Hua Su

Global Gene Expression Profiling and Validation in Esophageal Squamous Cell Carcinoma and Its Association with Clinical Phenotypes

Purpose: Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with poor prognosis. Understanding molecular changes in ESCC will enable identification of molecular subtypes and provide potential targets for early detection and therapy. Experimental Design: We followed up a previous array study with additional discovery and confirmatory studies in new ESCC cases by using alternative methods. We profiled global gene expression for discovery and confirmation, and validated selected dysregulated genes with additional RNA and protein studies. Results: A total of 159 genes showed differences with extreme statistical significance (P < E-15) and 2-fold differences or more in magnitude (tumor/normal RNA expression ratio, N = 53 cases), including 116 upregulated and 43 downregulated genes. Of 41 genes dysregulated in our prior array study, all but one showed the same fold change directional pattern in new array studies, including 29 with 2-fold changes or more. Alternative RNA expression methods validated array results: more than two thirds of 51 new cases examined by real-time PCR (RT-PCR) showed 2-fold differences or more for all seven genes assessed. Immunohistochemical protein expression results in 275 cases which were concordant with RNA for five of six genes. Conclusion: We identified an expanded panel of genes dysregulated in ESCC and confirmed previously identified differentially expressed genes. Microarray-based gene expression results were confirmed by RT-PCR and protein expression studies. These dysregulated genes will facilitate molecular categorization of tumor subtypes and identification of their risk factors, and serve as potential targets for early detection, outcome prediction, and therapy. Clin Cancer Res; 17(9); 2955–66. ©2011 AACR.

Comprehensive characterization of Annexin I alterations in esophageal squamous cell carcinoma

Purpose: The purpose is to characterize alterations of the annexin I gene, its mRNA, and protein expression in esophageal squamous cell carcinoma. Experimental Design: Fifty-six cases of esophageal squamous cell carcinoma were analyzed using four microsatellite markers flanking the annexin I gene (9q11-q21) to identify loss of heterozygosity. In addition, we performed (a) single-strand conformation polymorphism and DNA sequencing along the entire promoter sequence and coding region to identify mutations, (b) real-time quantitative reverse transcription-PCR of RNA from frozen esophageal squamous cell carcinoma tissue (n = 37) and in situ hybridization (n = 5) on selected cases to assess mRNA expression, and (c) immunohistochemistry (n = 44) to evaluate protein expression. The prevalence of the allelic variants identified in the first 56 patients was refined in 80 additional esophageal squamous cell carcinoma patients and 232 healthy individuals. Results: Forty-six of 56 (82%) esophageal squamous cell carcinoma patients showed loss of an allele at one or more of the four microsatellite markers; however, only one (silent) mutation was seen. Two intragenic variants were identified with high frequency of allelic loss (A58G, 64%; L109L, 69%). Thirty of 37 (81%) esophageal squamous cell carcinoma patients showed reduced annexin I mRNA expression, which was confirmed by in situ hybridization, whereas annexin I protein expression was reduced in 79% of poorly differentiated tumor cell foci but in only 5% of well-differentiated tumor foci, although allelic loss on chromosome 9 was found in both tumor grades. Conclusions: Allelic loss of annexin I occurs frequently, whereas somatic mutations are rare, suggesting that annexin I is not inactivated in esophageal squamous cell carcinoma via a two-hit mechanism. A decrease in annexin I protein expression was confirmed, consistent with a quantitative decrease in mRNA expression, and appeared to be related to tumor cell differentiation. We conclude that annexin I is not the tumor suppressor gene corresponding to the high levels of loss of heterozygosity observed on chromosome 9 in esophageal squamous cell carcinoma; however, dysregulation of mRNA and protein levels is associated with this tumor type.

Genetic variants in DNA repair pathway genes and risk of esophageal squamous cell carcinoma and gastric adenocarcinoma in a Chinese population

The DNA repair pathways help to maintain genomic integrity and therefore genetic variation in the pathways could affect the propensity to develop cancer. Selected germline single nucleotide polymorphisms (SNPs) in the pathways have been associated with esophageal cancer and gastric cancer (GC) but few studies have comprehensively examined the pathway genes. We aimed to investigate associations between DNA repair pathway genes and risk of esophageal squamous cell carcinoma (ESCC) and GC, using data from a genome-wide association study in a Han Chinese population where ESCC and GC are the predominant cancers. In sum, 1942 ESCC cases, 1758 GC cases and 2111 controls from the Shanxi Upper Gastrointestinal Cancer Genetics Project (discovery set) and the Linxian Nutrition Intervention Trials (replication set) were genotyped for 1675 SNPs in 170 DNA repair-related genes. Logistic regression models were applied to evaluate SNP-level associations. Gene- and pathway-level associations were determined using the resampling-based adaptive rank-truncated product approach. The DNA repair pathways overall were significantly associated with risk of ESCC (P = 6.37 × 10(-4)), but not with GC (P = 0.20). The most significant gene in ESCC was CHEK2 (P = 2.00 × 10(-6)) and in GC was CLK2 (P = 3.02 × 10(-4)). We observed several other genes significantly associated with either ESCC (SMUG1, TDG, TP53, GTF2H3, FEN1, POLQ, HEL308, RAD54B, MPG, FANCE and BRCA1) or GC risk (MRE11A, RAD54L and POLE) (P < 0.05). We provide evidence for an association between specific genes in the DNA repair pathways and the risk of ESCC and GC. Further studies are warranted to validate these associations and to investigate underlying mechanisms.

iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophages

Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin‐protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of ≤ 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF‐α, IL‐6, IL‐10, GM‐CSF, RANTES and IFN‐β when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells.

A Gene Expression Signature from Peripheral Whole Blood for Stage I Lung Adenocarcinoma

Affordable early screening in subjects with high risk of lung cancer has great potential to improve survival from this deadly disease. We measured gene expression from lung tissue and peripheral whole blood (PWB) from adenocarcinoma cases and controls to identify dysregulated lung cancer genes that could be tested in blood to improve identification of at-risk patients in the future. Genome-wide mRNA expression analysis was conducted in 153 subjects (73 adenocarcinoma cases, 80 controls) from the Environment And Genetics in Lung cancer Etiology study using PWB and paired snap-frozen tumor and noninvolved lung tissue samples. Analyses were conducted using unpaired t tests, linear mixed effects, and ANOVA models. The area under the receiver operating characteristic curve (AUC) was computed to assess the predictive accuracy of the identified biomarkers. We identified 50 dysregulated genes in stage I adenocarcinoma versus control PWB samples (false discovery rate ≤0.1, fold change ≥1.5 or ≤0.66). Among them, eight (TGFBR3, RUNX3, TRGC2, TRGV9, TARP, ACP1, VCAN, and TSTA3) differentiated paired tumor versus noninvolved lung tissue samples in stage I cases, suggesting a similar pattern of lung cancer–related changes in PWB and lung tissue. These results were confirmed in two independent gene expression analyses in a blood-based case–control study (n = 212) and a tumor–nontumor paired tissue study (n = 54). The eight genes discriminated patients with lung cancer from healthy controls with high accuracy (AUC = 0.81, 95% CI = 0.74–0.87). Our finding suggests the use of gene expression from PWB for the identification of early detection markers of lung cancer in the future. Cancer Prev Res; 4(10); 1599–608. ©2011 AACR.

Genome-wide loss of heterozygosity and copy number alteration in esophageal squamous cell carcinoma using the Affymetrix GeneChip Mapping 10 K array

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide. Comprehensive genomic characterization of ESCC will further our understanding of the carcinogenesis process in this disease.ResultsGenome-wide detection of chromosomal changes was performed using the Affymetrix GeneChip 10 K single nucleotide polymorphism (SNP) array, including loss of heterozygosity (LOH) and copy number alterations (CNA), for 26 pairs of matched germ-line and micro-dissected tumor DNA samples. LOH regions were identified by two methods – using Affymetrixs genotype call software and using Affymetrixs copy number alteration tool (CNAT) software – and both approaches yielded similar results. Non-random LOH regions were found on 10 chromosomal arms (in decreasing order of frequency: 17p, 9p, 9q, 13q, 17q, 4q, 4p, 3p, 15q, and 5q), including 20 novel LOH regions (10 kb to 4.26 Mb). Fifteen CNA-loss regions (200 kb to 4.3 Mb) and 36 CNA-gain regions (200 kb to 9.3 Mb) were also identified.ConclusionThese studies demonstrate that the Affymetrix 10 K SNP chip is a valid platform to integrate analyses of LOH and CNA. The comprehensive knowledge gained from this analysis will enable improved strategies to prevent, diagnose, and treat ESCC.

Comparison of Global Gene Expression of Gastric Cardia and Noncardia Cancers from a High-Risk Population in China

Objective To profile RNA expression in gastric cancer by anatomic subsites as an initial step in identifying molecular subtypes and providing targets for early detection and therapy. Methods We performed transcriptome analysis using the Affymetrix GeneChip U133A in gastric cardia adenocarcinomas (n = 62) and gastric noncardia adenocarcinomas (n = 72) and their matched normal tissues from patients in Shanxi Province, and validated selected dysregulated genes with additional RNA studies. Expression of dysregulated genes was also related to survival of cases. Results Principal Component Analysis showed that samples clustered by tumor vs. normal, anatomic location, and histopathologic features. Paired t-tests of tumor/normal tissues identified 511 genes whose expression was dysregulated (P<4.7E-07 and at least two-fold difference in magnitude) in cardia or noncardia gastric cancers, including nearly one-half (n = 239, 47%) dysregulated in both cardia and noncardia, one-fourth dysregulated in cardia only (n = 128, 25%), and about one-fourth in noncardia only (n = 144, 28%). Additional RNA studies confirmed profiling results. Expression was associated with case survival for 20 genes in cardia and 36 genes in noncardia gastric cancers. Conclusions The dysregulated genes identified here represent a comprehensive starting point for future efforts to understand etiologic heterogeneity, develop diagnostic biomarkers for early detection, and test molecularly-targeted therapies for gastric cancer.

Overexpression of CDC25B and LAMC2 mRNA and protein in esophageal squamous cell carcinomas and premalignant lesions in subjects from a high-risk population in China.

Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood but likely hold the key to effective early detection approaches for this almost invariably fatal cancer. CDC25B and LAMC2 are two promising early detection candidates emerging from new molecular studies of ESCC. To further elucidate the role of these two genes in esophageal carcinogenesis, we did a series of studies to (a) confirm RNA overexpression, (b) establish the prevalence of protein overexpression, (c) relate protein overexpression to survival, and (d) explore their potential as early detection biomarkers. Results of these studies indicated that CDC25B mRNA was overexpressed (≥2-fold overexpression in tumor compared with normal) in 64% of the 73 ESCC cases evaluated, whereas LAMC2 mRNA was overexpressed in 89% of cases. CDC25B protein expression was categorized as positive in 59% (144 of 243) of ESCC cases on a tumor tissue microarray, and nonnegative LAMC2 patterns of protein expression were observed in 82% (225 of 275) of cases. Multivariate-adjusted proportional hazard regression models showed no association between CDC25B protein expression score and risk of death [hazard ratio (HR) for each unit increase in expression score, 1.00; P = 0.90]; however, several of the LAMC2 protein expression patterns strongly predicted survival. Using the cytoplasmic pattern as the reference (the pattern with the lowest mortality), cases with a diffuse pattern had a 254% increased risk of death (HR, 3.52; P = 0.007), cases with no LAMC2 expression had a 169% increased risk of death (HR, 2.69; P = 0.009), and cases with a peripheral pattern had a 130% greater risk of death (HR, 2.30; P = 0.02). CDC25B protein expression scores in subjects with esophageal biopsies diagnosed as normal (n = 35), dysplastic (n = 23), or ESCC (n = 32) increased significantly with morphologic progression. For LAMC2, all normal and dysplastic patients had a continuous pattern of protein expression, whereas all ESCCs showed alternative, noncontinuous patterns. This series of studies showed that both CDC25B and LAMC2 overexpress RNA and protein in a significant majority of ESCC cases. The strong relation of LAMC2 pattern of protein expression to survival suggests a role in prognosis, whereas the association of CDC25B with morphologic progression indicates a potential role as an early detection marker. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1424–35)

High frequency of CDKN2A alterations in esophageal squamous cell carcinoma from a high‐risk Chinese population

Because previous studies have shown that loss of heterozygosity (LOH) is common on chromosome arm 9p in esophageal squamous cell carcinoma (ESCC) and that genetic alterations in CDKN2A and CDKN2B on 9p are also common, we sought to determine whether LOH and these genetic alterations are related. We performed LOH studies on chromosome bands 9p21–p22 and searched for genetic alterations of CDKN2A and CDKN2B in 56 ESCCs from a high‐risk Chinese population. Seventy‐three percent of patients were found to have LOH at one or more loci on chromosome bands 9p21–p22, and LOH occurred more frequently in patients with a family history of upper gastrointestinal cancer than in those with a negative family history (P = 0.01, global permutation test). CDKN2A mutations (point mutations, deletions, insertions) were observed in 25% (14 of 56) of cases, and the LOH pattern was significantly different for individuals with and without a CDKN2A mutation (P = 0.01, global test). Three new single nucleotide polymorphisms (SNPs) and 2 previously reported SNPs were identified in this group of patients. Intragenic allelic loss at polymorphic sites in CDKN2A was detected in 32% (18 of 56) of patients. Seven of the 56 (13%) cases exhibited what is considered classic evidence (n = 4) or showed potential evidence (n = 3) of biallelic inactivation. Only one alteration was observed in CDKN2B, G171A in the 5′ untranslated region. Both mutation and intragenic allelic loss in CDKN2A appear to play a role in the development of ESCC. ©2003 Wiley‐Liss, Inc.

Allelic loss on chromosome 13q14 and mutation in deleted in cancer 1 gene in esophageal squamous cell carcinoma.

Previous studies have shown frequent allelic loss on chromosome 13 in esophageal squamous cell carcinoma (ESCC). We assessed the frequency of allelic loss on chromosome 13q14 and mutations of deleted in cancer 1 (DICE1) (also found on 13q14) in ESCC patients to determine if this candidate tumor suppressor gene has a role in the development of ESCC, and whether this gene was an inactivation target for allelic loss on chromosome 13q14. Initially, we examined allelic loss at five markers flanking DICE1 in 56 ESCC patients from Shanxi Province, China, and then examined the entire coding sequence of this gene for mutations using polymerase chain reaction–single-strand confirmation polymorphism (PCR–SSCP) analysis and DNA sequencing. Subsequently, we extended our evaluation to an additional 80 ESCC patients and 232 healthy individuals to confirm the germline variant found in the initial 56 ESCC patients. The frequencies of allelic loss were 71, 58, and 75% for D13S325, D13S757, and D13S887, respectively, in the initial 56 ESCC patients studied. Overall, 73% of informative patients had loss of heterozygosity (LOH) for at least one of these three markers. Somatic mutations were identified in three patients (3/56, 5%), and one novel polymorphism was identified in 3% of ESCC patients (4/136) and 3% of healthy individuals (6/232). We conclude that DICE1 mutations occur in ESCC but are infrequent. The candidate tumor suppressor gene corresponding to the frequent allelic loss on chromosome 13q14 in ESCC remains unknown.