Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE).

PURPOSE: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. EXPERIMENTAL DESIGN: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. RESULTS: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. CONCLUSIONS: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications.See related commentary by McMullen et al., p. 5271.

Preparation and characterization of erythrocyte membrane cloaked PLGA/arsenic trioxide nanoparticles and evaluation of their in vitro anti-tumor effect.

Arsenic trioxide (ATO) is used for acute promyelocytic leukemia (APL) that is resistant to all-trans-retinoic acid, but its direct intravenous injection sometimes induces severe toxic side effects. Here, we developed a delivery system of red blood cell membrane (RBCM) cloaked poly (lactic-co-glycolic) acid (PLGA)-ATO nanoparticles (RPANs) to reduce the toxicity. PLGA was used to entrap the ATO, and the PLGA-ATO nanoparticles (PANs) were prepared by the emulsification method. Then RBCMs were employed to cloak the PANs using ultrasonication, to develop the RPANs delivery system. The prepared RPANs had a uniform size of around 233.6 nm with an obvious core-shell structure, as observed by TEM. The completeness of the membrane proteins was confirmed by SDS-PAGE and an in vitro release time of 65 h was determined for the RPANs. The RPANs also exhibited low cytotoxicity against the 293k kidney cell line (84.6% cell viability rate), which suggested that the ATO toxicity was reduced by RBCM cloaking. Moreover, the anti-tumor effects of the RPANs against the HL60 cell line were comparable to those of ATO solution. Our study demonstrated that the RPANs system has anti-tumor potential and could be developed into a safe and sustained release delivery system for ATO.

Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.