ABSTRACT
Introduction: The purpose of this feasibility study was to select targeted therapies according to "ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)". Data interpretation was further supported by a browser-based Treatment Decision Support platform (MH Guide, Molecular Health, Heidelberg, Germany). Patients: We applied next generation sequencing based whole exome sequencing of tumor tissue and peripheral blood of patients with metastatic breast cancer (n = 44) to detect somatic as well as germline mutations. Results: In 32 metastatic breast cancer patients, data interpretation was feasible. We identified 25 genomic alterations with ESCAT Level of Evidence I or II in 18/32 metastatic breast cancer patients, which were available for evaluation: three copy number gains in HER2 , two g BRCA1 , two g BRCA2 , six PIK3CA, one ESR1 , three PTEN , one AKT1 and two HER2 mutations. In addition, five samples displayed Microsatellite instability high-H. Conclusions: Resulting treatment options were discussed in a tumor board and could be recommended in a small but relevant proportion of patients with metastatic breast cancer (7/18). Thus, this study is a valuable preliminary work for the establishment of a molecular tumor board within the German initiative "Center for Personalized Medicine" which aims to shorten time for analyses and optimize selection of targeted therapies.
ABSTRACT
The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking. VPS4 genes are present in virtually all eukaryotes. Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c. Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b. Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c). Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b. Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced. The chromosomal loci of all four VPS4 genes were determined by independent methods. A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence. Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions. Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence. To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells. All tested VPS4 fusion proteins were found in the cytosol. Expression of dominant-negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region. Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins. A physical interaction between the mouse paralogues was also supported by two-hybrid analyses.
Subject(s)
Adenosine Triphosphatases/genetics , Isoenzymes/genetics , Repressor Proteins , 3T3 Cells , ATPases Associated with Diverse Cellular Activities , Amino Acid Sequence , Animals , Blotting, Northern , Chromosome Mapping , Chromosomes, Human, Pair 16/genetics , Chromosomes, Human, Pair 18/genetics , Cloning, Molecular , Conserved Sequence/genetics , DNA/chemistry , DNA/genetics , DNA/isolation & purification , Endosomal Sorting Complexes Required for Transport , Gene Expression , Gene Expression Regulation, Enzymologic , Humans , Introns/genetics , Mice , Molecular Sequence Data , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Synteny , Vesicular Transport ProteinsABSTRACT
The aim of this study was to investigate whether a Staphylococcus aureus strain that carried an inducibly expressed erm(A) gene might exhibit resistance to the non-inducers telithromycin, ABT-773, clindamycin, quinupristin, dalfopristin or the combination quinupristin-dalfopristin after incubation in the presence of inhibitory concentrations of any of these compounds. Whenever resistant mutants were obtained, these were investigated for the molecular basis of the altered resistance phenotype. Resistant mutants were not selected with dalfopristin or quinupristin-dalfopristin, but were obtained with the other four agents. Irrespective of which drug was used for selection, all mutants were cross-resistant to clindamycin, quinupristin, telithromycin and ABT-773, and exhibited structural alterations in the erm(A) translational attenuator. The structural alterations observed included deletions of 14, 83, 121, 131, 147 or 157 bp, three different tandem duplications of 23, 25 or 26 bp, two different types of point mutation, as well as the insertion of IS256. All these alterations either completely prevented the formation of mRNA secondary structures in the erm(A) regulatory region or favoured the formation of those mRNA secondary structures that allowed translation of the erm(A) transcripts. Deletions, which were observed in almost two-thirds of the mutants, might be explained by illegitimate recombination between different parts of the erm(A) regulatory region.