ESMO Recommendations on clinical reporting of genomic test results for solid cancers.

BACKGROUND: Genomic tumour profiling has a crucial role in the management of patients with solid cancers, as it helps selecting and prioritising therapeutic interventions based on prognostic and predictive biomarkers, as well as identifying markers of hereditary cancers. Harmonised approaches to interpret the results of genomic testing are needed to support physicians in their decision making, prevent inequalities in precision medicine and maximise patient benefit from available cancer management options. METHODS: The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group assembled a group of international experts to propose recommendations for preparing clinical genomic reports for solid cancers. These recommendations aim to foster best practices in integrating genomic testing within clinical settings. After review of available evidence, several rounds of surveys and focused discussions were conducted to reach consensus on the recommendation statements. Only consensus recommendations were reported. Recommendation statements were graded in two tiers based on their clinical importance: level A (required to maintain common standards in reporting) and level B (optional but necessary to achieve ideal practice). RESULTS: Genomics reports should present key information in a front page(s) followed by supplementary information in one or more appendices. Reports should be structured into sections: (i) patient and sample details; (ii) assay and data analysis characteristics; (iii) sample-specific assay performance and quality control; (iv) genomic alterations and their functional annotation; (v) clinical actionability assessment and matching to potential therapy indications; and (vi) summary of the main findings. Specific recommendations to prepare each of these sections are made. CONCLUSIONS: We present a set of recommendations aimed at structuring genomics reports to enhance physician comprehension of genomic profiling results for solid cancers. Communication between ordering physicians and professionals reporting genomic data is key to minimise uncertainties and to optimise the impact of genomic tests in patient care.

Influence of plant severing on movement of Ostrinia nubilalis larvae in Zea mays hybrid seed production fields.

Genetically engineered corn hybrids that contain a cry gene from the bacterium Bacillus thuringiensis Berliner (Bt) are gaining popularity for controlling the corn pest Ostrinia nubilalis (Hübner). Continuous use of Bt corn, however, could select for O. nubilalis that are resistant to this corn. Monitoring for insect resistance is important, because it could help maintain the Bt technology. A possible monitoring method is to collect larval insects in commercial drying bins after harvest from Bt seed production fields. A drawback to this method is that these collections may be contaminated by insects that moved as later instars from severed non-Bt male rows into the adjacent Bt female rows. These larvae have little to no exposure to Bt toxin, resulting in possible "false positives." The objectives of this study were to first find which combination of planting and severing dates produces the least number of larvae that move from non-Bt male plants to Bt female plants and to assess O. nubilalis larval movement from severed non-Bt male rows to Bt female rows. Field studies in 2002 and 2003 were designed to simulate a hybrid seed production field. Results suggest that movement of O. nubilalis larvae from male corn is minimized when corn is planted early and male plants are severed by 2 wk post-anthesis. This reduces the likelihood of false positives by reducing the number of susceptible larvae moving between Bt and non-Bt plants. Also, larvae moved to all four female rows that were adjacent to the severed rows, but there were significantly more larvae found in the closest row compared with the other three. These results could be used to develop a monitoring program to find O. nubilalis larvae with resistance to Bt corn in field populations of O. nubilalis.

Early-season colonization patterns of the boll weevil (Coleoptera: Curculionidae) in Central Texas cotton.

It is commonly believed that colonization of early-season cotton, Gossypium hirsutum L., by overwintered boll weevils, Anthonomus grandis grandis Boheman, is concentrated on field margins. However, supporting experimental evidence is not available. In 1999 and 2000, we examined colonization patterns of overwintered boll weevils in Central Texas cotton on the bases of adult collections by a pneumatic sampler and hand collections of abscised infested squares. Samples were taken from sites arranged in a grid that extended inward >70 m from the field margin. Adults were collected from shortly after seedling emergence until the flowering stage, and infested squares were collected during the one-third grown square stage. Despite numerical trends, the numbers of adult weevils collected were not significantly different between years or sexes, or among plant phenological stages. Field-to-field variation among collections was considerable and likely prevented detection of differences among these factors. Spatial patterns represented by adult weevil and infested square collections were examined by logistic regressions fitted to the respective probabilities of weevil detection at each designated sample site. Although we observed trends for slightly decreased probability of weevil detection with increased distance from the field margin, these trends were too weak to be demonstrated statistically. Our results indicate the boll weevil does not consistently exhibit a strong edge-oriented colonization pattern, and that management tactics that are predicated on these patterns, such as border sprays, should be used with caution.

Growth, development, and survival of Nosema pyrausta-infected European corn borers (Lepidoptera: Crambidae) reared on meridic diet and Cry1Ab.

Transgenic corn, Zea mays L., hybrids expressing crystal protein endotoxin genes from Bacillus thuringiensis Berliner are an increasingly popular tactic for managing the European corn borer, Ostrinia nubilalis (Hübner), in North America. O. nubilalis populations also are often vulnerable to the ubiquitous entomopathogenic microsporidium Nosema pyrausta (Paillot). We examined the effect of feeding meridic diet incorporated with purified Cry1Ab on growth, development, and survival of Nosema-infected and uninfected neonate O. nubilalis. Infected larvae developed more slowly than uninfected larvae. Increasing the concentration of Cry1Ab in diet reduced larval development, and this effect was amplified by microsporidiosis. Infected larvae weighed significantly less than uninfected larvae. The relationship among Nosema infection, Cry1Ab concentration, and larval weight was fitted to an exponential function. The LC50 of infected larvae was one-third that of uninfected larvae, indicating that infected larvae are more vulnerable to toxin. This work has implications for resistance management of O. nubilalis and demonstrates that it is important to determine whether N. pyrausta is present when testing susceptibility of larvae to transgenic corn hybrids.

Factors related to the self-concept of institutionalized, white, male, adolescent drug abusers.

The main purpose of this research was to examine the relationship between selected drug-related, familial, and demographic variables and the self-concepts of institutionalized, white, male, adolescent drug abusers in a large midwestern city. In addition, the self-concepts of the subjects in the study were compared with the self-concepts of Pulliam et al.'s (1971) non-drug using adolescent sample. The TSCS, a family questionnaire, an index of socioeconomic status, and an interview were used to collect data from 140 subjects. The mean self-concept score of this sample was significantly lower than the mean self-concept score of Pulliam et al.'s sample. Results of a stepwise multiple regression yielded four statistically significant predictors of the subjects' self-concepts: both mother/adolescent relationship and father/adolescent relationship when the adolescent was between age thirteen and his present age; the number of the subject's prior status offenses and the subject's preference for non-depressant drugs. These variables were positively correlated with the subjects' self-concepts. This study demonstrated the importance of differentiating among institutionalized adolescents' self-concepts based on type of drug abused, prior criminal record, and parent/adolescent relationships.

Fibroblast growth factor-18 stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and a primary cause of disability, however, there are no treatments that can slow disease progression or repair damaged joint cartilage. Fibroblast growth factor-18 (FGF18) has been reported to have significant anabolic effects on cartilage. We therefore examined its effects on repair of cartilage damage in a rat meniscal tear model of OA. DESIGN: Surgical damage to the meniscus in rats leads to joint instability and significant damage to the articular cartilage at 3 weeks post-surgery. At this time, animals received bi-weekly intra-articular injections of FGF18 for 3 weeks, and the knee joints were then harvested for histologic examination. RESULTS: FGF18-induced dose-dependent increases in cartilage thickness of the tibial plateau, due to new cartilage formation at the articular surface and the joint periphery. The generation of new cartilage resulted in significant reductions in cartilage degeneration scores. The highest dose of FGF18 also induced an increase in chondrophyte size and increased remodeling of the subchondral bone. CONCLUSIONS: The results of this study demonstrate that FGF18 can stimulate repair of damaged cartilage in a setting of rapidly progressive OA in rats.

Drosophila Rrp1 domain structure as defined by limited proteolysis and biophysical analyses.

Drosophila Rrp1 is a DNA repair nuclease whose C-terminal region shares extensive homology with Escherichia coli exonuclease III, has nuclease activity, and provides resistance to oxidative and alkylating agents in repair-deficient E. coli strains. The N-terminal 421 amino acid region of Rrp1, which binds and renatures homologous single-stranded DNA, does not share homology with any known protein. Proteolysis by endoproteinase Glu-C (protease V8) reduces the Rrp1 protein to a single, cleavage-resistant peptide. The peptide (referred to as Rrp1-C274) begins with the sequence TKTTV, corresponding to cleavage between Glu-405 and Thr-406 of Rrp1. We determined that nuclease activity is intrinsic to Rrp1-C274 although altered when compared with Rrp1; 3'-exonuclease activity is reduced 210-fold, 3'-phosphodiesterase activity is reduced 6.8-fold, and no difference in apurinic/apyrimidinic endonuclease activity is observed. Rrp1 and Rrp1-C274 are both monomers with frictional coefficients of 2.2 and 1.4, respectively. Circular dichroism results indicate that Rrp1-C274 is predominantly alpha-helical, while the N-terminal 399 amino acids is predominantly random coil. These results suggest that Rrp1 may have a bipartite structural organization; a highly organized, globular C-terminal domain; and an asymmetric, protease-sensitive random coil-enriched N-terminal region. A shape model for this bipartite structure is proposed and discussed.

DNA binding properties of the Saccharomyces cerevisiae DAT1 gene product.

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein (Dat1p) that specifically recognizes the minor groove of non-alternating oligo(A).oligo(T) tracts. Sequence-specific recognition requires arginine residues found within three perfectly repeated pentads (G-R-K-P-G) of the Dat1p DNA binding domain [Reardon, B. J., Winters, R. S., Gordon, D., and Winter, E. (1993) Proc. Natl. Acad. Sci. USA 90, 11327-1131]. This report describes a rapid and simple method for purifying the Dat1p DNA binding domain and the biochemical characterization of its interaction with oligo(A).oligo(T) tracts. Oligonucleotide binding experiments and the characterization of yeast genomic Dat1p binding sites show that Dat1p specifically binds to any 11 base sequence in which 10 bases conform to an oligo(A).oligo(T) tract. Binding studies of different sized Dat1p derivatives show that the Dat1p DNA binding domain can function as a monomer. Competition DNA binding assays using poly(I).poly(C), demonstrate that the minor groove oligo(A).oligo(T) constituents are not sufficient for high specificity DNA binding. These data constrain the possible models for Dat1p/oligo(A).oligo(T) complexes, suggest that the DNA binding domain is in an extended structure when complexed to its cognate DNA, and show that Dat1p binding sites are more prevalent than previously thought.

A peptide motif that recognizes A.T tracts in DNA.

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein that specifically interacts with nonalternating oligo(A).oligo(T) tracts (A.T tracts). Deletion analysis of DAT1 coding information showed that the amino-terminal 36 residues are sufficient for specific DNA binding activity. Furthermore, a 35-residue synthetic peptide corresponding to amino acids 2-36 bound to A.T tracts with an equilibrium dissociation constant of 4 x 10(-10) M. Within this region the pentad Gly-Arg-Lys-Pro-Gly is repeated three times. Mutational analysis revealed that the Arg side chains are required for high-affinity binding, whereas the other pentad side chains are dispensable. Chemical interference experiments showed that the DAT1 protein interacts with the minor groove of the double helix. The data suggest that the pentad arginines interact in a cooperative manner with a repeated minor groove feature of A.T tract DNA to achieve high-affinity recognition. Amino acid similarities with other DNA binding proteins suggest that the DAT1 protein pentad represents a specialized example of a widespread motif used by proteins to recognize A.T base pairs.

High-throughput isolation of Caenorhabditis elegans deletion mutants.

The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information permits a broad-based gene inactivation approach in C. elegans, in which chemically mutagenized nematode populations are screened by PCR for deletion mutations in a specific targeted gene. By handling mutagenized worm growth, genomic DNA templates, PCR screens, and mutant recovery all in 96-well microtiter plates, we have scaled up this approach to isolate deletion mutations in >100 genes to date. Four chemical mutagens, including ethyl methane sulfonate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylpsoralen, induced detectable deletions at comparable frequencies. The deletions averaged approximately 1400 bp in size when using a approximately 3 kb screening window. The vast majority of detected deletions removed portions of one or more exons, likely resulting in loss of gene function. This approach requires only the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organism that can be handled in high density arrays.