PITUITARY ADENOMA PITUITARY ADENOMA HORMONAL AND MEDICAL MANAGEMENT.
Su1892 Sept9 DNA Methylation in Healthy, Adenoma and Colorectal Cancer Patients: A Comparison...
Transcript of Su1892 Sept9 DNA Methylation in Healthy, Adenoma and Colorectal Cancer Patients: A Comparison...
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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.
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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.
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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.
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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.
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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
determination of total DNA and mSEPT9 was performed using the Epi proColon real time-PCR assay. Septin9 protein expression in biopsy tissuewas evaluated by immunhistochemistryusing a polyclonal antibody for Septin9. Results: mSEPT9 PMR (percent methylation refer-ence) values larger than 1% were detected in 7.7% (1/13) NED, 100% (10/10) adenomaand 100% (16/16) CRC biopsy tissues. In plasma from the same patients, however, mSEPT9PMR values larger than 0.01% were detected in 10% (2/20) NED, 25% (5/20) adenomaand 75% (15/20) CRC. Immunhistochemical detection of Septin9 protein decreased withincreasing disease pathology, with Septin9 expression detected in 100% NED, 50-60%adenoma and 10-20% CRC tissues. Conclusions: mSEPT9 was confirmed as a highly sensitivebiomarker for the detection of CRC in blood. While the high level of mSEPT9 in CRC tissuecorrelated strongly with sensitive detection of these cancers in plasma, the strong correlationwas not maintained in tissue and plasma from adenoma patients. Varying degrees of tissuevascularization might underlie this observation, among other explanations. Finally, mSEPT9in biopsy tissue was inversely correlated with expression of the Septin9 protein.
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Circulating Free Tumor DNA as a Promising Marker for the Prediction ofSurvival and Monitoring of Remission in Colorectal Cancer After RadicalSurgeryGabriela Veprekova, Petra Minarikova, Marek Minarik, Lucie Benesova, BarboraBelsanova, Ludmila Lipska, Miroslav Levy, Miroslav Zavoral
Background The process of colorectal cancer (CRC) development is associated with tumorsuppressor genes inactivation and oncogenes activation. Tumor cell DNA is released as aresult of genome instability and tumor necrosis and can be detected as circulating cell-freeDNA (cfDNA) in a patients' plasma. The aim of our study was to evaluate the presence ofcirculating tumor cfDNA in patients before and after radical surgery. Methods In a groupof 201 patients with verified CRC (at stages I to IV), tumorous tissue was tested for thefollowing oncogenes KRAS, BRAF, PIK3A and tumor suppressor genes APC and TP53. Weanalyzed blood samples for circulating cfDNA at the time of diagnosis in all patients witheither of the above mutation present. Blood samples were obtained and tested for the samegenes in a 3 months interval. In patients undergoing surgery additional blood samples weretaken on the 1st and 7th postoperative day. The median value for dispensarisation was 13.5months, range 3-30 months. The detected cfDNA results were compared with clinicaloutcomes. Results In 71.6 % (144 of 201 cases) at least one mutation was detected in aprimary tumor. In 24.3 % of those (35 of 144 cases) circulating cfDNA was detected. Thedetection rate of cfDNA correlated with a particular clinical stage (Fig. 1). 75 patients witha known mutation underwent radical surgery, from those in only 10 patients cfDNA wasdetected prior to surgery. In stage I no cfDNA was detected (0 of 10), in stage II 4.5 % wasdetected (2 of 44), in stage III 27.7 % was detected (5 of 18), in stage IV 100 % was detected(3 of 3). No positive sample was detected in samples obtained on the 1st and 7th postoperativeday. 8 patients (80 %) maintained negative results completely. Mutated cfDNA was detectedin one case 4 months after R0 surgery - this correlated with the progression of a primarydisease. In another case cfDNA positivity was detected 17 months after R0 surgery with aCT scan revealing CRC relapse, however, after undergoing oncological treatment “genetic”remission was achieved since December 2010. Conclusion Circulating tumor cfDNA correl-ates with the clinical stage of colorectal cancer. If detected, it has the potential to assess theradicality of surgery as well the monitoring of remission in CRC patients. The recurrenceof cfDNA most likely indicates the relapse of a primary disease. Supported by grant IGAMZ NS9809.
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Nanoarchitectural Changes of Chromatin by Histone Deacetylase (HDAC)Dysregulation Occurs in Colorectal Field CarcinogenesisYolanda E. Stypula, Dhwanil Damania, Dhananjay Kunte, Hariharan Subramanian, MartDeLaCruz, Amir C. Patel, Hemant K. Roy, Vadim Backman
Introduction: Our group has focused on using the novel optics technology, partial wavespectroscopic microscopy (PWS), to assess field carcinogenesis for colorectal cancer (CRC)via analysis of endoscopically normal rectal brushings (reviewed in Gastro 2011). Themechanisms for these light scattering signatures remain unclear but since PWS targets thenano-architecture and our data suggests a strong nuclear component, this implicates highorder chromatin (Biophysics J 2010). To investigate the biological underpinnings, we consid-ered the histone deacetylase (HDAC) family of proteins since they are critical regulators ofhigh order chromatin and dysregulated in many cancers. In this study, we investigate whetherHDAC expression occurs during field carcinogenesis (via rectal biopsies) and also if HDACis responsible for altered PWS parameters. Methods: For PCR analysis, total RNA fromhuman rectal biopsy samples (n=24) was isolated using Trizol reagent. Gene expression wasanalyzed using custommade Taqman LowDensity Arrays (TLDA). Pharmacological inhibitionof HDACs was performed using increasing concentrations of valproic acid, VPA (0.1-1.5mM).We used HT29 colon cancer cell lines displaying varying tumorigenicity: HT29 control and
S-529 AGA Abstracts
CSK (C-terminal Src Kinase) shRNA knockdown, which was previously created and is amore aggressive cell line (Mol Cancer Ther 2008) with concomitant increase in Ld (PNAS2008). The effect of HDAC inhibition on cell viability was established by WST-1 cellproliferation assay. PWS was performed as previously described (Cancer Res 2009) withchromatin abnormalities confirmed by transmission electron microscopy (TEM). Results:We found HDAC4 and HDAC7 to be significantly upregulated (20-40%) in human rectalbiopsies (P-value < 0.05). Upon treatment of VPA, cell viability decreased. Interestingly, theeffect of HDAC inhibition was greater in the more tumorigenic colon cancer cell line (CSKconstructs). The PWS results also showed that the effect of VPA on nuclear disorder strengthwas greater in the CSK constructs (Table 1). TEM images of control and treated cell linesalso show reduced chromatin clumping and altered distribution following VPA treatment(Figure 1). These results support the hypothesis that HDAC inhibitors target more tumorig-enic cell types and that this is related to the higher-order chromatin structure. Conclusions:We demonstrate for the first time, that HDAC4 and HDAC7 are upregulated in humancolorectal field carcinogenesis. Furthermore, we note that HDAC may be responsible forthe PWS nanoscale changes. These novel findings may have implications for screening,chemoprevention (via targeting HDACs) and cancer biology (role of high order chromatinalterations in early carcinogenesis).Disorder strength differences following HDAC inhibitor (VPA) treatment.
Representative TEM images of untreated CSK construct nucleus and CSK construct nucleusfollowing 0.5mM VPA treatment.
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Plasma Methylated Sept9 is a Screening Marker in Both Left- and Right-SidedColon Cancer. Comparison to FOBT and CEA ResultsKinga Tóth, Jürgen K. Beck, Kerstin Buser, Zsolt Tulassay, Robert Stöhr, HenrietteGolcher, Vera Schellerer, Béla Molnár
Background: Methylated SEPT9 (mSEPT9) DNA is a sensitive and specific biomarker forthe detection of colorectal cancer (CRC) from peripheral blood. Many aspects of the biologyof mSEPT9 in the plasma of CRC patients have yet to be explored, including relationshipto CRC location, FOBT positivity and other blood-based tumor markers. Aims: 1) Determinethe sensitivity of mSEPT9 for CRC detection in the left- and right-sided CRC2) ComparemSEPT9 and FOBT positivity rates in individuals with no evidence of disease (NED) andCRC patients. 3) Compare mSEPT9 and another blood-based tumor marker, carcinoem-bryonic antigen (CEA), in NED and CRC patients. Materials and Methods: Plasma samplesfor mSEPT9 analysis were collected from NED (n = 92) and CRC (n = 92) after colonoscopy.Total DNAwas prepared and bisulfite-converted using the Epi proColon kit 2.0 (EpigenomicsAG, Germany). Qualitative determination of total DNA and mSEPT9 was performed usingthe Epi proColon 2.0 real time-PCR assay (Roche LC480). Samples for FOBT were collectedfrom NED (n = 17) and CRC (n = 22) prior to colonoscopy. Serum samples for CEA analysiswere collected from NED (n = 27) and CRC (n = 27). Results: mSEPT9 values were detectedin 15.2 % (14/92) NED and 95.6 % (88/92) CRC. FOBT was positive for 29.4 % (5/17)NED and 68.2 % (15/22) CRC. Positive CEA results were detected in 14.8 % (4/27) of NEDand 51.8 % (14/27) CRC. When analyzed according to location within the colon, mSEPT9was positive in 96.4 % (54/56) of left-sided CRC and 94.4 % (34/36) of right-sided CRC.FOBT was positive in 66.7 % (10/15) of left-sided CRC and 33.3 % (5/15) of right-sidedCRC. CEA was positive in 64.3 % (9/14), of left-sided CRC and 35.7 % (5/14) of right-sided CRC. Conclusion: mSEPT9 was confirmed as a highly sensitive biomarker for thedetection of CRC in blood. SEPT9 methylation level has no difference between left and rightside colon cancer. mSEPT9 showed higher sensitivity for CRC compared to FOBT in CRC,specially in right sided cancers. Plasma mSEPT9 showed higher sensitivity in both sides ofCRC then blood CEA.
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Serum Glucose Enhances the Ability of Carbohydrate Antigen 19-9 (CA 19-9)to Discriminate Cases of Pancreatic Adenocarcinoma From Matched ControlsNeeraj Sardana, Gary J. Badger, Richard Zubarik
Purpose: Pancreatic adenocarcinoma (PAC) is a disease for which no effective screeningmodality exists. A majority of patients with PAC are diagnosed with diabetes either at thetime of diagnosis or within two years prior. The pathophysiological relationship betweenPAC and impaired glucose tolerance remains poorly understood. The serum tumor markercarbohydrate antigen 19-9 (CA 19-9) is often elevated in patients with PAC but has limitationswhen used alone as a screening test. We set out to determine whether fasting serum glucose(FSG) can enhance the ability of serum CA 19-9 alone to discriminate patients with and
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