancer has been reported, especially in metastaticstatus. Therefore, we examined whether the NLR can be used as a marker for predictingchemotherapeutic response and prognosis for metastatic AGC who are receiving palliativechemotherapy. Method Total 269 patients diagnosed with metastatic AGC were enrolledbetween January 2006 and November 2009. NLR was calculated from lymphocyte andneutrophil counts on complete blood cell count taken before 1st chemotherapy. Patientswere divided into two groups: higher than median N/L ratio and lower than median. Then,we compared two groups by chemotherapeutic response rate, progression-free survival (PFS)and overall survival (OS). Results The median follow-up period was 340 days (range 72~1796 days) and median N/L ratio was 3.06 (range 0.18~18.16). 139 patients (NLR ≥ 3.0)were placed in the high NLR group, and 129 patients (NLR < 3.0) were placed in the lowNLR group. Low NLR group patients had a significantly higher chemotherapeutic responserate than high NLR group patients (89.9% versus 80.7%, p=0.034). Univariate analysesreveled that there is a significant relationship between the NLR and patients survival. (bothPFS and OS) Patients in low NLR group had longer PFS and OS than high NLR grouppatients. (186 vs. 150 days; p= 0.001; 414 vs. 285 days; p=0.001, respectively). Inmultivariateanalysis with Cox-regression model, NLR was shown a significant relationship with PFS(HR 1.466, 95% CI 1.137-1.889) and OS (HR 1.492, 95% CI 1.163-1.914). ConclusionThese results suggest that the elevated pre-treatment NLR can predict poor chemotherapeuticresponse rate, progression-free survival and overall survival in metastatic advanced gastriccancer patients.
S-528AGA Abstracts
Su1890
Colorectal Cancer Screening by Breath Analysis: A Specific Pattern of VolatileOrganic Compounts (VOCs) Can Discriminate Between Patients and HealthyControlsMaria Di Lena, Francesca Porcelli, Livia Trizio, Simona Giuratrabocchetta, ElisabettaTravaglio, Maria Tutino, Gianluigi De Gennaro, Donato F. Altomare
Introduction: early diagnosis of colorectal cancer by non-invasive and sensitive screeningmethod is still a major goal in oncological research. Recent investigations have evidencedthe presence of specific pattern of Volatile Organic Compounds (VOCs) in the breath ofpatients with lung and liver cancer and with mesotelioma, as a consequence of an increasedoxidative stress andmetabolic derangements of cancer cells. Aim of this preliminary prospect-ive comparative trial is to investigate whether colorectal cancer patients have a disease specificpatter of VOCs in their breath compared with healthy population. Patients and Methods:exhaled breath was collected in an inert bag (Tedlar®) according to a validate samplingprotocol using a dedicated device from non-smoker patients with histologically provedcolorectal cancer and non-smoker healthy controls (subjects negative at colonoscopy per-formed for colorectal cancer screening). The exhaled breath was processed offline in athermal desorber gas chromatography mass spectrometry (TDGSMS) in order to evaluatethe spectrum of the VOCs which were identified using an international library of compounds.For each spectrum spike the area was calculated. A training phase was carried out to setup the predictive model which was tested prospectively in a blind fashion in the prospectivetrail. Statistics: VOCs produced by the bags and these with extreme variability within eachgroup were screened out. The Support vector machine (SVM)methods validated by the“leave one out” method were used to identify the pattern of VOC which better discriminatethe two groups. Results: the VOCs of 34 patients and 36 controls, well matched for sex,but not for age, were examined. 58 VOCs were detected, 53 of which were identified bythe library. 23 VOCs (mainly long chain aldeids) were selected in the training phase to setup the prognostic specific pattern able to discriminate patients from controls. Using theSVM method 100% of the cases were correctly allotted and 86% of them were still correctlyrecognized in the internal validation test with the leave-one-out. The sensitivity and specificityof the breath test was 83% and 88% respectively and the area under the ROC curve 0.944.In the prospective blind trial phase further 10 colorectal cancer patients and 10 healthycontrols were tested confirming an accuracy of about 80%. Discussion: analysis of VOCsin oncology is a new promising diagnostic method and, although our data needs furtherconfirms, it seems to be a very powerful method for the screening of colorectal cancer.
Su1891
Identification of Methylome Signatures in Cancer and Adenoma vs. NormalColon Tissues by Next Generation SequencingHassan Ashktorab, Sudhir Varma, Hassan Brim
Background: Colorectal adenoma (CRA) to cancer (CRC) progression involves epigeneticchanges including DNA, miRNA, LINE, and SINE methylation. Here we performed wholegenome Bisulfite Next-Generation Sequencing (WGBNGS) on a normal, a tubular adenomaand a tumor patient's tissues DNA to elucidate epigenetic drivers in normal to cancerprogression. Patients and Methods: Genomic DNA was isolated from fresh frozen tissuesfrom a patient with normal colon, from a tubular adenoma lesion (<0.5 cm) and from acarcinoma . A WGBNGS at >20X reads depth was performed on these DNA samples.Alignment,mapping and CpGmethylation analyses were done. Pyrosequencing and immuno-histochemistry (IHC) were used to confirm selected methylation targets . Results: We identi-fied 94 unique CpG sites hypermethylated in 4 novel genes in adenoma compared to normaland 180 unique CpG sites in 7 genes in cancer. Ingenuity pathway analysis (IPA) showedthat the methylated genes including a novel gene ATXN7L1 were involved in WNT pathway,ubiquitination process and cross-talk with B-catenin, in the adenoma. Three of the hyperme-thylated genes in cancer were; Loc100506436: A novel gene with 17 unique CpG sites(EGFR pathway, cRNA CpG site hypermethylated is part of MEK1/2), GPNMB with 7 uniqueCpG sites (membrane protein likely in complexes with integrins) and TNFAIP2 with 8unique CpG sites (its role was noted in Head and neck cancer). IHC confirmed the lack ofGPNMB expression in cancer tissues. We also identified several miRNA including miR-192that, along with miR215, influence resistance to 5FU treatment. SINE and LINE hypermethyl-ation also identified in CRC vs. adenoma. Conclusion: Comparing methylomes of CRC,adenoma and normal revealed a subset of differentially methylated promoters and strikinglydivergent methylation in LINE, SINE and miRNA subfamilies, with an apparent carcinogenicimpact that is evident in the underlying methylom/genomic sequence. Thus, our studypotentially will reveal DNA methylation features with therapeutic and prognostic value.
Su1892
Sept9 DNA Methylation in Healthy, Adenoma and Colorectal Cancer Patients:A Comparison Between Tissue and Plasma SpecimensKinga Tóth, Orsolya Galamb, Ferenc Sipos, Katalin Leiszter, Gabor Valcz, ReinholdWasserkort, Zsolt Tulassay, Béla Molnár
Background: Colorectal cancer (CRC) is one of the most frequent causes of cancer-relateddeath worldwide. Colonoscopy is the gold standard for early detection of CRC, but patientcompliance in CRC screening programs is low. Blood-based tests are thought to increasepatient compliance for cancer screening. Epi proColon, a product based on detection ofSEPT9 DNA methylation (mSEPT9), has shown to be suitably sensitive and specific for thedetection of CRC in blood. Aims: 1) Correlation of mSEPT9 in matched peripheral bloodand biopsy tissue samples. 2) Evaluation of mSEPT9 for adenoma detection in blood. 3)Correlation betweenmSEPT9 and Septin9 protein expression in tissue. Material andMethods:Plasma samples were collected and analyzed from patients with no evidence of disease(NED), adenomas and CRC (n = 20 per class). Matching biopsy tissues were available from13 NED, 10 adenoma and 16 CRC patients. Total DNA from tissue was prepared usingQIAamp DNA extraction followed by custom bisulfite treatment. Plasma samples wereprocessed using the Epi proColon kit (Epigenomics AG, Berlin, Germany). Quantitative
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