Cynthia Villarroel

Reprimo as a Potential Biomarker for Early Detection in Gastric Cancer

Purpose: Gastric cancer is a curable disease if diagnosed at early stage. However, most cases are diagnosed at advanced stage because of the lack of screening programs. Therefore, the identification of plasma biomarkers for early detection is necessary. Experimental Design: To search for these biomarkers, we evaluated the DNA methylation patterns of 24 genes by Methylation-specific PCR in primary tissues from 32 retrospectively collected gastric cancer cases (testing group). Correlation between methylation and gene expression was evaluated in the MKN-45 cell line after treatment with 5-aza-2′-deoxycytidine. The most frequently hypermethylated genes were next evaluated in primary tissues and plasma samples from 43 prospectively collected gastric cancer cases as well as plasma samples from 31 asymptomatic age- and gender-matched controls (validation group). Results: In the testing group, 11 genes were hypermethylated in at least 50% of cases (APC, SHP1, E-cadherin, ER, Reprimo, SEMA3B, 3OST2, p14, p15, DAPK, and p16). Eight genes (BRCA1, p73, RARβ, hMLH1, RIZI, RUNX3, MGMT, and TIMP3) were statistically associated with a particular variant of gastric cancer, the signet-ring cell type (P = 0.03). Seven genes (APC, SHP1, E-cadherin, ER, Reprimo, SEMA3B, and 3OST2) were next evaluated in the validation group. We confirm the high frequency of methylation in primary tumors for all seven genes. However, only APC and Reprimo were frequently methylated in pair plasma samples. In asymptomatic controls, only Reprimo was infrequently methylated in comparison with plasma from gastric cancer cases (P < 0.001). Conclusion: Our results identified specific methylation profile associated to signet-ring cell-type histology and aberrant hypermethylation of Reprimo as a potential biomarker for early detection of gastric cancer.

In Silico analysis of Gastric carcinoma Serial Analysis of Gene Expression libraries reveals different profiles associated with ethnicity

Worldwide gastric carcinoma has marked geographical variations and worse outcome in patients from the West compared to the East. Although these differences has been explained by better diagnostic criteria, improved staging methods and more radical surgery, emerging evidence supports the concept that gene expression differences associated to ethnicity might contribute to this disparate outcome. Here, we collected datasets from 4 normal and 11 gastric carcinoma Serial Gene Expression Analysis (SAGE) libraries from two different ethnicities. All normal SAGE libraries as well as 7 tumor libraries were from the West and 4 tumor libraries were from the East. These datasets we compare by Correspondence Analysis and Support Tree analysis and specific differences in tags expression were identified by Significance Analysis for Microarray. Tags to gene assignments were performed by CGAP-SAGE Genie or TAGmapper. The analysis of global transcriptome shows a clear separation between normal and tumor libraries with 90 tags differentially expressed. A clear separation was also found between the West and the East tumor libraries with 54 tags differentially expressed. Tags to gene assignments identified 15 genes, 5 of them with significant higher expression in the West libraries in comparison to the East libraries. qRT-PCR in cell lines from west and east origin confirmed these differences. Interestingly, two of these genes have been associated to aggressiveness (COL1A1 and KLK10). In conclusion we found that in silico analysis of SAGE libraries from two different ethnicities reveal differences in gene expression profile. These expression differences might contribute to explain the disparate outcome between the West and the East.

Activating PIK3CA somatic mutation in congenital unilateral isolated muscle overgrowth of the upper extremity

Congenital unilateral overgrowth of the upper extremity affecting only the muscle tissue is rare. We describe on the clinical, histopathological, and neuroimaging findings in a 6‐year‐old girl with a congenital, non‐progressive muscle enlargement of the entire left upper limb with an ipsilateral hand deformity. No cutaneous stigmata or additional features were detected. Sanger sequencing for the AKT1, PIK3CA, and PTEN genes identified an activating c.3140A>G, p.H1047R mutation in the PIK3CA gene from the affected muscle DNA. We demonstrate that isolated congenital muscular upper limb overgrowth with aberrant hand muscles is another condition related genetically to the PIK3CA‐related overgrowth spectrum.

Caracterización de pacientes con cáncer colorrectal esporádico basado en la nueva subclasificación molecular de consenso

BACKGROUND Colorectal cancer (CRC) is an heterogeneous disease. Three carcinogenic pathways determine its molecular profile: microsatellite instability (MSI), chromosomal instability (CIN) and CpG island methylator phenotype (CIMP). Based on the new molecular classification, four consensus CRC molecular subtypes (CMS) are established, which are related to clinical, pathological and biological characteristics of the tumor. AIM To classify Chilean patients with sporadic CRC according to the new consensus molecular subtypes of carcinogenic pathways. MATERIAL AND METHODS Prospective analytical study of 53 patients with a mean age of 70 years (55% males) with CRC, operated at a private clinic, without neoadjuvant treatment. From normal and tumor tissue DNA of each patient, CIN, MSI and CIMP were analyzed. Combining these variables, tumors were classified as CMS1/MSI-immune, CMS2/canonical, CMS3/metabolic and CMS4/mesenchymal. RESULTS CMS1 tumors (19%) were located in the right colon, were in early stages, had MMR complex deficiencies and 67% had an activating mutation of the BRAF oncogene. CMS2 tumors (31%) were located in the left colon, had moderate differentiation, absence of vascular invasion, lymphatic and mucin. CMS3 tumors (29%) were also left-sided, with absence of vascular and lymphatic invasion, and 29% had an activating mutation of the KRAS oncogene. CMS4 tumors (21%) showed advanced stages and presence of metastases. CONCLUSIONS This new molecular classification contributes to understanding the heterogeneity of tumors. It is possible to differentiate molecular subgroups of a single pathological diagnosis of adenocarcinoma, opening the door to personalized medicine.

EGFR pathway subgroups in Chilean colorectal cancer patients, detected by mutational and expression profiles, associated to different clinicopathological features

Colorectal cancer is a multistep process affecting several signaling pathways including EGFR (epidermal growth factor receptor), a therapeutic target for metastatic disease. Our aim was to characterize the mutational and expression profiles of the EGFR pathway in colorectal tumors and to integrate these results according to five previously defined groups. We screened seven genes for mutations (KRAS-BRAF-PIK3CA-PIK3R1-AKT1-MAP2K1-PTEN) and six proteins (EGFR-p110α-p85α-PTEN-phosphoAKT-phosphoMEK1) by immunohistochemistry, PTEN deletion, and MSI. At least one mutated gene was observed in 68% of tumors (KRAS 45%, PIK3CA 21%, BRAF 14%, and PTEN 7%). PTEN deletion was observed in 10.7% of tumors and 19.6% were MSI-High. In all, 54% of tumors showed a high EGFR expression, 48% p110α, 4.4% phosphoAKT, and 22% phosphoMEK1; and 43% showed low PTEN expression and 22% p85α. In total, five groups of tumors were defined based on MSI, BRAF, and KRAS mutations. Three groups gather mainly early-stage tumors, whereas a fourth group is mostly conformed by advanced tumors. We described here that 71.4% of tumors from one group have a mutated PI3K/PTEN pathway, in comparison to other groups having 32%, 27%, and 25%. In addition, the five groups are differentiated by molecular features such as EGFR, p85α, p110α, and PTEN, showing variable expression among tumor groups. In conclusion, alterations on the EGFR pathway were found in a high percentage of colorectal cancer patients. Using the integration of diverse molecular markers, we ratified previous classification in an ethnic group having relevant genetic differences and living in a different environmental background, adding complementary molecular targets related to therapy.

Abstract 5167: Expression levels of orexin receptor 1 in different stages of colorectal cancer

Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer death in Chile. Currently, treatment is surgical and adjuvance is based on chemotherapy mainly. One of the risk factors of CRC is obesity and metabolic syndrome. In search of factors that induce cell apoptosis, orexins were identified. Orexins are neuropeptides that regulate appetite, inhibit satiety and increase energy expenditure. The altered expression of orexin or its dysregulation has been associated with a wide range of human diseases such as narcolepsy, obesity, drug addiction and cancer. In rats and mice lacking orexin receptor, an increase in body mass index (BMI) was observed. Orexin Receptor 1 (OX1R) is overexpressed in CRC, but is not detected in normal colon tissue. Its activation by Orexin-A promotes apoptosis in cancer cell lines, and can be modulated by diet. Aim: To determine OX1R expression in the adenoma-carcinoma progression of CRC as possible therapeutic target, and its correlation with BMI. Material and Methods: Patients with neoplastic colorectal lesions undergoing surgical or endoscopic treatment between 2014 and 2015 were prospectively enrolled. Tissue samples of 29 patients were divided into 4 groups. Group A: control (n = 5), Group B: low-grade adenoma (n = 5), Group C: high-grade adenoma (n = 7), Group D: adenocarcinoma (n = 12). Formalin-fixed-paraffin-embedded samples were used to perform immunohistochemistry (IHC) of OX1R. Fresh tissues from Group D were used to assess protein and mRNA expression of OX1R by Western-blot and quantitative RT-PCR, respectively. Primary cultures were established from fresh tumor tissue of patients with adenocarcinoma. To compare OX1R expression levels we used the t-student test. Results: IHC expression of OX1R was detected both in the luminal membrane of colorectal epithelium and in the cytoplasm. We observed stained cells in 0-1% of the total area in Group A, B and C. In Group D, 7 samples showed staining 0-1%, 2 samples 1-10% and 3 samples 10-20%. High mRNA and protein expression was detected in stage III- IV adenocarcinoma samples compared to stage 0, I and II tumor samples. Normal adyacent tissue of CRC patients does not express OX1R. BMI is inversely associated with OX1R expression. Orexin A was able to induce apoptosis in primary cell cultures from advanced tumors in a dose dependent manner. Conclusions: OX1R is expressed scarcely in early stages of carcinogenesis. Its expression is induced significantly only in the most advanced adenocarcinomas, both at protein and mRNA levels, and is inversely associated with BMI. In primary cultures of patients with CRC, orexin A is able to induce apoptosis in a dose dependent manner. FONDECYT 1140012. Citation Format: Ana M. Wielandt, Cynthia Villarroel, Claudia Hurtado, Kento Inada, Hiroshi Kawachi, Daniela Simian, Maria T. Vial, Marcela Figueroa, Magdalena Castro, Udo Kronberg, Francisco Lopez-Kostner. Expression levels of orexin receptor 1 in different stages of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5167.

Abstract 2230: Gene mutations and deletions inactivates PTEN tumor suppressor in Chilean colon cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction. Tumor suppressor PTEN acts as a negative regulator of the PI3K/AKT/mTOR pathway, thus controlling cell survival and proliferation. Mutations or deletions inactivating PTEN function lead to over-activation of the PI3K/AKT/mTOR pathway in cancer. PTEN gene alterations have been described in different types of tumors. In colon cancer these mutations range from 1% to 29% of the cases. The aim of this study was to determine the prevalence of PTEN mutations and/or deletions in Chilean colon cancer patients. Methods: ninety-one colon cancer patients were recruited for this study. Genomic DNA from normal and tumor tissue was obtained, and the deletion of PTEN locus was determined by PCR amplification of three STR markers flanking PTEN, and two intragenic markers. To search point mutations we amplified exons 7 and 8 by PCR, and sequenced. PTEN expression was evaluated through immunohistochemistry. Results: We observed deletion events in 9.8% (9/91) of tumors, and inactivating mutations in 5.5% (5/91). Two tumors presented a deletion in one allele and a mutation in the other. Immunohistochemistry detected a very weak expression of PTEN in 55/76 tumors. Conclusions: 15.4 % (12/91) of tumors have PTEN gene inactivated. We observed that deletion was a frequent mechanism for PTEN inactivation in Chilean colon cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Gonzalo Encina, Karin Alvarez, Paulina Orellana, Ana Maria Wielandt, Cynthia Villarroel, Daniela Simian, Luis Contreras, Udo Kronberg, Francisco Lopez, Pilar Carvallo. Gene mutations and deletions inactivates PTEN tumor suppressor in Chilean colon cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2014-2230

Abstract 2232: Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients

Introduction: The Epidermal Growth Factor Receptor (EGFR) signaling pathway regulates key cell functions like proliferation and survival. This pathway presents two signaling cascades, RAS/RAF/MAPK and PIK3/PTEN/AKT/mTOR. Mutations in different components of the EGFR pathway have been described in different cancers. This pathway is a therapeutic target for monoclonal antibodies and tyrosine kinase inhibitors based therapies. Additionally, in advanced colorectal cancer patients, somatic KRAS mutations are response predictors for biological therapy with anti-EGFR antibody. The sensitivity of the mutations detection methods is key to a proper application of personalized therapeutic strategies. Traditionally, somatic mutations are detected by Sanger sequencing and real-time PCR. We determined the presence of mutations in specific genes of the EGFR pathway in colon cancer tumors from Chilean patients. Methods: DNA was extracted from twenty-eight fresh frozen tumor samples. DNA libraries were created using SureSelect (Agilent) designed for the analysis of EGFR, KRAS, BRAF, MAP2K1, PIK3CA, PIK3R1 and PTEN genes. Next generation sequencing was performed in MiSeq sequencer (Illumina) at Sistemas Genomicos (Spain). Results: Sequencing coverage varied between 100 and 250 readings between tumors, which assures the real presence of the changes. This technique allows detecting mutations that are present in a smaller percentage of tumoral cells, as low as 20% in the sample. Pathogenic mutations were identified in 19 out of the 28 tumors. Mutations were found in KRAS (p.Gly12Asp, p.Gly12Arg, p.Gly12Val and p.Gly12Cys), BRAF (p.Val600Glu and p.Gly606Glu), PIK3CA (p.Glu542Lys, p.Glu545Lys, p.Glu545Gln, p.Ala1035Val, p.Asp1045Val and p.His1047Arg) and PTEN (p.Lys267ArgfsX9). Five tumors presented mutations in both branches of the EGFR signaling pathway (KRAS-PIK3CA, BRAF-PIK3CA and BRAF-PTEN). Three allelic variants were identified in MAP2K1 and PIK3R1. Mutations that were identified in KRAS, BRAF and PIK3CA genes corresponded to gain of function mutations while a change found in PTEN gene corresponded to a loss of function mutation. Finally, mutations in PIK3CA and KRAS of 6 tumors were not previously detected by Sanger sequencing. Discussion: Mutations were found in 68% of analyzed tumors. Eighteen percent of the tumors presented mutations in two genes. Forty-six percent of mutations were identified in KRAS gene. Three allelic variants of unknown significance were detected which have not been described in cancer mutations nor in SPNs databases. Therefore, it is necessary to perform functional studies to determine the effect on protein function. Next generation sequencing allowed us to obtain more reliable and reproducible results, and was able to detect mutations present in a low percentage of tumoral cells in the sample, maybe due to tumoral heterogeneity or contamination with normal cells. FONDECYT 1111020 Citation Format: Karin Alvarez, Paulina Orellana, Cynthia Villarroel, Gonzalo Encina, Daniela Simian, Camila Estay, Maki Kobayashi, Hiroshi Kawashi, Udo Kronberg, Francisco Lopez, Pilar Carvallo. Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2014-2232

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.