Adjacent Mucosa Tissues Research Articles

Variability in HER2 expression between primary colorectal cancer and corresponding metastases.

This study aimed to investigate the HER2 status of primary colorectal cancer (CRC) and corresponding metastases, and determine the correlation between HER2 and clinicopathological characteristics. We collected the clinicopathological features of 98 CRC patients and 66 patients which were previously evaluated for the KRAS status. The tissue samples of primary CRC tumors (n = 98), noninvolved colorectal mucosa (n = 98), paired lymph nodes (n = 98, 69 patients had positive metastatic nodes), and liver metastases (n = 22) were evaluated using immunohistochemistry for HER2. The kappa test was used to evaluate the concordance rate of HER2 status. Survival analysis was established according to the Kaplan-Meier method. HER2 overexpression was more common in primary tumors among the younger patients (P < 0.05). No survival significance was revealed in the HER2 status. The HER2-positive rate was 11.2% for the primary CRC tumors, 0% for the normal adjacent mucosa tissues, 10.1% for the matched positive lymph nodes, and 31.8% for the corresponding metastasis. Seven of sixty-nine cases (10.1%) exhibited biomarker discordance in nodal metastases compared with primary tumors (κ = 0.48, P < 0.05); 6 out of 22 cases (27.3%) exhibited biomarker discrepancy in liver metastases compared with primary tumors (κ = 0.32, P > 0.05). Compared with lymph nodes, HER2 overexpression in the primary tissue was more common in KRAS wild-type patients (P < 0.05). The present study showed a high rate of discordance in matched pairs of primary tumors and metastases, suggesting that the accurate evaluation of HER2 status is essential before any therapeutic decision.

Abstract B39: Understanding the association of gut microbiota and tumor microenvironment in gastric and esophageal cancer

Abstract Gastric and esophageal cancers remain the second and the sixth leading cause of cancer related deaths respectively. Dysbiosis of the gut microbiome remains a strong risk factor for gastrointestinal cancer, likely related to altered mucosal immunity and a pro-inflammatory immune microenvironment. However, the molecular interactions between host human cells and the microbiome are poorly understood as it pertains to gastric and esophageal cancer. Our main goal is to understand how alteration of regional mucosal microbiome alters the tumor microenvironment, thus creating conditions conducive to tumorigenesis. Understanding these changes at the molecular level will help contribute to our knowledge of the disease pathophysiology. Using a novel customized computational pipeline to identify and characterize bacteria from low microbial content endoscopic samples (Zhang et al., Genome Biol 2015), we used low-pass (10-30x) whole genome sequencing (WGS) to examine the microbiome composition of 15 gastric cancer samples along with their adjacent non-cancer mucosal tissue (n=30). We found a higher overall bacterial content in tumor samples compared to that of their corresponding matching normal tissues. Furthermore, we observed a dysbiosis in samples positive for Helicobacter pylori, consistent with previous findings that H. pylori alters composition of the gastric microbiome. Although a wide variety of other microbes were detected, we found specific enrichment of organisms like Veilonella Parvula (12/15) and Prevotella melaninogenica (10/15) in tumor tissues, both of which have been implicated in tumorigenicity. A subset of these samples (10 gastric cancer and 12 adjacent normal) underwent parallel RNAseq analysis and using a gene expression signature panel of 176 genes compiled from several published series, we examined immune cell types, specifically B cells, CD8+ T cells, Treg cells, CD4+ Th1, Th2 and Th17 helper cells and macrophages, to determine an association with tumor versus the normal mucosa. We found an overall higher immune response, and more specifically a higher association of Th1 and Th2 helper cells as well as macrophages, with tumors as compared to normal mucosa. We further corroborated these findings using a single cell RNA sequencing approach on three tumor and matching normal samples. Using ELISA, we detected a significantly increased expression of pro-inflammatory cytokines such as TNFα, IL-8, GRO, MCP-1 and IL-1a in tumor samples than in normal mucosa. Increased expression of TNF-α and IL-8 has been directly correlated to tumor invasion and metastasis, and tumor vascularity respectively. Additionally, MCP-1 has been shown to be important in recruitment of macrophages and is secreted by gastric epithelial cells in response to pro-inflammatory cytokines upon H. pylori infection. We used a similar approach, of performing WGS and RNAseq analysis in parallel, on 9 normal squamous cell carcinoma, 8 non-dysplastic Barrett’s esophagus and 6 esophageal adenocarcinoma samples (n = 23 samples). WGS analysis revealed a higher microbial diversity in normal squamous cell carcinomas compared to the non-dysplastic Barrett’s esophagus with high abundance of Veilonella Parvula, Prevotella melaninogenica and Streptococcus parasanguinis. Unsupervised clustering analysis of the entire data set revealed high abundance of Fusobacterium nucleatum in adenocarcinoma samples, which has been associated with a shorter survival in Esophageal cancer. Unsupervised clustering analysis of RNAseq results revealed a high enrichment of CD4+ Th1 and Th2 helper cell as well as CD8+ T cell associated immune response in some adenocarcinoma and non-dysplastic Barrett’s esophageal samples. Together, our data are derived from endoscopic biopsy samples from patients, and suggest that both gastric and esophageal cancers display distinct microbial patterns associated with chronic inflammation and a tumor-promoting pro-inflammatory microenvironment. Citation Format: Prashant V. Thakkar, Chao Zhang, Prateek Sharma, Sreekar Vennelaganti, Doron Betel, Manish A. Shah. Understanding the association of gut microbiota and tumor microenvironment in gastric and esophageal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B39.

High expression of microRNA‑31 and its host gene LOC554202 predict favorable outcomes in patients with colorectal cancer treated with oxaliplatin.

The expression levels of microRNA‑31 (miR‑31) and LOC554202 have been previously investigated in colorectal cancer (CRC) and their oncogenic and/or tumor suppressive roles have been described. The aim of the present study was to examine the role of miR‑31 and its host gene LOC554202 in the prognosis of patients with CRC. Patients with CRC treated with oxaliplatin‑based chemotherapy between June 2005 and March 2010 were recruited to the First Affiliated Hospital of China Medical University. Tumor and adjacent mucosal tissues were collected. The detection of miR‑31 and/or LOC554202 was performed with probe hybridization targeting. Correlation analysis was performed among the expression levels of miR‑31, LOC554202, and their association with clinicopathological parameters and/or survival rates. miR‑31 and LOC554202 were expressed at high levels in CRC (P<0.01) compared with adjacent intestinal mucosa. A linear correlation was noted for the two markers in CRC tissues (P<0.01). The expression of miR‑31 was significantly higher in adenocarcinoma than in the adjacent intestinal mucosa (P<0.01), whereas the expression of LOC554202 was significantly higher in the adenocarcinoma and the rectal cancer tissue regions (P<0.01). The high expression levels of miR‑31 and LOC554202 were associated with high disease‑free survival (DFS) and overall survival (OS) rates (P<0.05). Associations between the increase in DFS and OS and the elevated expression levels of miR‑31 and LOC554202 were present in patients with colon cancer but not in patients with rectal cancer (P<0.05). These data indicated that miR‑31 and LOC554202 may be potential markers for evaluation of the prognosis of patients treated with oxaliplatin‑based chemotherapy.

Robust prediction of gene regulation in colorectal cancer tissues from DNA methylation profiles

ABSTRACTDNA methylation is recognized as one of several epigenetic regulators of gene expression and as potential driver of carcinogenesis through gene-silencing of tumor suppressors and activation of oncogenes. However, abnormal methylation, even of promoter regions, does not necessarily alter gene expression levels, especially if the gene is already silenced, leaving the exact mechanisms of methylation unanswered. Using a large cohort of matching DNA methylation and gene expression samples of colorectal cancer (CRC; n = 77) and normal adjacent mucosa tissues (n = 108), we investigated the regulatory role of methylation on gene expression. We show that on a subset of genes enriched in common cancer pathways, methylation is significantly associated with gene regulation through gene-specific mechanisms. We built two classification models to infer gene regulation in CRC from methylation differences of tumor and normal tissues, taking into account both gene-silencing and gene-activation effects through hyper- and hypo-methylation of CpGs. The classification models result in high prediction performances in both training and independent CRC testing cohorts (0.92<AUC<0.97) as well as in individual patient data (average AUC = 0.82), suggesting a robust interplay between methylation and gene regulation. Validation analysis in other cancerous tissues resulted in lower prediction performances (0.69<AUC<0.90); however, it identified genes that share robust dependencies across cancerous tissues. In conclusion, we present a robust classification approach that predicts the gene-specific regulation through DNA methylation in CRC tissues with possible transition to different cancer entities. Furthermore, we present HMGA1 as consistently associated with methylation across cancers, suggesting a potential candidate for DNA methylation targeting cancer therapy.

Krüpple like lactor 12 expression in colorectal cancer tissues and its relationship with epithelial mesenchymal transition

Objective To explore the mechanism of krupple like lactor 12 (KLF12) in epithelial mesenchymal transition (EMT) of colorectal cancer. Methods Immunohistochemistry technique was utilized to detect the expression of KLF12, E-cadherin, N-cadherin, Vimentin and Snail in cancer tissues and adjacent mucosa tissues of 85 colorectal cancer patients, and clinical and follow-up data were also collected. KLF12-small interfering RNA (siRNA) was synthesized and transfected into HT-29 cells. Methyl thiazol tetrazolium (MTT) assay was applied to detect cell viability. Wound assay and Transwell assay were used to test invasion and migration of cells. The mRNA and protein expression of related genes was detected by real time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Results Results of immunohistochemistry demonstrated that the positive rate of KLF12, N-cadherin, Vimentin and Snail proteins in cancer tissues was higher, and that of E-cadherin was lower than that in adjacent mucosa tissues (χ2=43.724, 26.145, 35.788, 10.985, 59.225 respectively, P=0.000). The expression of KLF12 was associated with depth of tumors (χ2=7.773, P=0.005), and patients positive for KLF12 had poor prognosis (χ2=8.724, P=0.003). Strongest KLF12 protein was found in HT-29 cells (P=0.000). Cell viability, invasion and migration decreased after KLF12 was inhibited (F=22.541, 15.002 and 21.908 respectively, P=0.000). The expression of N-cadherin and Snail decreased, and E-cadherin increased after KLF12 was inhibited (P=0.000). Conclusion KLF12 might be involved in EMT of colorectal cancer to enhance invasion and metastasis. Key words: Kruppel-like transcription factor 12; Colorectal cancer; Epithelial mesenchymal transition; Invasion; Metastasis; Prognosis

A Novel Predictive Marker of Resistance to Neoadjuvant Chemoradiation Therapy in Young Rectal Cancer Patients

The aim of the study was to delineate the gene expression profile of LGR5 in rectal cancer (RC) tissues among young Egyptian patients. The study aimed at investigating the possible link between this cancer stem cells (CSCs) related gene and clinical outcome, including response to neoadjuvant chemo-radiotherapy (CRT). The study was conducted on 30 young Egyptian RC patients, who were recommended to undergo neoadjuvant CRT. Paired tumor and non-tumor adjacent mucosal tissues were obtained from patients by routine biopsy techniques. Total RNA was extracted followed by reverse transcription, then quantitative PCR was performed using SYBR green. Expression levels of several CSCs related gene including LGR5 in tumor relative to adjacent non-tumor tissues were calculated using the comparative Cq method after normalization for the expression of ACTB. Patients were followed up for assessment of response to neoadjuvant CRT based on revised RECIST 1.1. Among CSCs genes that are tested, only Overexpression of LGR5 in human RC tissues compared to non- tumor tissues was detected. Furthermore, correlation analysis showed that higher expression of LGR5 was correlated with more depth of tumor invasion, LN metastasis, advanced cTNM stage and mesorectal fascia involvement. On the other hand, it was not correlated with gender, age, tumor site nor pathological features. These results suggest that LGR5 may not only play an important role in the progression of RC but also serve as a potential unfavorable prognostic biomarker for RC. In addition, high LGR5 expression level was associated with poor response to CRT.ROC curve analysis showed that LGR5 expression is an excellent predictor for response to neoadjuvant therapy. 1- High LGR5 expression is, most likely, indicative of poor prognosis among young Egyptian RC patients. 2- LGR5 expression level is proposed to be a novel predictive marker of resistance to neoadjuvant CRT in young Egyptian RC patients.

Blockage of glycolysis by targeting PFKFB3 suppresses tumor growth and metastasis in head and neck squamous cell carcinoma

BackgroundMany cancers including head and neck squamous cell carcinoma (HNSCC) are characterized by a metabolic rewiring with increased glucose uptake and lactate production, termed as aerobic glycolysis. Targeting aerobic glycolysis presents a promising strategy for cancer therapy. This study investigates the therapeutic potential of glycolysis blockage by targeting phosphofructokinase-2/fructose-2, 6-bisphosphatase 3 (PFKFB3) in HNSCC.Methods1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) was used as a selective antagonist of PFKFB3. Glycolytic flux was determined by measuring glucose uptake, lactate production and ATP yield. PFKFB3 expression was examined using HNSCC tissue arrays. Cell proliferation, apoptosis and motility were analysed. HNSCC xenograft mouse model and metastasis mouse model were established to examine the therapeutic efficacy of PFK15 in vivo.ResultsHNSCC showed an increased PFKFB3 expression compared with adjacent mucosal tissues (P < 0.01). Targeting PFKFB3 via PFK15 significantly reduced the glucose uptake, lactate production and ATP generation in HNSCC cell lines. PFK15 suppressed cell proliferation, halted cell cycle progression and induced cell apoptosis. The invadopodia of HNSCC cells was markedly reduced after PFK15 treatment, thereby impairing cell motility and extracellular matrix degradation ability. The in vivo data from the xenograft mice models proved that PFK15 administration suppressed the tumor growth. And the results from the metastatic mice models showed administration of PFK15 alleviated the lung metastasis of HNSCC and extended the life expectancy of mice.ConclusionsThe pharmacological inhibition of PFKFB3 via PFK15 suppressed tumor growth and alleviated metastasis in HNSCC, offering a promising strategy for cancer therapy.

Molecular profile of 5-fluorouracil pathway genes in colorectal carcinoma

BackgroundThis study addresses involvement of major 5-fluorouracil (5-FU) pathway genes in the prognosis of colorectal carcinoma patients.MethodsTesting set and two validation sets comprising paired tumor and adjacent mucosa tissue samples from 151 patients were used for transcript profiling of 15 5-FU pathway genes by quantitative real-time PCR and DNA methylation profiling by high resolution melting analysis. Intratumoral molecular profiles were correlated with clinical data of patients. Protein levels of two most relevant candidate markers were assessed by immunoblotting.ResultsDownregulation of DPYD and upregulation of PPAT, UMPS, RRM2, and SLC29A1 transcripts were found in tumors compared to adjacent mucosa in testing and validation sets of patients. Low RRM2 transcript level significantly associated with poor response to the first-line palliative 5-FU-based chemotherapy in the testing set and with poor disease-free interval of patients in the validation set irrespective of 5-FU treatment. UPP2 was strongly methylated while its transcript absent in both tumors and adjacent mucosa. DPYS methylation level was significantly higher in tumor tissues compared to adjacent mucosa samples. Low intratumoral level of UPB1 methylation was prognostic for poor disease-free interval of the patients (P = 0.0002). The rest of the studied 5-FU genes were not methylated in tumors or adjacent mucosa.ConclusionsThe observed overexpression of several 5-FU activating genes and DPYD downregulation deduce that chemotherapy naïve colorectal tumors share favorable gene expression profile for 5-FU therapy. Low RRM2 transcript and UPB1 methylation levels present separate poor prognosis factors for colorectal carcinoma patients and should be further investigated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2826-8) contains supplementary material, which is available to authorized users.

Abstract 3127: Small but lethal, miR-137 acts as a tumor suppressive microRNA in colorectal cancer

Abstract Colorectal cancer is the 3rd most common cause of cancer-related deaths for men and women in the United States. Understanding the mechanisms associated with the initiation and progression of colorectal cancer is needed to develop precise therapies. Multiple regulatory processes are responsible for maintaining cellular homeostasis, including microRNA (miRNA) regulation. We previously demonstrated that miR-137 expression was significantly decreased in 84% of tumors as compared to adjacent mucosal tissue from patients with rectal cancer (n = 68). Restoration of miR-137 reduced colon cancer growth, measured by colony formation and tumorsphere assays. Induction of miR-137 expression significantly decreased tumor growth in our xenograft tumor model by approximately 50% (n = 10). To understand the mechanism associated with the tumor suppressive function of miR-137, we transfected miR-137 mimic into HCT-116 cells and performed microarray analysis to identify changes in gene expression and identify key regulatory pathways effected by miR-137 restoration. Multiple miR-137 targets were down regulated, as compared to cells treated with a negative control mimic. The data was validated using quantitative real-time PCR (qPCR). Finally, the expression of miR-137 target genes was analyzed in a panel of colon cancer cell lines using qPCR. In summary, miR-137 acts as a tumor suppressive microRNA by negatively regulating multiple targets that are overexpressed in colorectal cancer. Citation Format: Amber Rae Smith, Rebecca Marquez, Bryan Tsao, Alexandria Roy, Bailey Wilkerson, Surajit Pathak, Kristi Neufeld, Xiao-Feng Sun, Liang Xu. Small but lethal, miR-137 acts as a tumor suppressive microRNA in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2015-3127

EGR‐1 Regulates Radiation‐Induced Apoptosis in Human Head and Neck Squamous Cell Cancer

Objectives: (1) Investigate that early growth response-1 (EGR-1) expression is expressed in human head and neck squamous cancer (human HNSCC). (2) Evaluate whether EGR-1 affects radiation-induced apoptosis in human HNSCC and would thus serve as the proper prognostic biomolecular marker of radiation therapy. Methods: The protein expression of EGR-1 by immunohistochemistry was investigated in human HNSCC tissues. EGR-1 expression was evaluated at different time points after irradiation in human HNSCC cell lines. To evaluate the impact of EGR-1 knock-down on radiation-induced apoptosis of human HNSCC cell lines, cell apoptosis assays using small-interfering RNA were performed. Western blot analysis was used to assess alteration in apoptosis related protein expression after irradiation in human HNSCC cell lines. Results: EGR-1–positive immunoreactions were observed relative to adjacent mucosal tissue in 15 tissues (58.6%) of 28 human HNSCC tissues. After 5Gy and 8Gy irradiation, EGR-1 increased with peak activation at 2 hours. The cleaved caspase 3, cleaved caspase 7, and cleaved PARP were increased at 2 hours after irradiation. Proapoptotic protein Bax was increased at 2 hours after irradiation. EGR-1 knock-down cells displayed decreased radiation-induced apoptosis, compared with control cells in cell apoptosis assay. Cleaved caspase 3, cleaved caspase 7, cleaved PARP, and Bax activation was decreased by EGR-1 knock-down after irradiation. Conclusions: EGR-1 had abundant expression in human HNSCC tissue. EGR-1 knock-down decreased radiation-induced apoptosis through caspase 3, caspase, 7, PARP, and Bax. EGR-1 may play an important role in treatment response after radiation therapy in human HNSCC.

Molecular characteristics of mismatch repair genes in sporadic colorectal tumors in Czech patients

BackgroundMismatch repair (MMR) genes are known to be frequently altered in colorectal cancer (CRC). Both genetics and epigenetics modifications seems to be relevant in this phenomenon, however it is still not clear how these two aspects are interconnected. The present study aimed at characterizing of epigenetic and gene expression profiles of MMR genes in sporadic CRC patients from the Czech Republic, a country with one of the highest incidences of this cancer all over Europe.MethodsExpression levels and CpG promoter methylation status of all MMR genes were evaluated in DNA from tumor and adjacent mucosal samples of 53 incident CRC patients.ResultsWe have found significantly increased transcription levels in EXO1 gene in tumor tissues (P = 0.05) and significant over-expression of MSH3 gene in colon tumors when compared to adjacent mucosal tissues (P = 0.02). Interestingly, almost all MMR genes were differently expressed when localization of tumors was compared. In particular, colon tumors showed an up-regulation of EXO1, MSH2, MSH3, MSH6, and PMS2 genes in comparison to rectal tumors (P = 0.02). Expression levels of all MMR genes positively correlated between each other. The promoter methylation of MLH1 gene was observed in 9% of CRC tissues only.ConclusionsIn our study, we have observed different pattern of MMR genes expression according to tumor localization. However, a lack of association between methylation in MMR genes and their corresponding expressions was noticed in this study, the relationship between these two aspects is worthy to be analyzed in larger population studies and in pre-malignant stages.

MiR-139 targets CXCR4 and inhibits the proliferation and metastasis of laryngeal squamous carcinoma cells

Our previous studies have showed that chemokine receptor 4 (CXCR4) was over-expressed in laryngeal squamous cell carcinoma (LSCC). However, the mechanism underlying aberrant CXCR4 expression remains unclear. To investigate the roles played by miRNAs in CXCR4 over-expression in LSCC, putative miR-139 was predicted through computational algorithms, including TargetScan, PicTar and miRBase, and luciferase reporter assay was explored to confirm that whether CXCR4 was directly regulated by miR-139. Then, quantitative real-time PCR, immunohistochemistry and in situ hybridization methods were employed to detect the expression of miR-139 and CXCR4 in primary LSCC tissues, normal adjacent mucosal tissues and metastatic lesions derived from 40 LSCC patients in the Second Hospital, Xi'An JiaoTong University. Finally, gain- and loss-of-function assays were adopted to explore the effects of miR-139 and CXCR4 on proliferation, invasion and metastasis of the human LSCC cell line Hep-2 in vitro and in vivo. Our results showed that miR-139 dampened CXCR4 expression, and CXCR4 was directly targeted by miR-139. Additionally, the expression of miR-139 was reduced in alignment with the progression of primary to metastatic LSCC. Moreover, an inverse correlation was observed between miR-139 and CXCR4 protein levels in LSCC specimens. Functional analyses demonstrated that ectopic expression of miR-139 inhibited cell proliferation, migration and metastasis of Hep-2 cells in vitro and in vivo. Similar to the observations seen in restoring miR-139 expression, dampening of CXCR4 expression inhibited cell growth, migration and invasion, whereas miR-139 over-expression reversed the pro-metastatic effect of CXCR4. Taken together, we conclude that miR-139 targets CXCR4 and inhibits proliferation and metastasis of LSCC.

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.

Expression and clinical significance of vascular endothelial growth factor-C and nm23-H1 in stage II and III colorectal carcinomas

To discuss the expression and clinical significance of VEGF-C and nm23-H1 in stage II and III colorectal carcinomas. SP immunohistochemical staining was employed to determine the expression of vascular endothelial growth factor-C (VEGF-C) and nm23-H1 in the tumor tissues of 110 cases of stage II and III colorectal carcinomas and in the adjacent mucosal tissues of 53 cases as control, and analyze their correlation with cliniopathological features and prognosis. The positive expression of VEGF-C in the carcinoma tissues was 71.8%, significantly higher than that in the adjacent mucosal tissues (22.6%, P < 0.001). The positive expression of nm23-H1 in the carcinoma tissues was 57.3%, significantly lower than that in the adjacent mucosal tissues (90.6%, P < 0.001). The expression of VEGF-C was significantly correlated with lymph node metastasis (P < 0.05), and the nm23-H1 expression was significantly correlated with lymph node metastasis and pathological type (P < 0.05). The expression of VEGF-C and nm23-H1 did not show a significant correlation with age, gender, primary tumor site, tumor size and depth of invasion (P > 0.05). The VEGF-C expression was negatively related with nm23-H1 expression in colorectal carcinoma (r = -0.361, P < 0.001). The median overall survival (MOS) and median disease free survival (MDFS) of 110 patients with colorectal carcinoma were 55 and 48 months, respectively. The colorectal patients with different VEGF-C and nm23-H1 expression showed significant differences in the 5-year OS rate and 5-year DFS rate (P < 0.001). The patients with negative VEGF-C expression and positive nm23-H1 expression had a better prognosis. The joint detection of VEGF-C and nm23-H1 expression is very promising in prediction of the prognosis of patients with stage II and III colorectal carcinoma. However, whether it can be used as a marker in prognosis judgment needs further investigation.

RON Is Associated with Tumor Progression in Head and Neck Squamous Cell Cancer

Objective: To investigate that the recepteur d’origine nantais (RON) expression is expressed in human head and neck squamous cell cancer (HNSCC) cells and to evaluate whether RON affects tumor cell behavior in head and neck cancer cell lines, thus serving as a target for molecular therapy in a preclinical model. Method: The protein expression of RON by immunohistochemistry and western blot analysis was investigated in human head-neck cancer tissues. To evaluate the impact of RON knock down on behavior of human head-neck cancer cell lines, invasion and migration assays using small-interfering RNA were performed. Western blot analysis was used to assess alteration in protein expression. Results: RON positive immunoreactions were observed relative to adjacent mucosal tissue in all 28 cases, while 15 cases (53.6%) were over-expressed. RON over-expression was significantly associated with lymph node metastasis. RON protein expression was increased in cervical metastatic lymph node tissue by western blot analysis. Treatment of RON ligand, macrophage stimulating proetin (MSP) increased cell migration and invasion in human head-neck cancer cell lines (SNU 1041, PCI 50). The knockdown of RON significantly suppressed tumor cell migration, invasion. The Erk, p38 phosphorylation was reduced by knockdown of RON in PCI 50. Conclusion: RON expression might play an important role in the progression, especially lymphatic metastasis of head and neck squamous cell cancer. Further investigations are needed to assess the potential of RON-directed molecular therapy in head and neck squamous cell cancer.

Increased Intratumoral Neutrophil in Colorectal Carcinomas Correlates Closely with Malignant Phenotype and Predicts Patients' Adverse Prognosis

BackgroundSubstantial evidence suggests that the presence of inflammatory cells plays a critical role in the development and/or progression of human tumors. Neutrophils are the common inflammatory cells in tumors; however, the infiltration of intratumoral neutrophils in colorectal carcinoma (CRC) and its effect on CRC patients' prognosis are poorly understood.Methodology/Principal FindingsIn this study, the methods of tissue microarray and immunohistochemistry (IHC) were used to investigate the prognostic significance of intratumoral CD66b+ neutrophil in CRC. According to receiver operating characteristic curve analysis, the cutoff score for high intratumoral CD66b+ neutrophil in CRC was defined when the mean counts were more than 60 per TMA spot. In our study, high intratumoral CD66b+ neutrophil was observed in 104/229 (45.4%) of CRCs and in 29/229 (12.7%) of adjacent mucosal tissues. Further correlation analysis showed that high intratumoral neutrophil was positively correlated with pT status, pM status and clinical stage (P<0.05). In univariate survival analysis, a significant association between high intratumoral neutrophil and shortened patients' survival was found (P<0.0001). In different subsets of CRC patients, intratumoral neutrophil was also a prognostic indicator in patients with stage II, stage III, grade 2, grade 3, pT1, pT2, pN0 and pN1 (P<0.05). Importantly, high intratumoral neutrophil was evaluated as an independent prognostic factor in multivariate analysis (P<0.05).Conclusions/SignificanceOur results provide evidence that increased intratumoral neutrophil in CRC may be important in the acquisition of a malignant phenotype, indicating that the presence of intratumoral neutrophil is an independent factor for poor prognosis of patients with CRC.

Decreased expression of Beclin 1 correlates with a metastatic phenotypic feature and adverse prognosis of gastric carcinomas

Beclin 1 plays a critical role in the regulation of autophagy, apoptosis, differentiation and the development and progression of cancer. The clinicopathological significance of Beclin 1 expression in patients with gastric carcinoma (GC) has not been yet elucidated. Immunohistochemistry (IHC) was performed to investigate the Beclin 1 expression in GCs and normal mucosal tissues. Receiver operating characteristic curve analysis, spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. The highly expressed Beclin 1 was observed in 90/155 (58.1%) of GCs, in 24/60 (40.0%) adjacent mucosal tissues and in 13/30 (43.3%) of normal gastric mucosa tissues (P = 0.036). Decreased expression of Beclin 1 in cancer cells was significantly correlated with poor differentiation, nodal and distant metastasis, advanced TNM stage, and tumor relapse. More importantly, Decreased expression of Beclin 1 was associated with shorter survival as evidenced by univariate and multivariate analysis. Our findings provide a basis for the concept that decreased expression of Beclin 1 in GC may be important in the acquisition of a metastatic phenotype, suggesting that decreased Beclin 1 expression, as examined by IHC, is an independent biomarker for poor prognosis of patients with GC.

Abstract 4646: Evaluation of EGFR gene amplification status, mRNA, protein, and phosphoprotein levels expression in head and neck cancer patient tissues

Abstract Purpose: Evaluate EGFR gene amplification status and EGFR mRNA and protein/phosphoprotein levels in tumors from patients with head and neck squamous cell carcinoma (HNSCC) surgically treated with curative intent from 2000-4 to define relationships between and assess the prognostic values of these biomarkers. Background: EGFR has been reported to be overexpressed in HNSCC, and high levels of EGFR protein and EGFR gene amplification have been independently reported to be associated with poorer prognosis. To our knowledge, tumor levels of the known site-specific phosphorylated EGFR (Y992, Y1068) have not been reported as prognostic indicators for HNSCC. Methods: 58 tumor specimens and 50 paired adjacent mucosal tissues from patients surgically treated for HNSCC were collected through an Early Detection Research Network (EDRN) study. Tissue microarrays (TMAs) were constructed using these paired patient samples and 30 oral mucosal specimens from cancer-free controls. Three cores from each specimen were arrayed on two TMAs and evaluated for EGFR gene amplification by dual-color FISH and for EGFR by IHC (EGFR levels in these IHC specimens have been previously reported). Corresponding fresh-frozen tumor specimens were evaluated by reverse-phase protein array (RPPA) for total EGFR, EGFR P-Y992 and P-Y1068 levels and by quantitative RT-PCR for EGFR mRNA levels. Progression-free survival (PFS) was calculated using the University of Pittsburgh Head and Neck Cancer Registry data as months from date of surgery to date of first cancer recurrence, new primary, or death. Hazard Ratios were determined using Cox proportional hazards models. Results: Tumors with high level EGFR gene amplification expressed significantly higher levels of EGFR protein as assessed by IHC than tumors without EGFR gene amplification (P&amp;lt;0.001). Increased EGFR levels were also detected in 11.4% of tumors without EGFR gene amplification. EGFR gene amplification was not associated reduced PFS (H.R.=1.64, P=0.16). High EGFR protein levels and elevated tumor levels of EGFR P-Y992 as determined by RPPA were not associated with reduced PFS. However, high tumor levels of EGFR P-Y1068 were significantly associated with reduced PFS (H.R.=3.34, P=0.008). Conclusions: Similar to previous findings, we find that patients with tumor EGFR gene amplification tend to have shorter PFS than do patients without EGFR gene amplification. We find that tumors with EGFR gene amplification have significantly higher EGFR protein levels as assessed by IHC. Though increased EGFR protein levels in tumors are not always associated with gene amplification, this may be an important mechanism for increased EGFR expression. Differential levels of site-specific EGFR phosphorylation at Y992 and Y1068 indicate that signaling pathways associated with these sites may play different roles in disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4646.

R412 – Role of KITENIN in Progression and Metastasis of SCC

Problem The expression of KAI1 (a metastatic suppressor gene) in cancer cells results in reduced cell motility and invasiveness. A cDNA clone of VANGL1, a member of the tetraspanin protein family that specifically interacts with the COOH-terminal cytoplasm domain of KAI1, was isolated and renamed KITENIN (KAI1 COOH-terminal interacting tetraspanin). The purpose of this study was to investigate the role of KITENIN on the progression and metastasis of transfected squamous cell carcinoma using in vivo and in vitro experiments. Methods Locally advanced squamous cell carcinoma tissues from five patients were obtained for investigation of KITENIN expression. Malignant tumors, normal adjacent mucosa tissues, metastatic lymph nodes, and non-metastatic lymph nodes were collected. KITENIN or vector only (control) was transfected into SCC (squamous cell carcinoma) VII, a mouse squamous cell carcinoma cell line, using FuGENE 6. An in vitro assay (invasion, migration, and proliferation) for KITENIN and the vector-transfected group was studied. The KITENIN or vector-transfected SCC VII cells were injected subcutaneously into 12 C3H/HeJ syngeneic mice (6 mice for each group). The tumor size was measured daily for 4 weeks. During the fifth week after injection, the presence of metastasis in the lung and liver tissue was evaluated for each mouse with a tumor mass on the back; the tissues were assessed by gross and microscopic examination. Results KITENIN was highly expressed in tumors and metastatic lymph nodes from patients. KITENIN-transfected cells showed significantly increased invasion, migration, and proliferation compared with the vector-transfected cells. Tumor volume was more increased in the KITENIN-transfected cells-injected mice. Lung metastasis was found in the KITENIN-group (3/6 mice), while no metastasis in the vector-group. Conclusion KITENIN participates in the progression and metastasis of SCC. Significance An antisense KITENIN strategy may be a useful method to inhibit progression and metastasis in squamous cell carcinoma. Support This study was financially supported by Chonnam National University, 2006.

The ubiquitin‐proteasome pathway and enhanced activity of NF‐κB in gastric carcinoma

NF-kappa B, ubiquitin and proteasome have been shown be important factors in oncogenesis. The aim of this study was to determine whether NF-kappa B could be a sensitive biomarker for gastric carcinoma. Tumor and adjacent mucosal tissue specimens in 92 patients with gastric carcinoma were studied. The expression of NF-kappa B was evaluated by immunohistochemistry. The expression of I kappa B alpha, ubiquitin in cytoplasm and NF-kappa B in nucleoplasm was assayed by Western blot. DNA binding-activity of NF-kappa B was confirmed by electrophoretic mobility shift assay (EMSA). Fluorogenic technique was performed to measure the 26S proteasome activity. NF-kappa B positive expression in tumor tissues (82.4%) was significant higher than that in adjacent mucosal tissues (32.7%, P < 0.05). The increase of NF-kappa B activation was accompanied by the increases of ubiqutin, 26S proteasome activation and a degradation of I kappa B alpha but not the ubiquitin-conjugated I kappa B alpha/NF-kappa B complex in gastric carcinoma. NF-kappa B expression was significantly increased in patients with lymph node metastasis, TNM stage III/IV or with the habit of high intake of pickled vegetables. This study demonstrates that the constitutive activation of NF-kappa B is likely due to the activation of ubiquitin-proteasome pathway and NF-kappa B can be used as a prognostic biomarker.