Alterations In Cervical Cancer Research Articles

Some Polyphenolic Compounds as Potential Therapeutic Agents in Cervical Cancer: The Most Recent Advances and Future Prospects.

The leading causes of cancer include gradual changes in regulatory proteins, dysregulated cell-signaling pathways, dysfunction of apoptosis, and oxidative stress. Consuming polyphenols from food sources has been proven to have strong connections with ameliorating specific physiological biomarkers along with other elements concerning cancer. Recent studies have focused on polyphenols' molecular mechanisms of action and anticancer and chemopreventive properties and effects in the treatment of different types of cancer. Polyphenols participate in the regulation of numerous cellular mechanisms alongside signaling pathways through their effects on inflammation, cellular proliferation, apoptosis, and partially via epigenetic alterations in cervical cancer. A number of animal models and cell and human studies have indicated the use of polyphenols to be safe and tolerable. Thus, it would be fair to state that, with their advantages vis-à-vis lack of toxicity, cost, and access, and with the positive clinical results, polyphenols have a potential to make a difference in cancer treatment. The present review examined the chemical and physical properties, analogs, metabolites, and mechanisms of physiological activities of various polyphenols and how they may affect the incidence rate and management of cervical cancer. Therefore, this review constitutes a starting point to examine the potential applications for cervical cancer.

Identification of Cross-Pathway Connections via Protein-Protein Interactions Linked to Altered States of Metabolic Enzymes in Cervical Cancer.

Metabolic reprogramming is one of the emerging hallmarks of cancer cells. Various factors, such as signaling proteins (S), miRNA, and transcription factors (TFs), may play important roles in altering the metabolic status in cancer cells by interacting with metabolic enzymes either directly or via protein-protein interactions (PPIs). Therefore, it is important to understand the coordination among these cellular pathways, which may provide better insight into the molecular mechanism behind metabolic adaptations in cancer cells. In this study, we have designed a cervical cancer-specific supra-interaction network where signaling pathway proteins, TFs, and microRNAs (miRs) are connected to metabolic enzymes via PPIs to investigate novel molecular targets and connections/links/paths regulating the metabolic enzymes. Using publicly available omics data and PPIs, we have developed a Hidden Markov Model (HMM)-based mathematical model yielding 94, 236, and 27 probable links/paths connecting signaling pathway proteins, TFs, and miRNAs to metabolic enzymes, respectively, out of which 83 paths connect to six common metabolic enzymes (RRM2, NDUFA11, ENO2, EZH2, AKR1C2, and TYMS). Signaling proteins (e.g., PPARD, BAD, GNB5, CHECK1, PAK2, PLK1, BRCA1, MAML3, and SPP1), TFs (e.g., KAT2B, ING1, MED1, ZEB1, AR, NCOA2, EGR1, TWIST1, E2F1, ID4, RBL1, ESR1, and HSF2), and miR (e.g., mir-147a, mir-593-5p, mir-138-5p, mir-16-5p, and mir-15b-5p) were found to regulate two key metabolic enzymes, EZH2 and AKR1C2, with altered metabolites (L-lysine and tetrahydrodeoxycorticosterone, THDOC) status in cervical cancer. We believe, the biology-based approach of our system will pave the way for future studies, which could be aimed toward identifying novel signaling, transcriptional, and post-transcriptional regulators of metabolic alterations in cervical cancer.

Genome-wide somatic copy number alteration analysis and database construction for cervical cancer.

Cervical cancer is a common gynecological malignancy with high incidence and mortality. Somatic copy number alterations (CNAs) play an important role in identifying tumor suppressor genes and oncogenes and are a useful diagnostic indicator for many cancer types. However, the genomic landscape of CNAs in cervical cancer has not yet been comprehensively characterized. In the present study, we collected 974 cervical cancer samples from different data sources. All samples were analyzed by genomic arrays to obtain high-resolution CNAs. Focal genomic regions with CNA events and potential cancer driver genes were identified by GISTIC2.0. Meanwhile, we constructed a comprehensive cervical cancer database by PHP and self-written Perl and R scripts. In total, 54 recurrent regions of amplification and deletion were detected. Frequently altered tumor suppressor genes were found in these regions, including PIK3CA, ERBB2, EP300 and FBXW7. CNA hotspots and related enriched functional categories were also identified. The incidence of chromothripsis in cervical cancer was estimated to be 6.06%, and the chromosome pulverization hotspot regions were detected. Based on the curated data, we developed CNAdbCC (http://cailab.labshare.cn/CNAdbCC/), a comprehensive database for copy number alterations in cervical cancer. We provide a user-friendly Web interface for data mining and visualization. It is the most comprehensive public database devoted exclusively to genomic alterations in cervical cancer. These results extend our molecular understanding of cervical cancer. The database will enable researchers to explore specific CNA patterns in this lethal cancer and facilitate the discovery of therapeutic candidates.

Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.

Clinical factors associated with comprehensive genomic variation profiling of cervical cancer.

62 Background: To better understand the mechanism contributing to cervical carcinogenesis, we perform a comprehensive analysis of genomic alterations in cervical cancer (CC). Methods: We conducted whole-exome sequencing of 43 CCs and established strict integrated workflow of genomic analysis. Correlation analysis was performed to measure the relationships between gene mutations and clinical characteristics of CC patients. Results: Genomic analysis revealed 28 genes frequently mutated in CC including BRD4 (4/43), AXL (4/43), MED12 (4/43), which are undetermined genomic features in CC, and identified recurrent genetic mutations in PIK3CA (16/43), KMT2D (11/43), KMT2C (6/43), FBXW7 (5/43), FAT1 (5/43), ERBB2 (4/43), EP300 (3/43) and NFE2L2 (3/43). Intriguingly, CC patients harbored high frequent mutations in BRCA2 (7/43), but not in BRCA1. The relationship between BRCA2 mutations and clinical factors is yet to be determined. The identified mutations predominately resulted in the dysregulation of the PI3K/AKT/mTOR pathway. Interestingly, we found that AXL mutations were positively correlated with FIGO stage ( P = 0.028), regional lymph node metastasis ( P = 0.011) and distant metastasis ( P = 0.022). KMT2C mutations were positively correlated with FIGO stage ( P = 0.004) and distant metastasis ( P = 0.009). SF3B1 mutations were positively correlated with regional lymph node metastasis ( P = 0.027) and distant metastasis ( P< 0.001). NFE2L2, ERBB2, RAC1, MED12, MET, BAP1, PTPRD and HNF1Amutations were positively correlated with distant metastasis ( P = 0.045, P = 0.004, P = 0.022, P = 0.001, P< 0.001, P< 0.001, P = 0.014, P< 0.001, respectively). POLD1 mutations were positively associated with regional lymph node metastasis ( P = 0.029). However, NOTCH1 mutations were negatively associated with regional lymph node metastasis ( P = 0.047). STK11 mutations were negatively associated with FIGO stage ( P = 0.013). Conclusions: Our results highlight the application of deep sequencing for understanding the molecular mechanisms and uncovering potential diagnostic and therapeutic targets of CC.

Hypomethylation of the lncRNA SOX21-AS1 has clinical prognostic value in cervical cancer

AimsCervical cancer seriously affects women's health. The function of methylated alterations in the long non-coding RNAs (lncRNAs) promote the progression and metastasis of cancer. Our study aims to identify the functional effects of lncRNA methylation in cervical carcinogenesis. Main methodsGenome-wide DNA methylation of 6 samples was assessed using the Illumina Infinium MethylationEPIC BeadChip. RNA sequencing (RNA-seq) data and survival follow-up time of 307 samples from The Cancer Genome Atlas (TCGA) dataset were enrolled in this study. The statistical analysis and graphical work were mainly realized by R language. Key findingsMethylation map identified 3962 hypermethylated CpG sites and 4484 hypomethylated CpG sites in cervical cancer (|Δβ| ≥ 0.20). Bioinformatic analysis of the lncRNA expression identified 363 upregulated and 664 downregulated lncRNAs with log2 (fold change) ≥ 1.00 in squamous cervical carcinoma (SCC) samples. Weighted gene co-expression network analysis (WGCNA) and Venn diagram revealed that lncRNA MAGI2 antisense RNA 3 (lncRNA MAGI2-AS3), lncRNA WT1 antisense RNA (lncRNA WT1-AS) and lncRNA SOX21 antisense divergent transcript 1 (lncRNA SOX21-AS1) were important methylation changed lncRNAs. Kaplan-Meier survival curves showed only lncRNA SOX21-AS1 had clinical prognostic value in cervical cancer. Gene set enrichment analysis (GSEA) suggest that lncRNA SOX21-AS1 involve in the multiple cellular processes and might significantly suppress cervical tumorigenesis. SignificanceThese insights into the functional role of lncRNA SOX21-AS1 DNA methylome alterations in cervical cancer might promote clinically new applicable in diagnosis and prognosis.

MGL Ligand Expression Is Correlated to Lower Survival and Distant Metastasis in Cervical Squamous Cell and Adenosquamous Carcinoma.

Cervical cancer is the fourth most common cancer type in women worldwide and is characterized by a highly immune-suppressive microenvironment. Here, we describe aberrant glycosylation as a factor mediating this immunosuppressive microenvironment. Expression of a specific carbohydrate ligand for the immune-regulatory C-type lectin MGL was correlated to poor disease-specific survival and distant recurrences in squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC), the most common histological subtypes of cervical cancer. MGL ligand expression was also associated with lymph node metastasis, the absence of CD14+ myeloid cells and the presence of CD14−CD163+ myeloid cells. Indeed, expression of the MGL receptor itself could be detected on CD163+ cells, suggesting that MGL+ myeloid cells are able to interact locally with MGL ligand+ tumor cells. Additionally, MGL ligand expression correlated to the occurrence of PIK3CA mutations, the most frequently observed oncogenic alteration in cervical cancer. In conclusion, we present prognostic value for MGL ligand expression in SCC/ASC patients, which further supports an immune evasive role for the C-type lectin MGL in the tumor immune compartment.

Integrated analysis of HPV-mediated immune alterations in cervical cancer

ObjectiveHuman papillomavirus (HPV) infection is the primary cause of cervical cancer. HPV-mediated immune alterations are known to play crucial roles in determining viral persistence and host cell transformation. We sought to thoroughly understand HPV-directed immune alterations in cervical cancer by exploring publically available datasets. Methods130 HPV positive and 7 HPV negative cervical cancer cases from The Cancer Genome Atlas were compared for differences in gene expression levels and functional enrichment. Analyses for copy number variation (CNV) and genetic mutation were conducted for differentially expressed immune genes. Kaplan-Meier analysis was performed to assess survival and relapse differences across cases with or without alterations of the identified immune signature genes. ResultsGenes up-regulated in HPV positive cervical cancer were enriched for various gene ontology terms of immune processes (P=1.05E-14~1.00E-05). Integrated analysis of the differentially expressed immune genes identified 9 genes that displayed either CNV, genetic mutation and/or gene expression changes in at least 10% of the cases of HPV positive cervical cancer. Genomic amplification may cause elevated levels of these genes in some HPV positive cases. Finally, patients with alterations in at least one of the nine signature genes overall had earlier relapse compared to those without any alterations. The altered expression of either TFRC or MMP13 may indicate poor survival for a subset of cervical cancer patients (P=1.07E-07). ConclusionsWe identified a novel immune gene signature for HPV positive cervical cancer that is potentially associated with early relapse of cervical cancer.

Identification of SEC62 as a potential marker for 3q amplification and cellular migration in dysplastic cervical lesions.

BackgroundChromosome 3 amplification affecting the 3q26 region is a common genomic alteration in cervical cancer, typically marking the transition of precancerous intraepithelial lesions to an invasive phenotype. Though potential 3q encoded target genes of this amplification have been identified, a functional correlation of potential oncogenic function is still missing. In this study, we investigated copy number changes and the expression level of SEC62 encoded at 3q26.2 as a new potential 3q oncogene in dysplastic cervical lesions and analyzed its role in cervical cancer cell biology.MethodsExpression levels of Sec62 and vimentin were analyzed in liquid based cytology specimens from 107 women with varying grades of cervical dysplasia ranging from normal cases to cancer by immunofluorescence cytology. Additionally, a subset of 20 representative cases was used for FISH analyses targeting SEC62. To further explore the functional role of Sec62 in cervical cancer, HeLa cells were transfected with a SEC62 plasmid or SEC62 siRNA and analyzed for their proliferation and migration potential using real-time monitoring and trans-well systems as well as changes in the expression of EMT markers.ResultsFISH analyses of the swabbed cells showed a rising number of SEC62 gains and amplifications correlating to the grade of dysplasia with the highest incidence in high grade squamous intraepithelial lesions and squamous cell carcinomas. When analyzing the expression level of Sec62 and vimentin, we found a gradually increasing expression level of both proteins according to the severity of the dysplasia. In functional analyses, SEC62 silencing inhibited and SEC62 overexpression stimulated the migration of HeLa cells with only marginal effects on cell proliferation, the expression level of EMT markers and the cytoskeleton structure.ConclusionsOur study suggests SEC62 as a target gene of 3q26 amplification and a stimulator of cellular migration in dysplastic cervical lesions. Hence, SEC62 could serve as a potential marker for 3q amplification, providing useful information about the dignity and biology of dysplastic cervical lesions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2739-6) contains supplementary material, which is available to authorized users.

Abstract 434: Rational molecular assessment and innovative drug selection (RAIDs): exome data from cervical cancer

Abstract Background Recent retrospective data1,2 identified major molecular alterations in cervical cancer (CC), but so far there has been no prospective assessment on patient outcome using a complete molecular profiling with quality control evaluation of treatment. The Cetuxicol (phase 2) clinical trial showed that the addition of Cetuximab over a 6 week period, did not improve DFS. PI3K pathway mutations in the tumor in the Cetuximab treatment arm led to a worse DFS3. We are lacking prognostic and predictive biomarkers for CC treatment and there is a growing need for the development of biomarkers to follow up the course of the disease. Methodology RAIDs (http://www.raids-fp7.eu) is a multidisciplinary co-operation between academic clinical centers, SMEs and translational research platforms in seven European countries. It includes: 1) a cognitive cohort study (BioRAIDs)4, one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 500 patients enrolled in BioRAIDs combine Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor (ct) DNA at ERASMUS (The Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France). Bioinformatics pipelines to detect somatic mutations and clustering methods were developed in order to stratify the patients into different subtypes. Following quality control, the pharmacological profiling of a panel of 20 CC cell lines using a panel of drugs which may potentially synergize with “standard treatment” has been completed. Drugs were chosen so as to interfere with different signaling pathways. Results Molecular profiles including exome sequencing and ctDNA analyses on 48 quality controlled samples from the BioRAIDs patients and 20 CC cell lines will be presented. Preliminary results on HPV insertion sites in tumor and serum will be reported. Somatic and DNA copy number alterations from exome sequencing profiles confirm PI3K pathway mutations to be a dominant feature in CC. Stratifications of patients’ tumors based on their mutation profiles show that some cell lines cluster similarly to patients’ tumors, suggesting that they will be helpful for the prediction of response to drugs for patients within the same cluster. Conclusions The identification of predictive tumor/blood based biomarkers will permit the definition of new strategies for precision medicine in CC. A bioinformatics analysis which will assess drug responsiveness in CC cell lines in relation to specific genomic alterations is ongoing. Its results should permit to select treatments according to genetic “constellations” of the tumors. Citation Format: Suzy Scholl, Maud Kamal, Els Berns, Balzs Balint, Attila Kereszt, Leanne de Koning, Emmanuelle Jeannot, Windy Luscap-rondof, Vonick Sibut, Philippe Hupe, Gemma Kenter, Sanne Samuels, Katja Jordanova, Sandrine Blanchet, Laurence Lafanachere, Marc Billaud, RAIDs consortium. Rational molecular assessment and innovative drug selection (RAIDs): exome data from cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 434.

New molecular targets against cervical cancer.

Cervical cancer is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer death in women. Major advances but still insufficient achievements in the treatment of locally advanced and high-risk early stage patients have occurred in the last decade with the incorporation of concurrent cisplatin with radiation and, lately, gemcitabine added to cisplatin chemoradiation. Despite a number of clinical studies incorporating molecular-targeted therapy as radiosensitizers being in progress, so far, only antiangiogenic therapy with bevacizumab added to cisplatin chemoradiation has demonstrated safety and shown encouraging results in a Phase II study. In advanced disease, cisplatin doublets do not have a great impact on the natural history of the disease with median survival rates not exceeding 13 months. The first Phase III study of bevacizumab, added to cisplatin or a non-cisplatin-containing doublet, showed significant increase in both overall survival and progression-free survival. Further studies are needed before bevacizumab plus chemotherapy can be considered the standard of care for advanced disease. Characterization of the mutational landscape of cervical cancer has already been initiated, indicating that, for now, few of these targetable alterations match with available agents. Progress in both the mutational landscape knowledge and developments of novel targeted therapies may result in more effective and individualized treatments for cervical cancer. The potential efficacy of knocking down the key alterations in cervical cancer – E6 and E7 human papillomavirus oncoproteins – must not be overlooked.

Genome-wide DNA methylation assay reveals novel candidate biomarker genes in cervical cancer

The oncogenic human papilloma viruses (HPVs) are associated with precancerous cervical lesions and development of cervical cancer. The DNA methylation signatures of the host genome in normal, precancerous and cervical cancer tissue may indicate tissue-specific perturbation in carcinogenesis. The aim of this study was to identify new candidate genes that are differentially methylated in squamous cell carcinoma compared with DNA samples from cervical intraepithelial neoplasia grade 3 (CIN3) and normal cervical scrapes. The Illumina Infinium HumanMethylation450 BeadChip method identifies genome-wide DNA methylation changes in CpG islands, CpG shores and shelves. Our findings showed an extensive differential methylation signature in cervical cancer compared with the CIN3 or normal cervical tissues. The identified candidate biomarker genes for cervical cancer represent several types of mechanisms in the cellular machinery that are epigenetically deregulated by hypermethylation, such as membrane receptors, intracellular signaling and gene transcription. The results also confirm extensive hypomethylation of genes in the immune system in cancer tissues. These insights into the functional role of DNA methylome alterations in cervical cancer could be clinically applicable in diagnostics and prognostics, and may guide the development of new epigenetic therapies.

HPV Pathway Profiling: HPV Related Cervical Dysplasia and Carcinoma Studies

Cervical cancer is the second most common malignancy among women worldwide. While more than 90% of cervical cancers carry one or more high risk HPV types, the exact relationship between HPV oncoproteins and signaling pathways alterations in cervical cancer remains to be clarified. Vaccines and targeted therapies are today considered of great potential for the improvement of patients' outcome. This review will focus on the interaction of HPV with cervical cancer pathway activations. Next generation high throughput screening technologies for molecular and protein profiling as well as innovative logistics, necessary for the implementation of the personalized care of cervical cancer patients will be discussed.

HPV Pathway Profiling: HPV Related Cervical Dysplasia and Carcinoma Studies

Cervical cancer is the second most common malignancy among women worldwide. While more than 90% of cervical cancers carry one or more high risk HPV types, the exact relationship between HPV oncoproteins and signaling pathways alterations in cervical cancer remains to be clarified. Vaccines and targeted therapies are today considered of great potential for the improvement of patients’ outcome. This review will focus on the interaction of HPV with cervical cancer pathway activations. Next generation high throughput screening technologies for molecular and protein profiling as well as innovative logistics, necessary for the implementation of the personalized care of cervical cancer patients will be discussed.

Mutations of the TP53 gene in adenocarcinoma and squamous cell carcinoma of the cervix: A systematic review

ObjectiveMutations of the tumor suppressor gene TP53 are the most significant events in several human cancers. Few studies have analyzed the frequency of TP53 alterations in squamous cell carcinoma and adenocarcinoma of the cervix with controversial results. This study provides a detailed analysis of TP53 mutation spectra in cervical squamous cell carcinoma and adenocarcinoma from different geographical regions. MethodsThe analysis of TP53 mutational profiles was performed in 1353 cervical cancers retrieved from the IARC p53 mutation database (R15, 2010) and the COSMIC data along with the literature review of related studies identified by PubMed searching. ResultsThis analysis showed a significant higher mutation frequency of TP53 gene in cervical adenocarcinoma (32 of 241; 13.3%) compared to squamous cell carcinoma (39 of 657; 5.9%; P=0.0003, χ2 test). The proportion of adenocarcinoma with mutated TP53 varied from 4% in North America to 19% in Asia. Among the six hot-spot codons of TP53 gene, three codons (175, 248 and 273) were the most commonly mutated in both types of cervical cancer, one codon (249) mainly in squamous cell carcinoma and one codon (282) only in adenocarcinoma. The G to A and C to T transitions were the prevalent type of mutations in both squamous cell carcinoma and adenocarcinoma (48.7% and 53.5% of all mutations, respectively). The frequency of C to A transversion was relatively high only in adenocarcinoma (25%), while the mirror mutation G to T was comparatively frequent in squamous cell carcinoma (14.6%). ConclusionsDifferent patterns of TP53 mutations occur in squamous cell carcinoma and adenocarcinoma of the cervix in different regions of the world. The highest frequency of mutated TP53 has been observed in cervical adenocarcinoma from Asia. Further studies are needed to better define the role of TP53 alterations in cervical cancer and possibly to understand the impact of mutations on cancer prognosis and outcomes.

Abstract 3202: Decreased Dkk3 protein expression is associated with a poor prognosis of cervical cancer

Abstract Dkk3 has tumor suppressor properties in various cancers. Recently, we reported that the mRNA level of Dkk3 gene was down-regulated by epigenetic alteration in cervical cancer and that forced expression of Dkk3 resulted in the growth inhibition of cervical cancer cells. The aim of this study was to assess the expression of Dkk3 protein in cervical cancer tissue and determine potential clinical correlations. Immunohistochemical analysis was performed on 94 cervical cancer tissues. Clinical and pathological parameters were obtained from medical record. Survival data were estimated using Kaplan-Meier estimates and compared with log-rank test where indicated. Out of 94 tissue samples, negative, weak, moderate and high expression of Dkk3 were observed in 29 (30.9%), 31 (33.0%), 18 (19.1%) and 16 (17.0%) tissue samples. Dkk3 expression was negatively correlated with FIGO clinical stage (p<0.03, Mann-Whitney test). The 5-year disease free survival of patients with a negative or weak expression was significantly lower than that of those with moderate or high expression (70.6% versus 86.3%, respectively, p=0.04, log-rank test). The result of this study showed that down-regulation of Dkk3 is associated with advanced FIGO clinical stage and poor survival outcome of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3202. doi:10.1158/1538-7445.AM2011-3202

Abstract LB-270: Exploration of TCGA generated DNA copy number data in ovarian cancer points to interesting proapaptotic gene for survival prediction

Abstract Exploration of TCGA generated DNA copy number data in ovarian cancer points to interesting proapaptotic gene for survival prediction Using data generated by The Cancer Genome Atlas (TCGA) project, we investigated the use of copy number changes in prediction of both response to therapy as well as identification of possible prognostic survival bio-markers. The analysis was performed using an initial data set of 557 arrays (68 Affymetrix 6.0 and 489 Agilent 244K arrays). The raw data was processed using Nexus Copy Number version 5.1 software (BioDiscovery, El Segundo CA) and regions of copy number change were identified using the built-in FASST and SNP-FASST algorithms. As previously reported, using only the Agilent data set, we had identified a number of regions with statistically significant differences across between sample groups that responded to therapy as compared to those with progressive disease1. Performing gene enrichment analysis on these regions indicated a relationship with apoptosis. In our current study, we have also considered survival and have identified FAM105A located on the short arm of chromosome 5 as a possible component of Ovarian Cancer. A number of studies have identified the small 728Kb region between TRIO and ANKH gained in various cancers and hypothesized that these genes are possible oncognes2–3. Using Kaplan-Mier survival analysis, we have in fact been able to show that the FAM105A gene located between TRIO and ANKH is highly predictive of survival (p=0.00024) with the deletion causing a much poorer prognosis. This result was validated using the Affymetrix data on a different set of samples. Very little is known about this gene except the fact that using functional screening, it has demonstrated strong proapaptotic behavior which is in concordance with our findings here4. We are in the process of expanding the validation data set to several hundred samples. 2 Judith N Kloth, Jan Oosting, Tom van Wezel, Karoly Szuhai, Jeroen Knijnenburg, Arko Gorter, Gemma G Kenter, Gert Jan Fleuren, and Ekaterina S Jordanova, Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer, BMC Genomics. 2007; 8: 53. Published online 2007 February 20. doi: 10.1186/1471–2164–8-53. 3 Coe BP, Henderson LJ, Garnis C, Tsao MS, Gazdar AF, Minna J, Lam S, Macaulay C, Lam WL., High-resolution chromosome arm 5p array CGH analysis of small cell lung carcinoma cell lines, Genes Chromosomes Cancer. 2005 Mar;42(3):308–13. 4 Otto Mannherz, Daniel Mertens, Meinhard Hahn, Peter Lichter, Functional screening for proapoptotic genes by reverse transfection cell array technology, Genomics, Volume 87, Issue 5, May 2006, Pages 665–672, ISSN 0888–7543, DOI: 10.1016/j.ygeno.2005.12.009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-270. doi:10.1158/1538-7445.AM2011-LB-270

Expression of nuclear Notch3 in cervical squamous cell carcinomas and its association with adverse clinical outcomes

Objective The aim of this study was to clarify the functional role of Notch3 in human cervical carcinomas. Methods Notch3 expression in cervical cancer was assessed by immunohistochemistry, and data on clinical variables were collected by retrospective chart review. We used dual-color fluorescence in situ hybridization (FISH) to analyze DNA copy number alterations in cervical cancer. Inactivation of Notch3 and knocking down Notch3 gene were done using γ-secretase inhibitor and Notch 3 specific SiRNA to asses Notch3 function in cervical cancer either in vivo or in vitro. Results Immunohistochemical analysis revealed that Notch3 was significantly overexpressed in cervical squamous cell carcinomas compared with adenocarcinomas. In contrast to normal cervical tissue and cervical intraepithelial neoplasms [CINs], squamous cell carcinomas demonstrated higher nuclear Notch3 immunoreactivity. Notch3 amplification was not found in any cervical carcinomas using FISH analysis. Notch3 nuclear expression was significantly correlated with Jagged-1, a putative Notch3 ligand, and Pbx1b, a potential Notch3 downstream target ( P < 0.05).Patients with cervical carcinomas positive for nuclear Notch3 expression had significantly shorter overall survival than their peers whose tumors did not express nuclear Notch3. Inactivation of Notch3 decreased cell proliferation and induced apoptosis in ME180 and SKGIIIb cell lines that overexpressed Notch3. Injection of a γ-secretase inhibitor into ME180 cell tumors established on mice, demonstrated a reduction in tumor growth. Conclusion Our findings suggest that Notch3 might play important role for the proliferation and survival of Notch3 overexpressing tumors and that inactivation of Notch3 may represent a new therapeutic avenue for cervical squamous cell carcinomas.

Expression of Smad2 and Smad4 in cervical cancer: absent nuclear Smad4 expression correlates with poor survival

Alterations in transforming growth factor-Β signaling, due to a decrease in Smad2 and especially Smad4 expression, has primarily been reported in pancreatic and colorectal cancers, although loss of the chromosomal region 18q21.1, containing the loci of Smad2 and Smad4, is among the most frequent molecular alterations in cervical cancer. The aim of our study was to investigate whether decreased Smad2 and Smad4 protein expression in primary cervical cancers is associated with molecular alterations at 18q21.1, mutations in the functional domains of Smad2 and Smad4 or hypermethylation, and to assess the biological relevance of decreased Smad2 and Smad4 expression. Subsequently, Smad2, Smad4 and p21 protein expression was determined by immunohistochemistry in 117 primary cervical carcinomas, assembled in a tissue array. Smad signaling was shown to be associated with p21 mRNA expression. All the tumors expressed Smad2 or Smad4. Weak cytoplasmic Smad2 or weak cytoplasmic Smad4 expression could not be attributed to loss of heterozygosity at 18q21.1. Despite weak/moderate Smad2 expression and absent nuclear Smad4 expression, the coding regions of the functional MH1 and MH2 domains of Smad2 and Smad4 were unchanged, as assessed by sequence analysis. The Smad4 promoter region was unmethylated in tumor samples with weak/moderate cytoplasmic Smad4 expression. Remarkably, both weak cytoplasmic Smad4 expression and absent nuclear Smad4 expression significantly correlated with poor disease-free (P=0.003 and P=0.003, respectively) and overall 5-year survival (P=0.003 and P=0.010, respectively). Our findings support the hypothesis that Smad4 is a target molecule for functional inactivation in cervical cancer.

Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer

BackgroundCervical carcinoma develops as a result of multiple genetic alterations. Different studies investigated genomic alterations in cervical cancer mainly by means of metaphase comparative genomic hybridization (mCGH) and microsatellite marker analysis for the detection of loss of heterozygosity (LOH). Currently, high throughput methods such as array comparative genomic hybridization (array CGH), single nucleotide polymorphism array (SNP array) and gene expression arrays are available to study genome-wide alterations. Integration of these 3 platforms allows detection of genomic alterations at high resolution and investigation of an association between copy number changes and expression.ResultsGenome-wide copy number and genotype analysis of 10 cervical cancer cell lines by array CGH and SNP array showed highly complex large-scale alterations. A comparison between array CGH and SNP array revealed that the overall concordance in detection of the same areas with copy number alterations (CNA) was above 90%. The use of SNP arrays demonstrated that about 75% of LOH events would not have been found by methods which screen for copy number changes, such as array CGH, since these were LOH events without CNA. Regions frequently targeted by CNA, as determined by array CGH, such as amplification of 5p and 20q, and loss of 8p were confirmed by fluorescent in situ hybridization (FISH). Genome-wide, we did not find a correlation between copy-number and gene expression. At chromosome arm 5p however, 22% of the genes were significantly upregulated in cell lines with amplifications as compared to cell lines without amplifications, as measured by gene expression arrays. For 3 genes, SKP2, ANKH and TRIO, expression differences were confirmed by quantitative real-time PCR (qRT-PCR).ConclusionThis study showed that copy number data retrieved from either array CGH or SNP array are comparable and that the integration of genome-wide LOH, copy number and gene expression is useful for the identification of gene specific targets that could be relevant for the development and progression in cervical cancer.