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1.  MicroRNA-224 negatively regulates p21 expression during late neoplastic progression in inflammatory bowel disease 
Inflammatory bowel diseases  2013;19(3):471-480.
Background
The development of colon cancer represents a major complication in patients with inflammatory bowel disease (IBD). The importance of microRNAs (miRs) in carcinogenesis is becoming clearer, as miRs have been implicated in the regulation of cancer-related cellular processes to include apoptosis, differentiation, cell cycle progression, and immune function. In the current study, we sought to identify miR dysregulation specific to progression along the normal-inflammation-cancer axis in colonic specimens from IBD patients.
Methods
MiR microarrays and quantitative RT-PCR were used to detect and confirm dysregulated miRs. Receiver-operating characteristic curve analysis was applied to evaluate the potential utility of miR-224 as a neoplastic disease marker in IBD. For miR-224 target mRNA identification, mRNA microarrays were employed in combination with bioinformatic analyses, Western blotting, and luciferase activity measurements.
Results
We identified 30 miRs that were differentially expressed between chronically inflamed mucosae and cancers arising in IBD. MiR-224 levels increased successively at each stage of IBD progression and accurately discriminated cancers from normal or chronically inflamed IBD tissues. Moreover, mRNA arrays combined with bioinformatic analyses suggested participation of miR-224 in cell cycle regulation. Subsequently, cell cycle experiments indicated that miR-224 regulates the G1/S checkpoint. Finally, in silico prediction analyses, confirmed by Western blotting and luciferase assays, identified p21 as a specific direct mRNA target of miR-224.
Conclusions
These findings reveal miR dysregulation specific to IBD-associated colorectal carcinoma. MiR-224 is overexpressed in IBD-cancers and targets p21, a key cell cycle regulator. Moreover, these results establish the participation of miR-224 in IBD-carcinogenesis.
doi:10.1097/MIB.0b013e31827e78eb
PMCID: PMC4259288  PMID: 23399735
microRNA; inflammatory bowel disease; colon cancer
3.  MicroRNA-21 inhibits Serpini1, a gene with novel tumour suppressive effects in gastric cancer 
Digestive and Liver Disease  2012;44(7):589-596.
Background
A thorough understanding of gastric cancer at the molecular level is urgently needed. One prominent oncogenic microRNA, miR-21, was previously reported to be upregulated in gastric cancer.
Methods
We performed an unbiased search for downstream messenger RNA targets of miR-21, based on miR-21 dysregulation, by using human tissue specimens and the MKN-28 human gastric carcinoma cell line. Molecular techniques include microRNA microarrays, cDNA microarrays, qRT-PCR for miR and mRNA expression, transfection of MKN-28 with miR-21 inhibitor or Serpini1 followed by Western blotting, cell cycle analysis by flow cytometry and luciferase reporter assay.
Results
This search identified Serpini1 as a putative miR-21 target. Luciferase assays demonstrated direct interaction between miR-21 and Serpini1 3’UTR. MiR-21 and Serpini1 expression levels were inversely correlated in a subgroup of gastric cancers, suggesting a regulatory mechanism that included both of these molecules. Furthermore, Serpini1 induced growth retardation of MKN28 and induced vigorous G1/S arrest suggesting its potential tumour-suppressive function in the stomach.
Conclusion
Taken together, these data suggest that in a subgroup of gastric cancers, miR-21 is upregulated, inducing downregulation of Serpini1, which in turn releases the G1-S transition checkpoint, with the end result being increased tumour growth.
doi:10.1016/j.dld.2012.02.016
PMCID: PMC3360813  PMID: 22464652
microRNA-21; Serpini1 gene; gastric cancer
5.  Unique patterns of CpG island methylation in inflammatory bowel disease-associated colorectal cancers 
Inflammatory Bowel Diseases  2011;18(4):641-648.
Objective
CpG island (CGI) hypermethylation at discrete loci is a prevalent cancer-promoting abnormality in sporadic colorectal carcinomas (S-CRCs). We investigated genome-wide CGI methylation in inflammatory bowel disease (IBD)-associated CRCs (IBD-CRCs).
Design
Methylation microarray analyses were conducted on 7 IBD-CRCs, 17 S-CRCs, and 8 normal control colonic tissues from patients without CRC or IBD. CGI methylator phenotype (CIMP), a surrogate marker for widespread cancer-specific CGI hypermethylation, was examined in 30 IBD-CRCs and 43 S-CRCs.
Results
The genome-wide CGI methylation pattern of IBD-CRCs was CIMP status-dependent. Based on methylation array data profiling of all autosomal loci, CIMP+ IBD-CRCs grouped together with S-CRCs, while CIMP− IBD-CRCs grouped together with control tissues. CIMP− IBD-CRCs demonstrated less methylation than did age-matched CIMP− S-CRCs at all autosomal CGIs (z-score −0.17 vs. 0.09, p=3×10−3) and CRC-associated hypermethylation target CGIs (z-score −0.43 vs. 0.68, p=1×10-4). Age-associated hypermethylation target CGIs were significantly overrepresented in CGIs that were hypermethylated in S-CRCs (p=1×10−192), but not in CGIs that were hypermethylated in IBD-CRCs (p=0.11). In contrast, KRAS mutation prevalence were similar between IBD-CRCs and S-CRCs. Notably, CIMP+ prevalence was significantly higher in older than in younger IBD-CRC cases (4.2% vs. 50.0%, p=0.02), but not in S-CRC cases (16.7% vs. 9.7%, p=0.92).
Conclusions
Cancer-specific CGI hypermethylation and age-associated CGI hypermethylation are diminished in IBD-CRCs relative to S-CRCs, while KRAS mutation rate is comparable between these cancers. CGI hypermethylation appears to play only a minor role in IBD-associated carcinogenesis. We speculate that aging, rather than inflammation per se, promotes CIMP+ CRCs in IBD patients.
doi:10.1002/ibd.21826
PMCID: PMC3214229  PMID: 21830278
ulcerative colitis; Crohn’s disease; inflammatory bowel disease; colorectal cancer; DNA methylation microarray
6.  LARP7 is a potential tumor suppressor gene in gastric cancer 
We previously reported frequent truncating mutation of the RNA binding protein gene, La ribonucleoprotein domain family, member 7 (LARP7) in gastric cancers (GC) with frequent microsatellite instability. LARP7 negatively regulates positive transcription elongation factor-b (p-TEFb) by binding to and stabilizing 7sk RNA. p-TEFb has been linked to proliferation and de-differentiation in various tissues. Therefore, we reasoned that loss of LARP7 may contribute to gastric tumorigenesis. In the current study, we evaluated LARP7 mRNA expression in 18 GCs, their corresponding non-neoplastic gastric tissues (NGC), and 18 normal gastric tissues from healthy individuals (NN).We also assessed the effects of transient siRNA-mediated LARP7 knockdown in immortalized non-neoplastic gastric epithelial cells. LARP7 mRNA was significantly decreased in GCs (median 2.5) relative to NNs (median 14.9, p<0.01) as well as relative to their corresponding NGCs (median 8.1, p<0.01). Transfection of a siRNA directed against LARP7 (anti-LARP7 siRNA) decreased 7sk levels by 72% relative to a control siRNA (p<0.01). Furthermore, anti-LARP7 siRNA transfection increased cell proliferation by 23% (p<0.01) and cell migration by 22% (p<0.001) relative to control siRNA transfection. Taken together, these findings suggest that LARP7 downregulation occurs early during gastric tumorigenesis and may promote gastric tumorigenesis via p-TEFb deregulation.
doi:10.1038/labinvest.2012.59
PMCID: PMC3446825  PMID: 22488152
LARP7; gastric cancer; RNA binding protein; 7sk snRNA; p-TEFb
7.  A microRNA downregulated in human cholangiocarcinoma controls cell cycle through multiple targets involved in the G1/S checkpoint 
Hepatology (Baltimore, Md.)  2011;54(6):2089-2098.
Background and rationale
MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study, we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be downregulated in CCA, participates in cancer homeostasis. miR-494 was identified as downregulated in CCA based on miR arrays. Its expression was verified with quantitative real time RT-PCR (qRT-PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR-494.
Main Results
Upregulation of miR-494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR-494, followed by pathway analysis, suggested that miR-494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR-494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR-494 downregulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR-494 and the 3’-Untranslated Region (UTR) of Cyclin-dependent-kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR-494 induces a significant cancer growth retardation in-vivo.
Conclusions
Our findings demonstrate that miR-494 is downregulated in CCA and that its upregulation induces cancer cell growth retardation through multiple targets involved in G1-S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR-494 emerges as an important regulator of cholangiocarcinoma growth and its further study may lead to the development of novel therapeutics.
doi:10.1002/hep.24591
PMCID: PMC3212606  PMID: 21809359
cancer; cell cycle
8.  Polo-like Kinase 1 regulates cell proliferation and is targeted by miR-593* in esophageal cancer 
Polo-like kinase 1 (PLK1) is overexpressed in various human cancers. However, the biological functions and the post-transcriptional regulations of PLK1 in esophageal cancer (EC) are still unknown. The purposes of this study are to determine whether PLK1 can be a molecular target of EC therapy, and to identify a microRNA targeting PLK1. We performed loss-of- and gain-of-function experiments regarding cell proliferation, cell cycle, apoptosis, in vivo tumor formation, and luciferase reporter assays, using siRNAs against PLK1 and microRNA. PLK1 protein was expressed in all 11 EC cell lines, but not in normal esophageal epithelial cells (HEEpiC). Knock-down of PLK1 in EC cells induced G2/M arrest (p<0.001) in cell cycle assay, and reduced cell proliferation (p=0.019) and tumor formation ability in vivo (p<0.0001). MiR-593*, identified as a microRNA targeting PLK1 by a database search, was less expressed especially in six EC cell lines than HEEpiC cells. Moreover, miR-593* expression level was inversely correlated with PLK1 mRNA level in 48 clinical tissue specimens of EC (p=0.006). Introduction of synthetic miR-593* suppressed PLK1 expression by 69–73%, reduced cell proliferation (p=0.008), and increased cell proportion of G2/M phase (p=0.01) in HSA/c (an EC cells), whereas a miR-593* inhibitor up-regulated PLK1 expression by 11–55%. Additionally, luciferase assay demonstrated that miR-593* interacted two binding sites in the PLK1 3′-UTR and reduced 56.8–71.5% of luciferase activity by degrading luciferase mRNA in HSA/c cells. In conclusion, PLK1 is post-transcriptionally regulated by miR-593*, and could be a promising molecular target for EC treatment.
doi:10.1002/ijc.25874
PMCID: PMC3176391  PMID: 21170987
esophageal cancer; PLK1; microRNA; miR-593*
9.  Novel Candidate Colorectal Cancer Biomarkers Identified by Methylation Microarray-Based Scanning 
Endocrine-related cancer  2011;18(4):465-478.
DNA hypermethylation is a common epigenetic abnormality in colorectal cancers (CRCs) and a promising class of CRC screening biomarkers. We conducted a genome-wide search for novel neoplasia-specific hypermethylation events in the colon.
We applied methylation microarray analysis to identify loci hypermethylated in 17 primary CRCs relative to 8 non-neoplastic colonic tissues (NCs) from neoplasia-free subjects. These CRC-associated hypermethylation events were then individually evaluated for their ability to discriminate neoplastic from non-neoplastic cases, based on real-time quantitative methylation-specific PCR (qMSP) assays in 113 colonic tissues: 51 CRCs, 9 adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs).
A strict microarray data filtering identified 169 candidate CRC-associated hypermethylation events. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from age-matched control NCs (p<0.05 by ROC curve analysis); methylation of VSX2 achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity, p<1E-6), followed by BEND4, NPTX1, ALX3, miR-34b, GLP1R, BTG4, HOMER2, ZNF583, and GJC1. Adenomas were significantly discriminated from control NCs by hypermethylation of VSX2, BEND4, NPTX1, miR-34b, GLP1R, and HOMER2 (p<0.05). CRC-NCs were significantly distinguished from control NCs by methylation of ALX3 (p<1E-4).
In conclusion, systematic, methylome analysis has identified ten novel methylation events in neoplastic and non-neoplastic colonic mucosae from CRC patients. These potential biomarkers significantly discriminate CRC patients from controls. Thus, they merit further evaluation in stool- and circulating DNA-based CRC detection studies.
doi:10.1530/ERC-11-0083
PMCID: PMC3464012  PMID: 21636702
Colorectal cancer; colorectal adenoma; methylation; microarray; real-time methylation-specific PCR; biomarker
10.  Dynamic changes in the expression of microRNA-31 during inflammatory bowel disease-associated neoplastic transformation 
Inflammatory bowel diseases  2011;17(1):221-231.
Background
Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miRs) expression has been linked to carcinogenesis, however no reports document a relationship between IBD-related neoplasia (IBDN) and altered miR expression. In the current study we sought to identify specific miR dysregulation along the normal-inflammation-cancer axis.
Methods
miR microarrays and quantitative RT-PCR were used to detect dysregulated miRs. Receiver operating characteristic curve analysis was employed to test for potential usefulness of miR-31 as a disease marker of IBDNs. In silico prediction analysis, Western blot, and luciferase activity measurement were employed for target identification.
Results
Several dysregulated miRs were identified between chronically inflamed mucosae and dysplasia arising in IBD. MiR-31 expression increases in a stepwise fashion during progression from normal to IBD to IBDN and accurately discriminated IBDNs from normal or chronically inflamed tissues in IBD patients. Finally, we identified factor inhibiting hypoxia inducible factor 1 as a direct target of miR-31.
Conclusions
Our study reveals specific miR dysregulation as chronic inflammation progresses to dysplasia. MiR-31 expression levels increase with disease progression and accurately discriminates between distinct pathological entities that co-exist in IBD patients. The novel effect of miR-31 on regulating factor inhibiting hypoxia inducible factor 1 expression provides a new insight on the pathogenesis of IBDN.
doi:10.1002/ibd.21359
PMCID: PMC3006011  PMID: 20848542
Inflammatory bowel disease; IBD-related neoplasia; microRNA; microRNA factor inhibiting hypoxia inducible factor 1
11.  MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3 
Hepatology (Baltimore, Md.)  2009;49(5):1595-1601.
Cholangiocarcinomas (CCA) are aggressive cancers, with a high mortality and poor survival rate. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of the late diagnosis secondary to relatively poor accuracy diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on 5 primary CCAs and 5 normal bile duct specimens (NBD). Several miRs were dysregulated, and miR-21 was overexpressed, in CCAs. miR-21 differential expression in these 10 specimens was verified with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the Receiver Operating Characteristic (ROC) curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA (mRNA) levels of TIMP3 were significantly lower in CCAs than in normals.
Conclusions
MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree.
doi:10.1002/hep.22838
PMCID: PMC3124086  PMID: 19296468
PDCD4; TIMP3; oncomiR
12.  The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim 
Gastroenterology  2009;136(5):1689-1700.
Background & Aims
Barrett’s esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known.
Methods
Esophageal cultured cells (HEEpiC, QhTRT, ChTRT, GihTRT and OE-33) and esophageal tissues (22 normal epithelia, 24 BE and 22 EAC) were studied. MiR microarrays and quantitative RT-PCR were employed to explore and verify differentially expressed miRs. Quantitative genomic PCR was performed to study genomic copy number variation at the miR-106b-25 polycistron and MCM7 gene locus on chromosome 7q22.1. In vitro cell proliferation, cell cycle, and apoptosis assays and in vivo tumorigenesis experiments were performed to elucidate biological effects of the miR-106b-25 polycistron. Western blotting and luciferase assays were performed to confirm direct mRNA targeting by miR-106b-25 polycistron.
Results
The miR-106b-25 polycistron exerted potential proliferative, anti-apoptotic, cell cycle-promoting effects in vitro and tumorigenic activity in vivo. MiRs -93 and -106b targeted and inhibited p21, while miR-25 targeted and inhibited Bim. This polycistron was upregulated progressively at successive stages of neoplasia, in association with genomic amplification and overexpression of MCM7. In addition, miRs -93 and -106b decreased p21 mRNA, while miR-25 did not alter Bim mRNA, suggesting the following discrete miR effector mechanisms: 1) for p21, mRNA degradation; 2) for Bim, translational inhibition.
Conclusions
The miR-106b-25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression and proliferation via suppression of two target genes, p21 and Bim.
PMCID: PMC2887605  PMID: 19422085
13.  A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett’s esophagus 
Cancer research  2009;69(10):4112-4115.
Esophageal adenocarcinoma risk in Barrett’s esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of 8 BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors (NPs) and 50 progressors (Ps) using real-time quantitative methylation-specific PCR. Ps were significantly older than NPs (70.6 vs. 62.5 years, p < 0.001). We evaluated a linear combination of the 8 markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUCs) were high in the 2-, 4-year and combined data models (0.843, 0.829 and 0.840; p<0.001, p<0.001 and p<0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age vs. age alone were substantial (Δ-AUC = 0.152, 0.114 and 0.118, respectively) in all three models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia.
doi:10.1158/0008-5472.CAN-09-0028
PMCID: PMC2752375  PMID: 19435894
14.  Silencing of Claudin-11 Is Associated with Increased Invasiveness of Gastric Cancer Cells 
PLoS ONE  2009;4(11):e8002.
Background
Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11 (CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region.
Methodology/Principal Findings
Levels of CLDN11 methylation and mRNA expression were measured in primary gastric cancer tissues, noncancerous gastric mucosae, and cell lines of gastric origin using quantitative methylation-specific PCR (qMSP) and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2′-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness.
Conclusions/Significance
These data suggest that hypermethylation of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.
doi:10.1371/journal.pone.0008002
PMCID: PMC2776495  PMID: 19956721
15.  Beyond Field Effect: Analysis of Shrunken Centroids in Normal Esophageal Epithelia Detects Concomitant Esophageal Adenocarcinoma 
Background and Aims:
Because of the extremely low neoplastic progression rate in Barrett’s esophagus, it is difficult to diagnose patients with concomitant adenocarcinoma early in their disease course. If biomarkers existed in normal squamous esophageal epithelium to identify patients with concomitant esophageal adenocarcinoma, potential applications would be far-reaching. The aim of the current study was to identify global gene expression patterns in normal esophageal epithelium capable of revealing simultaneous esophageal adenocarcinoma, even located remotely in the esophagus.
Methods:
Tissues comprised normal esophageal epithelia from 9 patients with esophageal adenocarcinoma, 8 patients lacking esophageal adenocarcinoma or Barrett’s, and 6 patients with Barrett’s esophagus alone. cDNA microarrays were performed, and pattern recognition in each of these subgroups was achieved using shrunken nearest centroid predictors.
Results:
Our method accurately discriminated normal esophageal epithelia of 8/8 patients without esophageal adenocarcinoma or Barrett’s esophagus and of 6/6 patients with Barrett’s esophagus alone from normal esophageal epithelia of 9/9 patients with Barrett’s esophagus and concomitant esophageal adenocarcinoma. Moreover, we identified genes differentially expressed between the above subgroups. Thus, based on their corresponding normal esophageal epithelia alone, our method accurately diagnosed patients who had concomitant esophageal adenocarcinoma.
Conclusions:
These global gene expression patterns, along with individual genes culled from them, represent potential biomarkers for the early diagnosis of esophageal adenocarcinoma from normal esophageal epithelia. Genes discovered in normal esophagus that are differentially expressed in patients with vs. without esophageal adenocarcinoma merit further pursuit in molecular genetic, functional, and therapeutic interventional studies.
PMCID: PMC2323355  PMID: 18425214
16.  Rarity of Somatic Mutation and Frequency of Normal Sequence Variation Detected in Sporadic Colon Adenocarcinoma Using High-Throughput cDNA Sequencing 
We performed high-throughput cDNA sequencing in colorectal adenocarcinoma and matching normal colorectal epithelium. All six hundred three genes in the UCSC database that were expressed in colon cancers and contained open reading frames of 1000 nucleotides or less were selected for study (total basepairs/bp, 366,686). 304,350 of these 366,686 bp (83.0%) were amplified and sequenced successfully. Seventy-eight sequence variants present in germline (i.e. normal) as well as matching somatic (i.e. tumor) DNA were discovered, yielding a frequency of 1 variant per 3,902 bp. Fifty-one of these sequence variants were homozygous (26 synonymous, 25 non-synonymous), while 27 were heterozygous (11 synonymous, 16 non-synonymous). Cancer tissue contained only one sequence-altered allele of the gene ATP50, which was present heterozygously alongside the wild-type allele in matching normal epithelium. Despite this relatively large number of bp and genes sequenced, no somatic mutations unique to tumor were found. High-throughput cDNA sequencing is a practical approach for detecting novel sequence variations and alterations in human tumors, such as those of the colon.
PMCID: PMC2287164  PMID: 18389087
17.  Beyond Field Effect: Analysis of Shrunken Centroids in Normal Esophageal Epithelia Detects Concomitant Esophageal Adenocarcinoma 
Background and Aims
Because of the extremely low neoplastic progression rate in Barrett’s esophagus, it is difficult to diagnose patients with concomitant adenocarcinoma early in their disease course. If biomarkers existed in normal squamous esophageal epithelium to identify patients with concomitant esophageal adenocarcinoma, potential applications would be far-reaching. The aim of the current study was to identify global gene expression patterns in normal esophageal epithelium capable of revealing simultaneous esophageal adenocarcinoma, even located remotely in the esophagus.
Methods
Tissues comprised normal esophageal epithelia from 9 patients with esophageal adenocarcinoma, 8 patients lacking esophageal adenocarcinoma or Barrett’s, and 6 patients with Barrett’s esophagus alone. cDNA microarrays were performed, and pattern recognition in each of these subgroups was achieved using shrunken nearest centroid predictors.
Results
Our method accurately discriminated normal esophageal epithelia of 8/8 patients without esophageal adenocarcinoma or Barrett’s esophagus and of 6/6 patients with Barrett’s esophagus alone from normal esophageal epithelia of 9/9 patients with Barrett’s esophagus and concomitant esophageal adenocarcinoma. Moreover, we identified genes differentially expressed between the above subgroups. Thus, based on their corresponding normal esophageal epithelia alone, our method accurately diagnosed patients who had concomitant esophageal adenocarcinoma.
Conclusions
These global gene expression patterns, along with individual genes culled from them, represent potential biomarkers for the early diagnosis of esophageal adenocarcinoma from normal esophageal epithelia. Genes discovered in normal esophagus that are differentially expressed in patients with vs. without esophageal adenocarcinoma merit further pursuit in molecular genetic, functional, and therapeutic interventional studies.
PMCID: PMC2323355  PMID: 18425214
18.  Rarity of Somatic Mutation and Frequency of Normal Sequence Variation Detected in Sporadic Colon Adenocarcinoma Using High-Throughput cDNA Sequencing 
We performed high-throughput cDNA sequencing in colorectal adenocarcinoma and matching normal colorectal epithelium. All six hundred three genes in the UCSC database that were expressed in colon cancers and contained open reading frames of 1000 nucleotides or less were selected for study (total basepairs/bp, 366,686). 304,350 of these 366,686 bp (83.0%) were amplified and sequenced successfully. Seventy-eight sequence variants present in germline (i.e. normal) as well as matching somatic (i.e. tumor) DNA were discovered, yielding a frequency of 1 variant per 3,902 bp. Fifty-one of these sequence variants were homozygous (26 synonymous, 25 non-synonymous), while 27 were heterozygous (11 synonymous, 16 non-synonymous). Cancer tissue contained only one sequence-altered allele of the gene ATP50, which was present heterozygously alongside the wild-type allele in matching normal epithelium. Despite this relatively large number of bp and genes sequenced, no somatic mutations unique to tumor were found. High-throughput cDNA sequencing is a practical approach for detecting novel sequence variations and alterations in human tumors, such as those of the colon.
PMCID: PMC2287164  PMID: 18389087

Results 1-18 (18)