Concordance among gene expression-based predictors for ER-positive breast cancer treated with adjuvant tamoxifen.

BACKGROUND: ER-positive (ER+) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. In this study, we evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER+ tumors treated with adjuvant tamoxifen only. METHODS: Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (OncotypeDX), Mammaprint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapse-free survival (DRFS) was estimated by Kaplan-Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance. RESULTS: All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, OncotypeDX, Mammaprint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%-100%). CONCLUSIONS: Most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative disease who might be considered suitable candidates for adjuvant endocrine therapy alone.

Cytokeratin profiles of male breast cancers.

AIMS: The prognostic factors and expression of molecular markers in male breast carcinomas are similar to those in female breast cancers. The identification of distinct cytokeratin (CK) profiles (basal as opposed to luminal cells) helps to identify subsets of tumours with different clinical behaviour. The aim of this study was to investigate CK expression in male breast cancer. METHODS AND RESULTS: Thirty-two cases of male breast cancer were studied. The panel of CKs studied by immunohistochemistry included: 5/6, 14, 17, 18 and 19. Pathological findings and CK expression were analysed in all cases. Histological patterns included ductal carcinoma in situ, invasive ductal carcinoma and mixed patterns. Four cases were positive for CK5/6 and CK14, identifying a basal-like phenotype. CK17 was negative in all but two cases. All cases expressing either CK5/6 or CK14 were invasive carcinomas of high nuclear and histological grade and were also larger compared with the tumours not expressing CK5/6 and CK14. All tumours except three (also negative for CK5/6) expressed CK18 and CK19. The four basal-like tumours were negative for Her-2 expression. CONCLUSIONS: Male breast carcinomas have a basal-like phenotype that is similar in frequency to that of female breast carcinomas. The expression of CK5/6 and CK14 identifies a subset of pathologically aggressive male breast cancers.

Turbulent flow properties of large-scale vortex systems.

Large-scale computations of dynamically interacting vortex tubes forming filaments are performed with a view toward investigating their relationship to turbulent fluid flow. It is shown that the statistical properties of the tubes are consistent with commonly accepted observations about turbulence such as the Kolmogorov inertial range spectrum and lognormality of the vorticity distribution. A loop-removal algorithm is demonstrated to reduce the nominally exponential growth rate in the number of tubes to linear growth without apparent harm to the underlying physics. In this form, a vortex tube method may become a practical means for simulating high Reynolds number turbulent flows.

The LightTyper: high-throughput genotyping using fluorescent melting curve analysis.

Instrumentation, chemistry, and software for high-throughput genotyping using fluorescent melting curves are described. The LightTyper system provides post-amplification genotyping within 10 min using samples in 96- or 384-well microplate formats. The system is homogenous because all reagents are added at the beginning of the reaction and there is no sample manipulation between amplification and genotyping. High-resolution melting curves are achieved by slow and steady heating. As samples are heated, panels of blue light-emitting diodes excite the probes, and fluorescence emission is acquired with a cooled charge-coupled device camera. A variety of probe chemistries are compatible for genotyping on the LightTyper, including dsDNA dyes, single-labeled probes, and fluorescence resonance energy transfer systems. Genotyping is performed automatically, and each sample is given a score reflecting the similarity of the genotype to the standards provided. Standard genotypes can be selected from within the run or imported from other files. Samples and genotypes can be grouped to allow multiple-allele detection on one or many samples. The utility of the LightTyper is illustrated by genotyping samples for the Factor V Leiden mutation and for mutations in the CFTR gene.

Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping.

Fluorescent hybridization probes were multiplexed for color genotyping of the apolipoprotein E locus using model oligonucleotide targets. Fluorescence resonance energy transfer was observed during adjacent hybridization of 3'-fluorescein-labeled "donor" probes paired with 5'-labeled "acceptor" probes with different emission spectra reporting at codons 112 and 158. The acceptor dyes emitted at either 640 nm (LightCycler Red 640) or 705 nm (LightCycler Red 705) and were monitored with a LightCycler, a thermal cycler with an integrated fluorimeter. The color of the acceptor dye identified each site and the characteristic melting temperatures of the fluorescein-labeled probes identified single base changes within each codon. Color compensation of temperature-dependent spectral overlap was applied to completely separate each channel. Competition between the probes and the complementary strand for the target sequence decreased resonance energy transfer, indicating an advantage of single-stranded target. Hybridization probes of the same length, but different GC content are T(m) shifted by the same amount during A:C mismatch duplex melting. Genotyping was optimal at both sites if melting curve analysis was preceded by a slow (1 degrees C/s) annealing phase. Although each site preferred different concentrations of Mg(2+) and target strand for optimal genotyping, conditions for multiplexing were found. This method, along with an appropriate amplification technique, should allow real-time multiplex genotyping from genomic DNA.

Rapid F508del and F508C assay using fluorescent hybridization probes.

Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The hybridization of adjacent fluorescent probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent hybridization temperature (Tm) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent Tm and the true equilibrium Tm. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein hybridization probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator gene of chromosome 7. While genotyping for the F508del, an allele with the F508C base change was detected. For both F508del and F508C variants, the Tm shift from wild type was greater with a 24-mer probe than with a 35-mer probe. Fluorescent monitoring of hybridization probes is a versatile technique that can detect unexpected sequence alterations.

Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes.

Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing by Tm for the first time. The new probe design uses a 3'-fluorescein-labeled probe and a 5'-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations.

Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves.

A microvolume fluorimeter integrated with a rapid thermal cycler allows both amplification and point mutation detection from genomic DNA in approximately 30 min. This homogeneous method combines rapid cycle DNA amplification with allele-specific fluorescent probe melting profiles for product genotyping. The amplification reaction includes a primer internally labeled with Cy5 and a 3'-fluorescein-labeled probe that spans the region of interest. During asymmetric amplification, the probe hybridizes to excess Cy5-labeled strand and is observed as fluorescence resonance energy transfer. Resonance energy transfer increases each cycle as product accumulates during amplification. When fluorescence is monitored as the temperature increases through the Tm of the probe/product duplex, a characteristic melting profile for each genotype is obtained. Fluorescence genotyping of the common C677T base substitution in the methylenetetrahydrofolate reductase gene in 110 DNA samples correlated perfectly with genotyping by restriction enzyme digestion and gel electrophoresis. The relatively stable G:T mismatch of this example gave a 3 degrees C difference in Tm from complete Watson-Crick pairing, suggesting that this homogeneous fluorescence method can be used for all single-base mismatches.

The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake.

S. cerevisiae accumulate iron by a process requiring a ferrireductase and a ferrous transporter. We have isolated a mutant, fet3, defective for high affinity Fe(II) uptake. The wild-type FET3 gene was isolated by complementation of the mutant defect. Sequence analysis of the gene revealed the presence of an open reading frame coding for a protein with strong similarity to the family of blue multicopper oxidoreductases. Consistent with the role of copper in iron transport, growth of wild-type cells in copper-deficient media resulted in decreased ferrous iron transport. Addition of copper, but not other transition metals (manganese or zinc), to the assay media resulted in the recovery of Fe(II) transporter activity. We suggest that the catalytic activity of the Fet3 protein is required for cellular iron accumulation.

Synthesis and benzodiazepine binding activity of a series of novel [1,2,4]triazolo[1,5-c]quinazolin-5(6H)-ones.

Investigation of tricyclic heterocycles related to the 2-arylpyrazolo[4,3-c]quinolin-3(5H)-ones, structures with high affinity for the benzodiazepine (BZ) receptor, led to the synthesis of 2-phenyl-[1,2,4]triazolo[1,5-c]quinazolin-5(6H)-one, a compound with 4 nM binding affinity to the BZ receptor. Analogues were prepared to assess the importance of the 2-substituent and ring substitution in modifying activity. Several novel synthetic routes were designed to prepare the target compounds, including a two-step synthesis beginning with an anthranilonitrile and a hydrazide. Of the 34 compounds screened in this series, three compounds were found to be potent BZ antagonists in rat models. The leading compound, 9-chloro-2-(2-fluorophenyl) [1,2,4]triazolo[1,5- c]quinazolin-5(6H)-one (CGS 16228), showed activity comparable to that of CGS 8216 from the pyrazolo[4,3-c]quinoline series.

CGS 19755: a novel competitive N-methyl-D-aspartate (NMDA) receptor antagonist with anticonvulsant, anxiolytic and anti-ischemic properties.

CGS 19755 is a potent and selective competitive antagonist at NMDA receptors in the brain. In preclinical animal tests, the compound produces anticonvulsant, anxiolytic and anti-ischemic effects. CGS 19755 is not very active when administered orally, and intravenous administration is the most practical for clinical application. The anti-ischemic potential of CGS 19755 provides the most attractive application for clinical investigation.

4-(Phosphonoalkyl)- and 4-(phosphonoalkenyl)-2-piperidinecarboxylic acids: synthesis, activity at N-methyl-D-aspartic acid receptors, and anticonvulsant activity.

A series of 4-(phosphonoalkyl)- and 4-(phosphonoalkenyl)-2-piperidinecarboxylic acids were synthesized, and their biological activity was assessed as competitive ligands for the NMDA receptor, both in vitro by using a receptor binding assay ([3H]CGS 19755 binding) and in vivo by using an NMDA seizure model in mice. The analogues were also evaluated in [3H]AMPA and [3H]kainate binding to assess their affinity for non-NMDA excitatory amino acid receptor subtypes. A number of these analogues show potent and selective NMDA antagonistic activity both in vitro and in vivo. Most notable are 4-(phosphonomethyl)-2-piperidinecarboxylic acid (1a) (CGS 19755) and the phosphonopropenyl analogue 1i, both of which show anticonvulsant activity in the 1-2 mg/kg ip range. With the aid of computer-assisted modeling, a putative bioactive conformation for AP-5 is hypothesized from the SAR data presented and a preliminary model for the antagonist-preferring state of the NMDA receptor is presented.

Behavioral pharmacological profile of CGS 19755, a competitive antagonist at N-methyl-D-aspartate receptors.

CGS 19755 (cis-4-phosphonomethyl-2-piperidine-carboxylic acid), a competitive antagonist at N-methyl-D-aspartate (NMDA)-preferring receptors, blocked both NMDA-induced convulsions in normal CF1 mice and sound-induced wild running in seizure-prone DBA/2 mice. The ED50 values for CGS 19755 to produce these effects (in the range of 2 mg/kg i.p.) were at least 3-fold lower than those which impaired the traction reflex, an index of motor coordination. When administered p.o. by gavage, CGS 19755 had little or no effect in these test procedures. In an experimental model of anxiety in rats, CGS 19755 significantly increased conflict responding within a relatively narrow dose range (minimum effective dose, 1.73 mg/kg i.p.). At higher doses of CGS 19755, this effect appeared to be obscured by drug-induced reductions in overall responding. Potential muscle relaxant effects were also suggested by the generalization of CGS 19755 to diazepam discriminative stimuli (ED50 = 9.0 mg/kg i.p.) and by impaired rotorod performance (ED50 = 6.2 mg/kg i.p.) in rats. Although some resemblances were apparent between the behavioral effects of CGS 19755 and those of phencyclidine-type drugs, the phencyclidine-like behaviors appeared only at considerably higher doses of CGS 19755 than those associated with anticonflict activity, and only partial generalization of CGS 19755 to dexoxadrol was observed at high doses. CGS 19755 promises to be an important new research tool for investigating the function of brain NMDA receptors.

A comparison of PCP-like compounds for NMDA antagonism in two in vivo models.

The PCP-like compounds ketamine and dexoxadrol were evaluated in two behavioral test procedures known to be sensitive to competitive N-methyl-D-aspartate (NMDA) receptor antagonists. In the NMDA-induced convulsion test in mice, ketamine and dexoxadrol blocked convulsant activity only at doses that also induced nonspecific effects of PCP-like behaviors, thereby confounding the interpretation of results. These compounds also blocked NMDA-induced discriminative stimuli in rats; however, this effect was produced at doses lower than those which induced the nonspecific behavioral effects. These results provide evidence that in behavioral procedures, PCP-like compounds may block excitatory amino acid receptor stimulation by NMDA. The NMDA discrimination identifies these interactions without the influence of motor deficit or other behavioral motor effects.

CGS 8216, a benzodiazepine antagonist, reduces food intake in food-deprived rats.

CGS 8216, a benzodiazepine receptor antagonist with weak inverse agonist properties, reduced food intake in food-deprived rats when administered orally or intraperitoneally at doses that antagonize diazepam. This effect was sustained when CGS 8216 was administered daily for five days, indicating no rapid tolerance to the anorectic effect. Ro 15-1788 did not reduce feeding when administered orally, and was active only at high intraperitoneal doses (54 and 100 mg/kg). CGS 9896, a close analog of CGS 8216 but a benzodiazepine partial agonist with anxiolytic properties, did not reduce food intake at doses as high as 100 mg/kg IP or PO. These results support prior suggestions that benzodiazepine receptors may modulate feeding behavior, and suggest that CGS 8216 may have appetite suppressant properties.

CGS 9896: agonist-antagonist benzodiazepine receptor activity revealed by anxiolytic, anticonvulsant and muscle relaxation assessment in rodents.

CGS 9896, a pyrazoloquinoline that potently binds to benzodiazepine receptors, has been reported to have anticonflict activity in conventional footshock paradigms and to antagonize pentylenetetrazol-induced seizures. In the present experiments, the pentylenetetrazol discriminative cue was blocked by CGS 9896 with a potency comparable to that of diazepam. CGS 9896 also selectively lengthened the latency to terminate self-initiated brain stimulation reward. These procedures extend the anxiolytic activity of CGS 9896 to models that do not rely upon footshock-induced conflict. CGS 9896 did not impair the traction reflex in mice, did not impair rotorod performance in rats, did not reduce unpunished operant responding and decreased motor activity only slightly, indicating no distinguishable sedation or muscle relaxation in rodent models. In fact, diazepam-induced rotorod impairment was blocked by CGS 9896. The anticonvulsant effects of CGS 9896, as indicated by audiogenic seizure and pentylenetetrazol-induced seizure studies, were substantial but were weaker than those of diazepam, possibly because of the muscle relaxant component of diazepam. Ethanol-induced motor impairment was potentiated more markedly by diazepam than by CGS 9896. Mixed agonist-antagonist properties of CGS 9896 therefore emerge when a comprehensive battery of behavioral assessments is utilized. CGS 9896 may have clinical anxiolytic activity without sedation or muscle relaxation.

The neuropharmacology of various diazepam antagonists.

Recently, compounds which bind avidly to benzodiazepine binding sites have been shown to possess diazepam antagonist properties. For example, the benzodiazepine RO 15-1788 and the pyrazoloquinoline CGS 8216 can antagonize the anxiolytic, sedative, muscle relaxant and anticonvulsant properties of diazepam. The beta-carbolines have also been shown to antagonize several actions of diazepam. Other compounds including physostigmine, naloxone, bicuculline, picrotoxin, caffeine and theophylline, lack appreciable affinity for benzodiazepine binding sites but do antagonize at least some of the behavioral actions of diazepam. Their antagonist properties are probably the result of opposing pharmacological actions rather than direct receptor antagonism. Clinically, a potent safe diazepam antagonist could be used to reverse effects of diazepam overdose and to speed recovery of diazepam-treated patients after various out-patient procedures.