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Supplementary Tables S1 & S2 from Reprimo Methylation Is a Potential Biomarker of Barrett's-Associated Esophageal Neoplastic Progression

Supplementary Figure 2 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Supplementary Table 3 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Supplementary Table 1 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Supplementary Table 4 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Supplementary Figure 1 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Supplementary Table 2 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

The identification of new molecular pathways supporting the growth of myelodysplastic syndrome (MDS) stem and progenitor cells is needed to understand clinical variation and develop targeted therapies. Within myeloid malignancies, men have worse outcomes compared to women, suggesting male sex hormone driven effects in malignant hematopoiesis. The androgen receptor promotes the expression of five granulocyte-colony factor receptor regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor that regulates cytokine signaling in differentiated granulocytes but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is upregulated in stem and progenitor cells from patients with MDS and secondary acute leukemia. CCRL2 knockdown suppressed the growth and clonogenicity of MDS92 and MDS-L cells in vitro and in vivo. Moreover, CCRL2 knockdown significantly suppressed the phosphorylation of JAK2, STAT3, and STAT5 in MDS cells. CCRL2 co-precipitated with JAK2 and its suppression decreased the interaction of JAK2 with STAT proteins. Cell lines expressing JAK2V617F showed less effect of CCRL2 knockdown on growth and clonogenicity compared to those expressing wild type. However, the selective JAK2 inhibitor fedratinib potentiated the effects of CCRL2 knockdown in MDS and leukemia cells expressing both wild type JAK2 and JAK2V617F. In conclusion, our results implicate CCRL2 as a mediator of MDS and secondary acute leukemia cell growth, in part through JAK2/STAT signaling.

Subsets of non-acute promyelocytic leukemia (APL) acute myelogenous leukemia (AML) exhibit aberrant retinoid signaling and demonstrate sensitivity to retinoids in vitro. We present the results of a phase 1 dose-escalation study that evaluated the safety, pharmacodynamics, and efficacy of IRX195183, a novel retinoic acid receptor α agonist, in patients with relapsed or refractory myelodysplastic syndrome (MDS) or AML. In this single center, single arm study, eleven patients with relapsed or refractory MDS/AML were enrolled and treated. Oral IRX195183 was administered at two dose levels: 50 mg daily or 75 mg daily for a total of two 28-day cycles. Patients with stable disease or better were allowed to continue on the drug for four additional 28-day cycles. Common adverse events included hypertriglyceridemia, fatigue, dyspnea, and edema. Three patients at the first dose level developed asymptomatic Grade 3 hypertriglyceridemia. The maximally tolerated dose was not reached. Four of the eleven patients had (36%) stable disease or better. One had a morphological complete remission with incomplete hematologic recovery while on the study drug. Two patients had evidence of in vivo leukemic blast maturation, as reflected by increased CD38 expression. In a pharmacodynamics study, plasma samples from four patients treated at the lowest dose level demonstrated the capacity to differentiate leukemic cells from the NB4 cell line in vitro. These results suggest that IRX195183 is safe, achieves biologically meaningful plasma concentrations and may be efficacious in a subset of patients with MDS/AML. Clinical Trial Registration: clinicaltrials.gov, identifier NCT02749708.

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.

Mesothelin (MSLN) may be the most “dramatic” of the tumor markers, being strongly overexpressed in nearly one-third of human malignancies. The biochemical cause is unclear. We previously ascribed this cancer-specific overexpression to an element, Canscript, residing around 50 bp 5′ of the transcription start site in cancer (Hucl, T., Brody, J. R., Gallmeier, E., Iacobuzio-Donahue, C. A., Farrance, I. K., and Kern, S. E. (2007) Cancer Res. 67, 9055–9065). Herein, we found a Canscript promoter activity elevated over 100-fold in cancer cells. In addition to a highly conserved TEAD1 (TEA domain family member 1)-binding MCAT motif, nucleotide substitution revealed the consensus core sequence (WCYCCACCC) of an SP1-like motif in Canscript. The unknown transcription factor binding to the SP1-like motif may hold the key for the cancer specificity of Canscript. SP1, GLI1, and RUNX1, -2, and -3 appeared unlikely to be the direct transcription factors acting at the SP1-like motif, but KLF6 had some features of such a candidate. YAP1, a TEAD1-binding protein, appeared necessary, but not sufficient, for Canscript activity; knockdown of YAP1 by small interfering RNAs greatly reduced MSLN levels in MSLN-overexpressing cells, but overexpressing YAP1 in MSLN-negative cells did not induce MSLN expression. Cansript-like sequences were found in other genes up-regulated in pancreatic cancer; reporters driven by the sequences from FXYD3, MUC1, and TIMP1 had activities more than 2 times that of the control. This suggested that the cause of MSLN overexpression might also contribute mechanistically to the overexpression of other tumor markers.

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.

Cholangiocarcinomas (CCAs) are aggressive cancers, with high mortality and poor survival rates. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of late diagnosis secondary to relatively poor accuracy of 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 five primary CCAs and five normal bile duct specimens (NBDs). Several miRs were dysregulated and miR-21 was overexpressed in CCAs. miR-21 differential expression in these 10 specimens was verified by 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 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 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. (HEPATOLOGY 2009.)

Although the CDH13 gene has been shown to undergo epigenetic silencing by promoter methylation in many types of tumors, hypermethylation of this gene in Barrett's-associated esophageal adenocarcinogenesis has not been studied. Two hundred fifty-nine human esophageal tissues were therefore examined for CDH13 promoter hypermethylation by real-time methylation-specific PCR. CDH13 hypermethylation showed discriminative receiver-operator characteristic curve profiles, sharply demarcating esophageal adenocarcinoma (EAC) from esophageal squamous cell carcinoma (ESCC) and normal esophagus (NE) (p < 0.0001). CDH13 normalized methylation values (NMV) were significantly higher in Barrett's esophagus (BE), dysplastic BE (D) and EAC than in NE (p < 0.0000001). CDH13 hypermethylation frequency was 0% in NE but increased early during neoplastic progression, rising to 70% in BE, 77.5% in D and 76.1% in EAC. Both CDH13 hypermethylation frequency and its mean NMV were significantly higher in BE with than without accompanying EAC. In contrast, only 5 (19.2%) of 26 ESCCs exhibited CDH13 hypermethylation. Furthermore, both CDH13 hypermethylation frequency and its mean NMV were significantly higher in EAC than in ESCC, as well as in BE or D vs. ESCC. Interestingly, mean CDH13 NMV was significantly lower in short-segment than in long-segment BE, a known clinical risk factor for neoplastic progression. Similarly, BE segment length was significantly lower in specimens with unmethylated than with methylated CDH13 promoters. 5-aza-2'-deoxycytidine treatment of OE33 EAC and KYSE220 ESCC cells reduced CDH13 methylation and increased CDH13 mRNA expression. These findings suggest that hypermethylation of CDH13 is a common, tissue-specific event in human EAC, occurs early during BE-associated neoplastic progression, and correlates with known clinical neoplastic progression risk factors.

Human pituitary tumor-transforming 1 (PTTG1)/securin is a putative oncoprotein that is overexpressed in various tumor types. However, the involvement of PTTG1 in gastrointestinal cancer development and progression remains unclear. In this study, we investigated the clinical significance and biological effects of PTTG1 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical studies performed on 113 primary ESCC specimens revealed a high prevalence of PTTG1 overexpression (60.2%), which was significantly associated with lymph node metastasis (regional, P = 0.042; distant, P = 0.005), advanced tumor stage (P = 0.028), and poorer overall survival (P = 0.017, log-rank test; P = 0.044, Cox proportional hazard model). Eleven ESCC cell lines expressed PTTG1 protein at levels 2.4 to 6.6 times higher than those in normal esophageal epithelial cells (HEEpiC). PTTG1 protein expression was confined to the nucleus in HEEpiC cells but present in both the cytoplasm and nucleus in ESCC cells. Two small interfering RNAs (siRNA) inhibited PTTG1 mRNA and protein expression in three ESCC cell lines by 77% to 97%. In addition, PTTG1 down-regulation by these siRNAs significantly reduced cell motility in all three ESCC cell lines (P < 0.01) in vitro, as well as popliteal lymph node metastases of ESCC cells in nude mice (P = 0.020). Global gene expression profiling suggested that several members of the Ras and Rho gene families, including RRAS, RHOG, ARHGAP1, and ARHGADIA, represented potential downstream genes in the PTTG1 pathway. Taken together, these findings suggest that PTTG1 overexpression promotes cell motility and lymph node metastasis in ESCC patients, leading to poorer survival. Thus, PTTG1 constitutes a potential biomarker and therapeutic target in ESCCs with lymph node metastases. [Cancer Res 2008;68(9):3214–24]

The nel-like1 (NELL1) gene maps to chromosome 11p15, which frequently undergoes loss of heterozygosity in esophageal adenocarcinoma (EAC). NELL1 promoter hypermethylation was examined by real-time methylation-specific polymerase chain reaction in 259 human esophageal tissues. Hypermethylation of this promoter showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and EAC from normal esophagus (NE) (P

Background & Aims: Gene silencing via promoter hypermethylation is a central event in the pathogenesis of cancers. To identify novel methylation targets in colon cancer, we conducted a genome-wide, microarray-based, in silico, and epigenetic search. Methods: Complementary DNA microarray experiments were first performed to identify genes down-regulated in primary colon cancers and up-regulated in colon cancer cell lines after global DNA demethylation by 5-aza-2′-deoxycitidine. Candidate methylation targets were then identified by combining these microarray data with in silico genetic and functional searches. Candidate genes recognized by these searches were further investigated for promoter hypermethylation in colon cancer using methylation-specific polymerase chain reaction. Results: We identified 51 novel and 3 known candidate methylation targets. Subsequent epigenetic analysis revealed that primary colon cancers demonstrated frequent methylation of somatostatin (SST, 30 of 34 cases, 88%) and the substance P precursor gene tachykinin-1 (TAC1; 16 of 34 cases, 47%). TAC1 methylation intensity was significantly higher in Dukes A/B than in Dukes C/D cancers (P = .01). SST methylation intensity was significantly higher in low-level microsatellite instability (MSI-L) than in non-MSI-L cancers (P = .02). Methylation was associated with messenger RNA down-regulation for both SST and TAC1. Furthermore, we isolated 5 additional novel promoter methylation targets: NELL1, AKAP12, caveolin-1, endoglin, and MAL. Conclusions: These data strongly suggest that SST and TAC1 are involved in colon carcinogenesis. Further studies are now indicated to elucidate mechanisms underlying their involvement in colon cancer and their values as clinical biomarkers. NELL1, AKAP12, caveolin-1, endoglin, and MAL are also promising tumor suppressor gene candidates deserving of further study.

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.

The greatest interpretive challenge of modern medicine may be to functionally annotate the vast variation of human genomes. Demonstrating a proposed approach, we created a library of BRCA2 exon 27 shotgun-mutant plasmids including solitary and multiplex mutations to generate human knockin clones using homologous recombination. This 55-mutation, 13-clone syngeneic variance library (SyVaL) comprised severely affected clones having early-stop nonsense mutations, functionally hypomorphic clones having multiple missense mutations emphasizing the potential to identify and assess hypomorphic mutations in novel proteomic and epidemiologic studies, and neutral clones having multiple missense mutations. Efficient coverage of nonessential amino acids was provided by mutation multiplexing. Severe mutations were distinguished from hypomorphic or neutral changes by chemosensitivity assays (hypersensitivity to mitomycin C and acetaldehyde), by analysis of RAD51 focus formation, and by mitotic multipolarity. A multiplex unbiased approach of generating all-human SyVaLs in medically important genes, with random mutations in native genes, would provide databases of variants that could be functionally annotated without concerns arising from exogenous cDNA constructs or interspecies interactions, as a basis for subsequent proteomic domain mapping or clinical calibration if desired. Such gene-irrelevant approaches could be scaled up for multiple genes of clinical interest, providing distributable cellular libraries linked to public-shared functional databases.

Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun–mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.

Background: Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miR) 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 reverse-transcriptase polymerase chain reaction (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 coexist 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. (Inflamm Bowel Dis 2011;)

BACKGROUND: Colorectal cancer (CRC) is 1 of the leading causes of death in the Western world. CRC develops from premalignant lesions, chiefly colorectal adenomas. Currently, the most accurate and recommended screening method for finding colorectal adenomas is colonoscopy performed on all individuals aged >50 years. However, the costs and risks associated with this procedure are relatively high. The objectives of the current study were to correlate epigenetic alterations that occur in normal rectal mucosa, smoking status, and age with the presence or absence of concomitant colorectal adenomas and to assess the potential clinical value of methylation in normal rectal biopsies as a screening assay for the presence of polyps and, hence, the need for a full colonoscopy. METHODS: One hundred thirteen normal rectal mucosal biopsies from 113 patients were studied. DNA was extracted, modified with sodium bisulfite, and subjected to real-time quantitative, methylation-specific polymerase chain reaction analysis for the following genes: adenomatous polyposis coli (APC); cadherin 1, type 1, E-cadherin (epithelial) (CDH1); estrogen receptor 1 (ESR1); cytokine high in normal 1 (HIN1); hyperplastic polyposis protein 1 (HPP1); O-6 methylguanine-DNA methyltransferase (MGMT); neural epidermal growth factor-like 1 (NELL1); splicing factor 3B, 14-kDa subunit (p14); cyclin-dependent kinase (CDK) inhibitor 2B (inhibits CDK4) (p15); retinoic acid receptor beta (RARβ); somatostatin (SST); tachykinin, precursor 1 (TAC1); and tissue inhibitor of metalloproteinase (TIMP) metallopeptidase inhibitor 3 (TIMP3). Data were then analyzed using several proprietary software programs. RESULTS: By using several sets of genes, clinical characteristics, and multivariate analyses, the authors developed a prediction model for the presence of concomitant colorectal adenomas at the time of rectal biopsy. They also observed strong correlations between smoking status and rectal methylation pattern and between smoking status and the presence or risk of concomitant adenomas. CONCLUSIONS: A prediction model was developed for the presence of colorectal adenomas based on gene methylation patterns in the normal rectum. The results indicated that these genes may be involved in early stages of adenoma formation. The observed epigenetic alterations in these markers may be caused in part by the effects of smoking and/or age. Normal rectal methylation may be useful as a biomarker for narrowing the population in need of screening colonoscopy. Cancer 2010. © 2010 American Cancer Society.

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 our study are to determine whether PLK1 can be a molecular target of EC therapy and to identify a microRNA (miRNA) targeting PLK1. We performed loss-of-function and gain-of-function experiments regarding cell proliferation, cell cycle, apoptosis, in vivo tumor formation and luciferase reporter assays, using siRNAs against PLK1 and miRNA. PLK1 protein was expressed in all 11 EC cell lines, but not in normal esophageal epithelial cells (HEEpiC). Knockdown 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 miRNA 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 upregulated 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.

This systematic review was designed to determine postoperative complication rates of radical surgery for rectal cancer (abdominal perineal resection and anterior resection).Lack of accepted complication rates for rectal cancer surgery may hinder quality improvement efforts and may impede the conception of future studies because of uncertainty regarding the expected event rates.All prospective studies of rectal cancer receiving radical surgery published between 1990 and August 2008 were obtained by searching Ovid MEDLINE, EMBASE, as well as ASCO GI, CAGS, and ASCRS meeting abstracts between 2004 and 2008. There was no language restriction. The outcomes extracted were anastomotic leak, pelvic sepsis, postoperative death, wound infection, and fecal incontinence. Summary complication rates were obtained using a random effects model; the Z-test was used to test for study heterogeneity.Fifty-three prospective cohort studies and 45 randomized controlled studies with 36,315 patients (24,845 patients had an anastomosis) were eligible for inclusion. Most of the studies found were based in continental Europe (58%), followed by Asia (25%), United Kingdom (10%), North America (5%), and Australia/New Zealand. The anastomotic leak rate, reported in 84 studies, was 11% (95% CI: 10, 12); the pelvic sepsis rate, in 29 studies, was 12% (9, 16); the postoperative death rate, in 75 studies, was 2% (2, 3); and the wound infection rate, in 50 studies, was 7% (5, 8). Fecal incontinence rates were reported in too few studies and so heterogeneously that numerical summarization was inappropriate. Year of publication, use of preoperative radiation, use of laparoscopy, and use of protecting stoma were not significant variables, but average age, median tumor height, and method of detection (clinical vs. radiologic) showed significance to explain heterogeneity in anastomotic leak rates. Year of publication, study origin, average age, and use of laparoscopy were significant, but median tumor height and preoperative radiation use were not significant in explaining heterogeneity among observed postoperative death rates. With multivariable analysis, only average age for anastomotic leak and year of publication for postoperative death remained significant.Benchmark complication rates for radical rectal cancer surgery were obtained for use in sample size calculations in future studies and for quality control purposes. Postoperative death rates showed improvement in recent years.

BackgroundCoding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3'-untranslated regions (3'UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3'UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae.Methodology/principal findingsIn order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r=0.86, p=7.2x10(-13)). 3'UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3'UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3'UTR MSI (9.0-fold; p = 3.6x10(-4)).ConclusionsThis mutational survey of well-characterized short 3'UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3'UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.

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 reverse-transcription polymerase chain reaction (RT-PCR) were employed to explore and verify differentially expressed miRs. Quantitative genomic PCR was performed to study 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 biologic effects of the miR-106b-25 polycistron. Western blotting and luciferase assays were performed to confirm direct messenger RNA (mRNA) targeting by the miR-106b-25 polycistron. Results The miR-106b-25 polycistron exerted potential proliferative, antiapoptotic, cell cycle-promoting effects in vitro and tumorigenic activity in vivo. MiRs-93 and -106b targeted and inhibited p21, whereas miR-25 targeted and inhibited Bim. This polycistron was up-regulated progressively at successive stages of neoplasia, in association with genomic amplification and overexpression of MCM7 . In addition, miRs-93 and -106b decreased p21 mRNA, whereas 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 2 target genes: p21 and Bim.

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 eight 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 and 50 progressors using real-time quantitative methylation-specific PCR. Progressors were significantly older than nonprogressors (70.6 versus 62.5 years; P < 0.001). We evaluated a linear combination of the eight markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUC) were high in the 2-year, 4-year, and combined data models (0.843, 0.829, and 0.840; P < 0.001

Cancers have been revealed to be extremely heterogenous in terms of the frequency and types of mutations present in cells from different malignant tumors. Thus, it is likely that uniform clinical treatment is not optimal for all patients, and that the development of individualized therapeutic regimens may be beneficial. We describe the generation of multiple, unique small peptides nine to thirty-four amino acids in length which, when labeled with the radioisotope (32)P, bind with vastly differing efficiencies to cell lines derived from different colon adenocarcinomas. In addition, the most effective of these peptides permanently transfers the (32)P radioisotope to colorectal cancer cellular proteins within two hours at a rate that is more than 150 times higher than in cell lines derived from other cancers or from the normal tissues tested. Currently, the only two FDA-approved radioimmunotherapeutic agents in use both employ antibodies directed against the B cell marker CD20 for the treatment of non-Hodgkin's lymphoma. By using the method described herein, large numbers of different (32)P-labeled peptides can be readily produced and assayed against a broad spectrum of cancer types. This report proposes the development and use of (32)P-labeled peptides as potential individualized peptide-binding therapies for the treatment of colon adenocarcinoma patients.

The A-kinase anchoring protein 12 (AKAP12) is a kinase scaffold protein with known tumor suppressor activity. Recently, AKAP12 promoter hypermethylation was reported in gastric and colorectal cancers. We examined AKAP12 promoter hypermethylation using real-time methylation-specific PCR in 259 human esophageal tissues. AKAP12 hypermethylation showed highly discriminative receiver-operator characteristic (ROC) curve profiles, clearly distinguishing esophageal adenocarcinoma (EAC) from esophageal squamous cell carcinoma and normal esophagus (P < 0.0001). AKAP12-normalized methylation values were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's, and EAC than in normal esophagus (P < 0.0000001). AKAP12 hypermethylation frequency was zero in normal esophagus but increased early during neoplastic progression, to 38.9% in BE from patients with Barrett's alone, 52.5% in dysplastic Barrett's metaplasia, and 52.2% in EAC. AKAP12 hypermethylation levels were significantly higher in normal esophageal epithelia from patients with EAC (mean = 0.00082) than in normal esophagi from patients without Barrett's or esophageal cancer (mean = 0.00007; P = 0.006). There was a significant correlation between AKAP12 hypermethylation and BE segment length, a known clinical neoplastic progression risk factor. In contrast, only 2 (7.7%) of 26 esophageal squamous cell carcinomas exhibited AKAP12 hypermethylation. Treatment of BIC and OE33 EAC cells with 5-aza-2'-deoxycytidine reduced AKAP12 methylation and increased AKAP12 mRNA expression. AKAP12 mRNA levels in EACs with unmethylated AKAP12 (mean = 0.1663) were higher than in EACs with methylated AKAP12 (mean = 0.0668). We conclude that promoter hypermethylation of AKAP12 is a common, tissue-specific event in human EAC, occurs early during Barrett's-associated esophageal neoplastic progression, and is a potential biomarker for the early detection of EAC. (Cancer Epidemiol Biomarkers Prev 2008;17(1):111–7)

Purpose: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation.Experimental Design: TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution.Results: TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barrett's metaplasia alone, 57.5% in dysplastic Barrett's esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression.Conclusions: TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.

Purpose: Reprimo, a candidate tumor-suppressor gene, regulates p53-mediated cell cycle arrest at G2 phase, and tumor-suppressor gene methylation is involved in the pathogenesis and progression of esophageal cancer. Our aim was to determine whether and at what phase of neoplastic progression Reprimo methylation occurs in Barrett's adenocarcinogenesis, as well as its columnar or squamous cell-type specificity. We also sought to determine whether Reprimo expression could be restored in vitro by the demethylating agent 5-aza-deoxycytidine (5AzaC). Experimental Design: Quantitative methylation-specific PCR for Reprimo was done using an ABI7700 (Taqman) apparatus on 175 endoscopic biopsy specimens. In addition, reverse transcription-PCR and quantitative methylation-specific PCR were done on esophageal carcinoma cells before and after treatment with 5AzaC. Results: In Barrett's esophagus (BE; P = 0.001), high-grade dysplasia (HGD; P = 0.001), and esophageal adenocarcinoma (EAC; P = 0.00003), the level and frequency of Reprimo methylation were significantly higher than in normal esophagus (NE). There was no statistically significant difference between BE and EAC, HGD and EAC, or NE and esophageal squamous cell carcinoma (ESCC). Reprimo methylation occurred in 0 of 19 NE samples, 6 (13%) of 45 ESCC, 9 (36%) of 25 BE, 7 (64%) of 11 HGD, and 47 (63%) of 75 EAC. Analysis of Reprimo methylation in EAC versus NE revealed an area under the receiver-operator characteristic curve of 0.812 (P < 0.00001; 95% confidence interval, 0.73-0.90). In vitro 5AzaC treatment of OE33 EAC cells reduced Reprimo methylation and increased Reprimo expression. Conclusions: Reprimo methylation occurs significantly more frequently in BE, HGD, and EAC than in NE or ESCC, suggesting that this epigenetic alteration is a specialized columnar, cell-specific early event with potential as a biomarker for the early detection of esophageal neoplasia.

Background & Aims: Multiple studies have shown that promoter methylation of tumor suppressor genes underlies esophageal carcinogenesis. Hypothetically, methylation resulting in tumor suppressor gene inactivation might result in tumors that are unresponsive to chemotherapy and radiation. Accordingly, our aim was to find methylation markers that could be used to predict response to chemoradiation. Methods: Tumor specimens were obtained before treatment from 35 patients enrolled in a uniform chemoradiation treatment protocol. Methylation-specific quantitative polymerase chain reaction was performed on all samples. Pathology reports from esophagectomy specimens were used to define response to treatment. Results: Thirteen (37%) of 35 patients were responders, and 22 (63%) of 35 patients were nonresponders. The number of methylated genes per patient was significantly lower in responders than in nonresponders (1.4 vs 2.4 genes per patient; Student t test, P = .026). The combined mean level of promoter methylation of p16, Reprimo, p57, p73, RUNX-3, CHFR, MGMT, TIMP-3, and HPP1 was also lower in responders than in nonresponders (Student t test, P = .003; Mann-Whitney test, P = .001). The frequency (15% of responders vs 64% of nonresponders; Fisher exact test, P = .01) and level (0.078 in responders vs 0.313 in nonresponders; Mann-Whitney test, P = .037) of Reprimo methylation was significantly lower in responders than in nonresponders. Conclusions: Reprimo methylation occurred at significantly lower levels and less frequently in chemoradioresponsive than in nonresponsive esophageal cancer patients, suggesting potential clinical application of this single-gene biomarker in defining prognosis and management. In addition, increased methylation of a 9-gene panel correlated significantly with poor responsiveness to chemoradiation.

To investigate the relationship between Barrett's 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 BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. 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 BE's-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.

For developing successful cancer gene therapy strategies, tumor-specific gene delivery is essential. In this study, we used esophageal cancer (EC) cells to identify and evaluate esophageal tumor-specific gene promoters. Four genes (polo-like kinase-1/PLK, survivin/BIRC5, karyopherin alpha 2/KPNA2, and pituitary tumor transforming gene protein 1/PTTG1) were identified by a microarray analysis as highly expressed in EC cell lines vs. five normal organ tissues (liver, lung, kidney, brain, and heart). By quantitative RT-PCR, the average mRNA expression levels of these four genes in 20 primary ECs were 2.7-fold (PLK), 6.1-fold (survivin), 2.6-fold (KPNA2), and 2.4-fold (PTTG1) higher than that of each gene in 24 different normal organs. By dual luciferase assay, the promoter activity of PLK and survivin in EC cell lines was 18.9-fold and 28.5-fold higher, respectively, than in normal lung and renal cells. The promoters of PLK and survivin could be useful tools for developing EC-specific gene therapy vectors.

Background. Cholangiocarcinoma (CCA) is an aggressive epithelial cancer of the biliary tree with survival measured in months. The only potentially curative treatment for CCA is surgical resection. Unfortunately, the vast majority of patients are diagnosed at late stages, when surgery is not an option. MicroRNAs (miRs) are short single stranded RNA sequences, recently shown to have major regulatory functions in cancer. TIMP3 is one of 4 members of the metalloproteinases family and its role was associated with cancer invasion and metastasis. We aimed at elucidating the regulatory mechanisms of TIMP3 in CCA. Methods. Fifteen histologically confirmed CCAs and 9 normal liver specimens were collected at surgery at Johns Hopkins Hospital after informed consent was obtained from all patients. The NLs were obtained at the time of surgery for CCA from normal liver. Quantitative RT-PCR (qRTPCR) for messenger RNA (mRNA) and miRs and quantitative Methylation Specific PCR (qMSP) were performed as previously described. Results. We measured the qRT-PCR level of TIMP3 mRNA and found it to be underexpressed in CCA vs. NL specimens, suggesting a tumor-suppressive role for TIMP3 in the biliary tree. In contrast, our previous work revealed that miR-21 is overexpressed in CCA vs. NL, suggesting a possible negative regulation of TIMP3 by miR-21. In silico searches suggested that TIMP3 is indeed a putative target of miR-21. The CCA cell lines CAK1, TFK1 and HuCCT1 were transfected with a miR-21 inhibitor and TIMP3 protein levels were measured. TIMP3 was significantly elevated in all 3 cell lines transfected with the inhibitor, strongly suggesting that miR-21 downregulates TIMP3 protein levels. Interestingly, miR-21 does not directly bind to TIMP3 mRNA, as evinced by luciferase assays, suggesting the presence of an intermediary molecule or a different mechanism of miR-21 regulation of TIMP3. To investigate a potential co-regulation of TIMP3 in CCA by hypermethylation, we measured the promoter methylation in all 24 specimens. TIMP3 promoter was found to be hypermethylated in only 1/15 CCA (6.6%), 0/3 CCA cell lines and 0/9 NL specimens. Conclusions: TIMP3 mRNA is elevated in NL and decreased in CCA specimens implicating TIMP3 as a tumor suppressor gene in the biliary tree. Our data also suggests that TIMP3 promoter hypermethylation does not appear to play a major role in its downregulation in human CCA specimens. In contrast, miR-21 is a major regulator of TIMP3 in CCA. Furthermore, miR-21 may exert at least part of its oncogenic, pro-invasive effects in CCA by inhibiting TIMP3.

Background: Barrett’s esophagus predisposes to esophageal adenocarcinoma. However, the value of endoscopic surveillance in Barrett’s esophagus has been debated because of the low incidence of esophageal adenocarcinoma in Barrett’s esophagus. Moreover, high inter-observer and sampling-dependent variation in the histologic staging of dysplasia make clinical risk assessment problematic. In this study, we developed a 3-tiered risk stratification strategy, based on systematically selected epigenetic and clinical parameters, to improve Barrett’s esophagus surveillance efficiency. Methods and Findings: We defined high-grade dysplasia as endpoint of progression, and Barrett’s esophagus progressor patients as Barrett’s esophagus patients with either no dysplasia or low-grade dysplasia who later developed high-grade dysplasia or esophageal adenocarcinoma. We analyzed 4 epigenetic and 3 clinical parameters in 118 Barrett’s esophagus tissues obtained from 35 progressor and 27 non-progressor Barrett’s esophagus patients from Baltimore Veterans Affairs Maryland Health Care Systems and Mayo Clinic. Based on 2-year and 4-year prediction models using linear discriminant analysis (area under the receiver-operator characteristic (ROC) curve: 0.8386 and 0.7910, respectively), Barrett’s esophagus specimens were stratified into high-risk (HR), intermediate-risk (IR), or low-risk (LR) groups. This 3-tiered stratification method retained both the high specificity of the 2-year model and the high sensitivity of the 4-year model. Progression-free survivals differed significantly among the 3 risk groups, with p=0.0022 (HR vs. IR) and p,0.0001 (HR or IR vs. LR). Incremental value analyses demonstrated that the number of methylated genes contributed most influentially to prediction accuracy. Conclusions: This 3-tiered risk stratification strategy has the potential to exert a profound impact on Barrett’s esophagus surveillance accuracy and efficiency.