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Although the diagnostic and therapeutic modalities of esophageal squamous cell carcinoma (ESCC) have been improved considerably, the five-year survival rate is still not satisfied. To detect the numberial aberrations of the chromo-somes in ESCC, fluorescence in situ hybridization (FISH) was performed on interphase nuclei prepared from 220 esophag-eal carcinoma tissues with specific centromeric probes for chromosomes 3, 8, 10, 20 and Y. The main aberrations of the euchromosomes was chromosome gain, including trisome, tetrasome, and polysome. The gain rates of the four euchromo-some were 84.9%, 77.5%, 63.7% and 83.2%, and the frequencies of polysome for each euchromosome were 24.6%, 34.9%, 23.4% and 31.7%, respectively. Loss of chromosome Y was observed in 61.2% of male patients. The combination of the four chromosome probes 3, 8, 10 and 20 detected 74.5% of ESCC and the combination of 3, 8, 20 and Y detected 85.0%.These results indicated that both sets of the four centromeric probe combinations provide candidate biomarkers for the di-agnosis of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer in Asia, characterized by high incidence and mortality rate. Although significant progress has been made in surgery and adjuvant chemoradiotherapy, the prognosis of the patients with this cancer still remains poor. Investigation into protein alterations that occurred in tumors can provide clues to discover new biomarkers for improving diagnosis and guiding targeted therapy. Hundreds of papers have appeared over the past several decades concerning protein alterations in ESCC. This review summarizes all the dysregulated proteins investigated in the disease from 187 published papers and analyzes their contributions to tumor development and progression. We document protein alterations associated with tumor metastasis and the transition from normal esophageal epithelia to dysplasia in order to reveal the most useful markers for prediction of clinical outcome, early detection, and identification of high-risk patients for targeted therapies. In particular, we discuss the largest and most rigorous studies on prognostic implications of proteins in ESCC, in which cyclin D1, p53, E-cadherin and VEGF appeared to have the strongest evidence as independent predictors of patient outcome.

The gene of SKP2, located on chromosome 5p13, plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins including p27kip1, p21cip1, p57kip2, p130, cyclin E, and c-Myc. Previous studies in this laboratory revealed that gain of chromosome 5p was often seen in esophageal squamous cell carcinoma (ESCC). In the present study, we examined the amplification status and expression level of SKP2 in ESCC and investigated its clinicopathologic significance. Amplification and elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). The SKP2 protein expression level as determined by immunohistochemical staining showed a significant inverse correlation with p27 protein. In vivo assay showed that inhibition of SKP2 expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, knockdown of SKP2 by RNA interference inhibited cell migration and invasion ability. Knockdown of SKP2 expression sensitized cancer cells to anoikis, and a wobble mutant of SKP2 that is resistant to SKP2 small interfering RNA can rescue this effect. Expression level of pAkt decreased after SKP2 knockdown. Treatment of cells with phosphoinositidyl 3-kinase inhibitor (LY294002) and constitutively activator (insulin-like growth factor I) had significant effects on the anoikis of SKP2 RNA interference cells. These results show for the first time that SKP2 is amplified and overexpressed in ESCC. Elevated expression of SKP2 protected cancer cells from anoikis, and this effect was mediated, at least in part, by the phosphoinositidyl 3-kinase-Akt pathway. (Mol Cancer Res 2009;7(1):12–22)

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Background Esophageal cancer is one of the most common malignancies in the world. In order to identify the proteins associated with esophageal squamous cell carcinomas (ESCC), we analyzed the protein profiles of ESCC cases with tumor and matched adjacent normal tissues. Methods Two-dimensional electrophoresis (2-DE) was carried out to analyze the protein profiles. Dysregulated protein spots were identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) and verified by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC-ESI-IT MS). RT-PCR and immunohistochemistry on tissue microarray were performed to confirm the gene dysregulation in esophageal cancerous tissues. RNA interference (RNAi) was used to knock down the gene expression in ESCC cell lines. Apoptosis assay with annexin V-FITC/PI staining was conducted and cells were analyzed by flow cytometry. Results 2-DE showed that two protein spots with approximate molecular weights and different pI were elevated in 12 out of 18 ESCCs as compared to the corresponding normal tissues. Both the two spots were identified as MnSOD by MALDI-TOF and were verified by LC-ESI-IT MS. MnSOD overexpression was detected in 14 tumors out of 24 cases by RT-PCR and 52 tumors out of 116 cases by immunohistochemistry comparing to normal epithelia. siRNA-mediated silencing of MnSOD in KYSE450 and KYSE150 cell lines revealed that MnSOD protected ESCC cells from apoptosis induced by ultraviolet (UV) and doxorubicin (DOX). Conclusions These findings suggest that there existed two isoforms of MnSOD protein in normal and tumor esophageal tissues. MnSOD was overexpressed in ESCC and its up-regulation in esophageal cancer cells was associated with apoptosis resistance.

Esophagin/SPRR3 is one of the cornified-envelope structural precursor proteins, which is expressed during epithelia cell differentiation. In 1996, another research group discovered, and our own laboratory subsequently confirmed, frequent and dramatic decreased Esophagin/SPRR3 expression in esophageal squamous cell carcinoma (ESCC). However, the role of Esophagin/SPRR3 in tumorigenesis of esophageal epithelium remains undetermined. In this study, we demonstrate that expression of Esophagin/SPRR3 is frequently downregulated in ESCC. In contrast, no correlations between downregulation of Esophagin/SPRR3 expression and clinicopathologic characteristics were observed. Diminished Esophagin/SPRR3 expression was present in dysplastic epithelia, suggesting that Esophagin/SPRR3 alteration could represent an early event in squamous carcinogenesis of the esophagus. Exogenous expression of Esophagin/SPRR3 significantly suppressed the ability of ESCC cells to form colonies in plastic and soft agar, as well as tumor formation in vivo. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end label assay and immunofluorescence analysis of the active form of Caspase 3 indicated that dysregulated apoptosis might contribute to reduced tumorigenicity. In particular, upregulation of CDK11p46 protein was observed in ESCC cells expressing Esophagin/SPRR3, but not in control cells, indicating that Esophagin/SPRR3-induced apoptosis may be due, at least in part, to increased expression of CDK11p46 protein. These findings suggest that Esophagin/SPRR3 may play a role in the maintenance of normal esophageal epithelial homeostasis, and that aberrant expression of Esophagin/SPRR3 may contribute to the tumorigenesis of ESCC.

Sensitive and spatial exploration of the metabolism of tumors at the metabolome level is highly challenging. In this study, we developed an in situ metabolomics method based on ambient mass spectrometry imaging using air flow-assisted desorption electrospray ionization (AFADESI), which can spatially explore the alteration of global metabolites in tissues with high sensitivity. Using this method, we discovered potential histopathological diagnosis biomarkers (including lipids, amino acids, choline, peptides and carnitine) from 52 postoperative lung cancer tissue samples and then subsequently used these biomarkers to generate images for rapid and label-free histopathological diagnosis. These biomarkers were validated with a sensitivity and a specificity of 93.5% and 100%, respectively. Moreover, a single imaging analysis of a cryosection that visualized all these biomarkers, taking tens of minutes, revealed the type and subtype of the cancer. This method could potentially be used as a molecular pathological tool for rapid clinical lung cancer diagnosis and immediate image-guided surgery.

Cell invasion and migration significantly contribute to tumor metastasis. Microtubule-associated protein 4 (MAP4) protein is one member of microtubule-associate proteins family. It is responsible for stabilization of microtubules by modulation of microtubule dynamics. However, there is little information about the involvement of MAP4 in human cancer. Here we show that MAP4 serves as a regulator of invasion and migration in esophageal squamous cancer cells. By activating the ERK-c-Jun-vascular endothelial growth factor A signaling pathway, MAP4 promotes cell invasion and migration in vitro, tumor growth and metastasis in mouse models. Immunohistochemical staining of operative tissues indicated that MAP4 expression was associated with tumor stage, lymph node metastasis and shorter survival of the patients with esophageal squamous cell carcinoma (ESCC). Multivariate Cox regression analysis showed that MAP4 is an independent prognostic indicator. In the serial sections of ESCC tissues, there was a positive correlation between MAP4 and vascular endothelial growth factor A expression. Notably, an intratumoral injection of MAP4-small interfering RNA (siRNA) remarkably inhibited the growth of the tumors that formed by the MAP4-expressing ESCC cells in nude mice, and a combination of MAP4-siRNA and Bevacizumab significantly enhanced the inhibition effect. Our data suggest that MAP4 is probably a useful prognostic biomarker and a potential therapeutic target for the disease.

Calreticulin (CRT) is an endoplasmic reticulum Ca 2+ -binding protein that is involved in regulation of multiple cellular functions. We previously showed that CRT is over-expressed in esophageal squamous cell carcinoma (ESCC) and contributes to metastatic phenotypes through several pathways. Here we discuss the role of CRT in the progression of ESCC and the potentiality of CRT as a drug target and biomarker for ESCC.

Under some special conditions, the quality of the inquired image is low, and the image has blurred characteristic, in order to improve the quality of the blurred image, this paper proposes a blurred image enhancement algorithm based on improved multi-scale Retinex. The shortcomings of the traditional methods are analyzed, by the idea of constraining weighted average to optimize traditional algorithm, and enhance detail information of blurred image, and multiple blurred image are utilized on the algorithm performance simulation test. The simulation results show that the blurred image enhancement algorithm of improved multi-scale Retinex, solves the problem of the traditional Retinex algorithm, speed up the running speed of the blurred image enhancement, makes the blurred image clearer, and obtain better visual effect. Introduction In the blurred conditions for image shooting, due to the impact of atmospheric scattering effects, the color and contrast of captured image degenerate, image quality is lower, adversely affect is produced to the outdoor video surveillance system, so the blurred image have to be processed, so as to improve the quality of the image, which has very important significance [1]. The main purpose of blurred image enhancement is to improve the image contrast and saturation, and keep chroma unchanged. The blurred image enhancement algorithm is divided into physical methods and image processing method currently [2]. The physical method requires the prior knowledge of sunny image at same scene, and some physical device, which is not convenient in practical applications [3]. Therefore, independence on the physical device, has become the main research direction of blurred image enhancement, the common methods include: histogram equalization, wavelet transform, homomorphic filtering and Retinex algorithm [4-7]. The histogram equalization method by increasing the detail contrast of the image, to improve the quality of the image, but the original image color is distorted; homomorphic filtering method can have better processing for the image of not uniform illumination, but has poor effect on blurred image [8]. The Retinex theory of color constancy proposed by Edwin Land, can enhance the contrast of the image, and provide a new enhancement method to solve the blurred image quality problems [9]. However, a large number of studies show that, the image output by Retinex theory have some defects, like halo and over enhancement, the complexity of the algorithm is high, and can lead to RGB color offset to gray [10]. Then, some scholars have put forward a series of Retinex image improved enhancement algorithm, to some extent improve the blurred image quality, the enhancement effect is superior to the traditional Retinex algorithm, but the algorithm complexity is much higher [11]. Zhao Quanyou etc. obtained better multi-scale enhancement effect with the nonlinear Sigmoid, but it can only enhance dark region, and also not suitable for blurred image enhancement [12]. The traditional Retinex image enhancement algorithm and defects In the traditional method, dark channel enhancement method is utilized for a scene image shoot under the fog environment, but the enhancement effect is not good. Therefore, this paper proposes International Conference on Automation, Mechanical Control and Computational Engineering (AMCCE 2015) © 2015. The authors Published by Atlantis Press 1221 blurred video image enhancement algorithm of improved multi-scale weighted average Retinex algorithm. The multi-scale weighted average Retinex algorithm is to determine a preferred enhanced block from many blocks need to be enhanced with regarding the edge pixels as the center, the image texture structure information in unit time is ( ) , ; G x y t , intuitionistic blurred sets of image texture subspace are defined as conduction function: 0 ( ) ( , ) ( , ) lim[ ] x u u u u x t p x t x x σ σ ∆ → − + ∆ ∂ = = − ∆ ∂ (1) Adopting multi-scale weighted average Retinex algorithm to process adaptive enhancement for gray pixels value of image, as: 2

Gain of chromosome 11q13 is a common event in esophageal squamous cell carcinoma (ESCC). The cortactin gene (CTTN, also EMS1), located at 11q13, plays a pivotal role in coupling membrane dynamics to cortical actin assembly. This gene has been implicated in the motility of several types of cells. In the present study, we found that the amplification and overexpression of the CTTN gene was associated with lymph node metastasis in ESCC. Functional analysis by small interfering RNA–mediated silencing of CTTN revealed that in addition to the effect on cell migration, CTTN influenced cell invasiveness by anoikis resistance. In vivo assay showed that inhibition of CTTN expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, we showed for the first time that the protective role of CTTN in anoikis resistance was correlated with the activation of the phosphatidylinositol 3-kinase/Akt pathway. Overall, the data suggest that CTTN is an oncogene in the 11q13 amplicon and exerts functions on tumor metastasis in ESCC. (Cancer Res 2006; 66(24): 11690-9)

The genetic bases underlying esophageal tumorigenesis are poorly understood. Our previous studies have shown that coordinated deletion of the Smad4 and PTEN genes results in accelerated hair loss and skin tumor formation in mice. Herein, we exemplify that the concomitant inactivation of Smad4 and PTEN accelerates spontaneous forestomach carcinogenesis at complete penetrance during the first 2 months of age. All of the forestomach tumors were invasive squamous cell carcinomas (SCCs), which recapitulated the natural history and pathologic features of human esophageal SCCs. A small population of the SCC lesions was accompanied by adenocarcinomas at the adjacent submucosa region in the double mutant mice. The rapid progression of forestomach tumor formation in the Smad4 and PTEN double knockout mice corresponded to a dramatic increase in esophageal and forestomach epithelial proliferation. The decreased expression of p27, p21, and p16 together with the overexpression of cyclin D1 contributed cooperatively to the accelerated forestomach tumorigenesis in the double mutant mice. Our results point strongly to the crucial relevance of synergy between Smad4 and PTEN to suppress forestomach tumorigenesis through the cooperative induction of cell cycle inhibitors. (Cancer Res 2006; 66(14): 6972-81)

P53 codon 72 polymorphisms have been reported to be associated with cancers of the lung, esophagus and cervix. However, there have been no reports on the interaction of select risk factors and p53 codon 72 polymorphisms in gastric cancer susceptibility. 155 gastric cancer cases and 134 cancer-free controls were enrolled at the Memorial Sloan Kettering Cancer Center (MSKCC) from November 1992 to November 1994. The crude odds ratio (OR1) associated with the (Pro/Pro) polymorphism and the risk of gastric cancer was 1.27 (0.70–2.33). Adjusting for age, sex, race and education (OR2) and further adjusting for BMI, calories, sodium, smoking, vitamin C, fiber, alcohol, fat, and H. pylori status (OR3) did not yield significant results. Significant joint effects were associated with high fat consumption (OR1 = 2.61 (95% CI:1.13–6.06); OR2 = 2.85 (95% CI:1.14–7.15) for total cancers and for proximal tumors (OR1 = 2.56 (95%CI:1.00–6.54)). The low vitamin C intake/high-risk polymorphism group (Pro/Pro) had an OR1 of 4.82 (95% CI: 1.72–13.45) and the OR2 was 6.19 (95% CI: 2.08–18.40) for distal tumors. The point estimates were increased for interaction odds ratios but not statistically significant (OR1 = 4.25 (95% CI: 0.66–27.50); OR2 = 4.73 (95% CI: 0.67–33.43); OR3 = 5.55 (95% CI: 0.66–46.47)). Further studies specifically looking at proximal and distal tumors are required to confirm any potential interaction between the p53 codon 72 polymorphisms an environmental risk, in particular low dietary vitamin C and high fat consumption.

Allelic loss on chromosome 3p occurs frequently in esophageal cancer. The human ribosomal protein L14 gene (RPL14) is located on chromosome 3p21.3. In the present study, we investigated alteration of RPL14 at both the genomic DNA and RNA levels in 129 Chinese esophageal squamous cell carcinomas (ESCC) and 17 dysplasia adjacent to tumor tissues by a combination of tissue microdissection, microsatellite analysis of the intragenic marker, reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing. In the tested informative cases, loss of heterozygosity (LOH) of RPL14 was observed in 29 out of 68 (43%) tumors. Decreased expression of the gene was detected in 31 out of 49 (63%) carcinomas. No mutation was found in the remaining RPL14 allele of the tumors with LOH. We examined subsequently the allelic status of RPL14 in the dysplasia (preneoplastic lesions) between malignant tissues and histologically normal epithelia. Of 17 tested dysplasia in which the tumors showed LOH, eight (47%) displayed the same allelic loss as their corresponding tumors, seven (41%) exhibited microsatellite instability (MSI), and only two retained both the RPL14 alleles. The data suggest that alteration of RPL14 occurred frequently in ESCC and might be an earlier event in the tumorigenesis of the esophagus. Analysis to RPL14 gene may contribute to the early detection of ESCC as a potential molecular marker.

Our previous work has shown that a number of genes locating on 1q21 were down-regulated in esophageal squamous cell carcinomas (ESCC) and they may involve in carcinogenesis. To determine whether chromosome 1q LOH occurs in ESCC, we analyzed LOH in 61 ESCCs using 18 microsatellite markers on chromosome 1q. Forty-six of 61 (75.4%) tumors presented LOH at one or more loci. A significant association was found between chromosome 1q LOH and histopathological grade. LOH on D1S3466 had a negative correlation with family history, whereas LOH on D1S2777 positively correlated with smoking. These results suggest this region harbor putative tumor suppressor gene(s) contributing to tumorigenesis and differentiation in ESCC.

Gadd45a is an important player in cell cycle G2-M arrest in response to genotoxic stress. However, the underlying mechanism(s) by which Gadd45a exerts its role in the control of cell cycle progression remains to be further defined. Gadd45a interacts with Cdc2, dissociates the Cdc2-cyclin B1 complex, alters cyclin B1 nuclear localization, and thus inhibits the activity of Cdc2/cyclin B1 kinase. These observations indicate that Gadd45a nuclear translocation is closely associated with its role in cell cycle G2-M arrest. Gadd45a has been characterized as a nuclear protein, but it does not contain a classical nuclear localization signal, suggesting that Gadd45a nuclear translocation might be mediated through different nuclear import machinery. Here we show that Gadd45a associates directly with B23 (nucleophosmin), and the B23-interacting domain is mapped at the central region (61-100 amino acids) of the Gadd45a protein using a series of Myc tag-Gadd45a deletion mutants. Deletion of this central region disrupts Gadd45a association with B23 and abolishes Gadd45a nuclear translocation. Suppression of endogenous B23 through a short interfering RNA approach disrupts Gadd45a nuclear translocation and results in impaired Gadd45a-induced cell cycle G2-M arrest. These findings demonstrate a novel association of B23 and Gadd45a and implicate B23 as an important regulator in Gadd45a nuclear import.

Migration inhibitory factor-related protein 14 (MRP14) is one of calcium-binding proteins, referred as S100A9. The heterodimeric molecule formed by MRP14 with its partner MRP8 (S100A8) is the major fatty acid carrier in neutrophils. The MRP8/14 complex has been also implicated in the intracellular transport of arachidonic acid and its precursors in keratinocytes. We show here the involvement of MRP14 in human esophageal cancer. In an initial study, mRNA differential display-reverse transcription polymerase chain reaction (DD-PCR) was performed with two esophageal carcinomas, one esophageal adenocarcinoma and matched normal adjacent mucosa. DD-PCR with the arbitrary primer OPA3 showed that one cDNA band was highly expressed in normal tissues, but disappeared or substantially decreased in tumor counterparts. It was later identified to be the 3'-end of migration inhibitory factor-related protein 14 (MRP14). Northern blotting, RT-PCR and Western blotting corroborated the down-regulation of MRP14 in 58/64 squamous cell carcinomas and 2/2 adenocarcinomas as compared with adjacent normal epithelia of the esophagus. MRP14 was undetectable in 3/3 esophageal-carcinoma cell lines. Immunochemistry demonstrated that expression of MRP14 was restricted to normal esophageal epithelia. No mutation was found in the genomic DNA of the MRP14 gene by PCR and directed DNA sequencing. Our finding suggested that the reduction of MRP14 expression is a frequent event in Chinese human esophageal cancer.

Allelic loss on chromosome 13 occurs frequently in esophageal squamous cell carcinomas. To define minimal deletion intervals and find candidate tumor suppressor gene(s), we conducted a study of loss of heterozygosity (LOH) in 59 esophageal squamous cell carcinomas from northern China using a panel of ten microsatellite markers on chromosome 13q. The results showed that 12 of 59 (20%) cases presented allelic loss in three or more consecutive adjacent chosen markers, suggesting loss of larger fragments on chromosome 13q. Two minimal deletion regions of overlap were found: one was located on band 13q12.3 from markers D13S171 to D13S267, and the other on band 13q14.1-q14.3 from markers D13S263 to D13S168. The latter was a new deletion region that was never reported in esophageal squamous cell carcinomas. More frequent LOH was observed in higher pathological grade of esophageal cancer at loci D13S171, D13S263 and in later stage of esophageal cancer at D13S263. The data suggest the possibility that one or more unknown tumor suppressor gene(s) on 13q may play an important role in the development of esophageal squamous cell carcinomas.

Purpose: We previously revealed that the expression of Annexin A2 (ANXA2) was increased in ESCC by two-dimensional electrophoresis and MALDI-TOF and LC-ESI-IT MS. This study aims to investigated the implication of Annexin A2 in ESCC. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription. Experimental Design: Immunohistochemistry was performed to analyze the expression of ANXA2 in esophageal squamous cell carcinomas (ESCC). siRNA knockdown, transwell assay and wound-healing assays were used to investigate the function of ANXA2 in cells. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis were used to seek the potential effector molecule of ANXA2. Chromatin immunoprecipitation, and reporter gene assays were used to confirm the relationship between c-Myc and HIF-1 alpha. Co-immunoprecipitation assay was performed to demonstrate the relationship between ANXA2 and c-Myc. Immunofluorescence was used to reveal the location of ANXA2. Results: Knockdown of Annexin A2 significantly inhibited the migration and invasion of ESCC cells. At the molecular level, VEGF, MMP-2, MMP-9, p-MEK and p-ERK were down-regulated in ANXA2-siRNA cells. The HIF-1 alpha mRNA and protein levels were significantly lowered after knocked down the Annexin A2. HIF-1 alpha is the effector molecule of ANXA2 in ESCC cells, and that ANXA2 affects the expression of HIF-1 alpha at the transcription level because when knocking down HIF-1 alpha, the level of VEGF, MMP-2, MMP-9, p-MEK and p-ERK reduced as well. c-Myc is a transcription factor for HIF-1 alpha. ANXA2 has a direct protein interaction with c-Myc and knockdown of ANXA2 down-regulated the expression of c-Myc. The Tyr 24 phosphorylation form of Annexin A2 (Y24D-ANXA2) was located in the nucleus, and the non-phosphorylation form (Y24A-ANXA2), in the cytoplasm and cell membrane. Transfection of pcDNA3.1-ANXA2-Y24D enhanced the migration and invasion as compared with that of pcDNA3.1-ANXA2-Y24A in esophageal cancer cells. A treatment with the Annexin A2 phosphorylation inhibitor, Dasatinib, changed the location of Annexin A2 and lowered the expression of c-myc and HIF-1 alpha. Conclusions: These results suggest that in esophageal cancer cells, phosphorylated Annexin A2 could enter into nucleus, which stabilizes c-myc and promotes the transcription of HIF-1 alpha, and the activates MAPK pathway, increases matrix metalloproteinase (MMP) activity and vascular endothelial growth factor (VEGF) expression, which results in the enhancement of cell invasion and migration. Citation Format: Sai Ma, Yan-Yi Jiang, Li-Fei Wu, Jia-Jie Hao, Yu Zhang, Xin Xu, Yan Cai, Ming-Rong Wang. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 891. doi:10.1158/1538-7445.AM2017-891

Platinum resistance is a critical barrier for clinicians to improve the survival of ovarian cancer. Our study evaluated the correlation between copy number variations (CNVs) of neurotrophic tyrosine receptor kinase 3 (NTRK3) and the prognosis of ovarian cancer, which might predict platinum resistance in ovarian cancer patients. Array comparative genomic hybridization (CGH) was used to test gene backgrounds between platinum-sensitive and platinum-resistant relapsed populations and CNVs of NTRK3 were indicated by cluster analysis. Fluorescence in situ hybridization (FISH) was adopted in 41 cases for further verification, which confirmed the results of array CGH. Spearman's rank correlation analysis and χ2 test were used to evaluate the accuracy of CNVs of NTRK3 which predicted platinum-sensitive or platinum-resistant recurrence. We detected CNVs of NTRK3 between 2 groups by array CGH, and amplification of NTRK3 was confirmed by FISH in the platinum-sensitive recurrence group with enlarged samples. The test concordance of 2 methods was 78.6%. Among 41 cases with satisfied FISH results, the median time to recurrence (TTR) of patients with amplified and nonamplified NTRK3 were respectively 18 and 5 months (P

BACKGROUND: We sought to investigate the role of the Bcl-2 translocation at the chromosomal and protein levels in minimal bone marrow (BM) infiltration by diffuse large B-cell lymphomas (DLBCL). METHODS: The presence of the Bcl-2/IgH fusion gene was detected in BM samples and paraffin-embedded lymph node (LN) samples from 103 patients with DLBCLs using FISH. Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using immunocytochemistry (ICC) and immunohistochemistry (IHC), respectively. RESULTS: Bcl-2/IgH translocation in paraffin-embedded LN tissue sections was observed in 43 (41.7%) patients by FISH. Of the 43 patients, the Bcl-2/IgH rearrangement in the bone marrow specimens occurred in 34 patients. The Bcl-2/IgH recombination rate in stage III cancers was not significantly different compared to the rate observed in stage I/II cancers (p = 0.101), respectively showing no statistical differences between stage IV and I/II (p = 0.179). In 64.7% (22/34) of the cases with t(14;18), Bcl-2 was detected based on ICC analysis. Positive Bcl-2 ICC staining and the t(14;18) translocation were positively correlated (p < 0.001). We then applied our FISH method to slides with at least one abnormal cell and were subjected to FISH analysis after staining. During the follow-up, no infiltration by cytomorphology for 16 DLBCL patients whose bone marrow presented Bcl-2/IgH gene rearrangement at diagnosis, and two cases were positive by morphology compared to FISH-positive results 6 months later; 9 out of 16 patients (56.3%) presented with positive Bcl-2/IgH results earlier than the morphology evaluation after 12 months. CONCLUSIONS: Utilizing both FISH and cytologic morphology, the assessment of Bcl-2/IgH translocation status could contribute to the better detection of minimal bone marrow infiltration and relapse receiving treatment by DLBCL cells.

Esophageal squamous cell carcinoma (ESCC) is a world-wide prevalent cancer, which is particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified novel significantly mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to previously discovered ones (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Moreover, our approaches uncovered many novel druggable candidates, and XPO1 was further explored as a therapeutic target because of its mutation and protein overexpression. Together, our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

To date, no robust biomarkers have been available in clinical practice that can provide an early diagnostic evaluation of lung cancer. The objective of this study was to identify potential biomarkers for the early detection of lung cancer using bronchial brushing specimens.Immunocytochemistry was used to investigate the expression of 35 proteins in 880 bronchial brushing specimens from both outpatients and inpatients who had either lung cancer or benign lung lesions. An optimal panel was identified that had high sensitivity and considerable specificity for detecting lung cancer. Associations between protein expression and clinicopathologic parameters were assessed.Tumor protein 53 (TP53), TP63, Ki67, epidermal growth factor receptor (EGFR), minichromosome maintenance complex component 6 (MCM6), MCM7, uncharacterized proteins KIAA1522 and KIAA0317, and ubiquitin-protein ligase UHR1 (ICBP90) frequently presented high expression in bronchial brushing specimens from patients who had lung cancer compared with patients who had benign lung lesions. A 6-protein panel consisting of TP53, Ki67, MCM6, MCM7, KIAA1522, and KIAA0317 was identified as the best combination, with sensitivity of 81.1% (309 of 381 specimens) for detecting nonsmall cell lung cancer (NSCLC) and 86.8% (145 of 167 specimens) for detecting small cell lung cancer (SCLC) (specificity, 83.3%; 65 of 78 specimens). The combination of cytology and the protein panel significantly improved the sensitivity of bronchial brushing examination for detecting lung cancer (P.00001), which increased from 49.1% to 81% in early stage NSCLC (stage I and II). In combined analyses, the protein panel was positively associated with patient sex (P=.00033), tumor type (P.00001), tumor location (P.00001), and lymph node metastasis (P=.028).The 6-protein panel is a potential biomarker for the early detection of lung cancer in bronchial brushings.

For advanced epithelial ovarian cancer (EOC), time to recurrence (TTR) is an important indicator to gauge the therapeutic efficacy of postoperative adjuvant chemotherapy. Our objective was to determine the genes that could potentially distinguish patients with short versus long TTR after initial administration of platinum-paclitaxel combination chemotherapy in advanced EOC. Tumor samples of 159 patients were obtained during the primary cytoreduction. Array comparative genomic hybridization (CGH) was carried with genomic DNA from 17 EOC samples (8 with TTR > 15 months and 9 with TTR ≤ 6 months) to screen candidate gene set, copy-number changes (CNC) of which were significantly different between early and late relapse cases. Seventeen candidate genes were identified by array CGH. The analysis of consistency between real-time PCR and array CGH revealed that 4 genes displayed consistent results, namely GSTT1, ISG20L1, STARD5 and FREM1. In a 142-case validation set, CNC of 4 candidate genes was evaluated and verified by real-time PCR. Sixty five point five percent of the patients were correctly divided into early (TTR ≤ 10 months) and late (TTR > 10 months) recurrent group by CNC of the 4 genes using discriminant analysis. The results showed that CNC of 4-gene set could potentially determine early (TTR ≤ 10 months) or late relapse (TTR > 10 months) after initial platinum-paclitaxel combination chemotherapy in advanced EOC.

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

Esophageal squamous cell carcinoma (ESCC) is a common cancer type in China. In this study, we aimed to develop aneuploidy markers for diagnosis and prognosis of ESCC. Chromosomal aneuploidies were detected in 493 primary tumors and 61 precancerous lesions by fluorescence in situ hybridization with chromosome enumeration probes (CEP), and cut-off values were set by receiver operating characteristic (ROC) curves. According to the cut-off values, chromosomes 3, 8, 10, 12, 17 and 20 presented frequent gains, with rates of 70.1, 69.7, 58.9, 66.9, 67.5 and 77.2 % in tumors and of 32.1, 26.8, 33.9, 41.2, 44.0 and 42.0 % in precancerous lesions. Loss of chromosome Y was detected in 72.0 % of male patients. An optimal four-probe panel CEP3/12/17/20 was established for detecting ESCC (sensitivity: 86.1 %), and CEP3/10/12/20 for precancerous lesions (sensitivity: 48.0 %). Gain of CEP8 was significantly correlated with lymph node metastasis (LNM) and late stages (P = 0.002 and 0.001), and loss of CEPY with age (P = 0.002, male). Kaplan–Meier survival curves indicated that patients with positive CEP10/17 (pT1 + T2, P = 0.041) and CEP8/17 (stages IIb + III + IV, P = 0.002) had poor overall survival. Combinations of LNM/stage and CEP panels could divide patients into more subgroups, including LNM + CEP3/17, LNM + CEP10/17, LNM + CEP3/10/17, stage + CEP3/17, stage + CEP10/17 and stage + CEP3/10/17 (P = 0.0004, 0.0003, 0.0001, 0.005, 0.001 and 0.0008, respectively). Multivariate Cox regression analysis confirmed that the above combinational models were independent prognostic factors. Our data suggest that the combinational probe sets may have potential for detection and prognostic prediction of ESCC.

BACKGROUND: Genomic aberration is a common feature of human cancers and also is one of the basic mechanisms that lead to overexpression of oncogenes and underexpression of tumor suppressor genes. Our study aims to identify frequent genomic changes in pancreatic cancer. MATERIALS AND METHODS: We used array comparative genomic hybridization (array CGH) to identify recurrent genomic alterations and validated the protein expression of selected genes by immunohistochemistry. RESULTS: Sixteen gains and thirty-two losses occurred in more than 30% and 60% of the tumors, respectively. High-level amplifications at 7q21.3-q22.1 and 19q13.2 and homozygous deletions at 1p33-p32.3, 1p22.1, 1q22, 3q27.2, 6p22.3, 6p21.31, 12q13.2, 17p13.2, 17q21.31 and 22q13.1 were identified. Especially, amplification of AKT2 was detected in two carcinomas and homozygous deletion of CDKN2C in other two cases. In 15 independent validation samples, we found that AKT2 (19q13.2) and MCM7 (7q22.1) were amplified in 6 and 9 cases, and CAMTA2 (17p13.2) and PFN1 (17p13.2) were homozygously deleted in 3 and 1 cases. AKT2 and MCM7 were overexpressed, and CAMTA2 and PFN1 were underexpressed in pancreatic cancer tissues than in morphologically normal operative margin tissues. Both GISTIC and Genomic Workbench software identified 22q13.1 containing APOBEC3A and APOBEC3B as the only homozygous deletion region. And the expression levels of APOBEC3A and APOBEC3B were significantly lower in tumor tissues than in morphologically normal operative margin tissues. Further validation showed that overexpression of PSCA was significantly associated with lymph node metastasis, and overexpression of HMGA2 was significantly associated with invasive depth of pancreatic cancer. CONCLUSION: These recurrent genomic changes may be useful for revealing the mechanism of pancreatic carcinogenesis and providing candidate biomarkers.

Objectives: In a population-based case–control study in Yangzhong, China, we investigated the relationship between genetic polymorphisms of GSTP1 and susceptibility to gastric cancer and its premalignant lesion, chronic gastritis. The possible gene–gene interactions between GSTP1 polymorphisms and GSTM1, GSTT1 genes were explored. Methods: Epidemiologic data were collected by standard questionnaire from 133 gastric cancer cases, 166 chronic gastritis cases, and 433 cancer-free population controls. Blood samples for Helicobacter pylori and molecular marker assays were collected from 84 gastric cancer cases, 146 chronic gastritis, and 429 population controls. GSTP1 polymorphisms were determined by the PCR-RFLP method and H. pylori infection was measured by the ELISA method. Associations between certain GSTP1 genotypes and both gastric cancer and chronic gastritis were assessed by odds ratios (ORs) and 95% confidence intervals (CIs) derived from logistic regression. Results: The distributions of three GSTP1 genotypes, Ile/Ile, Ile/Val, and Val/Val, were similar in gastric cancer cases, chronic gastritis, and controls. After adjusting for age, gender, education, body mass index, pack-year of smoking, alcohol drinking, H. pylori infection, salt and fruit intakes, the adjusted ORs of Val/Val were 1.3 (95% CI: 0.1–11.2) for gastric cancer and 0.9 (95% CI: 0.2–4.8) for chronic gastritis. Combining the Val alleles (Val/Val and Ile/Val) into one group, no association was observed between GSTP1 and both gastric cancer and chronic gastritis. In addition, the allelism at the GSTP1 locus did not increase gastric cancer and chronic gastritis risks associated with the GSTM1 or GSTT1 genotypes. Conclusion: Our data suggest that the GSTP1 genotype seems not to be associated with the risk of gastric cancer and chronic gastritis in a high-risk Chinese population.

DNAJB6 is a member of the heat shock protein 40 (Hsp40) family. We here investigated the clinical correlation and biological role of DNAJB6 overexpression in colorectal cancer (CRC). The expression of DNAJB6 protein was examined in 200 cases of colorectal adenocarcinomas by immunohistochemistry (IHC) technology. Gene transfection and RNA interference were performed to determine the effect of DNAJB6 expression on the invasion of CRC cells and to explore the underlying molecular mechanisms in vitro and in vivo. Overexpression of DNAJB6 was found in 39% (78/200) of the CRC tissues, especially in tumors at pT4 as compared with at pT1-3 (P = 0.02). A Kaplan-Meier survival analysis revealed a correlation between DNAJB6 expression and overall survival (OS) times (P = 0.003). Multivariate analysis confirmed that DNAJB6 overexpression was an independent prognostic factor for CRC (P = 0.002). RNA interference-mediated silencing of the DNAJB6 gene inhibited the invasion of CRC cells in vitro were accompanied by a significant reduction in the protein levels of IQ-domain GTPase-activating protein 1 (IQGAP1) and phosphorylated ERK (pERK). An in vivo assay showed that inhibition of DNAJB6 expression decreased the lung metastases of CRC cells. IHC analysis of serial sections showed that there was a positive correlation between DNAJB6 and IQGAP1 expression in primary CRC tissues (P = 0.013). The data suggest that DNAJB6 plays an important oncogenic role in CRC cell invasion by up-regulating IQGAP1 and activating the ERK signaling pathway and that DNAJB6 may be used as a prognostic marker for CRC.

To validate the feasibility for detecting EGFR and k-ras mutations using cytological specimens.Cytological specimens including fine-needle aspiration (FNA), pleural effusion (PLE) and fiberoptic bronchoscopic (FOB) brushing were collected from patients with non-small cell lung cancer (NSCLC ) from January 2011 to July 2011 at the Department of Cytology, Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS). Polymerase chain reaction (PCR) was carried out to amplify EGFR exons 18-21 and k-ras codons 12-13, and then the PCR products sequencing and analysis were performed.Fifty cytological specimens were collected including 19 cases of FOB, 9 cases of FNA, 22 cases of PLE. Of them DNA was successfully extracted in 43 cases, and specific PCR amplification products sequencing were performed in 42 cases. EGFR mutations were detected in 14 of 42 specimens (33.3%), the frequencies of EGFR mutations in exons 19, 20 and 21 were 16.7% (7/42), 4.8% (2/42) and 11.9% (5/42), respectively, and no mutation was found in exon 18. Higher frequencies of EGFR mutations were detected in exons 19 and 21 (85.7%). Mutations were identified in 38.7% (12/31) cases of adenocarcinoma. K-ras mutations were found in 2 of 42 specimens (4.8%). EGFR and K-ras mutations were not found in the same case.Cytological specimens are feasible for detecting EGFR and K-ras mutation. This is especially beneficial in patients in whom histological materials can not be obtained.

To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed.Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012).In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

Background At present no objective parameters to identify the risk of liver metastasis after surgery have been established in rectal cancer. Objective To identify the chromosomal aberrations that are correlated with liver metastasis of rectal cancer. Methods Primary tumor tissues of rectal carcinoma were analyzed by array-based comparative genomic hybridization (array-CGH). Genomic aberrations were identified by Genomic Workbench and MD-SeeGH. Results The most frequent gains in rectal cancer were at 20q11.21-q13.33, 8q11.21-q24.3, 13q12.11-q14.2 and losses in 5q13.2, 8p23.3-p22, 17p13.3-p13.2 and 18q11.2-q23. Seven amplifications at 6p21.1, 8q24.21, 8q24.3, 13q13.2 and 20q13.2-q13.32 and nine homozygous deletions at 1q31.3, 4q12-q13.1, 4q32.3-q33, 5q13.2, 8p23.2, 8q11.23, 16p13.2, 19p13.11 and 19q13.41 were identified. Both frequency plot comparison and SAM (Significance analysis of microarray) methods indicated that losses at 1p35.3, 4p14, 14q23.1-q32.11 and 18p11.32-p11.21 were more frequent in patients without liver metastasis. Conclusions These liver metastasis associated genomic changes may be useful to reveal the mechanism of metastasis and identify candidate biomarkers.

We have identified a novel human gene, designated C1orf10, using modified differential display PCR. The C1orf10 gene, which spans 5 kb in length, is composed of three exons. The deduced protein contains 495 amino acids with one transmembrane domain. The amino acid sequence of C1orf10 is characterized by the presence of a calcium-binding motif of about 90 amino acids at its N-terminal and a conserved consecutive repeat sequence of 60 amino acids that was identified previously only in bacterial ice nucleation proteins. In normal adult tissues, C1orf10 is highly expressed only in the esophagus and was undetectable in a total of 15 other tissues examined, suggesting its important role in esophageal cells. The expression of C1orf10 is either dramatically reduced or absent in esophageal cancer cell lines (3/3) as well as primary esophageal cancer tissues (35/37) compared with the corresponding normal esophageal mucosa. Using a radiation hybrid panel, C1orf10 was found to be located on chromosome 1q21. These findings suggest that expression of C1orf10 is unique to esophageal cells and that loss of its expression may play a role in the development of esophageal cancer.

Transglutaminase-3 (TGase-3) is an enzyme with the ability to catalyze the irreversible cross-linking of peptide-bound glutamine residues either with peptide-bound lysines or with primary amines. It has been implicated in the formation and assembly of the cornified cell envelope of the epidermis, hair follicle and perhaps other stratified squamous epithelia. We show here the involvement of TGase-3 in human esophageal cancer. In an initial study, mRNA differential display was performed with 3 pairs of esophageal cancer tissues and matched normal adjacent mucosa by a 10-mer arbitrary primer and mixed anchored primers (GT15N, N = A, C and G). Four differentially expressed cDNA bands were consistently observed in all 3 normal tissues but barely detected in their tumor counterparts. One of them was identified to be the 3; end of TGase-3. Northern blot and dot blot analyses of 14 samples confirmed the down-regulation of TGase-3 in malignant tissues compared with normal epithelia. RT-PCR revealed that TGase-3 expression was lost in 3 esophageal carcinoma cell lines and decreased in 35/38 tumors compared with adjacent normal mucosa. Taken together, 49/52 (94.2%) esophageal tumors presented down-regulation of the gene. Our data suggest that alteration of TGase-3 expression is a common event in the development of human esophageal cancer.

CTTN (EMS1) : an oncogene contributing to the metastasis of esophageal squamous cell carcinoma

Calreticulin (CRT) is a Ca2+-binding chaperone protein that alters cellular Ca2+-homeostasis in the endoplasmic reticulum (ER). Previously it was shown that CRT was overexpressed in esophageal squamous cell carcinoma (ESCC), and elevated CRT expression promoted the migration and invasion of ESCC cells. In the present study, the mechanisms underlying the role of CRT in esophageal carcinoma progression were investigated. Critically, depletion of CRT or protein-tyrosine phosphatase 1B (PTP1B) reduced ESCC cell migration and metastasis to the lung, whereas restoration of PTP1B protein levels rescued cell migration in CRT-silenced cells. Knockdown of CRT decreased PTP1B protein expression by reducing phosphorylation at the Y694 site of STAT5A, whereas knockdown of PTP1B reduced ERK1/2 phosphorylation at T204. Immunohistochemical analysis of CRT and PTP1B expression in ESCC patient tissues was strongly correlated. Importantly, PTP1B expression was associated with poor survival in patients with CRT overexpression. Overall, these data indicate a novel signaling pathway connecting CRT, STAT5A, PTP1B, and ERK1/2 in the regulation of ESCC cell migration. Implications: These findings suggest that PTP1B is a downstream effector of CRT signaling, promotes tumor progression, and can potentially be used as a new drug target for ESCC. Mol Cancer Res; 11(9); 986–94. ©2013 AACR.

The aim of this study is to evaluate the diagnostic values of the fluorescence in situ hybridization (FISH), NMP22 BladderChek, and liquid-based cytology (LBC) in the detection of bladder urothelial carcinoma (UC). Consecutive voided urine samples were collected from 138 in-house patients with a variety of urologic conditions and 37 healthy individuals as negative controls. FISH, NMP22 BladderChek, and LBC were performed on the specimens. All three tests were evaluated independently in a blinded fashion. In all, 104 out of the 175 patients enrolled in this study had histologically proven UC. LBC, FISH, and NMP22 BladderChek were successfully performed on 175, 149, and 119 cases, respectively. The three tests revealed overall sensitivities of 73.1%, 86.5%, and 67.6%, respectively. FISH was more sensitive than LBC (P=0.022) and NMP22 BladderChek (P=0.004). Combination of all the tests yielded a superior sensitivity of 96.7% compared with LBC (P

Although the majority of spontaneous tumors derive from a single cell, people have come to realize intra-tumor heterogeneity of individual tumors. Human cancers frequently display substantial difference in phenotypic features, such as the degree of differentiation, cell proliferation rate, invasion and metastatic potential, response to therapy and many other aspects. Molecular biology studies have confirmed the occurrence of new mutations during the process of tumor progression, which provide more powerful evidences to show the existence of intra-tumor genetic heterogeneity. This re-view will focus on recent major advances in the study of tumor genetic heterogeneity. Considering that genetic heterogene-ity analysis can provide important information to indicate how long normal cells transform into tumor cells and how to spread and migrate, we firstly describe experimental evidences of intra-tumor genetic heterogeneity. Then we discuss the research value of genetic diversity in the evolutionary history of human individual tumor, introduce the two modes of the genetic heterogeneity - cancer stem cell model and the clonal evolution model, and summarize the implications of in-tra-tumor heterogeneity studies in metastasis and therapy. In addition, the article presents the research methods of genetic heterogeneity, including specific gene and genome-wide level, pointing out their strengths and limitations.

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

The aim of the present study was to screen and identify the chromosomal aberrations that are correlated with clinicopathological characteristics of rectal cancer using array-based comparative genomic hybridization (array-CGH). Forty-eight fresh frozen tumor tissues of rectal carcinoma were analyzed by array-CGH. The results showed that most frequent gains included 8q24.3, 20q11.21-q13.32, 20q13.33 and losses in 8p23.3-p12, 17p13.1-p12 and 18q11.2-q23 were noted. Fourteen amplifications and seven homozygous deletions were identified in the rectal cancer samples. Losses of 4p16.1-p15.31, 8p21.1-p12 and gains of 7p12.3-p12.1 and 13q33.1-q34 were associated with positive lymph node status and advanced clinical stage (stages III and IV). The 17q24.2-25.3 gain was more frequent in patients with distant metastasis. Integrated analysis indicated that overexpression of PDP1, TRIB1, C13orf27, FOXA2, PMEPA1 and PHACTR3 was associated with gains, and underexpression of FHOD, SMAD4 and BCL2 was associated with losses. Pathway enrichment analysis showed that pathways of nitrogen metabolism, oxidative phosphorylation, cell cycle, maturity onset diabetes of young, cytokine-cytokine receptor interaction, MAPK signaling pathway and dentatorubropallidoluysian atrophy were influenced by copy number changes.

A bladder carcinoma cell line, RT112/84, has been cytogenetically characterized by classical techniques and fluorescence in situ hybridization. The RHG banding and FISH analysis revealed a mixture of two clones and multiple chromosome rearrangements.

Background Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC). Methods Using multiplex-fluorescence in situ hybridization (M-FISH) and oligo array-based comparative hybridization (array-CGH), we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. Results M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134) of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM) (P = 0.004 and 0.022) and advanced stages (P = 0.004 and 0.039). Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026). Conclusions The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.

Atypical protein kinase Cι (PKCι) has been identified as an oncoprotein in esophageal squamous cell carcinomas. However, the mechanisms underlying the role of PKCι in this disease remain unclear. In the present work, we found that inhibition of PKCι expression by RNAi induced apoptosis via the down-regulation of β-catenin in esophageal cancer cells. Furthermore, we found that PKCι regulated β-catenin in an autophagy dependent way. Since down-regulation of β-catenin induced by knockdown of PKCι could be rescued by autophagy inhibition; knockdown of PKCι activated autophagy and promoted the recruitment of β-catenin into autophagosome. These results suggested that PKCι positively regulated β-catenin through negatively regulated autophagy and depletion of PKCι promoted apoptosis via autophagic degradation of β-catenin in esophageal cancer cells. These data provide new insights into PKCι signaling in human cancer.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies with poor prognosis in China. Patients with ESCC may present with vague symptoms in early stage and most of the cases are diagnosed at advanced stage, without the chance of optimal therapy. In the development and progression of ESCC, cytogenetic and molecular aberrations are frequently observed. This review is to summarize the advances in the chromosomal and genomic alterations of ESCC reported recently.

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.