Metastatic Lymph Node Tissues Research Articles

Long non-coding RNA biomarker for human laryngeal squamous cell carcinoma prognosis

Long non-coding RNAs (lncRNA) were discovered in tumors. The regulation of lncRNA in human laryngeal squamous cell carcinoma (LSCC) remains incomplete. Uncovering the potential of lncRNA to stratify the prognosis of LSCC and streamline the vast amount of clinical information will affect medical interventions. The surgical resected LSCC tissues, adjacent non-cancerous tissues (ANCT) and lymph node metastatic tissue (LNM) were collected from 76 patients for lncRNA AC008440.10 expression assay. The stages of LSCC and LNM were classified accordingly. We integrated the epigenetic information with enhanced CT imaging and pathological evaluations to predict the patients' survival by comprehensive statistical algorithms using equal weighting. Significant downregulation of lncRNA AC008440.10 was detected in LSCC tumor and metastatic lymph node in advanced stage of patient samples compared with those in early stage. The pattern of differentially expressed AC008440.10 displayed a clear trend that significantly related to tumor progression. The downregulation of lncRNA AC008440.10 correlates with increasing risk of metastasis, poor prognosis and patient survival. The potential for lncRNA AC008440.10 to be developed as a novel biomarker for stratification of the prognosis was especially promising when clinic parameters were hybridized with equal weight, and using a panel of complementary parameters yielded a more powerful predictability of LSCC prognosis than any single parameter individually.

MP65-16 NOVEL BIOMARKER SIGNATURE FOR PREDICTING PATHOLOGIC RESPONSE OF UROTHELIAL CARCINOMA OF THE BLADDER TO CISPLATIN-BASED NEOADJUVANT CHEMOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-16 NOVEL BIOMARKER SIGNATURE FOR PREDICTING PATHOLOGIC RESPONSE OF UROTHELIAL CARCINOMA OF THE BLADDER TO CISPLATIN-BASED NEOADJUVANT CHEMOTHERAPY Patrick Hensley, Matthew Purdom, Natasha Kyprianou, Chi Wang, and Andrew James Patrick HensleyPatrick Hensley More articles by this author , Matthew PurdomMatthew Purdom More articles by this author , Natasha KyprianouNatasha Kyprianou More articles by this author , Chi WangChi Wang More articles by this author , and Andrew JamesAndrew James More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2082AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cisplatin-based neoadjuvant chemotherapy (NC) has become standard practice in the management of muscle invasive urothelial carcinoma of the bladder, but it confers only a modest survival advantage. Patients with pathologic progression on NC have poor outcomes and may benefit from upfront cystectomy. We previously demonstrated that phenotypic epithelial-mesenchymal transition (EMT) is associated with advanced stage and grade. This study pursued the predictive value of EMT signature effectors of bladder cancer therapeutic response to NC. METHODS A tissue microarray (TMA) was constructed with matched pre-NC transurethral resection (TUR) specimens and post-NC cystectomy specimens from the same patients (N=69) with specimens from patients managed with surgery alone serving as control (N=21). TMA sections were immunostained for the EMT biomarkers E-cadherin (epithelial marker), N-cadherin and Vimentin (mesenchymal markers) and β-catenin (EMT effector) and staining intensity was scored by two blinded reviewers. Data was statistically analyzed using 1-way ANOVA and T-tests. RESULTS Increased expression of the mesenchymal markers Vimentin and N-cadherin on TUR specimen was associated with nodal metastasis and extravesical disease (≥ypT3 or N+) at the time of cystectomy (P=0.019 and 0.002, respectively). Decreased Vimentin and N-cadherin expression independently predicted complete pathologic response to NC (ypT0, P<0.001 and 0.024, respectively). In post-NC cystectomy specimens, only N-cadherin expression was correlated with pathologic stage (P=0.004). In TUR specimens from patients managed with surgery alone, increased expression of β-catenin and N-cadherin predicted extravesical disease at the time of cystectomy (P=0.033 and 0.007, respectively). When comparing pre- and post-NC specimens, treatment with platinum-based NC induced a mixed EMT picture, with stable β-catenin and N-cadherin expression and decreased E-cadherin and Vimentin expression, relative to control patients. Expression patterns in metastatic lymph node tissue was inconsistent. CONCLUSIONS Expression of EMT markers have potential value in predicting pathologic response to cisplatin-based NC. Further validation is necessary to correlate these findings with clinical outcomes and establish these as novel biomarkers of adverse response to NC towards the goal of optimizing patient selection for NC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e864-e865 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Patrick Hensley More articles by this author Matthew Purdom More articles by this author Natasha Kyprianou More articles by this author Chi Wang More articles by this author Andrew James More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Clinicopathological features of lipid cell variant of urothelial carcinoma.

487 Background: Lipid cell variant (LCV) of urothelial carcinoma (UC) which was first described by Mostofi et al in 1999 is a very rare variant of UC. Because it has only been documented in occasional case reports and a small series about this variant since then, clinicopathological characteristics of LCV are not yet clarified. In this study, we assessed the clinicopathological characteristics of LCV experienced in our hospital. Methods: The medical records of patients with LCV, who were treated in our hospital between September 2015 and September 2017, were retrospectively reviewed and analyzed. Results: In this period, of 301 patients undergoing TURBT and 42 patients undergoing nephroureterectomy, 13 patients including 10 patients with bladder cancer and 3 with pelvic ureter cancer, were found to be diagnosed as LCV of UC at our hospital. The median observation period of these patients was 6 months (IQR, 4-9 months). Among 10 bladder cancer patients who had confirmed this variant in transurethral resection of bladder tumor (TURBT) specimens, all cases had concurrent high-grade UC and the most cases had tumor features including pT2 or more local stages and lympho-vascular invasion (LVI) positive. Co-existence of micropapillary and/or plasmacytoid variant were seen in 5 patients. Lymph node metastases were observed in 3 patients, and this variant was histologically confirmed within the lymph node metastatic tissue in one case. No patient had distant metastasis. Among 3 pelvic ureter cancer patients who had undergone nephroureterectomy, all cases had concurrent high-grade UC and the most cases had tumor features including pT3 local stage and LVI positive. Micropapillary variant was coexisted in one patient. No patient had distant metastasis. 7 patients were no evidence of disease and 6 were alive with disease. The estimated median time from surgery to recurrence was not reached. The 6-months disease-free survival rate was 59%. Conclusions: LCV is seen in the patients who have high-grade UC, and tends to be accompanied with other aggressive variants including micropapillary and plasmacytoid. All patients with this variant had advanced stage cancer at presentation with high recurrence rates.

Overexpression of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 is associated with the poor prognosis of patients with colorectal cancer.

Tumor necrosis factor receptor-associated protein-1 (TRAP-1), a mitochondrial chaperone, contributes significantly to the progression of cancer. However, the understanding of its involvement in the clinicopathological characteristics and prognosis of colorectal cancer (CRC) remains limited. The aim of the present study was to assess the significance of TRAP-1 expression in CRC. The expression of TRAP-1 was evaluated in corresponding cancerous, paracancerous, lymph node and distant metastatic tissues of 256 cases of CRC by immunohistochemistry. The associations between TRAP-1 expression and the clinicopathological parameters and survival rates of patients was assessed. Out of 256 patients with CRC, TRAP-1 expression was detected in 203 (79.3%). TRAP-1 expression was significantly increased in cancerous tissue compared with that in corresponding paracancerous tissues (P<0.001). Overexpression of TRAP-1 was significantly associated with differentiation (P=0.011), depth of invasion (P=0.006), lymph node metastasis (P<0.001) and tumor-node-metastasis stage (P<0.001). In patients with high TRAP-1 expression, the 5-year overall survival (OS) rate was 38.0%, in contrast to 56.5% in patients with low TRAP-1 expression (P=0.003). Similarly, the 5-year progression-free survival (PFS) was 26.6% for patients with high TRAP-1 expression and 53.3% for patients with low TRAP-1 expression (P<0.001). Multivariate analyses indicated the TRAP-1 expression is an independent prognostic factor for poorer OS [P=0.015; hazard ratio (HR), 1.914] and PFS (P<0.001; HR, 2.534). Thus, TRAP-1 may serve as a potential biomarker for predicting the prognosis of patients with CRC. Specifically, overexpression of TRAP-1 may predict progression and poor survival in cases of CRC.

Heterogeneity of COX-2 and multidrug resistance between primary tumors and regional lymph node metastases of gastric cancer.

Cyclooxygenase-2 (COX-2) is involved in the progression of gastric cancer; however, the role of COX-2 in multidrug resistance (MDR) is still unclear. This study aimed to elucidate the relationship between COX-2 and MDR so as to show the heterogeneity of gastric primary tumors and regional lymph node metastases. Between 2008 and 2009, 56 primary tumor samples and paired metastatic lymph node tissues from gastric cancer patients confirmed by surgery and pathological examination in our hospital were collected. The expression levels of COX-2 and MDR-associated factors such as P-glycoprotein (P-gp), glutathione S-transferase pi (GST-π) and topoisomerase II alpha (Topo-II-α) were determined by immunohistochemical staining. Tumor cells from these tissues were cultured and the cell chemosensitivities for 11 chemotherapeutic agents were measured by sulforhodamine B assay. The expression levels of COX-2, P-gp and GST-π were significantly higher in metastatic lymph node tissues than in primary tumors, while the expression level of Topo-II-α was lower in metastatic lymph node tissues than in primary tumors (all P <0.05). In primary tumors, COX-2 and GST-π were positively correlated and COX-2 and Topo-II-α were negatively correlated; in metastatic lymph node tissues, a positive correlation was found between COX-2 and P-gp (all P <0.05). The inhibition rates of eADM, VP-16, THP and MMC on cells from primary tumors were significantly lower than those on cells from metastatic lymph nodes, while the inhibition rates of HCPT, L-OHP and VCR on cells from metastatic lymph nodes were lower than those on cells from primary tumors. The expression of COX-2 and MDR-associated factors as well as cell chemosensitivities are different in primary tumors and regional lymph node metastases of gastric cancer, and this may be an indication of their heterogeneity.

Early Growth Response 1-Dependent Downregulation of Matrix Metalloproteinase 9 and Mouse Double Minute 2 Attenuates Head and Neck Squamous Cell Carcinoma Metastasis

Background/Aims: The functional relevance of early growth response-1 (EGR1) on cancer invasion remains controversial. The effect of EGR1 on the expression of MMP9, which is important for HNSCC invasion, is still disputed. There is no previous data showing the effect of EGR1 on mouse double minute 2 (MDM2), an enhancer of matrix metalloproteinase 9 (MMP9) expression. Our aim is to clarify the negative correlation between EGR1 expression and head and neck squamous cell carcinoma (HNSCC) metastasis. Methods: EGR1 mRNA and protein expressions were compared in normal and HNSCC tissues using The Cancer Genome Atlas (TCGA) dataset analysis or immunohistochemistry (IHC), respectively. In vitro cell invasion was evaluated Matrigel invasion assay. EGR1-dependent inhibition of MDM2 transcription was assessed by promoter–luciferase assay and chromatin immunoprecipitation (ChIP). Results: TCGA data showed that EGR1 mRNA levels are significantly higher in normal oral tissues as compared with HNSCC tumor tissues (adjusted P = 1.64x10^-16). In addition, nonmetastatic HNSCC tissues showed significantly higher EGR1 mRNA levels as compared with metastatic tissues (adjusted P = 0.023). IHC analysis showed that primary tumor tissues expressed significantly higher levels of nuclear EGR1 compared with paired metastatic lymph node tissues (P < 0.05). EGR1 overexpression downregulated MMP9 and MDM2 protein expression. Consistent with these observations, TCGA data analysis found significantly fewer metastatic patients among a subgroup of population presenting higher EGR1 expressions with lower MMP9 and/or MDM2. Conclusion: Our data suggests that EGR1 prevents HNSCC metastasis through downregulation of MMP9 and MDM2. EGR1 might be a potential candidate to attenuate HNSCC metastasis.

Derivation and basic characterization of colorectal carcinoma primary cell lines

Colorectal carcinoma is one of the most common malignancies in western countries. Among different approaches to its research, primary cancer cell lines can play an important role. The main purposes of this study were: 1) to establish an effective and reproducible method of colorectal cancer cell isolation and cultivation from primary tumours and lymph node metastases and 2) to elucidate the biological features of the tumours favouring successful cell cultivation. The tumour cells were obtained from colectomy specimens. Primary tumour and lymph node metastasis tissue was used for establishing the tissue cultures. Colectomy samples were further processed for routine histopathological assessment: tumour grade, stage, angioinvasion and perineural spread were evaluated. Features of tissue culture cells were assessed using phase contrast microscopy and immune-histochemical techniques. WST-1 assay and X-CELLigence real time analysis were carried out for viability and proliferation testing before and after treatment with irinotecan and oxaliplatin. Molecular features of the tumour including K-RAS/B-RAF/N-RAS mutations were tested using allele-specific PCR. Results of the cultivation process were compared to the histopathological and molecular features of the tumours. In total, we obtained 33 samples from the primary site of tumours and 20 samples from lymph node metastases; in total, 27 cell lines were successfully isolated. Morphologic features characteristic of tumour cells in primary cell lines and epithelial differentiation (positive for AE1/AE3 cytokeratin) were evaluated. Higher tumour stage, angioinvasion and presence of perineural spread in primary tumour correlated positively with successful cell isolation from lymph node metastasis. All samples tested were NRAS wild-type. No correlation was found between molecular phenotype and the cell culture features. A higher proliferation potential was observed in the primary tumour cells, whereas higher sensitivity to irinotecan was found in the lymph node metastatic cells. Using mechanical dissociation, we successfully derived and cultivated CRC cells from primary tumours and lymph node metastases with success rate 3 % and 70% respectively. Primary tumour features favouring successful establishment of cell cultures were identified.

MicroRNA-23a promotes pancreatic cancer metastasis by targeting epithelial splicing regulator protein 1.

miR-23a plays vital roles in various cancer metastases. Here, we found that miR-23a expression was significantly up-regulated in pancreatic cancer tissues compared with adjacent normal tissues. miR-23a up-regulation was significantly associated with differentiated degree, lymphoid nodal status, tumor invasion and poor survival rate in pancreatic cancer patients. We also found that miR-23a expression was significantly up-regulated in lymph node metastatic tissues and in pancreatic cancer cells that underwent epithelial-mesenchymal transition (EMT). miR-23a down-regulation blocked TGF-β1-induced EMT and reversed the phenotype of EMT in Panc-1 cells. Furthermore, miR-23a down-regulation inhibited Panc-1 cells migration and invasion in vitro and liver metastases in vivo. But the effect of miR-23a up-regulation in Aspc-1 cells was opposite to that of miR-23a down-regulation in Panc-1 cells. Epithelial splicing regulatory protein 1 (ESRP1) was identified as a direct target of miR-23a. Restoration of ESRP1 rescued the effect of miR-23a on pancreatic cancer cell progression. Moreover, miR-23a up-regulation in Aspc-1 cells induced a shift in CD44 expression from variant isoforms (CD44v) to the standard isoform (CD44s) together with increased FGFR2 IIIc mRNA levels, and decreased FGFR2 IIIb expression during EMT. But the effect of miR-23a down-regulation in Panc-1 cells was opposite to that of miR-23a up-regulation in Aspc-1 cells. In addition, the effect of miR-23a up-regulation was partly reversed by ESRP1 over-expression. Taken together, our findings indicated that miR-23a functions as an oncogene by regulating ESRP1 in pancreatic cancer.

Abstract 1243: FAM134B mutation in esophageal squamous cell carcinoma: Its clinical significance and quantification by electrochemical methods

Abstract Aim: The aim of this research was to detect novel sites of FAM134B mutations, copy number variations and their clinicopathological significance in esophageal squamous cell carcinoma (ESCC) patients. Also, this study was intended to develop a simple and inexpensive electrochemical detection method for the analysis of FAM134B mutations using a single-use and disposable screen-printed electrode. Method: Approximately 102 fresh tissue samples of ESCC and matched non-cancer adjacent tissues were recruited. The DNA copy numbers of FAM134B were initially studied by qRT-PCR. The FAM134B mutations were then quantified via high resolution melt curve (HRM) and Sanger sequencing analysis. In order to quantify the level of point mutation or SNPs in FAM134B gene, a new electrochemical method was also developed. The underlying working principle of the method is relied on the base dependent affinity interactions towards gold electrode. Since two DNA sequences with different DNA base compositions (i.e., amplified mutated sequences will be distinctly different than its wild type sequence) will have different adsorption affinity towards an unmodified gold electrode, accurate measurement of adsorbed DNA on the electrode surface will give the measure of point mutation or SNPs present in the DNA sequences. Target DNA sequences were first extracted from clinical samples and then PCR amplified and purified prior to adsorption on a single-use screen-printed gold electrode. The amount of mutation sites on a DNA sequence is quantified by monitoring the Faradaic current generated by the [Fe(CN)6]3-/4- system present in the electrolyte solution. Result: Amplification of FAM134B DNA was noted in 37% of ESCC tissues whereas 35% cases showed loss of FAM134B copies compared to matched non-tumor tissues. Overall, thirty-seven FAM134B mutations were documented in exons 4, 5, 7, 9 as well as introns 2, 4-8 of FAM134B. Also, FAM134B mutations were detected in all the metastatic ESCC cases and in 14% (8/57) of the primary ESCC. Using the new electrochemical method, we were able to detect mutations in 50 ng of target PCR-amplified product within 1 h with high reproducibility (% RSD= &amp;lt;2) and specificity. Conclusion: DNA copy number variations and frequent mutations of FAM134B in metastatic lymph node tissues in ESCC patients indicate its critical role in the pathogenesis of ESCCs. Also the mutation detection via electrochemical methods was successful distinguishing single point mutation in DNA from oesophageal cancer implying its potential application in point mutation detection in clinical diagnostics. Note: This abstract was not presented at the meeting. Citation Format: Md. Hakimul Haque, Vinod Gopalan, Muhammad J. A. Shiddiky, Alfred K. Lam. FAM134B mutation in esophageal squamous cell carcinoma: Its clinical significance and quantification by electrochemical methods [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 1243. doi:10.1158/1538-7445.AM2017-1243

Abstract 3948: Comprehensive characterization of genomic and transcriptomic heterogeneity in advanced bladder cancer by multiregional analysis

Abstract Background: Bladder cancer is a highly heterogeneous disease - both clinically, and at the genomic and the transcriptomic levels. Despite complete tumor resections, recurrent tumors share a high number of mutations with the initial/earlier tumors, indicating that new tumors arise from fields of transformed areas of cells in the urothelium (i.e. field disease). Furthermore, the high mutational load found in bladder cancer and the assumed high degree of heterogeneity may have huge implications on therapy response. Thus, detailed knowledge on tumor heterogeneity and mutational dynamics in adjacent normal urothelial cells is needed in order to understand field disease, disease development and disease progression. To address this, we performed comprehensive genomic and transcriptomic analysis of multiple samples of tumor and normal tissue procured from four cystectomy specimens and associated metastases. Methods: Multiple tumors, lymph node metastasis, relapse biopsies and adjacent normal tissue from four patients with muscle invasive disease were analyzed. A targeted panel (illumina TruSeq Custom Amplicon v1.5) was designed from whole exome sequencing of bulk tumor DNA and germline DNA. Targeted sequencing of DNA from small cellular regions procured by laser-microdissection from tumor biopsies and from adjacent normal tissue was used for generating a comprehensive genomic map of the bladder. Transcriptomic profiling (Fluidigm GE) was carried out using RNA from LMD procured tumor regions matching the regions used for targeted sequencing. Results: In total, whole exome sequencing was performed on DNA from 15 bulk tumors with an average coverage of 59.2X. We identified a total of 331, 862, 685 and 1344 mutations, respectively, in the four patients with 15-85% of the mutations being ancestral. Of all the mutations, 95, 380, 117 and 335, respectively, were estimated to have a functional impact. Targeted sequencing of 207, 595, 538 and 483 mutations in the four patients was performed on DNA from 137 small cellular regions, including DNA from 17 adjacent normal tissue areas. We observed low local heterogeneity while comparing more distant regions revealed significantly higher levels of heterogeneity. This observation was in agreement with the heterogeneity observed at the transcriptomic level, where some regions within the same bladder appeared to have basal characteristics while others appeared to have luminal characteristics. Conclusion: Large intra-patient genomic and transcriptomic heterogeneity can be observed in bladder cancer. However, even in multifocal tumors, all the regions shared a substantial number of mutations, indicating a common origin. Ongoing investigations indicate the presence of low frequency tumor associated mutations in normal appearing tissue. Citation Format: Mathilde B. Thomsen, Philippe Lamy, Iver Nordentoft, Søren Vang, Line Reinert, Søren Høyer, Torben F. Ørntoft, Jørgen B. Jensen, Lars Dyrskjøt. Comprehensive characterization of genomic and transcriptomic heterogeneity in advanced bladder cancer by multiregional analysis [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 3948. doi:10.1158/1538-7445.AM2017-3948

Abstract 3926: Is FGD3 a potentially prognostic marker for breast cancer

Abstract Background: Prognostic factors are capable of providing information on clinical outcomes at the time of diagnosis; they are usually indicators of growth, invasion, and metastatic potential (Gasparini G. et al. 1993; Hayes DF. et al. 1998). Tissue marker is one of the prognostic factors; to date, only a small proportion of markers are ultimately clinically useful, including Ki-67 and HER2. Faciogenital dysplasia 3 protein (FGD3), a putative regulator of cell morphology and motility, has been shown to regulate cell migration. In a study of 3,256 tumors, low expression of FGD3 mRNA indicates poor outcome (Hayakawa M. et al. 2008; Scooter W. et al. 2014). However, an immunohistochemistry (IHC) study to evaluate FGD3 protein expression has not been done. We hypothesize that the expression levels of FGD3 protein by IHC might improve the prediction of patient outcomes, and FGD3 might be a potentially prognostic marker of breast cancer. Materials and Methods: 322 cases of breast cancer Tissue Microarrays (TMA) (BR1504a, BR1505b, HBre-Duc068Bch-01, and BR20837) were purchased from US Biomax, Inc (Rockville, MD). Rabbit polyclonal antibody against FGD3 was purchased from Novus Biologicals, LLC (Littleton, CO). Tissue cores were stained for FGD3 with Dako’s EnVision + Dual Link System. Image acquisition was performed using an Olympus camera and software. FGD3 protein expression by IHC was quantitatively determined in the range of 0-4. Unpaired t-test was used for data analyses. Results: 1) Benign tumor and breast adenocarcinomas in lower stages showed strong expression of FGD3, whereas the breast adenocarcinomas in higher stages showed mild ~ weak expression. 2) Invasive breast cancer in stage IIA showed strong FGD3 expression compared to matched metastatic carcinoma which showed mild expression of FGD3. 3) FGD3 protein levels for tumors (n=135) with N0 indicating no regional lymph node metastasis were compared with tumors with lymph node metastasis (N1-3; n=98) and corresponding matched metastatic tissue (n=103). An unpaired t-test comparing N0 vs. N1-3 showed lymph node metastasis is associated with lower FGD3 protein levels (p&amp;lt;0.0001). Conclusion: FGD3 protein expression levels within breast tumors were different according to metastatic status. Our IHC results suggest the possibility of FGD3 to be a prognostic marker in patients with breast cancer. Citation Format: Yuliang Sun, Scooter Willis, Xiaoqian Lin, Justin Achua, Casey Williams, Brian Leyland-Jones. Is FGD3 a potentially prognostic marker for breast cancer [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 3926. doi:10.1158/1538-7445.AM2017-3926

GRIK3: A novel oncogenic protein related to tumor TNM stage, lymph node metastasis, and poor prognosis of GC.

Glutamate receptor, ionotropic, kainate 3 (GRIK3), as a member of the glutamate kainate receptor family, mainly participated in neuroactive ligand receptor interaction pathway. Other members of GRIK family were previously reported to regulate cellular migration, transformation, and proliferation in tumor. However, the mechanism of GRIK3 in tumor is still unclear. Therefore, the purpose of our study was to reveal the expression and clinical significance of GRIK3 in gastric cancer (GC). First, we performed the expression analysis and survival analysis of GRIK3 using The Cancer Genome Atlas (TCGA) database, and the results showed that the GRIK3 expressed differentially between gastric cancer tissues and the adjacent normal tissues and that higher expression of GRIK3 was associated with poor survival outcomes. And the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that GRIK3 mainly took part in cancer-related process. Subsequently, the validated immunohistochemistry showed that GRIK3 expressed higher in the GC tissues than in the matched normal tissues and the patients with overexpressed GRIK3 had worse survival outcomes. The univariate and multivariate analyses suggested that the expression of GRIK3 was an independent prognostic factor to predict GC prognosis. Furthermore, additional experiment showed that the lymph node metastasis tissues had higher GRIK3 expression than their matched primary GC tissues. These findings suggested that elevated GRIK3 expression could serve as an independent prognostic biomarker and a novel potential treatment target for patients with GC.

Estrogen promotes tumor metastasis via estrogen receptor beta-mediated regulation of matrix-metalloproteinase-2 in non-small cell lung cancer.

In non–small cell lung cancer (NSCLC), estrogen significantly promotes NSCLC cell growth via estrogen receptor beta (ERβ). However, the effects by which ERβ contributes to metastasis in NSCLC have not been previously reported. This study aims at defining whether the stimulation of ERβ promotes NSCLC metastasis in vitro and in vivo. Here, Our results showed that estrogen and ERβ agonist enhanced aggressiveness of two lung cancer cell lines (A549 and H1793) and promoted murine lung metastasis formation. ER-inhibitor Fulvestrant treatment or ERβ-knockdown significantly suppressed the migration, invasion and nodule formation of NSCLC cells. The expression level of ERβ protein was analyzed in matched samples of metastatic lymph node and primary tumor tissues from the same individuals, and we found significantly higher levels of ERβ were expressed in lymph node compared to primary tumor tissues. Moreover, Studies on both surgical biopsies and on lung cancer cells revealed that the expression level of ERβ and matrix-metalloproteinase-2 (MMP-2) were associated. Furthermore, inhibition of ERβ resulted in down-regulation of MMP-2 expression. Taken together, our results demonstrate that activation of ERβ in lung cancer cells promotes tumor metastasis through increasing expression of invasiveness-associated MMP-2. These results also highlight the therapeutic potential of inhibition of ERβin the treatment of advanced NSCLC.

Correlation of expression of epithelial cell adhesion molecule and lymph node metastasis of breast cancer

Objective To investigate the expression and significance of epithelial cell adhesion molecule (Ep-CAM) in breast cancer. Methods Twenty-three cases of breast cancer tissue samples and paired lymph node metastases confirmed pathologically were collected. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology was used to screen and identify the differentially expressed proteins between primary tumor and lymph node metastasis of breast cancer. The expression of Ep-CAM was detected by Western blotting in 4 cases of primary breast cancer tissues and paired lymph node metastases. And the expressions of Ep-CAM in 252 cases of breast lesions were detected by immunohistochemical method. Results Quantitative proteomic examination results showed that differentially expressed proteins existed in breast cancer primary tumor and lymph node metastasis, and the expression of Ep-CAM in metastatic lesions was higher than that in primary tumor. Western blotting results showed that the expression of Ep-CAM in metastatic lesions (1.46±0.22) was higher than that in primary tumor (1.16±0.09), which was consistent with the results of proteomic. The immunohistochemical results showed that the positive expression rate of Ep-CAM in lymph node metastasis tissues (93.16%, 109/117) was significantly higher than that in primary breast cancer without metastasis (72.73%, 64/88), and the difference was statistically significant (χ2=15.921, P=0.000). The positive expression rate of Ep-CAM in primary breast cancer with lymphatic metastasis (72.65%, 85/117) was lower than that in paired lymph node metastases (P=0.001). Conclusion Ep-CAM is diffe-rentially expressed in primary tumor and lymph node metastasis of breast cancer, which may be related to the lymphatic metastasis of breast cancer. Key words: Cell adhesion molecule; Breast neoplasms; Proteomics; Immunohistochenmistry

MicroRNA-144-3p suppresses gastric cancer progression by inhibiting epithelial-to-mesenchymal transition through targeting PBX3

MicroRNAs are aberrantly expressed in a wide variety of human cancers. The present study aims to elucidate the effects and molecular mechanisms of miR-144-3p that underlie gastric cancer (GC) development. It was observed that miR-144-3p expression was significantly decreased in GC tissues compared to that in paired non-tumor tissues; moreover, its expression was lower in tissues of advanced stage and larger tumor size, as well as in lymph node metastasis tissues compared to that in control groups. miR-144-3p expression was associated with depth of invasion (P = 0.030), tumor size (P = 0.047), lymph node metastasis (P = 0.047), and TNM stage (P = 0.048). Additionally, miR-144-3p significantly inhibited proliferation, migration, and invasion in GC cells. It also reduced F-actin expression and suppressed epithelial-to-mesenchymal transition (EMT) in GC cells. Furthermore, pre-leukemia transcription factor 3 (PBX3) was a direct target gene of miR-144-3p. PBX3 was overexpressed in GC tissues and promoted EMT in GC cells. The effects of miR-144-3p mimics or inhibitors on cell migration, invasion, and proliferation were reversed by PBX3 overexpression or downregulation respectively. These results suggest that miR-144-3p suppresses GC progression by inhibiting EMT through targeting PBX3.

MiR 1296-5p Inhibits the Migration and Invasion of Gastric Cancer Cells by Repressing ERBB2 Expression.

The metastasis of gastric cancer, one of the most common tumors, has a molecular mechanism that is still largely unclear. Here we investigated the role of possible tumor-suppressor miR-1296-5p in the cell migration and invasion of ERBB2-positive gastric cancer. It found that miR-1296-5p was significantly down-regulated in gastric cancer tissues. Moreover, it was down-regulated in lymph node metastatic gastric cancer tissues compared with non-metastatic gastric cancer tissues. The luciferase activity of ERBB2 3'-untranslated region-based reporters constructed in SNU-216 and NUGC-4 gastric cancer cells suggested that ERBB2 was the target gene of miR-1296-5p. Overexpressed miR-1296-5p reduced its target protein level and Rac1 activation, and inhibited the migration and invasion of SNU-216 and NUGC-4 gastric cancer cells. MiR-1296-5p was down-regulated in ERBB2-positive gastric cancer tissues compared with ERBB2-negative gastric cancer tissues. In ERBB2-positive gastric cancers, the miR-1296-5p expression was suppressed in a majority of metastatic lymph node tissues compared to non-metastatic gastric cancer samples. The migration and invasion of gastric cancer cells was inhibited by miR-1296-5p overexpression or herceptin treatment, and rescued by the overexpression of constitutively active Rac1-Q61L or ERBB2. Taken together, our findings first suggest that miR-1296-5p might be involved in the regulation on the migration and invasion of human gastric cancer cells at least in part via targeting ERBB2/Rac1 signaling pathway.

Identification of Nucleobindin-2 as a Potential Biomarker for Breast Cancer Metastasis Using iTRAQ-based Quantitative Proteomic Analysis.

Metastasis is a lethal step in the progression of breast cancer. None of the metastasis-associated biomarkers identified up to now has a definite prognostic value in breast cancer patients. This study was designed to identify biomarkers for breast cancer metastasis and predictors of the prognosis of breast cancer patients. The differentially expressed proteins between 23 paired primary breast tumor and metastatic lymph nodes were identified by quantitative iTRAQ proteomic analysis. Immunohistochemistry was applied to locate and assess the expression of NUCB2 in paired primary breast tumor and metastatic lymph node tissues (n = 106). The relationship between NUCB2 expression and the clinicopathological characteristics of breast cancer patients (n = 189) were analyzed by χ2 test. Kaplan-Meier analysis and Cox hazard regression analysis were utilized to investigate the relationship between its expression and prognosis of breast cancer patients. The iTRAQ proteomic results showed that 4,837 confidential proteins were identified, 643 of which were differentially expressed in the primary breast cancer tissues and the paired metastatic lymph nodes. NUCB2 protein was found decreased in paired metastatic lymph nodes (P = 0.000), with the positive expression rate being 82% in primary breast cancer tissues and 47% in paired metastatic lymph nodes, respectively. According to Kaplan-Meier analysis, the overall survival time of patients with positive expression of NUCB2 protein were shorter than those with negative NUCB2 expression (P = 0.004). Cox regression model suggested that NUCB2 was a risk factor of breast cancer patients (P = 0.045, RR = 1.854). We conclude that NUCB2 can be used as a potential biomarker for breast cancer metastasis and a prognostic predictor of breast cancer patients.

Therapeutic Outcomes of Patients with Multifocal Papillary Thyroid Microcarcinomas and Larger Tumors.

A retrospective review of 626 patients with multifocal papillary thyroid carcinoma (PTC) including 147 patients (23.5%) with multifocal papillary thyroid microcarcinoma (PTMC) from a total of 2,536 patients with PTC who visited the Chang Gung Medical Center in Linkou, Taiwan, was performed. A comparison of the clinical features between 626 multifocal and 1,910 solitary PTC cases showed that patients in the multifocal PTC group were older and had a smaller mean tumor size, a more advanced tumor-node-metastasis (TNM) stage, and a higher percentage of nonremission status compared to patients in the solitary PTC group. Of the 626 patients with multifocal PTC, the group with larger tumors showed a more advanced TNM stage, a higher percentage of lymph node metastasis and soft tissue invasion, and a higher nonremission rate compared to the multifocal PTMC group. Of the 626 patients with multifocal PTC, 25 patients (4%) died during a mean follow-up period of 7.1 ± 5.3 years. Kaplan-Meier survival curves showed a significantly lower survival rate associated with multifocal PTMC compared to that with solitary PTMC.

Identification of lymph node metastasis-related microRNAs in lung adenocarcinoma and analysis of the underlying mechanisms using a bioinformatics approach.

This study aimed to screen lymphatic metastasis-related microRNAs (miRNAs) in lung adenocarcinoma and explore their underlying mechanisms using bioinformatics. The miRNA expression in primary lung adenocarcinoma, matched adjacent non-tumorigenic and lymph node metastasis tissues of patients were profiled via microarray. The screened metastasis-related miRNAs were then validated using quantitative real-time PCR in a second cohort of lung adenocarcinoma patients with lymphatic metastasis. Significance was determined using a paired t-test. Target genes of the metastasis-related miRNAs were predicted using TargetScan, and transcription factors (TFs) were predicted based on the TRANSFAC and ENCODE databases. Furthermore, the related long non-coding RNAs (lncRNAs) were screened with starBase v2.0. The miRNA-TF-mRNA and lncRNA-miRNA-mRNA networks were constructed to determine the key interactions associated with lung adenocarcinoma metastasis. According to the miRNA microarray results, there were 10 miRNAs that were differentially expressed in metastatic tissues compared with primary tumor and adjacent non-tumorigenic tissues. Among them were increased levels of miR-146a-5p, miR-342-3p, and miR-150-5p, which were validated in the second cohort. Based on the miRNA-TF-mRNA network, vascular endothelial growth factor A and transcription factors (TFs) including TP53, SMAD4, and EP300 were recognized as critical targets of the three miRNAs. Interactions involving SNHG16-miR-146a-5p-SMAD4 and RP6-24A23.7-miR-342-3p/miR-150-5p-EP300 were highlighted according to the lncRNA-miRNA-mRNA network. miR-146a-5p, miR-342-3p, and miR-150-5p are lymphatic metastasis-related miRNAs in lung adenocarcinoma. Bioinformatics analyses demonstrated that SNHG16 might inhibit the interaction between miR-146a-5p and SMAD4, while RP6-24A23.7 might weaken miR-342-3p-EP300 and miR-150-5p-EP300 interactions in metastasis.

Abstract A141: Prognostic value of cancer stem cells (CSC) in head and neck squamous carcinoma (HNSCC)

Abstract Introduction: Cancer stem-cell (CSC) theory of tumorigenesis has been established during the last decade. There are phenotypically different subpopulations of cells in found in HNSCC tissue including diverse cancer cells, stromal cells and infiltrating inflammatory cells. According to a CSC theory only a small subpopulation of tumor cells can reproduce themselves and sustain the tumor growth. CSC subpopulation can retain some of the markers (surface epitopes) typical for normal stem cells, the most often mentioned being CD44+, CD29+, CD24+ and CD133+ markers. Isolated CSCs can be transplanted to the immunodeficient animal host and drive a tumor growth. Material and Methods: Peripheral blood samples together with primary tumor, metastatic and non-metastatic lymph node tissue samples were collected from 178 patients with primary HNSCC. We examined peripheral blood from all patients with the focus on lymphocyte subpopulation (CD3+, CD4+CD25+, CD4+/CD8+, CD19+, CD4+CD45RA+, CD8+CD28-, CD3-CD16+CD56+, CD4+CD25+Foxp3+, CD4+161+, CD8+161+) before the commencement of anti-tumor therapy. The level of the markers of cancer stem cells (CD44+, CD133+, CD29+) on the surface of the cancer cells, the infiltration of immunocompetent cells in the tumor microenvironment of specimens from primary tumors, from metastases to the neck lymph nodes and in control lymph nodes was measured, where samples were taken during the surgery. The correlation of cancer stem cells (CD44+, CD133+, CD29+) with the prognosis of the patient was analyzed with a special interest. Mean follow-up was 40 months. Results: A significant representation of cells expressing characteristics of CSCs has been identified in the tumor microenvironment of HNSCC. Subpopulation of CD44 + keratinocytes directly correlated with the presence of negative histological features (angioinvasion, lymphangioinvasion, perineural spread) (p = 0.007), i.e. factors significantly influencing the patient´s prognosis (p = 0.03). Conversely, CD133 + cells in the primary tumor negatively correlated with N stage (p = 0.04), thus suggesting that a higher proportion of CD133 + keratinocytes in the primary tumor yields lower metastatic potential. Presence of CD44 + and CD29 + keratinocytes does not correlate with N stage (p = 0.8 respectively. P = 0.4). Conclusion: Cells with CSCs characteristics can be shown in the HNSCC tumor tissue. Presence of certain subpopulations of keratinocytes (CD44+, CD133+) expressing the cell surface markers of CSCs can elucidate the patient´s prognosis. The therapy targeted to the cancer stem cells, in combination with immunotherapy, could increase the survival of the patients with HNSCC. Acknowledgements: The research was supported by AZV MZ CR (grant No. NV16-28594A and NV16-28600A) Note: This abstract was not presented at the conference. Citation Format: Michal Zabrodsky, Jan Bouček. Prognostic value of cancer stem cells (CSC) in head and neck squamous carcinoma (HNSCC) [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A141.