Santhoshi Bandla

Gastrointestinal Adenocarcinomas of the Esophagus, Stomach, and Colon Exhibit Distinct Patterns of Genome Instability and Oncogenesis

A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential use of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared with colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions.

Comparative Genomics of Esophageal Adenocarcinoma and Squamous Cell Carcinoma

BACKGROUND Esophageal cancer consists of two major histologic types: esophageal squamous cell carcinoma (ESCC), predominant globally, and esophageal adenocarcinoma (EAC), which has a higher incidence in westernized countries. Five-year overall survival is 15%. Clinical trials frequently combine histologic types although they are different diseases with distinct origins. In the evolving era of personalized medicine and targeted therapies, we hypothesized that ESCC and EAC have genomic differences important for developing new therapeutic strategies for esophageal cancer. METHODS We explored DNA copy number abnormalities in 70 ESCCs with publicly available array data and 189 EACs from our group. All data was from single nucleotide polymorphism arrays. Analysis was performed using a segmentation algorithm. Log ratio thresholds for copy number gain and loss were set at ±0.2 (approximately 2.3 and 1.7 copies, respectively). RESULTS The ESCC and EAC genomes showed some copy number abnormalities with similar frequencies (eg, CDKN2A, EGFR, KRAS, MYC, CDK6, MET) but also many copy number abnormalities with different frequencies between histologic types, most of which were amplification events. Some of these regions harbor genes for which targeted therapies are currently available (VEGFA, ERBB2) or for which agents are in clinical trials (PIK3CA, FGFR1). Other regions contain putative oncogenes that may be targeted in the future. CONCLUSIONS Using single nucleotide polymorphism arrays we compared genomic abnormalities in a large cohort of EACs and ESCCs. We report here the similar and different frequencies of copy number abnormalities in ESCC and EAC. These results may allow development of histology-specific therapeutic agents for esophageal cancer.

MicroRNA Prognostic Signature for Nodal Metastases and Survival in Esophageal Adenocarcinoma

BACKGROUND The incidence of esophageal adenocarcinoma is rapidly increasing and is now one of the leading causes of cancer death in the western world. MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of protein-encoding genes and are involved in the development, progression and prognosis of other malignancies. We hypothesized that global miRNA expression would predict survival and lymph node involvement in a cohort of surgically resected esophagus cancer patients. METHODS The miRNA analysis was performed using a custom Affymetrix microarray with probes for 462 known human, 2,102 predicted human, 357 mouse, and 238 rat miRNAs. Expression of miRNA was evaluated in 45 primary tumors, and the association of miRNA expression with patient survival and lymph node metastasis was assessed. The prognostic impact of identified unique miRNAs was verified with quantitative reverse transcriptase polymerase chain reaction. RESULTS Our data indicate that the expression of individual human miRNA species is significantly associated with postresection patient survival. Using data from five unique miRNAs, we were further able to generate a combined miRNA expression signature that is associated with patient survival (p=0.005; hazard ratio 3.6) independent of node involvement and overall stage. The expression of three miRNAs (miR-99b and miR-199a_3p and _5p) was also associated with the presence of lymph node metastasis. CONCLUSIONS These results suggest miRNA expression profiling could provide prognostic utility in staging esophagus cancer patients and treatment planning with endoscopic and neoadjuvant therapies. The alterations of specific miRNAs may further elucidate steps in the metastatic pathway and allow for development of targeted therapy.

HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of Barretts esophagus, 18 cases of low-grade dysplasia and 15 cases of high-grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and chromogenic in situ hybridization methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by chromogenic in situ hybridization and high-density microarrays. We further confirm the similar frequency of HER2 amplification by chromogenic in situ hybridization (18%; 21 out of 116) and SNP 6.0 microarrays (16%, 19 out of 116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12% (14 out of 116) of esophageal adenocarcinoma and 7% (1 out of 15) of high-grade dysplasia. No HER2 amplification or overexpression was identified in Barretts esophagus or low-grade dysplasia. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by chromogenic in situ hybridization than protein overexpression in esophageal adenocarcinoma (18 vs 12%). A modified two-step model for esophageal adenocarcinoma HER2 testing is recommended for clinical esophageal adenocarcinoma HER2 trial.

Early G1 cyclin-dependent kinases as prognostic markers and potential therapeutic targets in esophageal adenocarcinoma

Purpose: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear. Experimental Design: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored. Results: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway. Conclusions: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease. Clin Cancer Res; 17(13); 4513–22. ©2011 AACR.

Comparison of Cancer-Associated Genetic Abnormalities in Columnar-Lined Esophagus tissues with and without Goblet Cells

Objective:To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. Background:Barretts esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barretts esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. Methods:Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. Results:Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. Conclusions:This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

Clinicopathologic characteristics of high expression of Bmi-1 in esophageal adenocarcinoma and squamous cell carcinoma

BackgroundHigh expression of Bmi-1, a key regulatory component of the polycomb repressive complex-1, has been associated with many solid and hematologic malignancies including esophageal squamous cell carcinoma. However, little is known about the role of Bmi-1 in esophageal adenocarcinoma. The aim of this study is to investigate the amplification and high expression of Bmi-1 and the associated clinicopathologic characteristics in esophageal adenocarcinoma and squamous cell carcinoma.MethodsThe protein expression level of Bmi-1 was detected by immunohistochemistry (IHC) from tissue microarrays (TMA) constructed at the University of Rochester from using tissues accrued between 1997 and 2005. Types of tissues included adenocarcinoma, squamous cell carcinoma and precancerous lesions. Patients’ survival data, demographics, histologic diagnoses and tumor staging data were collected. The intensity (0–3) and percentage of Bmi-1 expression on TMA slides were scored by two pathologists. Genomic DNA from 116 esophageal adenocarcinoma was analyzed for copy number aberrations using Affymetrix SNP 6.0 arrays. Fisher exact tests and Kaplan-Meier methods were used to analyze data.ResultsBy IHC, Bmi-1 was focally expressed in the basal layers of almost all esophageal squamous mucosa, which was similar to previous reports in other organs related to stem cells. High Bmi-1 expression significantly increased from squamous epithelium (7%), columnar cell metaplasia (22%), Barrett’s esophagus (22%), to low- (45%) and high-grade dysplasia (43%) and adenocarcinoma (37%). The expression level of Bmi-1 was significantly associated with esophageal adenocarcinoma differentiation. In esophageal adenocarcinoma, Bmi-1 amplification was detected by DNA microarray in a low percentage (3%). However, high Bmi-1 expression did not show an association with overall survival in both esophageal adenocarcinoma and squamous cell carcinoma.ConclusionsThis study demonstrates that high expression Bmi-1 is associated with esophageal adenocarcinoma and precancerous lesions, which implies that Bmi-1 plays an important role in early carcinogenesis in esophageal adenocarcinoma.

Bile exposure inhibits expression of squamous differentiation genes in human esophageal epithelial cells.

Objective:This study aimed to identify pathways and cellular processes that are modulated by exposure of normal esophageal cells to bile and acid. Background:Barretts esophagus most likely develops as a response of esophageal stem cells to the abnormal reflux environment. Although insights into the underlying molecular mechanisms are slowly emerging, much of the metaplastic process remains unknown. Methods:We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile or acid exposure. Differentially expressed genes were classified into major biological functions using pathway analysis and interaction network software. Array data were verified by quantitative PCR and western blot both in vitro and in human esophageal biopsies. Results:Bile modulated expression of 202 genes, and acid modulated expression of 103 genes. Genes involved in squamous differentiation formed the largest functional group (n = 45) all of which were downregulated by bile exposure. This included genes such as involucrin (IVL), keratinocyte differentiation-associated protein (KRTDAP), grainyhead-like 1 (GRHL1), and desmoglein1 (DSG1) the downregulation of which was confirmed by quantitative PCR and western blot. Bile also caused expression changes in genes involved in cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling, and lipid metabolism. Analysis of human esophageal biopsies demonstrated greatly reduced expression of IVL, KRTDAP, DSG1, and GRHL1 in metaplastic compared to squamous epithelia. Conclusions:We report for the first time that bile inhibits the squamous differentiation program of esophageal epithelial cells. This, coordinated with induction of genes driving intestinal differentiation, may be required for the development of Barretts esophagus.

Cyclin E involved in early stage carcinogenesis of esophageal adenocarcinoma by SNP DNA microarray and immunohistochemical studies

BackgroundCyclin E is a cell cycle regulator which is critical for driving G1/S transition. Abnormal levels of cyclin E have been found in many cancers. However, the level changes of cyclin E in esophageal adenocarcinoma and its precancerous lesion have not been well studied. Here, we focus on the gene amplification and expression of cyclin E in these lesions, and aim to ascertain the relationship with clinicopathological characteristics.MethodsGenomic DNA was analyzed from 116 esophageal adenocarcinoma and 26 precancerous lesion patients using Affymetrix SNP 6.0 arrays. The protein overexpression of cyclin E was also detected using immunohistochemistry from tissue microarrays containing esophageal adenocarcinoma and precancerous lesions. Patient survival and other clinical data were collected and analyzed. The intensity and percentage of the cyclin E expressing cells in tissue microarrays were scored by two pathologists. Fisher exact tests and Kaplan-Meier methods were used to analyze data.ResultsBy genomic analysis, cyclin E was amplified in 19.0% of the EAC samples. By immunohistochemistry, high expression of cyclin E was observed in 2.3% of squamous mucosa tissues, 3.7% in columnar cell metaplasia, 5.8% in Barrett’s esophagus, 19.0% in low grade dysplasia, 35.7% in high grade dysplasia, and 16.7% in esophageal adenocarcinoma. The differences in cyclin E high expression between neoplastic groups and non-dysplasia groups are statistically significant (p < 0.05). The prognosis for patients with high cyclin E expression appeared slightly better than for those with low cyclin E expression although this was not statistically significant (p = 0.13).ConclusionsThe expression of cyclin E significantly increases from non-dysplasia esophageal lesion to low and high grade dysplasia, suggesting that cyclin E plays an important role in the early stage of carcinogenesis. Importantly, cyclin E is also amplified and highly expressed in a subset of esophageal adenocarcinoma patients, but this increase is not associated with worse prognosis.

Vitamin D receptor is highly expressed in precancerous lesions and esophageal adenocarcinoma with significant sex difference.

Bile acid reflux into the esophagus is important in the development of esophageal adenocarcinoma (EAC). Recently, vitamin D receptor (VDR) was recognized as a bile acid receptor as well as a vitamin receptor. Expression of VDR is reported to influence the development of various types of cancer, such as those of the breast, liver, and colon. However, little is known about the role of VDR in esophageal neoplasms. We investigated the clinicopathological role of VDR in esophageal tumors. We analyzed genomic DNA from 116 EACs for copy number aberrations. The VDR locus was amplified in 7% of EACs. Expression of the VDR protein was also detected by immunohistochemistry from tissue microarrays created from tissues of Barrett esophagus (BE), low-grade (LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CCM), squamous epithelium (SE), EAC, and esophageal squamous cell carcinoma (ESCC). The protein was highly expressed in 88% of CCM (58/66), 95% of BE (35/37), 100% of the 19 LGD, 94% of HGD (15/16), and 79% of EAC (86/109), but expression in SE and ESCC was rare. Female patients with EAC and CCM were significantly less likely to have high VDR expression than male patients. The overall survival rate was significantly different for patients with tumors exhibiting VDR amplification versus nonamplification. Our findings suggest that VDR plays a role in the early development of EAC through a bile acid ligand. The sex difference in VDR expression may help to explain why men have a high incidence of EAC.