Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-μL scale polymerase chain reaction (PCR) on microchips, has been integrated with DNA extraction and separation ...on a glass microchip in a fully integrated micro Total Analysis System by Easley et al., in 2006. IR-PCR has been demonstrated on both glass and PMMA microdevices where the fabrication (bonding) is not trivial. Polyester-toner (PeT) microfluidic devices have significant potential as cost-effective, disposable microdevices as a result of the ease of fabrication (∼$0.25 USD and <10 min per device) and availability of commercial substrates. For the first time, we demonstrate here the thermal cycling in PeT microchips on the IR-PCR system. Undesirable IR absorption by the black-toner bonding layer was eliminated with a spatial filter in the form of an aluminum foil mask. The solution heating rate for a black PeT microchip using a tungsten lamp was 10.1 ± 0.7 °C s−1 with a cooling rate of roughly −12 ± 0.9 °C s−1 assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the λ-phage genome (in 11 min) and a 1500 bp region of Azospirillum brasilense. Using a centrosymmetric chamber configuration in a multichamber PeT microchip, homogenous temperature distribution over all chambers was achieved with inter-chamber temperature differences at annealing, extension and denaturing steps of less than ±2 °C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human β-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human β-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of ∼10%. Development of PeT microchips for IR-PCR has the potential to provide rapid, low-volume amplification while also integrating PCR with extraction upstream and separation/detection downstream.
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•Polyester chips were used in an infra-red thermal control system for PCR.•Thermo-response of the chips to infra-red radiation was modified by a spatial mask.•PCR inhibition from the chip substrate was mitigated by dynamic surface passivation.•Chips with different sample throughput successfully delivered PCR results.
Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreductase was identified using metagenomic mining to catalyze a highly enantio- and diastereoselective ...dynamic kinetic resolution of a β-ketolactone. Subsequent lactone reduction with diisobutylaluminum hydride and phase transfer cyclization affords the bicyclic acetal fragment in 39% yield over four steps.
Transannular carbonyl-olefin metathesis reactions complement existing procedures for related ring-closing, ring-opening, and intermolecular carbonyl-olefin metathesis. This enables molecular editing ...of steroid-derived frameworks.
Transannular carbonyl–olefin metathesis reactions complement existing procedures for related ring-closing, ring-opening, and intermolecular carbonyl-olefin metathesis. We herein report the development and mechanistic investigation of FeCl
3
-catalyzed transannular carbonyl-olefin metathesis reactions that proceed
via
a distinct reaction path compared to previously reported ring-closing and ring-opening protocols. Specifically, carbonyl-ene and carbonyl-olefin metathesis reaction pathways are competing under FeCl
3
-catalysis to ultimately favor metathesis as the thermodynamic product. Importantly, we show that distinct Lewis acid catalysts are able to distinguish between these pathways to enable the selective formation of either transannular carbonyl-ene or carbonyl-olefin metathesis products. These insights are expected to enable further advances in catalyst design to efficiently differentiate between these two competing reaction paths of carbonyl and olefin functionalities to further expand the synthetic generality of carbonyl-olefin metathesis.
The development of new methods for forming carbon-carbon bonds is an important area of research in synthetic organic chemistry. Accomplishing the direct formation of new C-C bonds between a carbonyl ...and olefin starting material is a powerful transformation, enabling the synthesis of new olefin-containing molecules, especially for molecules containing small rings. Recently, the Schindler group has developed an operationally simple, mild, and environmentally benign strategy for carbonyl-olefin metathesis reactions relying on iron(III) as a Lewis acid. Rather than relying on stoichiometric alkylidenes as reagents, the Lewis acid catalyst induces oxetane formation and subsequent fragmentation, enabling catalyst turnover and producing acetone or aromatic aldehydes as waste products. This dissertation describes the application and extension of this method to a variety of new systems. Polycyclic aromatic hydrocarbons, important in material science and polymer chemistry, have been prepared under these conditions. Alkyl ketones, previously found to be problematic substrates, can undergo the carbonyl-olefin metathesis reaction in systems when iron(III) is able to form singly bridged homo-dimers that lead to much greater carbonyl activation than their monomeric counterparts. Finally, carbonyl-olefin metathesis is demonstrated in transannular systems. Ten membered rings derived from biologically important steroid molecules are shown to undergo divergent reactivity when treated with different Lewis acid catalysts. Total synthesis of terpene natural products is also an important and active area of research in synthesis as means of delivering useful quantities of biologically important, naturally occurring molecules as well as a driving force for the discovery and development of new methods. The tetracyclic meroterpenoid lingzhiol has potential reno-protective biologically activity and is a small but synthetically interesting natural product. This dissertation describes the completion of an enantioselective total synthesis of this compound. In the course of these studies, a metal-mediated reversal of enantioselectivity was discovered. This enables the enantioselective synthesis of both enantiomers. Finally, ent-kaurene diterpenoid natural products are a large class of complex natural products that are found in various traditional East Asian medicines. Over 600 compounds of this class are known and their biological activity has made them targets for synthetic chemistry over the last 60 years. Most of the existing synthetic strategies enable preparation of only one to three ent-kaurene compounds. Here is described studies toward a divergent synthetic strategy relying on synthesis of a common intermediate, which can be elaborated to numerous ent-kaurenes.
We recently defined a method for fabricating multilayer microdevices using poly(ethylene terephthalate) transparency film and printer toner, and showed these could be successfully applied to DNA ...extraction and amplification (Duarte
et al.
,
Anal. Chem
. 2011,
83
, 5182-5189). Here, we advance the functionality of these microdevices with flow control enabled by hydrophobic valves patterned using laser printer lithography. Laser printer patterning of toner within the microchannel induces a dramatic change in surface hydrophobicity (change in contact angle of DI water from 51° to 111°) with good reproducibility. Moreover, the hydrophobicity of the surface can be controlled by altering the density of the patterned toner
via
varying the gray-scale setting on the laser printer, which consequently tunes the valve's burst pressure. Toner density provided a larger burst pressure bandwidth (158 ± 18 Pa to 573 ± 16 Pa) than could be achieved by varying channel geometry (492 ± 18 Pa to 573 ± 16 Pa). Finally, we used a series of tuned toner valves (with varied gray-scale) for passive valve-based fluidic transfer in a predictable manner through the architecture of a rotating PeT microdevice. While an elementary demonstration, this presents the possibility for simplistic and cost-effective microdevices with valved fluid flow control to be fabricated using nothing more than a laser printer, a laser cutter and a laminator.
Exploiting in-expensive laser lithography to pattern commercial substrates, we provide the first demonstration for integrating and controlling passive valve function on poly(ethylene terephthalate) toner microdevices in a manner that streamlines a simple (print, assemble, laminate), cost-efficient fabrication process.
Abstract
Background
Tissue-based assessment of actionable mutations in pts with NSCLC is limited by invasive biopsies and adequacy of biopsied tumour material. Blood-based testing may overcome such ...limitations, allowing multiplex profiling in a single test. BFAST (NCT03178552) is an ongoing multicentre, open-label, multi-cohort study evaluating the relationship between blood-based next-generation sequencing (NGS) detection of actionable genetic alterations in cell-free DNA, and the activity of targeted therapies and immunotherapy in pts with treatment-naïve advanced NSCLC. We present first results from the ALK+ cohort.
Methods
Pts ≥18 years with stage IIIB/IV ALK+ NSCLC (detected by blood-based NGS) received oral alectinib 600mg twice daily. Asymptomatic/treated central nervous system (CNS) metastases were permitted. All pts (with/without CNS disease) had 8-weekly restaging and brain scans. Primary endpoint: confirmed investigator (INV)-assessed objective response rate (ORR; RECIST v1.1). Key secondary endpoints: independent review facility (IRF)-assessed ORR; INV- and IRF-assessed duration of response (DoR), progression-free survival (PFS), overall survival; and safety.
Results
Of 2,219 pts screened, blood-based NGS yielded results in 2,188 pts. Overall, 119 pts (5.4%) had ALK+ disease; 87 pts were enrolled and received alectinib. EML4 was the fusion partner in 73 (84%) pts, with TP53 mutations detected in 38 (44%) pts. Median blood-based tumour mutational burden was 2 (range, 0–21). Median follow-up: 12.6 months (range, 2.6–18.7). Confirmed ORR: 87.4% (95% CI 78.5–93.5) by INV and 92.0% (95% CI 84.1–96.7) by IRF. The 12-month INV-confirmed DoR was 75.9% (95% CI 63.6–88.2). In 35 (40%) pts with asymptomatic baseline CNS disease, ORR by INV was 91.4% (95% CI 76.9–98.2). Median PFS: not reached; 12-month PFS by INV was 78.4% (95% CI 69.1–87.7). Safety data were consistent with the known safety profile of alectinib.
Conclusions
Blood-based detection of ALK fusions results in high ORR and clinical benefit in pts receiving alectinib. These data validate the clinical utility of blood-based NGS as an additional method to inform clinical decision-making in ALK+ NSCLC.
Clinical trial identification
NCT03178552.
Editorial acknowledgement
Medical Writing support was provided by Nicola Griffin of Gardiner-Caldwell Communications and funded by F. Hoffmann-La Roche.
Legal entity responsible for the study
F. Hoffmann-La Roche Ltd.
Funding
F. Hoffman-La Roche Ltd.
Disclosure
S.M. Gadgeel: Honoraria (self), Advisory / Consultancy: Ariad, AstraZeneca, Bristol-Myers Squibb, Pfizer and F. Hoffmann-La Roche Ltd/Genentech. T.S.K. Mok: Leadership role: Sanonics Ltd; Honoraria (self), Advisory / Consultancy: ACEA Biosciences, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Chimed, Cirina, Fishawack Facilitate, Ignyta, Janssen, Lilly, Merck Serono, Merck Sharp & Dohme, Novartis, OncoGenex, Pfizer, F. Hoffmann-La Roche Ltd/Genentech, SFJ Pharm; Research grant / Funding (self): AstraZeneca, Bristol-Myers Squibb, Clovis Oncology, Merck Sharp & Dohme, Novartis, Pfizer, F. Hoffmann-La Roche Ltd, SFJ Pharmaceutical and XCovery. S. Peters: Honoraria (self), Advisory / Consultancy: AbbVie, Amgen, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Clovis, Eli Lilly, F. Hoffmann-La Roche Ltd, Janssen, Merck Sharp & Dohme, Merck Serono, Novartis, Pfizer, Regeneron and Takeda; Speaker Bureau / Expert testimony: AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, F. Hoffmann-La Roche Ltd, Merck Sharp & Dohme, Novartis and Pfizer. J.A.A. Alexander: Honoraria (self), Advisory / Consultancy: AstraZeneca, Boehringer Ingelheim Bristol-Myers Squibb, Pfizer and F. Hoffmann-La Roche Ltd/Genentech, Eli Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Takeda. N.B. Leighl: Research grant / Funding (self): F. Hoffmann-La Roche, Array, Guardant and AstraZeneca; Honoraria (self): AstraZeneca, Bristol-Myers Squibb, Merck Sharp & Dohme, F. Hoffmann-Lar Roche, Pfizer ; Advisory / Consultancy: Excovery. V. Sriuranpong: Honoraria (self), Advisory / Consultancy: AstraZeneca, Novartis, F. Hoffmann-La Roche Ltd, Pfizer, Sanofi, Eisai, Boehringer, Taiho, Merck Sharp & Dohme, Bristol-Myers Squibb ; Research grant / Funding (institution): AstraZeneca, Novartis, F. Hoffmann-La Roche Ltd, Pfizer, Boehringer, Eisai, Taiho, Lilly and Merck Sharp & Dohme. M. Perol: Honoraria (self): F. Hoffmann-La Roche Ltd, AstraZeneca, Bristol-Myers Squibb, Merck Sharp & Dohme, Pfizer, Takeda, Chugai, Boehringer Ingelheim, Eli Lilly, Amgen and AbbVie. G. De Castro Jr.: Advisory / Consultancy: AstraZeneca, Merck Sharp & Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche Ltd, Novartis, Boehringer Ingelheim, Pfizer, Bayer; Speaker Bureau / Expert testimony: Merck Sharp & Dohme, Bristol-Myers Squibb, Novartis, AstraZeneca, Pfizer, F. Hoffmann-La Roche Ltd, Bayer, TEVA ; Travel / Accommodation / Expenses: Merck Sharp & Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche Ltd, Bayer, Novartis, Boehringer Ingelheim, AstraZeneca, Pfizer and Bayer.. E. Nadal: Honoraria (self), Advisory / Consultancy: AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, F. Hoffmann-La Roche Ltd, Janssen, Merck Sharp & Dohme, Pfizer and Takeda. F. De Marinis: Honoraria (self): F. Hoffmann La Roche Ltd, Bristol-Myers Squibb, AstraZeneca and Merck. J. Han: Honoraria (self): Roche, AstraZeneca, BMS, MSD, Takeda; Advisory / Consultancy: AstraZeneca, BMS, MSD, Lilly, Novartis, Pfizer, Takeda; Research grant / Funding (self): Roche, Pfizer, ONO . M. Yan: Full / Part-time employment: F. Hoffmann-La Roche Ltd.. T. Riehl: Full / Part-time employment: Genentech, Inc. E. Schleifman: Full / Part-time employment: Genentech, Inc. S.M. Paul: Full / Part-time employment: Genentech, Inc. S. Mocci: Full / Part-time employment: Genentech, Inc. D. Shames: Full / Part-time employment: Genentech, Inc. M.S. Mathisen: Full / Part-time employment: Genentech, Inc. R. Dziadziuszko: Honoraria (self), Advisory / Consultancy: F. Hoffmann-La Roche Ltd, Pfizer, Boehringer Ingelheim, Clovis Oncology, Novartis, AstraZeneca and Tesaro..
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