Results of TRIO-15, a multicenter, open-label, phase II study of the efficacy and safety of ganitumab in patients with recurrent platinum-sensitive ovarian cancer.

BACKGROUND: IGF signaling has been implicated in the pathogenesis and progression of ovarian carcinoma (OC). Single agent activity and safety of ganitumab (AMG 479), a fully human monoclonal antibody against IGF1R that blocks binding of IGF1 and IGF2, were evaluated in patients with platinum-sensitive recurrent OC. METHODS: Patients with CA125 progression (GCIG criteria) or measurable disease per RECIST following primary platinum-based therapy received 18 mg/kg of ganitumab q3w. The primary endpoint was objective response rate (ORR) assessed per RECIST 1.1 by an independent radiology review committee (IRC) and/or GCIG CA125 criteria. Secondary endpoints included clinical benefit rate (CBR), progression free survival (PFS) and overall survival (OS). RESULTS: 61 pts. were accrued. Objective responses were seen in 5/61 patients (ORR 8.2%, 95% CI, 3.1-18.8) with 1 partial response (PR) by RECIST and 2 complete responses (CR) as well as 2 PR by CA125 criteria. CBR was 80.3% (95% CI, 67.8-89.0%). The median PFS according to RECIST by IRC was 2.1 months (95% CI, 2.0-3.1). The median PFS per RECIST IRC and/or CA125 was 2.0 months (95% CI, 1.8-2.2). The median OS was 21 months (95% CI, 19.5-NA). The most common overall adverse events were fatigue (36.1%) and hypertension (34.4%). Grade 1/2 hyperglycemia occurred in 30.4% of patients. Hypertension (11.5%) and hypersensitivity (8.2%) were the most frequent grade 3 adverse events. CONCLUSIONS: IGF1R inhibition with ganitumab was well-tolerated, however, our results do not support further study of ganitumab as a single agent in unselected OC patients.

Constant Isothiocyanate-Release Potentials across Biofumigant Seeding Rates.

Biofumigation is an integrated pest-management method involving the mulching of a glucosinolate-containing cover crop into a field in order to generate toxic isothiocyanates (ITCs), which are effective soil-borne-pest-control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest-control outcomes and develop best practices for choosing biofumigants, growth conditions, and mulching methods that allow the greatest potential isothiocyanate release. We measured the glucosinolate concentrations of six different commercial varieties of three biofumigant plant species: Brassica juncea (ISCI99, Vitasso, and Scala) Raphanus sativus (Diablo and Bento), and Sinapis alba (Ida Gold). The plants were grown in the range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, the highest ITC-release potentials were achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. The highest ITC-release potential occurred at the 50% flowering growth stage across the species. The seeding rate had a minor impact on the ITC-release potential of R. sativus but had no significant effects on the ITC-release potentials of the B. juncea or S. alba cultivars.

Characterisation by RNAi of pioneer genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines and the effects of combinatorial RNAi.

Changes in transcript abundance of 24 genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines encoding for putative secretions of unknown function were monitored by quantitative PCR (qPCR) at 0, 2, 7, 14 and 21 days post-invasion (pi) of soybean plantlets. Five groups of temporal patterns (A, B1, B2, C and D) were defined for the 24 genes plus data for two previously studied genes expressed in the same cell. Group D (two genes) showed no significant increase between 0 and 2 days pi and were the least abundantly expressed at 7-21 days pi. Transcripts of group C (nine genes including one studied previously) increased in abundance from 0 to 2 days pi but were the second least expressed for 7-21 days pi. Groups A (three genes), B1 (seven genes) and B2 (five genes including one studied previously) were all abundant at 7-21 days pi. B1 and B2 were discriminated by their relative abundance at 0 and 2 days pi. RNA interference (RNAi) targeting two genes of group A and one each of B1 and B2 in nematodes prior to invasion resulted in phenotypic effects on total parasites per plant and sexual fate at 10 days pi. Phenotype penetrance was reduced for three genes showing such effects and one with a strong effect in earlier work when two genes rather than one were concurrently targeted for RNAi. One gene (dg13) was more abundantly expressed after combinatorial RNAi than for either control nematodes or when targeting singly by RNAi. This work reports the unexpected elevation in mRNA expression after combinatorial RNAi that requires understanding before combinatorial RNAi can be advanced for highly effective cyst nematode control via plant biotechnology.

QPCR analysis and RNAi define pharyngeal gland cell-expressed genes of Heterodera glycines required for initial interactions with the host.

Changes in transcript abundance of genes expressed in the three pharyngeal gland cells of Heterodera glycines after host invasion were monitored by quantitative polymerase chain reaction (qPCR) and the consequences of disrupting their expression studied by RNAi treatment prior to invasion. Two transcripts were known to be expressed in the two subventral gland cells (hg-pel and hg-eng-1), a further two in the single dorsal gland cell only (hg-gp and hg-syv46), and a fifth transcript (hg-cm) was expressed by both gland cell types. The qPCR study established that transcripts of hg-syv46 and hg-gp increased in abundance by 2 days postinfection (dpi), with the former remaining the most abundant. The hg-cm transcript level showed minor changes from 0 to 14 dpi but did fall by 21 dpi. In contrast, hg-eng-1 and hg-eng-2 messenger (m)RNA declined by 7 dpi and hg-pel by 14 dpi before it increased at 21 dpi. RNAi-targeting of hg-eng-1 reduced the number of females present on the plants at 10 days. Targeting of hg-gp, hg-cm, and hg-pel caused a change in sexual fate favoring male development on roots. Both effects were evident after targeting hg-syv46. Suppression of hg-eng-1 mRNA levels in second-stage juveniles (J2i) by RNAi was transient, with a recovery by 15 days of incubation in water after treatment. Presoaking H. glycines J2 with double-stranded RNA has value for studying gene function during the nematode's early interaction with a plant.

Meloidogyne incognita: molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi.

RNA interference has been used to investigate the function of a cathepsin L cysteine proteinase Mi-cpl-1, in the plant-parasitic nematode Meloidogyne incognita. A reduction in gene transcript was observed and the number of nematodes infecting plants was reduced by almost 60% as was the number of established females producing eggs at 21 days post-infection. The cysteine proteinase activity of M. incognita, reported by the substrate GLUpNA, was inhibited by the cysteine proteinase inhibitor Oc-IDeltaD86. A reduction in cysteine proteinase activity was also seen following RNAi of Mi-cpl-1 in J2 stage nematodes. In situ hybridization analysis in young and mature female nematodes has shown that Mi-cpl-1 is expressed in the intestine, which suggests that its product is a digestive enzyme. The effects of knocking-out Mi-cpl-1gene function were consistent with a reduction in feeding efficiency. Here, we have shown a correlation between transcript abundance proteinase activity and parasitic success of M. incognita.

Multitrophic interactions involving genetically modified potatoes, nontarget aphids, natural enemies and hyperparasitoids.

Genetically modified (GM) potatoes expressing a cysteine proteinase inhibitor (cystatin) have been developed as an option for the management of plant parasitic nematodes. The relative impact of such plants on predators and parasitoids (natural enemies) of nontarget insects was determined in a field trial. The trial consisted of GM plants, control plants grown in soil treated with a nematicide and untreated control plants. The quantity of nontarget aphids and their quality as hosts for natural enemies were studied. Aphid density was significantly reduced by nematicide treatment and few natural enemies were recorded from treated potatoes during the study. In contrast, similar numbers of aphids and their more abundant predators were recorded from the untreated control and the GM potatoes. The size of aphids on GM and control plants was recorded twice during the study. During the first sampling period (2-9 July) aphids clip-caged on GM plants were smaller than those on control plants. During the second sampling period (23-30 July) there was no difference in aphid size between those from the GM and control plants. Host size is an important component of host quality. It can affect the size and fecundity of parasitoid females and the sex ratio of their offspring. However, neither the fitness of females of Aphidius ervi, the most prevalent primary parasitoid, nor the sex ratio of their progeny, were affected when the parasitoids developed on aphids feeding on GM plants. Two guilds of secondary parasitoid were also recorded during the study. The fitness of the most abundant species, Aspahes vulgaris, was not affected when it developed on hosts from GM plants. The transgene product, OC I Delta D86, was not detected in aphids that had fed on GM plants in the field, suggesting that there is minimal secondary exposure of natural enemies to the inhibitor. The results indicate that transgenic nematode resistance is potentially more compatible with aphid biological control than is current nematicide use.

A sequential approach to risk assessment of transgenic plants expressing protease inhibitors: effects on nontarget herbivorous insects.

Protease inhibitors expressed in transgenic plants can provide enhanced levels of resistance to important pest species. A sequential approach for testing the effects of protease inhibitor-expressing crops on nontarget herbivorous insects has been developed. The approach consists of five tiers. The first two tiers comprise the selection phase. In tier one, field surveys are used to characterise the nontarget invertebrate fauna of a crop. In tier 2, histochemical assays are used to identify the subset of herbivores with a particular class of digestive proteolytic enzymes. In the assessment phase a combination of laboratory 'worst-case scenario' studies (tier 3) and controlled environment or small-scale field trials (tier 4) are used to evaluate the impact of the protease inhibitor-expressing plants on the selected nontarget species. In the final tier, field trials are used to compare the relative effect of transgenic plants and current management practices, such as pesticide use, on selected species. The first four tiers of the approach are described using potatoes expressing cystatins, a family of cysteine proteinase inhibitors, as an example. Although the plants have enhanced levels of resistance to potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, the results establish that they have negligible impact on the nontarget herbivorous insect, Eupteryx aurata.

Transgenic potatoes with enhanced levels of nematode resistance do not have altered susceptibility to nontarget aphids.

Cysteine proteinase inhibitors (cystatins) confer resistance to plant-parasitic nematodes when expressed in transgenic plants. The survival and growth of nymphs of the peach-potato aphid, Myzus persicae, were adversely affected when cystatins were added to artificial diets. When aphids were clip-caged onto transgenic plants expressing chicken egg white cystatin (CEWc) there was no adverse effect on aphid fitness. Field populations of aphids on transgenic Desiree potatoes, expressing CEWc or a modified version of oryzacystatin I, were not significantly different from populations on control Desiree plants. The effect of other nematode management options on aphid numbers was also studied. A conventionally bred cultivar, with partial nematode resistance, supported higher populations of aphids than the transgenic lines at the beginning of the sampling period. Peak aphid densities on the untreated control and untreated transgenic lines were 7 and 5.2 aphids per plant. Aldicarb, commonly used to control nematodes on potatoes, reduced the value to less than 0.2 aphids per plant. The results demonstrate that levels of expression in the plant tissue actually consumed are important in determining the risk of cystatins to nontarget invertebrates. The study also highlights the importance of including currently used management options in any assessment of the impact of transgenic plants on nontarget organisms.

Neuronal uptake of pesticides disrupts chemosensory cells of nematodes.

Low doses of the acetylcholinesterase-inhibiting carbamate nematicides disrupt chemoreception in plant-parasitic nematodes. Fluorescein isothiocyanate (FITC)/dextran conjugates up to 12 kDa are taken up from the external medium by certain chemosensory neurons in Caenorhabditis elegans. Similar chemoreceptive neurons of the non-feeding infective stage of Heterodera glycines (soybean cyst nematode) fill with FITC and the nuclei of their cell bodies selectively stain with bisbenzimide. The widely used nematicide aldicarb disrupts the chemoreceptive response of H. glycines with 50% inhibition at very low concentrations (ca 1 pM), some 10(-6)-fold lower than required to affect locomotion. Similarly, the anthelmintic levamisole had this effect at 1 nM. Peptides selected as mimetics of aldicarb and levamisole also disrupt chemoreception in H. glycines and Globodera pallida at 10(-3)-fold or lower concentration than required to inhibit locomotion. We propose an uptake pathway for aldicarb, levamisole, peptide mimetics and other soluble molecules by retrograde transport along dendrites of chemoreceptive neurons to the cell bodies and synapses where they act. This may prove to be a general mechanism for the low-dose effects of some nematicides and anthelmintics.

The case for genetically modified crops with a poverty focus.

Recently seven National Academies of Science produced a report on transgenic plants and world agriculture. The report provides scientific perspectives to the ongoing public debate about the potential role for transgenic technology in world agriculture. In this article, we develop the themes of the report and emphasize the potential for future genetically modified (GM) crops with a poverty focus, emphasizing the potential of GM resistance to plant parasitic nematodes for subsistence potato farmers in Bolivia. We judge that a range of incremental gains to crop yields from many transgenes are valuable for future world security. We advocate the establishment of a standard that GM crops must achieve before they are both biosafe and appropriate for resource-poor farmers and we believe that the best interests of the poor require biotechnologists to work towards that objective.

An Algorithm for Optimizing Rotational Control of Globodera rostochiensis on Potato Crops in Bolivia.

The outline area of new cysts of Globodera rostochiensis was measured by image analysis. A linear regression of this value against egg content provided a basis for adjusting the egg number for cyst size. This adjusted egg content provides an estimate of the relative fullness of a cyst with eggs. This value showed an exponential decline in eggs over 3.5 years since the last potato crop. It corresponds to an average loss in the dormant egg population of 32.8 +/- 5.6%/year for 26 fields at Toralapa, Bolivia. This value compared well with a mean decline of 40 +/- 4%/year for 42 fields after measuring viable eggs/100 g soil on two occasions one year apart. The new approach allows declines to be estimated at one time point. The decline in lipid content of the dormant, unhatched second-stage juveniles (J2) was 17 +/- 6% per annum as measured by image analysis after Oil red O staining. This may be sufficient to compromise infectivity after 3 to 4 years of dormancy. A standard model was modified to consider the effect of both lipid depletion during dormancy and choice of susceptible potato on the population dynamics of G. rostochiensis under rotational control. It is concluded that a few cultivars may impose lower populations on G. rostochiensis in 3 to 4-year rotations than the majority used in Bolivia.

Parasitic nematodes, proteinases and transgenic plants.

Parasite proteinases have important functions in host-parasite interactions. Consequently, they have been investigated as targets for the control of both plant and animal parasites. Plant parasitic nematodes cause estimated annual losses to world agriculture of US$100 billion and, currently, their control often relies on highly toxic nematicides, with associated environmental risks. The potential of disrupting digestive proteinases for plant parasitic nematode control, via expression of proteinase inhibitors in transgenic plants, is summarized here by Catherine Lilley, Pauline Devlin, Peter Urwin and Howard Atkinson. They then consider whether the approach of expressing antinematode proteins in plants can be adapted for control of certain animal parasitic nematodes.

Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Müller).

Transgenic Arabidopsis thaliana has been developed which expresses the oryzacystatin mutant OC-I delta 86, which is an inhibitor of the major proteinase present in the digestive gland of the slug, Deroceras reticulatum. When fed on leaf tissue from plants expressing this inhibitor the growth of juvenile slugs was significantly reduced by 31% compared with those feeding on control leaf tissue. Furthermore, while surviving slugs did not individually consume less when feeding on leaf tissue expressing OC-I delta 86, the total amount of leaf tissue eaten was 50% less, due to reduced survival of slugs. The synthetic cysteine proteinase inhibitors E-64 and leupeptin also significantly reduced slug weight gain (by at least 40%) and digestive gland cysteine proteinase activity when administered in an artificial diet, indicating that their antimetabolic effects are due to direct inhibition of gut proteolytic activity. These results suggest that transgenic crop plants expressing phytocystatins could be used to suppress the growth rates of slug populations in the field.

Characterization of plant nematode genes: identifying targets for a transgenic defence.

Current control of plant parasitic nematodes often relies on highly toxic and environmentally harmful nematicides. As their use becomes increasingly restricted there is an urgent need to develop crop varieties with resistance to nematodes. The limitations surrounding conventional plant breeding ensure there is a clear opportunity for transgenic resistance to lessen current dependence on chemical control. The increasing use of molecular biology techniques in the field of plant nematology is now providing useful information for the design of novel defences to meet the new needs. Plant responses to parasitism are being investigated at the molecular level and nematode gene products that could be targets for a direct anti-nematode defence are being characterized. The potential of an anti-feedant approach to nematode control has been demonstrated. It is based on the transgenic expression of proteinase inhibitors. The rational development of this strategy involves characterization of nematode proteinase genes and optimization of inhibitors by protein engineering. Durability of the resistance can be enhanced by stacking transgenes directed at different nematode targets.

The Influence of Potato Cultivar on Lipid Content and Fecundity of Bolivian and British Populations of Globodera rostochiensis.

The influence of host cultivar on the lipid levels provided by a female to her progeny was investigated with Oil Red O stain and a quantitative image analyzer. A population of Globodera rostochiensis was multiplied at Toralapa Field Station in Bolivia on 25 different potato cultivars grown in that country. The mean neutral lipid content of newly formed second-stage juveniles varied significantly with cultivar over a 200% range. The corresponding range was only 18% and 28% for the same Bolivian and a UK population of G. rostochiensis, respectively, when both completed reproduction concurrently on 10 pot-grown European cultivars in the United Kingdom. Egg numbers per female varied with host for Bolivian cultivars that lack known partial resistance to Globodera spp. There was a 15-fold range between the most and least fecund nematode-host combinations (Kosi and Gendarme). The Bolivian G. rostochiensis population showed only a 2-fold range in mean eggs per cyst when grown on European cultivars in the UK. The fatty acid profiles of lipids from Bolivian G. rostochiensis cysts reared on Bolivian potato cultivars were dominated by C(20) (37-64%) and C(18) (28-46%) fatty acids and ranged from C(14) to C(22). The three major fatty acids detected were C(20:4:), C(20:1), and C(18:1). Few differences between cultivars were observed. For a UK population of G. rostochiensis reared on ssp. tuberosum, higher relative percentages of C(18) and monounsaturated fatty acids and lower relative percentages of C(20) and polyunsaturated fatty acids were found.

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs.

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.

Seasonal Changes in the Dorsal Pharyngeal Gland Nucleolus of Unhatched Second-Stage Juveniles of Globodera spp. in Bolivia.

Changes in the diameter of the nucleolus of the dorsal pharyngeal gland (DPGN) in unhatched second-stage juveniles (J2) of potato cyst nematodes, Globodera spp., were monitored for cysts recovered during two field experiments in the Bolivian Central Andes. In the first experiment, cysts were extracted from soil left fallow or supporting crops of potato, barley, lupin, or quinoa. The highest mean DPGN diameter for unhatched J2 occurred shortly after planting in January. The values were similar for individuals recovered from cysts associated with all cultivations. For cysts from potato plots, the lowest mean DPGN diameter of 2.26 +/- 0.05 mum occurred in March, but the value increased again by May to 2.53 +/- 0.05 mum. Similar seasonal changes were found for J2 under both nonhost crops and fallow with the smallest diameters recorded in May of 2.48 +/- 0.02 mum and 2.34 +/- 0.05 mum, respectively. Two possible factors might cause this significant seasonal change. First, some J2 may hatch early in the growing season, even in the absence of the host. This would enhance the proportion of dormant, unhatched J2 remaining in the cyst samples. Secondly, a seasonal change in the DPGN diameter may occur for most individuals with a transient fall value between January and March/May. A model defined by this study provides a good description of the observed effect, providing both factors are assumed to occur. The second experiment studied if changes in size of DPGN in response to a hatching stimulus are influenced by the cyst population age. The DPGN in unhatched J2 was measured for cysts recovered from soils that had supported potatoes that growing season or 2 or 4 years earlier. The unhatched J2 from the freshly cropped potato site showed the largest mean DPGN diameter of 3.66 +/- 0.05 mum after 7 days in potato root diffusate, whereas those from the 4-year sample had the smallest value of 3.20 +/- 0.05 mum. This significant difference may indicate a delayed response to the hatching stimulus with more prolonged J2 dormancy.

Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines.

Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.

Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin.

Plant nematodes are major pests of agriculture. Transgenic plant technology has been developed based on the use of proteinase inhibitors as nematode anti-feedants. The approach offers prospects for novel plant resistance and reduced use of environmentally damaging nematicides. A modified rice cystatin, Oc-I delta D86, expressed as a transgene in Arabidopsis thaliana, has a profound effect on the size and fecundity of females for both Heterodera schachtii (beet-cyst nematode) and Meloidogyne incognita (root-knot nematode). No females of either species achieved the minimum size they require for egg production. Ingestion of Oc-I delta D86 from the plant was correlated with loss of cysteine proteinase activity in the intestine thereby suppressing normal growth, as required of an effective antifeedant plant defence.