Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin.

Immunotoxins are antibody-toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.

Genotyping of Bacillus cereus strains by microarray-based resequencing.

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.

Influence of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) common polymorphisms on outcome in treatment of melanoma patients with CTLA-4 blockade.

Blockade of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a down-regulator of T-cell activation, can cause cancer regression in patients with metastatic melanoma. However, not all patients respond well to the therapy and some develop severe autoimmune reactions. We hypothesized that common genetic variation in the CTLA4 gene could contribute to response to CTLA-4 blockade and the occurrence of autoimmune reactions. We investigated 7 common single nucleotide polymorphisms, SNPs, (rs733618, rs4553808, rs11571317, rs5742909, rs231775, rs3087243, and rs7565213) in 152 white melanoma patients who received CTLA-4 blockade. Three SNPs were associated with response to therapy: proximal promoter SNPs, rs4553808 [P=0.002; odds ratio (OR) 3.39; 95% confidence interval (CI), 1.62-7.10] and rs11571327 (P=0.02; OR 2.89; 95% CI, 1.23-6.83) and the nonsynonymous SNP rs231775 (Thr17Ala, P=0.009; OR 0.39; 95% CI, 0.18-0.82). A haplotype analysis including the 7 SNPs suggested that the common haplotype, TACCGGG could be associated with no response (P=0.02) whereas the haplotype TGCCAGG (P=0.06; OR 4.13; 95% CI, 1.17-14.5) could be associated with response to the treatment. No significant association was observed for occurrence of severe autoimmune reactions (grade III/IV) either by single SNP or haplotype analyses. Our results suggest that genetic variation in CTLA4 could influence response to CTLA-4 blockade therapy in metastatic melanoma patients, but further studies are necessary to confirm the observed associations.

The Caenorhabditis elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated commissural motor axon guidance.

P21 activated kinases (PAKs) are major downstream effectors of rac-related small GTPases that regulate various cellular processes. We have identified the new PAK gene max-2 in a screen for mutants disrupted in UNC-6/netrin-mediated commissural axon guidance. There are three Caenorhabditis elegans PAKs. We find that each C. elegans PAK represents a distinct group previously identified in other species. Here we examine their roles in the postembryonic migration of the P cell neuroblasts and the axon guidance of the ventral cord commissural motoneurons (VCCMNs). We find that the two PAKs, max-2 and pak-1, are redundantly required for P cell migration and function with UNC-73/Trio and the rac GTPases (CED-10 and MIG-2). During axon guidance of the VCCMNs, PAK-1 also acts with the rac GTPases, CED-10 and MIG-2, and is completely redundant with MAX-2. Interestingly, we find that unlike MAX-2 activity during P cell migration, for motoneuron axon guidance max-2 is also required in parallel to this PAK-1 pathway, independent of rac GTPase signaling. Finally, we provide evidence that MAX-2 functions downstream of the UNC-6/netrin receptor UNC-5 during axon repulsion and is an integral part of its signaling.

Genetic variation, nucleotide diversity, and linkage disequilibrium in seven telomere stability genes suggest that these genes may be under constraint.

To maintain chromosomal integrity and to protect the ends of chromosomes against recognition as damaged DNA, end-to-end fusion, or recombination, a coordinated set of genes is required to stabilize the telomere. We surveyed common genetic variation in seven genes that are vital to telomere stability (TERT, POT1, TNKS, TERF1, TINF2, TERF2, and TERF2IP) and validated single nucleotide polymorphisms (SNPs) in four different ethnic groups (n=118 total). Overall, our data show limited degrees of nucleotide diversity in comparison with data from other gene families. We observed that these genes are highly conserved in sequence between species, and that for nearly all of the coding SNPs the most common allele is ancestral (i.e., it is observed in primate sequences). Our findings support the hypothesis that genetic variation in a pathway that is critical for telomere stability may be under constraint. These data establish a foundation for further investigation of these genes in population-genetics, evolution, and disease-association studies.

Pathology of lymph nodes from patients with breast implants: a histologic and spectroscopic evaluation.

There are only a few published reports describing the pathology of regional lymph nodes from patients with silicone breast implants. Systematic analytical chemical verification of foreign material has not previously been reported. In this study, biopsies of regional lymph nodes from 96 patients with breast implants were studied using conventional histology as well as laser-Raman microprobe spectroscopy and Fourier transform infrared microspectroscopy. Lymph nodes from 12 patients without implants served as negative controls. Foamy macrophages, ranging from rare scattered cells to confluent sheets, were observed in sections of lymph nodes from 91 patients with implants and only rare foamy macrophages were observed in sections from 4 patients without implants. Refractile material consistent with silicone was observed in sections from 86 patients with implants and in no sections from patients without implants. Fragments of foreign material consistent with polyurethane were observed in sections from 16 patients with implants and in no sections from patients without implants. Using spectroscopy, the presence of silicone was confirmed in 71 patients with implants, and the presence of polyurethane was confirmed in 2 patients with implants. Spectroscopy was negative for silicone and polyurethane in all patients without implants. In summary, regional lymph nodes from patients with breast implants often have histologic evidence of silicone migration. Characteristic histologic findings include foamy macrophages and refractile droplets of clear material. Polygonal fragments of polyurethane were observed in lymph nodes from a number of patients. This finding has not been previously reported. The presence of silicone and polyurethane was confirmed using confocal laser-Raman microprobe and Fourier transform infrared microspectroscopy. Other than two prior case reports, this is the first confirmatory evidence of silicone migration to lymph nodes in patients with breast implants and this is the first confirmatory report of polyurethane migration to lymph nodes.

SNP500Cancer: a public resource for sequence validation and assay development for genetic variation in candidate genes.

The SNP500Cancer Database provides sequence and genotype assay information for candidate single nucleotide polymorphisms (SNPs) useful in mapping complex diseases, such as cancer. The database is an integral component of the NCI's Cancer Genome Anatomy Project. SNP500Cancer provides bi-directional sequencing information on a set of control DNA samples derived from anonymized subjects (102 Coriell samples representing four self-described ethnic groups: African/African-American, Caucasian, Hispanic and Pacific Rim). All SNPs are chosen from public databases and reports, and the choice of genes includes a bias towards non-synonymous and promoter SNPs in genes that have been implicated in one or more cancers. The web site is searchable by gene, chromosome, gene ontology pathway and by known dbSNP ID. As of July 2003, the database contains over 3400 SNPs, 2490 of which have been sequenced in the SNP500Cancer population. For each analyzed SNP, gene location and over 200 bp of surrounding annotated sequence (including nearby SNPs) are provided, with frequency information in total and per subpopulation, and calculation of Hardy-Weinberg Equilibrium (HWE) for each subpopulation. Sequence validated SNPs with minor allele frequency > 5% are entered into a high-throughput pipeline for genotyping analysis to determine concordance for the same 102 samples. The website provides the conditions for validated genotyping assays. SNP500Cancer provides an invaluable resource for investigators to select SNPs for analysis, design genotyping assays using validated sequence data, choose selected assays already validated on one or more genotyping platforms, and select reference standards for genotyping assays. The SNP500Cancer Database is freely accessible via the web page at http://snp500cancer.nci.nih.gov/.