Andreea Olaru

Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk

Patients with Barretts esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC). Clinical neoplastic progression risk factors, such as age and the length of the esophageal BE segment, have been identified. However, improved molecular biomarkers predicting increased progression risk are needed for improved risk assessment and stratification. Using real-time quantitative methylation-specific PCR, we screened 10 genes (HPP1, RUNX3, RIZ1, CRBP1, 3-OST-2, APC, TIMP3, p16, MGMT, p14) for promoter hypermethylation in 77 EAC, 93 BE, and 64 normal esophagus (NE) specimens. A subset of genes manifesting significant differences in methylation frequencies between BE and EAC was then analysed in 20 dysplastic specimens. All 10 genes except p14 were frequently methylated in EACs, with RUNX3, HPP1, CRBP1, RIZ1, and OST-2 representing novel methylation targets in EAC and/or BE. p16, RUNX3, and HPP1 displayed increasing methylation frequencies in BE vs EAC. Furthermore, these increases in methylation occurred early, at the interface between BE and low-grade dysplasia (LGD). To demonstrate the silencing effect of hypermethylation, we selected the EAC cells BIC1, in which the HPP1 promoter is natively methylated, and subjected them to 5-aza-2′-deoxycytidine (Aza-C) treatment. Real-time RT–PCR indicated increased HPP1 mRNA levels after 3 days of Aza-C treatment, as well as decreased levels of methylated HPP1 DNA. Hypermethylation of a subset of six genes (APC, TIMP3, CRBP1, p16, RUNX3, and HPP1) was then tested in a retrospective longitudinal study of 99 BE and nine LGD specimens obtained from 53 BE patients undergoing surveillance endoscopy. Only high-grade dysplasia (HGD) or EAC were defined as progression end points. Two patient groups were compared: eight progressors (P) and 45 nonprogressors (NP), using Cox proportional hazards models to determine the relative progression risks of age, BE segment length, and methylation events. Multivariate analyses revealed that only hypermethylation of p16 (odds ratio (OR) 1.74, 95% confidence interval (CI) 1.33–2.20), RUNX3 (OR 1.80, 95% CI 1.08–2.81), and HPP1 (OR 1.77, 95% CI 1.06–2.81) were independently associated with an increased risk of progression, whereas age, BE segment length, and hypermethylation of TIMP3, APC, or CRBP1 were not independent risk factors. In combined analyses, risk was detectable up to, but not earlier than, 2 years preceding neoplastic progression. Hypermethylation of p16, RUNX3, and HPP1 in BE or LGD may represent independent risk factors for the progression of BE to HGD or EAC. These findings have implications regarding risk stratification, early EAC detection, and the appropriate endoscopic surveillance interval for patients with BE.

Application of cDNA microarrays to generate a molecular taxonomy capable of distinguishing between colon cancer and normal colon

In order to discover global gene expression patterns characterizing subgroups of colon cancer, microarrays were hybridized to labeled RNAs obtained from seventeen colonic specimens (nine carcinomas and eight normal samples). Using a hierarchical agglomerative method, the samples grouped naturally into two major clusters, in perfect concordance with pathological reports (colon cancer versus normal colon). Using a variant of the unpaired t-test, selected genes were ordered according to an index of importance. In order to confirm microarray data, we performed quantitative, real-time reverse transcriptase–polymerase chain reaction (TaqMan RT–PCR) on RNAs from 13 colorectal tumors and 13 normal tissues (seven of which were matched normal-tumor pairs). RT–PCR was performed on the gro1, B-factor, adlican, and endothelin converting enzyme-1 genes and confirmed microarray findings. Two hundred and fifty genes were identified, some of which were previously reported as being involved in colon cancer. We conclude that cDNA microarraying, combined with bioinformatics tools, can accurately classify colon specimens according to current histopathological taxonomy. Moreover, this technology holds promise of providing invaluable insight into specific gene roles in the development and progression of colon cancer. Our data suggests that a large-scale approach may be undertaken with the purpose of identifying biomarkers relevant to cancer progression.

Biomarkers of Esophageal Adenocarcinoma and Barrett’s Esophagus

The rising incidence and poor prognosis of esophageal adenocarcinoma in the Western world have intensified research efforts into earlier methods of detection of this disease and its relationship to Barrett’s esophagus. The progression of Barrett’s esophagus to adenocarcinoma has been the focus of particular scrutiny, and a number of potential tissue and serum-based disease biomarkers have emerged. The epidemiology and pathogenesis of esophageal adenocarcinoma are outlined. Tissue biomarkers allowing risk stratification of Barrett’s are reviewed as well as strategies currently being used to discover novel biomarkers that will facilitate the early detection of esophageal adenocarcinoma. Finally, the uses of biomarkers as predictive tests for targeted treatments and as surrogate endpoints in chemoprevention trials are considered.

Global gene expression profiling in Barrett's esophagus and esophageal cancer: a comparative analysis using cDNA microarrays

In order to identify and contrast global gene expression profiles defining the premalignant syndrome, Barretts esophagus, as well as frank esophageal cancer, we utilized cDNA microarray technology in conjunction with bioinformatics tools. We hybridized microarrays, each containing 8000 cDNA clones, to RNAs extracted from 13 esophageal surgical or endoscopic biopsy specimens (seven Barretts metaplasias and six esophageal carcinomas). Hierarchical cluster analysis was performed on these results and displayed using a color-coded graphic representation (Treeview). The esophageal samples clustered naturally into two principal groups, each possessing unique global gene expression profiles. After retrieving histologic reports for these tissues, we found that one main cluster contained all seven Barretts samples, while the remaining principal cluster comprised the six esophageal cancers. The cancers also clustered according to histopathological subtype. Thus, squamous cell carcinomas (SCCAs) constituted one group, adenocarcinomas (ADCAs) clustered separately, and one signet-ring carcinoma was in its own cluster, distinct from the ADCA cluster. We conclude that cDNA microarrays and bioinformatics show promise in the classification of esophageal malignant and premalignant diseases, and that these methods can be applied to small biopsy samples.

Transcriptional profiling suggests that Barrett's metaplasia is an early intermediate stage in esophageal adenocarcinogenesis

To investigate the relationship between Barretts esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BEs plus EAC (as a single combined tissue group) vs noncancer-associated BEs. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BEs-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.

Identification of Genes Uniquely Involved in Frequent Microsatellite Instability Colon Carcinogenesis by Expression Profiling Combined with Epigenetic Scanning

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5′untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type Ogene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.

Esophagin and proliferating cell nuclear antigen (PCNA) are biomarkers of human esophageal neoplastic progression

PCNA and esophagin have been implicated in the multistep process of carcinogenesis, but simultaneous characterization of these proteins in the early stages of esophageal neoplastic progression has yet to be undertaken. In morphologically normal esophageal epithelium, esophagin stains the granular layer cells, principally in their cell membrane portions. PCNA, in contrast, stains the nuclei of cells in the parabasal and basal layers. We examined 201 regions from 47 patients that represented different stages of esophageal neoplasia, comprising 34 areas of normal mucosa, 18 of dysplasia in squamous epithelium (DYS/SC), 39 squamous cell carcinoma (SCCA), 29 areas of Barretts esophagus, 48 of Barretts dysplasia (DYS/BAR) and 33 areas of adenocarcinoma (AC). The immunostaining patterns of esophagin and PCNA were evaluated and graded for level of expression. There was loss of esophagin expression in the high‐ and low‐grade dysplasias compared to normal epithelia. In the squamous dysplasias, there was more intense staining (of esophagin) in the atypical nuclei and superficial squamous epithelial cells than in the basal cells. PCNA staining was increased in intensity in the high‐grade dysplasias relative to normal basal layer cells. Combined analysis of esophagin and PCNA appears to reveal an inverse relationship between proliferation and differentiation during esophageal neoplastic progression. Moreover, this combined staining approach also offers promise for detecting esophageal cancer in early, precancerous stages.

Activin type II receptor restoration in ACVR2-deficient colon cancer cells induces transforming growth factor-beta response pathway genes

The activin type II receptor (ACVR2) gene is a putative tumor suppressor gene that is frequently mutated in microsatellite-unstable colon cancers (MSI-H colon cancers). ACVR2 is a member of the transforming growth factor (TGF)-β type II receptor (TGFBR2) family and controls cell growth and differentiation. SMAD proteins are major intracellular effectors shared by ACVR2 and TGFBR2 signaling; however, additional shared effector mechanisms remain to be explored. To discover novel mechanisms transmitting the ACVR2 signal, we restored ACVR2 function by transfecting wild-type ACVR2 (wt-ACVR2) into a MSI-H colon cancer cell line carrying an ACVR2 frameshift mutation. The effect of ACVR2 restoration on cell growth, SMAD phosphorylation, and global molecular phenotype was then evaluated. Decreased cell growth was observed in wt-ACVR2 transfectants relative to ACVR2-deficient vector-transfected controls. Western blotting revealed higher expression of phosphorylated SMAD2 in wt-ACVR2 transfectants versus controls, suggesting cells deficient in ACVR2 had impaired SMAD signaling. Microarray-based differential expression analysis revealed substantial ACVR2-induced overexpression of genes implicated in the control of cell growth and tumorigenesis, including the activator protein (AP)-1 complex genes JUND, JUN, and FOSB, as well as the small GTPase signal transduction family members, RHOB, ARHE, and ARHGDIA. Overexpression of these genes is shared with TGFBR2 activation. This observed similarity between the activin and TGF-β signaling systems suggests that activin may serve as an alternative activator of TGF-β effectors, including SMADs, and that frameshift mutation of ACVR2 may contribute to MSI-H colon tumorigenesis via disruption of alternate TGF-β effector pathways.

Mutational and LOH Analyses of the Chromosome 4q Region in Esophageal Adenocarcinoma

Objective: Mortality due to esophageal adenocarcinoma has risen markedly, but the molecular mechanisms underlying this carcinogenesis are still incompletely understood. Findings from loss of heterozygosity (LOH) studies have suggested that the long arm of chromosome 4 might harbor tumor suppressor genes relevant to esophageal adenocarcinoma. Methods: We performed LOH analysis of 4q in esophageal adenocarcinomas. Regions of LOH were further evaluated by studying two candidate tumor suppressor genes, hCDC4 and CARF, located within them. Results: 54% of the adenocarcinomas examined showed allelic deletion. LOH was observed in 53, 40, 32, 38, and 27% of tumors at positions D4S1554 (the locus of CARF), D4S1572, D4S1548, D4S2934, and D4S3021, respectively. An area of allelic deletion (spanning 3 million bases) was identified at 4q31.1–3 in 37% of tumors. This region harbors a candidate tumor suppressor gene: hCDC4. However, sequencing of the coding regions of CARF and hCDC4 at 4q35 and 4q31, respectively, did not identify mutations. Conclusions: Our findings demonstrate frequent LOH in esophageal adenocarcinoma at several loci including a novel area of allelic deletion at 4q31.1–3. The results imply that mutational or other alterations at these loci may be involved in the pathogenesis of esophageal adenocarcinoma. Candidate tumor suppressor genes located within these regions merit further study.

An Unsupervised Approach to Identify Molecular Phenotypic Components Influencing Breast Cancer Features

To discover a biological basis for clinical subgroupings within breast cancers, we applied principal components (PCs) analysis to cDNA microarray data from 36 breast cancers. We correlated the resulting PCs with clinical features. The 35 PCs discovered were ranked in order of their impact on gene expression patterns. Interestingly, PC 7 identified a unique subgroup consisting of estrogen receptor (ER); (+) African-American patients. This group exhibited global molecular phenotypes significantly different from both ER (−) African-American women and ER (+) or ER (−) Caucasian women (P < 0.001). Additional significant PCs included PC 4, correlating with lymph node metastasis (P = 0.04), and PC 10, with tumor stage (stage 2 versus stage 3; P = 0.007). These results provide a molecular phenotypic basis for the existence of a biologically unique subgroup comprising ER (+) breast cancers from African-American patients. Moreover, these findings illustrate the potential of PCs analysis to detect molecular phenotypic bases for relevant clinical or biological features of human tumors in general.