Identification of Novel Therapeutic Targets in Microdissected Clear Cell Ovarian Cancers

Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this, patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. All the arrays were analyzed using BRB ArrayTools and PathwayStudio software to identify the signaling pathways. Identified pathways validated using serous, clear cell cancer cell lines and RNAi technology. In vivo validations carried out using an orthotopic mouse model and liposomal encapsulated siRNA. Patient-derived clear cell and serous ovarian tumors were grafted under the renal capsule of NOD-SCID mice to evaluate the therapeutic potential of the identified pathway. We identified major activated pathways in clear cells involving in hypoxic cell growth, angiogenesis, and glucose metabolism not seen in other histotypes. Knockdown of key genes in these pathways sensitized clear cell ovarian cancer cell lines to hypoxia/glucose deprivation. In vivo experiments using patient derived tumors demonstrate that clear cell tumors are exquisitely sensitive to antiangiogenesis therapy (i.e. sunitinib) compared with serous tumors. We generated a histotype specific, gene signature associated with clear cell ovarian cancer which identifies important activated pathways critical for their clinicopathologic characteristics. These results provide a rational basis for a radically different treatment for ovarian clear cell patients

Identification of differentially expressed genes using an annealing control primer system in stage III serous ovarian carcinoma

<p>Abstract</p> <p>Background</p> <p>Most patients with ovarian cancer are diagnosed with advanced stage disease (<it>i.e</it>., stage III-IV), which is associated with a poor prognosis. Differentially expressed genes (DEGs) in stage III serous ovarian carcinoma compared to normal tissue were screened by a new differential display method, the annealing control primer (ACP) system. The potential targets for markers that could be used for diagnosis and prognosis, for stage III serous ovarian cancer, were found by cluster and survival analysis.</p> <p>Methods</p> <p>The ACP-based reverse transcriptase polymerase chain reaction (RT PCR) technique was used to identify DEGs in patients with stage III serous ovarian carcinoma. The DEGs identified by the ACP system were confirmed by quantitative real-time PCR. Cluster analysis was performed on the basis of the expression profile produced by quantitative real-time PCR and survival analysis was carried out by the Kaplan-Meier method and Cox proportional hazards multivariate model; the results of gene expression were compared between chemo-resistant and chemo-sensitive groups.</p> <p>Results</p> <p>A total of 114 DEGs were identified by the ACP-based RT PCR technique among patients with stage III serous ovarian carcinoma. The DEGs associated with an apoptosis inhibitory process tended to be up-regulated clones while the DEGs associated with immune response tended to be down-regulated clones. Cluster analysis of the gene expression profile obtained by quantitative real-time PCR revealed two contrasting groups of DEGs. That is, a group of genes including: <it>SSBP1</it>, <it>IFI6 DDT</it>, <it>IFI27</it>, <it>C11orf92</it>, <it>NFKBIA</it>, <it>TNXB</it>, <it>NEAT1 </it>and <it>TFG </it>were up-regulated while another group of genes consisting of: <it>LAMB2</it>, <it>XRCC6</it>, <it>MEF2C</it>, <it>RBM5</it>, <it>FOXP1</it>, <it>NUDCP2</it>, <it>LGALS3</it>, <it>TMEM185A</it>, and <it>C1S </it>were down-regulated in most patients. Survival analysis revealed that the up-regulated genes such as <it>DDAH2, RNase K and TCEAL2 </it>might be associated with a poor prognosis. Furthermore, the prognosis of patients with chemo-resistance was predicted to be very poor when genes such as <it>RNase K, FOXP1</it>, <it>LAMB2 </it>and <it>MRVI1 </it>were up-regulated.</p> <p>Conclusion</p> <p>The DEGs in patients with stage III serous ovarian cancer were successfully and reliably identified by the ACP-based RT PCR technique. The DEGs identified in this study might help predict the prognosis of patients with stage III serous ovarian cancer as well as suggest targets for the development of new treatment regimens.</p

MicroRNA Expression Variability in Human Cervical Tissues

MicroRNAs (miRNAs) are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Deregulation of miRNA expression has been discovered in a wide variety of tumours and it is now clear that they contribute to cancer development and progression. Cervical cancer is one of the most common cancers in women worldwide and there is a strong need for a non-invasive, fast and efficient method to diagnose the disease. We investigated miRNA expression profiles in cervical cancer using a microarray platform containing probes for mature miRNAs. We have evaluated miRNA expression profiles of a heterogeneous set of cervical tissues from 25 different patients. This set included 19 normal cervical tissues, 4 squamous cell carcinoma, 5 high-grade squamous intraepithelial lesion (HSIL) and 9 low-grade squamous intraepithelial lesion (LSIL) samples. We observed high variability in miRNA expression especially among normal cervical samples, which prevented us from obtaining a unique miRNA expression signature for this tumour type. However, deregulated miRNAs were identified in malignant and pre-malignant cervical tissues after tackling the high expression variability observed. We were also able to identify putative target genes of relevant candidate miRNAs. Our results show that miRNA expression shows natural variability among human samples, which complicates miRNA data profiling analysis. However, such expression noise can be filtered and does not prevent the identification of deregulated miRNAs that play a role in the malignant transformation of cervical squamous cells. Deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of this tumour type

High-resolution analysis of copy number alterations and associated expression changes in ovarian tumors

<p>Abstract</p> <p>Background</p> <p>DNA copy number alterations are frequently observed in ovarian cancer, but it remains a challenge to identify the most relevant alterations and the specific causal genes in those regions.</p> <p>Methods</p> <p>We obtained high-resolution 500K SNP array data for 52 ovarian tumors and identified the most statistically significant minimal genomic regions with the most prevalent and highest-level copy number alterations (recurrent CNAs). Within a region of recurrent CNA, comparison of expression levels in tumors with a given CNA to tumors lacking that CNA and to whole normal ovary samples was used to select genes with CNA-specific expression patterns. A public expression array data set of laser capture micro-dissected (LCM) non-malignant fallopian tube epithelia and LCM ovarian serous adenocarcinoma was used to evaluate the effect of cell-type mixture biases.</p> <p>Results</p> <p>Fourteen recurrent deletions were detected on chromosomes 4, 6, 9, 12, 13, 15, 16, 17, 18, 22 and most prevalently on X and 8. Copy number and expression data suggest several apoptosis mediators as candidate drivers of the 8p deletions. Sixteen recurrent gains were identified on chromosomes 1, 2, 3, 5, 8, 10, 12, 15, 17, 19, and 20, with the most prevalent gains localized to 8q and 3q. Within the 8q amplicon, <it>PVT1</it>, but not <it>MYC</it>, was strongly over-expressed relative to tumors lacking this CNA and showed over-expression relative to normal ovary. Likewise, the cell polarity regulators <it>PRKCI </it>and <it>ECT2 </it>were identified as putative drivers of two distinct amplicons on 3q. Co-occurrence analyses suggested potential synergistic or antagonistic relationships between recurrent CNAs. Genes within regions of recurrent CNA showed an enrichment of Cancer Census genes, particularly when filtered for CNA-specific expression.</p> <p>Conclusion</p> <p>These analyses provide detailed views of ovarian cancer genomic changes and highlight the benefits of using multiple reference sample types for the evaluation of CNA-specific expression changes.</p

The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

<p>Abstract</p> <p>Background</p> <p>The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking.</p> <p>Methods</p> <p>SOX11 expression and clinicopathological data was compared using χ²test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation.</p> <p>Results</p> <p>SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5'-Aza-2'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11.</p> <p>Conclusions</p> <p>SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours.</p

Complete Sequencing of pNDM-HK Encoding NDM-1 Carbapenemase from a Multidrug-Resistant Escherichia coli Strain Isolated in Hong Kong

BACKGROUND: The emergence of plasmid-mediated carbapenemases, such as NDM-1 in Enterobacteriaceae is a major public health issue. Since they mediate resistance to virtually all β-lactam antibiotics and there is often co-resistance to other antibiotic classes, the therapeutic options for infections caused by these organisms are very limited. METHODOLOGY: We characterized the first NDM-1 producing E. coli isolate recovered in Hong Kong. The plasmid encoding the metallo-β-lactamase gene was sequenced. PRINCIPAL FINDINGS: The plasmid, pNDM-HK readily transferred to E. coli J53 at high frequencies. It belongs to the broad host range IncL/M incompatibility group and is 88803 bp in size. Sequence alignment showed that pNDM-HK has a 55 kb backbone which shared 97% homology with pEL60 originating from the plant pathogen, Erwina amylovora in Lebanon and a 28.9 kb variable region. The plasmid backbone includes the mucAB genes mediating ultraviolet light resistance. The 28.9 kb region has a composite transposon-like structure which includes intact or truncated genes associated with resistance to β-lactams (bla(TEM-1), bla(NDM-1), Δbla(DHA-1)), aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD, ΔtnpATn1 and insL. Certain blocks within the 28.9 kb variable region had homology with the corresponding sequences in the widely disseminated plasmids, pCTX-M3, pMUR050 and pKP048 originating from bacteria in Poland in 1996, in Spain in 2002 and in China in 2006, respectively. SIGNIFICANCE: The genetic support of NDM-1 gene suggests that it has evolved through complex pathways. The association with broad host range plasmid and multiple mobile genetic elements explain its observed horizontal mobility in multiple bacterial taxa

Robotic-assisted laparoscopic prostatectomy

Prostate cancer remains a significant health problem worldwide and is the second highest cause of cancer-related death in men. While there is uncertainty over which men will benefit from radical treatment, considerable efforts are being made to reduce treatment related side-effects and in optimising outcomes. This article reviews the development and introduction of robotic-assisted laparoscopic radical prostatectomy (RALP), the results to date, and the possible future directions of RALP

Microarray Analysis Reveals Distinct Gene Expression Profiles Among Different Tumor Histology, Stage and Disease Outcomes in Endometrial Adenocarcinoma

Endometrial cancer is the most common gynecologic malignancy in developed countries and little is known about the underlying mechanism of stage and disease outcomes. The goal of this study was to identify differentially expressed genes (DEG) between late vs. early stage endometrioid adenocarcinoma (EAC) and uterine serous carcinoma (USC), as well as between disease outcomes in each of the two histological subtypes.Gene expression profiles of 20 cancer samples were analyzed (EAC = 10, USC = 10) using the human genome wide illumina bead microarrays. There was little overlap in the DEG sets between late vs. early stages in EAC and USC, and there was an insignificant overlap in DEG sets between good and poor prognosis in EAC and USC. Remarkably, there was no overlap between the stage-derived DEGs and the prognosis-derived DEGs for each of the two histological subtypes. Further functional annotation of differentially expressed genes showed that the composition of enriched function terms were different among different DEG sets. Gene expression differences for selected genes of various stages and outcomes were confirmed by qRT-PCR with a high validation rate.This data, although preliminary, suggests that there might be involvement of distinct groups of genes in tumor progression (late vs. early stage) in each of the EAC and USC. It also suggests that these genes are different from those involved in tumor outcome (good vs. poor prognosis). These involved genes, once clinically verified, may be important for predicting tumor progression and tumor outcome

CXCL12 expression by healthy and malignant ovarian epithelial cells

<p>Abstract</p> <p>Background</p> <p>CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value.</p> <p>Methods</p> <p>Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves.</p> <p>Results</p> <p>Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival.</p> <p>Conclusion</p> <p>Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00052468">NCT00052468</a></p