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BACKGROUND A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non–small cell lung cancer (NSCLC). METHODS Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists–accredited, Clinical Laboratory Improvement Amendments–certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. RESULTS Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. CONCLUSIONS NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing. Cancer 2015;121:631–639. © 2014 American Cancer Society.

Non-steroidal anti-inflammatory drugs (NSAIDs) specifically inhibit cyclooxygenase (COX) activity and are widely used as anti-arthritics, post-surgical analgesics, and for the relief of acute musculoskeletal pain. Recent studies suggest that non-specific NSAIDs, which inhibit both COX-1 and COX-2 isoforms, delay bone healing. The objectives of this study were 2-fold; first, to measure the relative changes in the normal expression of COX-1 and COX-2 mRNAs over a 42 day period of fracture healing and second, to compare the effects of a commonly used non-specific NSAID, ketorolac, with a COX-2 specific NSAID, Parecoxib (a pro-drug of valdecoxib), on this process. Simple, closed, transverse fractures were generated in femora of male Sprague-Dawley rats weighing approximately 450 g each. Total RNA was prepared from the calluses obtained prior to fracture and at 1, 3, 5, 7, 10, 14, 21, 35 and 42 days post-fracture and levels of COX-1 and COX-2 mRNA were measured using real time PCR. While the relative levels of COX-1 mRNA remained constant over a 21-day period, COX-2 mRNA levels showed peak expression during the first 14 days of healing and returned to basal levels by day 21. Mechanical properties of the calluses were then assessed at 21 and 35 days post-fracture in untreated animals and animals treated with either ketorolac or high or low dose parecoxib. At both 21 and 35 days after fracture, calluses in the group treated with the ketorolac showed a significant reduction in mechanical strength and stiffness when compared with controls (p

Epidemiological and dietary studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colon cancer, possibly through a mechanism involving inhibition of cyclooxygenase (COX)-2, which is overexpressed in premalignant adenomatous polyps and colon cancer. Because ultraviolet light (UV) can induce COX-2 and nonspecific NSAIDs can decrease UV-induced skin cancer, we evaluated the ability of two compounds, celecoxib (a specific COX-2 inhibitor) and indomethacin (a nonspecific NSAID), to block UV-induced skin tumor development in SKH:HR-1-hrBr hairless mice. Mice fed 150 or 500 ppm celecoxib showed a dose-dependent reduction (60% and 89%, respectively) in tumor yield. Indomethacin (4ppm) reduced tumor yield by 78%. Although both acute and chronic UV exposure increased cell proliferation and edema, neither compound reduced these parameters. In contrast, UV-induced prostaglandin synthesis in the epidermis was effectively blocked by both compounds. UV-induced increases in COX-2 expression in skin were also not altered in any of the treatment groups. Similarly, tumors that constitutively express high levels of COX-2 displayed no reduction by treatment with celecoxib or indomethacin. The dramatic protective effects of celecoxib suggests that specific COX-2 inhibitors may offer a way to safely reduce the risk of skin cancer in humans.

A series of methysulfonyl or sulfonamido substituted 4,5-diaryloxazole were prepared and evaluated for their ability to inhibit the inducible form of cyclooxygenase (COX-2) in vitro and in vivo. Several unique substitution patterns were identified that led to potent and selective inhibitors of COX-2. In general, 2-trifluoromethly-4,5-diaryloxazoles substituted with a methylsulfonyl or sulfonamido group were particularly potent inhibitors. One of the more potent compounds with a selectivity for COX-2 of about 800 fold was 4b (SC-299). SC-299, a highly fluorescent molecule, may be useful for spectroscopic studies on preferential inhibitor binding to COX-2. © 1999 John Wiley & Sons, Inc. Med Res Rev, 19, No. 3, 199–208, 1999.

Novel 4,5-diarylspiro[2.4]hept-5-enes, 6,7-diarylspiro[3.4]oct-6-enes, and 2,3-diarylspiro[4.4]non-2-enes have been synthesized and shown to be very potent inducible cyclooxygenase (COX-2) inhibitors with inhibition (IC 50 ) in the low nanomolar range and enzyme selectivity ratios as high as four orders of magnitude. The methyl sulfone spiro[2.4]hept-5-ene 1 (SC-58451) was found to be orally active (ED 50 = 0.3 mpk) in the rat adjuvant-induced arthritis model with no gastric lesions observed at 200 mpk.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure−activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a−c,k,l,n (COX-2, IC50 = 0.002−0.004 μM), in which all have in vitro COX-1/COX-2 selectivity >1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflamma...

A novel series of 5,6-diarylspiro[2.4]hept-5-enes was shown to provide highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. A study of structure-activity relationships in this series suggests that 3,4-disubstituted phenyl analogs are generally more selective than 4-substituted phenyl analogs and that replacement of the methyl sulfone group on the 6-phenyl ring with a sulfonamide moiety results in compounds with superior in vivo pharmacological properties, although with lower COX-2 selectivity. Several compounds have been shown to possess promising pharmacological properties in adjuvant-induced arthritis and edema analgesia models. The absence of gastrointestinal (GI) toxicity at 200 mpk of several selected compounds in rats and mice corresponds well with the weak potency for inhibition of COX-1 observed in the enzyme assay. Methyl sulfone 55 and sulfonamide 24 were shown to have superior in vivo pharmacological profiles, low GI toxicity, and good oral bioavailability and duration of action.

Series of 1,2-diarylpyrroles has been synthesized and found to contain very potent and selective inhibitors of the human cyclooxygenase-2 (COX-2) enzyme. The paper describes short and practical syntheses of the target molecules utilizing the Paal−Knorr reaction. Electrophilic substitution on 1 proceeds in a regioselective fashion, and the method was used to generate a number of tetrasubstituted pyrroles. Detailed SAR on the series has been studied by modifications of the aryl rings and the substituents in the pyrrole ring. Diarylpyrrole 1 is a very potent (COX-2, IC50 = 60 nm) and selective (COX-1/COX-2 = >1700) inhibitor whereas the isomeric 2 is completely inactive against COX-2. Modifications of the substituents on the fluorophenyl ring in 1 yields very potent inhibitors of COX-2 (IC50 = 40−80 nm) with excellent selectivity (1200 to >2500) vs COX-1. Analog 20 containing a sulfonamide group is an excellent inhibitor of COX-2 with an IC50 of 14 nm. Tetrasubstituted pyrroles containing groups such as COCF...

Series of 1,2-diarylimidazoles has been synthesized and found to contain highly potent and selective inhibitors of the human COX-2 enzyme. The paper describes a short synthesis of the target 1,2-diarylimidazoles starting with aryl nitriles. Different portions of the diarylimidazole (I) were modified to establish SAR. Systematic variations of the substituents in the aryl ring B have yielded very potent (IC50 = 10−100 nm) and selective (1000−12500) inhibitors of the COX-2 enzyme. The study on the influence of substituents in the imidazole ring established that a CF3 group at position 4 gives the optimum oral activity. A number of the diarylimidazoles showed excellent inhibition in the adjuvant induced arthritis model (e.g., ED50 = 0.02 mpk for 22 and 34). The diarylimidazoles are also potent inhibitors of carrageenan-induced edema (ED50 = 9−30 mpk) and hyperalgesia (ED50 = 11−40 mpk). Several orally active diarylimidazoles show no GI toxicity in the rat and mouse up to 200 mpk.

A series of sulfonamide-substituted 4,5-diarylthiazoles was prepared via three synthetic routes as selective COX-2 inhibitors. Recently in the synthesis of selective COX-2 inhibitors we have discovered that the sulfonamide moiety is a suitable replacement for the methylsulfonyl moiety yielding compounds with activity both in vitro and in vivo.

A series of novel sulfone substituted 4,5-diarylthiazoles have been synthesized and evaluated for their inhibition of the two isoforms of human cyclooxygenase (COX-1 and COX-2). This series displays exceptionally selective COX-2 inhibition.

Evidence from tissue culture studies suggests that glial cells are the principal source of prostaglandins in the brain. We have used immunohistochemistry, Western blot analysis, and enzyme activity assays to localize cyclooxygenase (COX), the enzyme responsible for the conversion of arachidonic acid to prostaglandins, in situ in the normal ovine brain. We observed very few immunoreactive glial cells. In contrast, an extensive distribution of COX-like immunoreactive (ir) neuronal cell bodies and dendrites and a corresponding pattern of COX enzyme activity were observed. COXir neurons were most abundant in forebrain sites involved in complex, integrative functions and autonomic regulation such as the cerebral cortex, hippocampus, amygdala, bed nucleus of the stria terminalis, substantia innominata, dorsomedial nucleus of the hypothalamus, and tuberomammillary nucleus. Moderate populations were observed in other regions of the central nervous system implicated in sensory afferent processing, including the dorsal column nuclei, spinal trigeminal nucleus, and superior colliculus, and in structures involved in autonomic regulation, such as the nucleus of the solitary tract, parabrachial nucleus, and the periaqueductal gray matter. We did not observe COXir axons or terminal fields, however. Our results suggest that neurons may use prostaglandins as intracellular or perhaps paracrine, but probably not synaptic, mediators in the normal brain.

The formation of new blood vessels by angiogenesis to provide adequate blood supply is a key requirement for the growth of many tumors. While normal blood vessels expressed the COX-1 enzyme, new angiogenic endothelial cells expressed the inducible COX-2. We evaluated the role of COX inhibitors in the mouse corneal micropocket assay in which angiogenesis is driven by the addition of a Hydron pellet containing basic fibroblast growth factor (bFGF). Neovascular areas were measured with a slit lamp five days after pellet implantation into the corneal stroma. All animals containing implants with bFGF (90 ng) developed intensive areas of neovascularization, whereas the controls implanted with the Hydron pellet alone did not. Indomethacin (a nonselective COX-1/COX-2 inhibitor) and SC-236 (a COX-2-selective inhibitor) inhibited angiogenesis in a dose-dependent manner. Importantly, the indomethacin-treated mice developed severe gastrointestinal toxicity at the efficacious dose of 3 mg/kg/day. By contrast, gastrointestinal lesions were not observed, and platelet COX-1 activity was unaffected, at anti-angiogenic doses of SC-236 (1-6 mg/kg/day). Furthermore, a COX-1-selective inhibitor, SC-560, was ineffective at doses up to 10 mg/kg, a dose that completely blocked platelet COX-1 activity in these mice. SC-236 was also effective in reducing angiogenesis driven by bFGF, vascular endothelium growth factor (VEGF), or carrageenan in the matrigel rat model. Finally, in several tumor models, SC-236 consistently and effectively inhibited tumor growth and angiogenesis. This novel antiangiogenic activity of COX-2 inhibitors indicates their potential therapeutic utility in several types of cancer.