A phase I trial of lenalidomide and radiotherapy in children with diffuse intrinsic pontine gliomas or high-grade gliomas.

PURPOSE: This study was performed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of the immunomodulatory agent, lenalidomide, when administered daily during 6 weeks of radiation therapy to children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) or high-grade glioma (HGG) PATIENTS & METHODS: Children and young adults < 22 years of age with newly diagnosed disease and no prior chemotherapy or radiation therapy were eligible. Children with HGG were required to have an inoperable or incompletely resected tumor. Eligible patients received standard radiation therapy to a prescription dose of 54-59.4 Gy, with concurrent administration of lenalidomide daily during radiation therapy in a standard 3 + 3 Phase I dose escalation design. Following completion of radiation therapy, patients had a 2-week break followed by maintenance lenalidomide at 116 mg/m2/day × 21 days of a 28-day cycle. RESULTS: Twenty-nine patients (age range 4-19 years) were enrolled; 24 were evaluable for dose finding (DIPG, n = 13; HGG, n = 11). The MTD was not reached at doses of lenalidomide up to 116 mg/m2/day. Exceptional responses were noted in DIPG and malignant glioma (gliomatosis cerebri) notably at higher dose levels and at higher steady state plasma concentrations. The primary toxicity was myelosuppression. CONCLUSION: The RP2D of lenalidomide administered daily during radiation therapy is 116 mg/m2/day. Children with malignant gliomas tolerate much higher doses of lenalidomide during radiation therapy compared to adults. This finding is critical as activity was observed primarily at higher dose levels suggesting a dose response.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Analysis of the DNA sequence and duplication history of human chromosome 15.

Here we present a finished sequence of human chromosome 15, together with a high-quality gene catalogue. As chromosome 15 is one of seven human chromosomes with a high rate of segmental duplication, we have carried out a detailed analysis of the duplication structure of the chromosome. Segmental duplications in chromosome 15 are largely clustered in two regions, on proximal and distal 15q; the proximal region is notable because recombination among the segmental duplications can result in deletions causing Prader-Willi and Angelman syndromes. Sequence analysis shows that the proximal and distal regions of 15q share extensive ancient similarity. Using a simple approach, we have been able to reconstruct many of the events by which the current duplication structure arose. We find that most of the intrachromosomal duplications seem to share a common ancestry. Finally, we demonstrate that some remaining gaps in the genome sequence are probably due to structural polymorphisms between haplotypes; this may explain a significant fraction of the gaps remaining in the human genome.

DNA sequence and analysis of human chromosome 18.

Chromosome 18 appears to have the lowest gene density of any human chromosome and is one of only three chromosomes for which trisomic individuals survive to term. There are also a number of genetic disorders stemming from chromosome 18 trisomy and aneuploidy. Here we report the finished sequence and gene annotation of human chromosome 18, which will allow a better understanding of the normal and disease biology of this chromosome. Despite the low density of protein-coding genes on chromosome 18, we find that the proportion of non-protein-coding sequences evolutionarily conserved among mammals is close to the genome-wide average. Extending this analysis to the entire human genome, we find that the density of conserved non-protein-coding sequences is largely uncorrelated with gene density. This has important implications for the nature and roles of non-protein-coding sequence elements.

Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

Genomic representations using concatenates of Type IIB restriction endonuclease digestion fragments.

We have developed a method for genomic representation using Type IIB restriction endonucleases. Representation by concatenation of restriction digests, or RECORD, is an approach to sample the fragments generated by cleavage with these enzymes. Here, we show that the RECORD libraries may be used for digital karyotyping and for pathogen identification by computational subtraction.

TOPO TA is A-OK: a test of phylogenetic bias in fungal environmental clone library construction.

TA cloning methods are widely used in analyses of environmental microbial diversity, yet the potential of TA methods to yield phylogenetically biased results has received little attention. To test for a TA bias, we constructed clone libraries of fungal amplicons spanning the ribosomal internally transcribed spacer (ITS) and partial large subunit (LSU) from 92 boreal forest soil DNA extracts using two contrasting methods: the Invitrogen TOPO-TA system and the Lucigen PCR-SMART system. The Lucigen system utilizes blunt-ended rather than TA cloning and transcription terminators to reduce biases due to toxicity of expressed inserts. We analysed 588 clone sequences from the two libraries. Species diversity estimators applied to operational taxonomical units (OTUs) were slightly higher for Invitrogen than Lucigen, but confidence intervals for accumulation curves overlapped. Abundances of OTUs were correlated between the libraries (r(2) = 0.5, P < 0.0001), but certain OTUs had contrasting abundances in the two libraries and a likelihood ratio test rejected homogeneity of the OTU counts. We constructed parsimony and Bayesian trees from aligned LSU regions, and the 'phylogenetic test' revealed that lineage representation was not significantly different between the two libraries. We conclude that characterization of this fungal community was fairly robust to cloning method and no biases due to TA cloning were found.

Strain characteristics of Streptococcus pneumoniae carriage and invasive disease isolates during a cluster-randomized clinical trial of the 7-valent pneumococcal conjugate vaccine.

Widespread use of 7-valent pneumococcal conjugate vaccine (PCV7) has led to significant reductions in disease while changing pneumococcal population dynamics via herd immunity and serotype replacement. We performed multilocus sequence typing (MLST) on 590 pneumococcal isolates obtained during the American Indian clinical trial of PCV7, in which communities were randomized for eligible children to receive either PCV7 or a meningococcal conjugate vaccine (MCV). Sequence types (STs) were analyzed to determine the impact of the vaccine on pneumococcal population structure and to assess the possible impact of pneumococcal genetic background on vaccine effects. One hundred forty-three STs were obtained, the most frequent being ST199, the only one that included vaccine serotypes (VTs), non-vaccine-associated nonvaccine serotypes (NVA/NVTs), and vaccine-associated serotypes (VATs). Serotype replacement observed in the PCV communities was due to a diverse population of STs, most of which also existed in the MCV communities. Possible capsular switching to create novel ST associations with NVA/NVTs was detected only once. Reductions in VTs and changes in VATs in PCV communities did not show evidence of variation by ST, after accounting for lower vaccine effectiveness against serotype 19F. These observations suggest the hypothesis that the vaccine acts as a "serotype filter": its effect on a particular strain can be predicted on the basis of the serotype of the strain, with little effect of genetic background (as assessed by MLST) over and above capsule. If sustained, such patterns provide some cause for optimism that rapid evolution of PCV escape strains with drug resistance or high virulence is unlikely.

Assembly of polymorphic genomes: algorithms and application to Ciona savignyi.

Whole-genome assembly is now used routinely to obtain high-quality draft sequence for the genomes of species with low levels of polymorphism. However, genome assembly remains extremely challenging for highly polymorphic species. The difficulty arises because two divergent haplotypes are sequenced together, making it difficult to distinguish alleles at the same locus from paralogs at different loci. We present here a method for assembling highly polymorphic diploid genomes that involves assembling the two haplotypes separately and then merging them to obtain a reference sequence. Our method was developed to assemble the genome of the sea squirt Ciona savignyi, which was sequenced to a depth of 12.7 x from a single wild individual. By comparing finished clones of the two haplotypes we determined that the sequenced individual had an extremely high heterozygosity rate, averaging 4.6% with significant regional variation and rearrangements at all physical scales. Applied to these data, our method produced a reference assembly covering 157 Mb, with N50 contig and scaffold sizes of 47 kb and 989 kb, respectively. Alignment of ESTs indicates that 88% of loci are present at least once and 81% exactly once in the reference assembly. Our method represented loci in a single copy more reliably and achieved greater contiguity than a conventional whole-genome assembly method.

The ner gene of Photorhabdus: effects on primary-form-specific phenotypes and outer membrane protein composition.

The nematode-bacterium complex of Heterorhabditis-Photorhabdus is pathogenic to insect larvae. The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes. The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries. Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as ner, a regulatory gene in gram-negative bacteria and their phages. Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form. These and other pieces of evidence indicate that the endogenous ner gene is not responsible for the secondary phenotype, but that ner, when overexpressed, can repress expression of primary phenotypes at stationary phase. Inactivation of the endogenous ner gene in the primary form affected the outer membrane protein profile. A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the ner primary strain.