Cancer spectrum and frequency among children with Noonan, Costello, and cardio-facio-cutaneous syndromes.

BACKGROUND: Somatic mutations affecting components of the Ras-MAPK pathway are a common feature of cancer, whereas germline Ras pathway mutations cause developmental disorders including Noonan, Costello, and cardio-facio-cutaneous syndromes. These 'RASopathies' also represent cancer-prone syndromes, but the quantitative cancer risks remain unknown. METHODS: We investigated the occurrence of childhood cancer including benign and malignant tumours of the central nervous system in a group of 735 individuals with germline mutations in Ras signalling pathway genes by matching their information with the German Childhood Cancer Registry. RESULTS: We observed 12 cases of cancer in the entire RASopathy cohort vs 1.12 expected (based on German population-based incidence rates). This corresponds to a 10.5-fold increased risk of all childhood cancers combined (standardised incidence ratio (SIR)=10.5, 95% confidence interval=5.4-18.3). The specific cancers included juvenile myelomonocytic leukaemia=4; brain tumour=3; acute lymphoblastic leukaemia=2; rhabdomyosarcoma=2; and neuroblastoma=1. The childhood cancer SIR in Noonan syndrome patients was 8.1, whereas that for Costello syndrome patients was 42.4. CONCLUSIONS: These data comprise the first quantitative evidence documenting that the germline mutations in Ras signalling pathway genes are associated with increased risks of both childhood leukaemia and solid tumours.

Radiosensitization by histone deacetylase inhibition in an osteosarcoma mouse model.

BACKGROUND: Osteosarcomas (OS) are highly malignant and radioresistant tumors. Histone deacetylase inhibitors (HDACi) constitute a novel class of anticancer agents. We sought to investigate the effect of combined treatment with suberoylanilide hydroxamic acid (SAHA) and radiotherapy in OS in vivo. METHODS: Clonogenic survival of human OS cell lines as well as tumor growth delay of OS xenografts were tested after treatment with either vehicle, radiotherapy (XRT), SAHA, or XRT and SAHA. Tumor proliferation, necrosis, microvascular density, apoptosis, and p53/p21 were monitored by immunohistochemistry. The CD95 pathway was performed by flow cytometry, caspase (3/7/8) activity measurements, and functional inhibition of CD95 death signaling. RESULTS: Combined treatment with SAHA and XRT markedly reduced the surviving fraction of OS cells as compared to XRT alone. Likewise, dual therapy significantly inhibited OS tumor growth in vivo as compared to XRT alone, reflected by reduced tumor proliferation, impaired angiogenesis, and increased apoptosis. Addition of HDACi to XRT led to elevated p53, p21, CD95, and CD95L expression. Inhibition of CD95 signaling reduced HDACi- and XRT-induced apoptosis. CONCLUSION: Our data show that HDACi increases the radiosensitivity of osteosarcoma cells at least in part via ligand-induced apoptosis. HDACi thus emerge as potentially useful treatment components of OS.

Analysis of surgical management of calvarial tumours and first results of a newly designed robotic trepanation system.

This study was performed to evaluate the surgical strategy in patients with calvarial tumours, in order to design and modify a robot-assisted trepanation system. A total of 75 patients underwent craniectomy for the treatment of calvarial tumours during the 10-year period from 1993 to 2002. The patients' complaints, the size, location and histology of the tumour, and the various cranioplasty techniques used were analysed retrospectively. In a second procedure several craniectomies at typical locations according to the study's results were performed in a laboratory setting using a hexapod robotic tool, constructed at the Helmholtz-Institute, RWTH Aachen University, and plastic model heads. The workflow was documented and the reproducibility and the accuracy of the procedure were registered. A total of 83 surgical procedures were performed on 75 patients. The majority (87 %) of lesions treated surgically were located in the frontal, temporal and anterior parts of the parietal region. Histological examination revealed benign lesions in 66 % of the patients and dural involvement in 46 %. According to these results craniectomies were performed using the robotic system. Mean positioning accuracy of the robotic system while milling was 0.24 mm, with a standard deviation of 0.04 mm, and maximum error under 1 mm. Craniectomies leaving a 1-mm layer of the tabula interna intact to ensure a healthy dura were performed in several regions successfully. The majority of calvarial tumours, requiring surgical treatment in our patients, were located in cosmetically relevant areas in which drilling can be carried out with the robotic trepanation system. Consequently, the surgical approach had to be planned carefully in order to achieve a good cosmetic outcome.

Dynamic cerebral autoregulation in patients with ruptured and unruptured aneurysms after induction of general anesthesia.

INTRODUCTION: Blood pressure management in patients undergoing surgery for clipping of aneurysms is demanding. More information about the ability of cerebral vessels to normally regulate cerebral blood flow may have a direct influence on the intraoperative management. In patients with subarachnoid hemorrhage (SAH) a disturbance of cerebral autoregulation has been reported and it correlated with the severity of the bleeding in these studies. The impairment of autoregulation was demonstrated using static measurements of cerebral pressure autoregulation. However, the dynamic component of the autoregulatory capacity seems to be of importance in the acute setting after SAH. The aim of this study was to evaluate dynamic pressure autoregulation in patients undergoing surgery for intracranial aneurysms. PATIENTS/MATERIAL AND METHODS: 36 patients with a mean age of 45 years were evaluated, 26 patients with acute SAH, 10 patients with unruptured aneurysms. Cerebral autoregulation in normocapnia was tested using thigh cuffs to alter arterial blood pressure and continuous registration of the blood flow velocities with transcranial Doppler sonography. After the induction of general anesthesia under normocapnia the autoregulatory index (ARI) was calculated (values between 0-9). Patient groups were compared using Wilcoxon- and Spearman's rank test. RESULTS: The two patient groups were comparable with regard to gender, age, PaCO(2), blood flow velocities and blood pressure. In patients with SAH mean ARI was 3.1/3.3 (right/left side) compared to 4.7/4.6 (right/left side) in patients without SAH. The difference was statistically significant (Wilcoxon p = 0.0399). The degree of impairment of the autoregulatory capacity increased significantly (p = 0.006) with the severity of the SAH (Hunt&Hess and Fisher scale). CONCLUSION: Dynamic pressure autoregulation is impaired in patients after SAH compared to patients without SAH and correlates with the severity of the SAH. We propose that autoregulation should be measured in all patients with SAH or that an impaired autoregulation should be taken into account in patients with SAH undergoing surgery in the acute phase.

Accidental dural tears occurring during supratentorial craniotomy -- a prospective analysis of predisposing factors in 100 patients.

OBJECT: Accidental dural tears during craniotomy constitute a possible source of CSF leakage and wound infection. This can turn an elective procedure into a complicated and cost-intensive problem. Only a few studies have addressed the incidence of dural tears, but there have been many studies dealing with various techniques that can be employed to repair dural tears. The present study was carried out to analyze predisposing factors for dural tears during trepanation in order to optimize the design of a robot-assisted trepanation system. PATIENTS: 100 patients were analyzed prospectively. An evaluation sheet was designed to document size and location of the lesion and the craniotomy, the geometry and number of burr holes, and the auxiliary tools used during bone flap removal. Furthermore, the suspected histology was noted and anatomical facts, including cranial vault thickness and the presence of hyperostosis frontalis interna, were documented. RESULTS: In 100 craniotomies performed, in the majority of cases (64 %), in order to gain access to intracerebral lesions, 30 dural tears were seen, involving both dural layers in 26 cases. There were 26 tears located under the margins of the craniotomy; the length was 0-3 cm in 18 patients (69 %). Significant predisposing factors were the thickness of the cranial vault and the presence of a hyperostosis frontalis. Furthermore, the location (frontal) and the diagnosis of an extracerebral pathology, including meningiomas, were significant factors for dural tears. Elderly patients and the use of the drill to complete the trepanation were also significant predisposing factors. Dural repair was done using suturing, in most of the cases combined with a free periostal flap. Central dural tears were integrated into the planned dural opening. A vascularized flap or muscle was used in the minority of cases. Postoperative cerebral fluid leakage was seen in two patients, wound infections in three. CONCLUSIONS: Dural tears occurring during craniotomy cannot be prevented, when predisposing factors are taken into account. The absence of brain damage may due to two factors: 1) in elderly patients with hyperostosis, an additional atrophy of the brain is present; 2) extracerebral tumors, with their space-occupying growth, shift the underlying brain away from the calvaria. Considering the design of a robot-assisted trepanation system, the following conclusions seem possible: dural tears cannot be avoided because predisposing factors are overriding. For improved safety, additional, specialized instrumentation is required.

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

Display of passenger proteins on the surface of Escherichia coli K-12 by the enterohemorrhagic E. coli intimin EaeA.

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides.

The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.

Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening.

The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22-25 comprising a beta-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22-25 tetrapeptide sequences and presented these proteins on the outer membrane of Escherichia coli cells via fusion to the Iga(beta) autotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1-5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in beta-turns. The folding pathway of two selected variants, EETI-beta(NEDE) and EETI-beta(TNNK), was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Comparative studies on the pharmacokinetics of hydrophilic prolinedithiocarbamate, sarcosinedithiocarbamate and the less hydrophilic diethyldithiocarbamate.

The pharmacokinetics of the antitoxic and anticarcinogenic compounds diethyldithiocarbamate, prolinedithiocarbamate and sarcosinedithiocarbamate were compared in rats. The bioavailability, the distribution in the organism, the oxidation to thiuramdisulfides, the cleavage to CS2 and the excretion in urine and bile were investigated. The results showed different behaviour of the three compounds. The more toxic diethyldithiocarbamate had a short in vivo half-life, was oxidized to tetraethylthiuramdisulfide in blood, and was metabolized to high yields of CS2 in 24 h. In contrast, prolinedithiocarbamate was more stable in vivo, was found predominantly in the urinary tract and was excreted in urine. The differences could not be explained by the presence of the carboxy group in the latter dithiocarbamate, since sarcosinedithiocarbamate, which also contains a carboxy group, behaved like diethyldithiocarbamate.

Genetic heterogeneity of prostatic carcinoma-derived cell lines as emphasized by DNA fingerprinting.

To investigate genetic heterogeneity during in vitro cultivation of human prostatic carcinomas following radical prostatectomy we performed DNA fingerprinting using the digoxygenin-labeled probes (GACA)4 and (GTG)5. DNA was isolated from fresh material stemming from different areas within one tumor and from cell cultures of the same material. The patterns which were obtained by the nucleolar organizer region (NOR)-specific probe (GACA)4 exhibit only a few prominent low molecular mass bands and no differences were observed between any of the tumors analyzed so far. Changes in the fingerprint pattern occurred between cell cultures derived from different areas within one tumor when the DNA was cleaved by HaeIII and signals detected with the (GTG)5 probe. The "area-specific" pattern was stable during several subcultivations of these cell lines, indicating genetic stability of these prostatic carcinoma cells in vitro. Thus individual cell lines derived from radical prostatectomy seem to represent a biological system very close to the situation in vivo.