Maria J. Maturana

Screening and Triage Test for Early Detection of Gastric Cancer (STEAD-GC): A noninvasive trial for early detection of gastric cancer.

17 Background: Gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. Previously, we identified a potential biomarker for non-invasive detection of GC, the DNA methylation of the promoter region of Reprimo, a p53-dependent G2 arrest mediator candidate (Clin Cancer Res 2008;14:6264-9). Furthermore, we developed a quantitative assay (MethyLight) for a mass screening of GC (DDW2011-1029128). Here we reported the preliminary findings of our ongoing prospective trial STEAD-GC (Screening and Triage test for Early Detection of Gastric Cancer) which is being conducted in Chile, a country with a high mortality rate for GC. METHODS Twenty GC cases (tumor, non-tumor tissues and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. RESULTS Concentrations of DNA were similar in both groups (Avg 32.2 ng/ml [range: 8.9-70.8 ng/ml]). The average DNA levels of Reprimo were higher in GC [964,215 copies/ml, 539,593 copies/ml and 80,113 copies/ml in tumor, non-tumor and plasma, respectively] but lower in symptomatic chronic gastritis [137,721 copies/ml and 8,387 copies/ml, tissue and plasma, respectively]. METHODS Twenty GC cases (tumor, non-tumor tissues, and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. CONCLUSIONS By using our previous cut-off of 15,125 copies/ml, our method correctly identified 10 out of 12 gastritis cases and 16 out of 20 GC cases (p value <0.001). Our data confirms our non-invasive method for early detection of GC may be suitable for a mass screening of GC.

Infecciones y alteraciones epigenéticas en cáncer

The role of epigenetics and infectious diseases at early stages of life influence pre-malignant lesions of cancer, in particular, gastric cancer, one of the most frequent tumours in Chile, Latin America, and worldwide. This article examines the role of H.pylori and epigenetic alterations (i.e. DNA methylation) at early stages of gastric cancer and proposes, from the paediatric point of view, strategies for prevention and early detection.

Combined use of methylated reprimo cell-free DNA and pepsinogens for noninvasive detection of early gastric cancer.

27 Background: Gastric cancer (GC) has been described as a multistep cascade of precursor lesions such as non-atrophic chronic gastritis (NACG), multiphocal atrophic gastritis (MAG), intestinal metaplasia (IM), low grade dysplasia (LGD) and high grade dysplasia (HGD) leading to early stages of GC (EGC). Currently, no non-invasive biomarkers for this progression are clinically available. We have previously identified a potential biomarker based on methylated Reprimo (RPRM) cell-free DNA (cfDNA) (Clin Cancer Res 2008;14:6264-9). In a cross-sectional study of 1,076 patients, we showed a sensitivity of 70.8% (95% CI: 60.3 to 81.3) and specificity of 74.3% (95% CI: 71.5 to 77) for methylated RPRM cfDNA, to distinguish NACG+MAG+IM+LGD vs HGD+EGC+AGC (Digestive Disease Week 2014 #108). However, the crude detection rate of EGC was only 46.6%. Here, we aim to explore the role of the combined use of methylated RPRM cfDNA and well stablished atrophy biomarkers such as pepsinogens, for non-invasive detection of EGC. ...

A novel potential biomarker to monitor treatment response in gastric cancer.

55 Background: Gastric cancer (GC) is the second leading cause of death from cancer worldwide. No clinical useful biomarkers that detect early gastric cancer and have prognostic/predictive value are available. DNA methylation of promoter region of Reprimo (RPRM), a p53-dependent G2 arrest mediator candidate gene, has been postulated as a potential biomarker for early diagnosis of GC (Clin Cancer Res. 2008;14:6264-9). Since RPRM has been found methylated in more than >90% of GC cases, we evaluate RPRM as a biomarker for monitoring response to treatment. METHODS We enrolled 28 patients with GC. Staging classification was carried out by AJCC system and evaluation of clinical response by RECIST criteria. DNA was obtained from plasma/serum by Proteinase K/quiagen and bisulfite converted by EZ DNA Methlyation Direct Kit (Zymo Research). Absolute quantification of DNA Methylation in serum/plasma of RPRM was performed by MethyLight technology. The IRB of PUC approved this study. All patients gave informed consent. RESULTS Among 28 patients with histological diagnoses of GC, average age was 64 y.o.(38-81 y.o.), male/female ratio 1.3/1, number of cases stage I 2, stage II 4, stage III 8, stage IV 13 and In situ or with preneoplastic lesions 3 patients. 13 patients underwent total gastrectomy and 19 cases received standard chemotherapy (CHM), 6 of them on a neoadjuvancy basis. At initial diagnoses, RPRM was detected in 67,8% (19/28) of patients (X 1049,7 copies per mL[49-11991]). 79% of patients had an objective response with one patient developing a complete response. In cases with clinical response, and at least two detections of RPRM, agreement was 81% with RPRM levels. At the end of study, one third of the patients finally died of gastric cancer. Interestingly, the average initial level of RPRM was higher on those with a fatal outcome (1822 vs 483 copies per mL). The two highest levels were seen on patients who developed a rapid progressive disease and died within months of diagnosis. CONCLUSIONS RPRM could be a potential biomarker to monitor treatment-response in GC. Levels of RPRM may anticipate clinical progression. All these findings should be evaluated in a large, prospective clinical trial.

Abstract 1188: Reprimo, a p53-dependent G2 arrest mediator, reduces tumorigenic capacity in human gastric cancer cell lines

Introduction: Gastric cancer (GC) is one of the leading cause of cancer-related deaths worldwide. The progression of GC is related to genetic as well as epigenetic changes, such as DNA methylation, that involves the downregulation of tumor suppressor genes. Recently we have identified Reprimo (RPRM), a p53-dependent G2 arrest mediator candidate as a putative biomarker for early diagnosis of GC (Clin Cancer Res 2008;14:6264-9). However, the role of RPRM in GC is unknown. Methods: The expression and sequence of the promoter and the coding region of RPRM were performed in five GC cell lines (AGS, NCI-N87, KATOIII, MKN28, MKN45). Two expression negative RPRM cell lines were stable transfected with a pCMV6 vector containing the full-length of the coding region of RPRM (pCMV6-RPRM). Cell growth and proliferation were analyzed by MTS, colony formation and soft agar assays. In addition, we analyzed the ability of migration through wound/healing assay. Results: Only two cell lines were negative for RPRM expression (AGS & MKN45). Bisulfite sequence analysis of the promoter region showed a dense CG-rich CpG island suggesting a role for methylation in regulation of gene expression. No mutations in the coding region were found. A significant decreased of cellular proliferation and anchorage independent growth were observed after the transfection with pCMV6-RPRM. The migration ability was also decreased in both cell lines. Conclusion: Our results suggest that RPRM reduces tumorigenic capacity in GC cell lines. Taken together we suggest that downregulation of RPRM is not only a putative biomarker for early diagnosis but also might play a role in the pathogenesis of GC. Grants-in-Aid for Scientific T 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1188. doi:1538-7445.AM2012-1188

Evaluation of reprimo, a p53-dependent G2 arrest mediator candidate, for monitoring response to treatment in gastric carcinoma.

e14503 Background: Gastric cancer (GC) is the second leading cause of cancer death worldwide. No clinically usefull biomarkers that detect early gastric cancer and have prognostic/predictive value are available. DNA methylation of promoter region of Reprimo (RPRM), a p53-dependent G2 arrest mediator candidate gene, has been postulated as a potential biomarker for early diagnosis of GC (Clin Cancer Res. 2008;14:6264-9). Since RPRM has been found methylated in more than >90% of GC cases, we evaluate RPRM for monitoring response to treatment. METHODS We enroll 14 patients with GC. Staging classification was carried out by AJCC system and evaluation of clinical response by RESIST criteria. DNA was obtained from plasma/serum by Proteinase K/quiagen and bisulfite converted by EZ DNA Methlyation Direct Kit (Zymo Research). Absolute quantification of DNA Methylation in serum/plasma of RPRM was performed by MethyLight technology. The IRB of PUC approved this study. All patients gave informed consent. RESULTS Among 14 patients with histological diagnoses of GC, average age was 64 y.o.(38-81 y.o.), male/female ratio 2.3/1, low/high-risk counties 1.6/1, stage III (3 cases), stage IV (10 cases) and 1 undefinded. Three patients underwent total gastrectomy and 11 cases received standard chemotherapy (CHM), 2 with neoadjuvant CHM. At initial diagnoses, RPRM was detected in 78% (11/14) of plasma/serum (967/471 copies per mL), respectively. All 3 negative RPRM cases suggest familial criteria of hereditary GC. In cases with clinical response, and at least three detections of RPRM, agreement was 100%. At 3 months of treatment, levels of RPRM were 226/0 copies per mL, plasma/serum, respectively. Currently, progression of disease has been found in 1 case. In this case, RPRM anticipate in 2 months this clinical progression. CONCLUSIONS RPRM could be a potential biomarker for monitoring response to treatment in GC. Both, plasma or serum could be used for this detection. Levels of RPRM anticipate clinical progression. RPRM might not be usefull for monitoring hereditary GC. All these findings should be evaluated in a large, prospective clinical trial.

S1928 Reprimo: A Novel Non-Invasive Biomarker for Gastric Cancer Detection

Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-invasive diagnosis have been described. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to noninvasive diagnosis of gastric cancer. We have previously shown high prevalence of aberrant hypermethylation of Reprimo (RPRM) a novel tumor suppressor gene, in plasma from gastric cancer but not in plasma from healthy donors (Clin Cancer Res. 2008;14:6264-9). This finding suggested that RPRM promoter methylation could be a sensitive and specific biomarker for the non-invasive detection of gastric cancer. Here we explore in a prospective and blind fashion specificity and sensitivity of RPRM for non-invasive diagnoses of gastric cancer. Methods. We prospectively collect 83 gastric cancer patients from two hospitals and 223 healthy donors from corresponding blood banks. Detection of RPRM was perform by methylation specific PCR (MSP). We perform endoscopy in healthy donors that were RPRM(+) in plasma. Results. Aberrant hypermethylation of RPRM was identified in 67/74 (90.5%) and 75/79 (94.9%) of tumor and plasma from gastric cancer patients, respectively. Among healthy donors, methylation of RPRM was identified in 29.1% (65 of 223) of tested cases. These differences were statistically significant (p< 0.001 by Fishers exact test). The sensibility and specificity of RPRM detection in plasma were 89% (95% CI: 81%-98%) and 67% (95% CI: 58%-75%) respectively. We perform endoscopy in 5 RPRM positive healthy donors, and no cancer was found. Only one healthy donor was diagnosed with chronic gastritis. Discussion. Our data indicate that detection of RPRM promoter methylation in cell free plasma DNA is a promissory method for non-invasive detection of gastric cancer. RPRM could be included in the serologic biopsy along with pepsinogen and gastrin 17 not only for the identification of gastric atrophy, the precursor lesion of gastric cancer but also for direct identification of gastric cancer. Supported by FONDECYT grant 1080563 from Government of Chile

Abstract 1992: In silico transciptome analysis and experimental validation of epigenetically regulated genes in gastric carcinoma

Gastric Cancer is one of the most common cancers worldwide and no biomarkers are available for early detection. To search for potential biomarkers we analysed in silico transcriptome followed by experimental validation of epigenetically regulated genes. Methods. In silico transcriptome analysis was performed after download 4 normal and 10 tumor gastric Serial Gene Expression (SAGE) libraries from CGAP/GEO domains to create a single file containing 121,409 tags. Clustering libraries were identified by PCA, COA and Support Clustering. Significance Analysis for Microarray (SAM) was performed to select differentially expressed tags and SAGE Genie/TAGmapper was applied for tag-gene association. CpG island in the promoter region of selected genes was interrogated by UCSC or EMBOSS CpGPlot browsers. Functional annotation was performed by Gene Ontology (GO) using Babelomics. Experimental validation of epigenetically regulated genes was performed by RT-PCR in five gastric cancer cell lines (NCI-N87, MKN-45, KATO-III, AGS, HSC-38) and other cell lines (MCF7, ZR-75-1, A549, Hep2, JURKAT, CASKI, FS and Hek293T]) after 5 uM of 5-azaC incubation for 72 hrs. Genes with restored expression were further analyzed by bisulfite sequencing and clinico-pathological significance was evaluated by in 91 early gastric cancers and 148 chronic gastritis cases by Tissue Microarray (TMA) methodology. Results. Two thousand and four hundred and thirty seven (2%) tags were expressed in all normal libraries and all tumor libraries. PCA, COA and Support Cluster analyses clearly identified normal and tumor libraries and were confirmed by Support clustering analysis. SAM revealed 90 tags differentially expressed between these two libraries with 78 down-regulated and 12 up-regulated tags in tumor libraries. Tag to gene association identified 59 genes. Functional annotation revealed that most of downregulated genes were negative regulators of apoptosis (GO:0016265) and overexpressed genes were cell cycle regulators (GO:0007049) or angiogenesis inducers (GO:0001525). Interestingly, 16 genes did not have any prior investigation in gastric carcinoma. Finally by bona-fide CpG island analysis, 38 genes contained CpG in their promoter regions, and 12 demonstrated epigenetic regulation after 5-azaC incubation including FLT1 and STAT1. Bisulfite sequence confirmed CpG methylation changes for FLT1 gene and IHC staining of STAT1 on TMAs revealed almost significant overexpression in cancer-associated gastritis vs chronic gastritis control cases (p=0.057). Discussion. Experimental validation of in silico analysis of gastric carcinoma transcriptomes reveal epigenetic regulation of several genes. Among these, STAT1 is a promissory candidate to distinguish chronic gastritis from cancer-associated gastritis. Supported by FONDECYT 1080563 from Government of Chile. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1992.