Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial.

Background: Lethal-7 (let-7) is a tumour suppressor miRNA which acts by down-regulating several oncogenes including KRAS. A single-nucleotide polymorphism (rs61764370, T > G base substitution) in the let-7 complementary site 6 (LCS-6) of KRAS mRNA has been shown to predict prognosis in early-stage colorectal cancer (CRC) and benefit from anti-epidermal growth factor receptor monoclonal antibodies in metastatic CRC. Patients and methods: We analysed rs61764370 in EXPERT-C, a randomised phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX plus or minus cetuximab in locally advanced rectal cancer. DNA was isolated from formalin-fixed paraffin-embedded tumour tissue and genotyped using a PCR-based commercially available assay. Kaplan–Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. Results: A total of 155/164 (94.5%) patients were successfully analysed, of whom 123 (79.4%) and 32 (20.6%) had the LCS-6 TT and LCS-6 TG genotype, respectively. Carriers of the G allele were found to have a statistically significantly higher rate of complete response (CR) after neoadjuvant therapy (28.1% versus 10.6%; P = 0.020) and a trend for better 5-year progression-free survival (PFS) [77.4% versus 64.5%: hazard ratio (HR) 0.56; P = 0.152] and overall survival (OS) rates (80.3% versus 71.9%: HR 0.59; P = 0.234). Both CR and survival outcomes were independent of the use of cetuximab. The negative prognostic effect associated with KRAS mutation appeared to be stronger in patients with the LCS-6 TT genotype (HR PFS 1.70, P = 0.078; HR OS 1.79, P = 0.082) compared with those with the LCS-6 TG genotype (HR PFS 1.33, P = 0.713; HR OS 1.01, P = 0.995). Conclusion: This analysis suggests that rs61764370 may be a biomarker of response to neoadjuvant treatment and an indicator of favourable outcome in locally advanced rectal cancer possibly by mitigating the poor prognosis of KRAS mutation. In this setting, however, this polymorphism does not appear to predict cetuximab benefit.

Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes.

Borderline ovarian tumors represent an understudied subset of ovarian tumors. Most studies investigating aberrations in borderline tumors have focused on KRAS/BRAF mutations. In this study, we conducted an extensive analysis of mutations and single-nucleotide polymorphisms (SNPs) in borderline ovarian tumors. Using the Sequenom MassArray platform, we investigated 160 mutations/polymorphisms in 33 genes involved in cell signaling, apoptosis, angiogenesis, cell cycle regulation and cellular senescence. Of 52 tumors analyzed, 33 were serous, 18 mucinous and 1 endometrioid. KRAS c.35G>A p.Gly12Asp mutations were detected in eight tumors (six serous and two mucinous), BRAF V600E mutations in two serous tumors, and PIK3CA H1047Y and PIK3CA E542K mutations in a serous and an endometrioid BOT, respectively. CTNNB1 mutation was detected in a serous tumor. Potentially functional polymorphisms were found in vascular endothelial growth factor (VEGF), ABCB1, FGFR2 and PHLPP2. VEGF polymorphisms were the most common and detected at four loci. PHLPP2 polymorphisms were more frequent in mucinous as compared with serous tumors (P=0.04), with allelic imbalance in one case. This study represents the largest and most comprehensive analysis of mutations and functional SNPs in borderline ovarian tumors to date. At least 25% of borderline ovarian tumors harbor somatic mutations associated with potential response to targeted therapeutics.

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Homozygous deletion mapping in myeloma samples identifies genes and an expression signature relevant to pathogenesis and outcome.

PURPOSE: Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes. EXPERIMENTAL DESIGN: Using single nucleotide polymorphism-based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome. RESULTS: We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the "cell death" network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets. CONCLUSIONS: We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma.

Integration of global SNP-based mapping and expression arrays reveals key regions, mechanisms, and genes important in the pathogenesis of multiple myeloma.

Multiple myeloma is characterized by genomic alterations frequently involving gains and losses of chromosomes. Single nucleotide polymorphism (SNP)-based mapping arrays allow the identification of copy number changes at the sub-megabase level and the identification of loss of heterozygosity (LOH) due to monosomy and uniparental disomy (UPD). We have found that SNP-based mapping array data and fluorescence in situ hybridization (FISH) copy number data correlated well, making the technique robust as a tool to investigate myeloma genomics. The most frequently identified alterations are located at 1p, 1q, 6q, 8p, 13, and 16q. LOH is found in these large regions and also in smaller regions throughout the genome with a median size of 1 Mb. We have identified that UPD is prevalent in myeloma and occurs through a number of mechanisms including mitotic nondisjunction and mitotic recombination. For the first time in myeloma, integration of mapping and expression data has allowed us to reduce the complexity of standard gene expression data and identify candidate genes important in both the transition from normal to monoclonal gammopathy of unknown significance (MGUS) to myeloma and in different subgroups within myeloma. We have documented these genes, providing a focus for further studies to identify and characterize those that are key in the pathogenesis of myeloma.

Enzyme-induced Aggregation of Plasmonic Nanoparticles for the Development of Label-free and Ultrasensitive Detection of Bacterial DNA

Sensitive detection of pathogens is critical to ensure the safety of food supplies and to prevent bacterial disease infection and outbreak at the first onset. While conventional techniques such as cell culture, ELISA, PCR, etc. have been used as the predominant detection workhorses, they are however limited by either time-consuming procedure, complicated sample pre-treatment, expensive analysis and operation, or inability to be implemented at point-of-care testing. Here, we present our recently developed assay exploiting enzyme-induced aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. In the experiments, AuNPs are first functionalized with specific, single-stranded RNA probes so that they exhibit high stability in solution even under high electrolytic condition thus exhibiting red color. When bacterial DNA is present in a sample, a DNA-RNA heteroduplex will be formed and subsequently prone to the RNase H cleavage on the RNA probe, allowing the DNA to liberate and hybridize with another RNA strand. This continuously happens until all of the RNA strands are cleaved, leaving the nanoparticles ‘unprotected’. The addition of NaCl will cause the ‘unprotected’ nanoparticles to aggregate, initiating a colour change from red to blue. The reaction is performed in a multi-well plate format, and the distinct colour signal can be discriminated by naked eye or simple optical spectroscopy. As a result, bacterial DNA as low as pM could be unambiguously detected, suggesting that the enzyme-induced aggregation of AuNPs assay is very easy to perform and sensitive, it will significantly benefit to development of fast and ultrasensitive methods that can be used for disease detection and diagnosis.

Ion-mediated conformational switches

Molecular switches are ubiquitous in Nature and provide the basis of many forms of transport and signalling. Single synthetic molecules that change conformation, and thus function, reversibly in a stimulus-dependent manner are of great interest not only to chemists but society in general; myriad applications exist in storage, display, sensing and medicine. Here we describe recent developments in the area of ion-mediated switching.

Redox-dependent conformational switching of diphenylacetylenes

Herein we describe the design and synthesis of a redox-dependent single-molecule switch. Appending a ferrocene unit to a diphenylacetylene scaffold gives a redox-sensitive handle, which undergoes reversible one-electron oxidation, as demonstrated by cyclic voltammetry analysis. ¹H-NMR spectroscopy of the partially oxidized switch and control compounds suggests that oxidation to the ferrocenium cation induces a change in hydrogen bonding interactions that results in a conformational switch.