PURPOSE The purpose of this study was to confirm our previous results that targeted agents matched with tumor molecular alterations were associated with improved outcomes compared to non-matched therapy in patients with advanced cancer. PATIENTS AND METHODS Outcomes of patients who were referred for treatment on phase I clinical trials at The University of Texas MD Anderson Cancer Center from 3/2011 to 1/2012 were compared between those who had received targeted therapy and those for whom no targeted therapy was available. Two-month landmark analyses for overall and progression-free survival (PFS) combining previously published and validation cohort patient data were performed. RESULTS In patients with 1 alteration, matched therapy (n=143) compared with treatment without matching (n=236) was associated with a higher objective response rate (12% vs. 5%; P<0.0001), longer PFS (median, 3.9 vs. 2.2 months; P=0.001), and longer survival (median, 11.4 vs. 8.6 months; P=0.04). In multivariate analysis, matched therapy was an independent factor predicting response (P<0.015) and PFS (P<0.004). Two-month landmark analyses in the matched therapy group demonstrated that the median survival of responders was 30.5 months compared to 11.3 months for non-responders (P=0.01); and the median PFS was 38.7 months compared to 5.9 months, respectively (P<0.0001). The respective values in the non-matched therapy group were 9.8 and 9.4 months (P=0.46) and 8.5 and 4.2 months (P=0.18). CONCLUSION This validation analysis confirms our previous observations. In the matched therapy group, 2-month landmark analyses demonstrated that responders have longer survival and PFS than non-responders.

Dear Editor:Clostridium acetobutylicum, a gram-positive, anaerobic, spore-forming bacterium, is capable of using a wide variety of carbon sources to produce acetone, butanol and ethanol. To improve solvent productivity of C. acetobutylicum, metabolic engineering is considered as a useful tool in developing strains with industrially desirable characteristics. However, to date, there are few useful methods for genetic manipulation of C. acetobutylicum, especially for gene disruption. To our knowledge, two types of vectors, including non-replicative and replicative integrative plasmids, have been developed for gene-inactivation in C. acetobutylicum. By using non-replicative integrative plasmids, buk and solR genes of C. acetobutylicum were inactivated [1,2]. However, due to their low frequencies of transformation and recombination, the non-replicative integrative plasmids are usually transformed at less than 1 integrative transformant per mg plasmid DNA. To obtain the integrative mutant, it may require higher transformation frequencies up to 10 5 , but the typical transformation frequencies were reported at 10 3 [3]. Harris et al. described the construction of a replicative integrative plasmid pETSPO and its application in the disruption of gene spo0A which could not be inactivated by using the non-replicative integrative plasmid [4]. With the functional replication origin in C. acetobutylicum, pETSPO increases opportunity for homologous recombination, but it is still time-consuming to screen for double crossover integration. Therefore, a more efficient tool for targeted gene inactivation in the C. acetobutylicum is much needed.Recently, a new strategy was developed to construct gene inactivation mutants by using group II intron-based Targetron technology. The mobile group II intron, originating from the Lactococcus lactis L1.LtrB intron, has been successfully used in a wide range of bacteria including Clostridium perfringens [5]. Without a proper replicon and/or promoter, the targetron plasmid pJIR750ai for C. perfringens from Sigma Aldrich was not applicable for gene disruption in the C. acetobutylicum directly (data not shown). Therefore, a modified targetron plasmid pSY6 was created by cloning the L1.LtrB group II intron fragment into the pIMP1-ptb, which was an E. coli-C. acetobutylicum shuttle vector containing a ptb (phosphotransbutyrylase) promoter [6].The gene buk, encoding the butyrate kinase, catalyzes the production of butyrate, and the gene solR located on the megaplasmid of the strain, encodes a putative repressor of solvent formation genes [7,8]. pSY6-buk and pSY6-solR vectors, constructed based on pSY6 ( Figure 1A and Supplementary information, Figure S1), were electroporated into C. acetobutylicum ATCC 824, respectively. Then, the cells were incubated overnight to induce the intron invasion (See Supplementary information, Materials and Methods). The overnight cultures were spread onto CGM medium (25 μg/ml erythromycin) and the transformants were analyzed

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Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

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Butanol is an important solvent and transport fuel additive, and can be produced by microbial fermentation. Attempts to generate a superior microbial producer of butanol have been made through different metabolic engineering strategies. However, to date, butanol bio-production is still not economically competitive compared to petrochemical-derived production because of its major drawbacks, such as, high cost of the feedstocks, low butanol concentration in the fermentation broth and the co-production of low-value by-products acetone and ethanol. Here we analyze the main bottlenecks in microbial butanol production and summarize relevant advances from recently reported studies. Further needs and directions for developing real industrially applicable strains in butanol production are also discussed.