Marc-Eric Halatsch

Abstract: OBJECT: The authors have previously reported that erlotinib, an EGFR tyrosine kinase inhibitor, exerts widely variable antiproliferative effects on 9 human glioblastoma multiforme (GBM) cell lines in vitro and in vivo. These effects were independent of EGFR baseline expression levels, raising the possibility that more complex genetic properties form the molecular basis of the erlotinib-sensitive and erlotinib-resistant GBM phenotypes. The aim of the present study was to determine candidate genes for mediating the cellular response of human GBMs to erlotinib. METHODS: Complementary RNA obtained in cell lines selected to represent the sensitive, somewhat responsive, and resistant phenotypes were hybridized to CodeLink Human Whole Genome Bioarrays. RESULTS: Expression analysis of 814 prospectively selected genes involved in major proliferation and apoptosis signaling pathways identified 19 genes whose expression significantly correlated with phenotype. Functional annotation analysis revealed that 2 genes (DUSP4 and STAT1) were significantly associated with sensitivity to erlotinib, and 10 genes (CACNG4, FGFR4, HSPA1B, HSPB1, NFATC1, NTRK1, RAC1, SMO, TCF7L1, and TGFB3) were associated with resistance to erlotinib. Moreover, 5 genes (BDNF, CARD6, FOSL1, HSPA9B, and MYC) involved in antiapoptotic pathways were unexpectedly found to be associated with sensitivity. Gene expressions were confirmed by quantitative polymerase chain reaction. CONCLUSIONS: Based on an analysis of gene expressions in cell lines with sensitive, somewhat responsive, and resistant phenotypes, the authors propose candidate genes for GBM response to erlotinib. The 10 gene candidates for conferring GBM resistance to erlotinib may represent therapeutic targets for enhancing the efficacy of erlotinib against GBMs. Five additional genes warrant further investigation into their role as putative cotargets of erlotinib.

Abstract: A newly-born infant with a congenital dural and bony defect and an associated short-segmented duplication of the superior sagittal sinus suffered from herniation and infarction of parietal brain tissue secondary to vacuum extraction. This ultimately led to the formation of a subgaleal cerebrospinal fluid (CSF) collection. Initial operative closure of the encephalocele was performed by attaching a galeal flap to the periostium surrounding the congenital defect. As the bony defect developed characteristics of a growing fracture later on, dural repair, transplantation of a split-bone flap and, finally, the insertion of a ventriculoperitoneal shunt became necessary. This case affirms that stringent indication and cautious usage of vacuum-assisted delivery is strongly recommended, especially in view of the possibility that undetected congenital cranial, vascular and/or cerebral alterations may be present.

Abstract: OBJECT: Thoracolumbar intraspinal subependymomas are very rare intramedullary low-grade tumors. The authors report on the clinical and morphological features of 2 cases of thoracolumbar intraspinal subependymomas and provide midterm follow-up data. METHODS: The clinical and radiological profiles of 2 patients with progressive spinal cord dysfunction due to thoracolumbar intraspinal subependymomas were retrospectively studied and compared with previously reported cases. RESULTS: Patients with intraspinal subependymomas initially presented with back pain and long-tract signs. The tumors were hyperintense on T2-weighted MR imaging, isointense on T1-weighted imaging, and noncontrast enhancing. Within 1 of the tumors, a medial septum was present on axial T2-weighted imaging. The tumors were intramedullary but grew exophytically and were amenable to gentle surgical separation from normal neural structures. Therefore, gross-total resection was feasible, and neurological outcome was good. No further adjuvant therapy was conducted. On follow-up (at 58 and 18 months, respectively), no tumor recurrence was observed. CONCLUSIONS: Symptomatic thoracolumbar intraspinal subependymomas with a distinct appearance on MR imaging are amenable to complete excision with favorable neurological outcome. In this study no tumor recurrence was observed at midterm follow-up in either patient, neither of whom underwent adjuvant therapy.

Abstract: Li-Fraumeni syndrome (LFS) represents an inherited tumor syndrome that is typically caused by germline mutations of the tumor suppressor gene TP53. TP53 dysfunction secondarily disturbs the genetic integrity of the cell. Here, we report a family with LFS harboring a germline TP53 mutation (R248W) located in the functional domain of the protein that binds to the minor groove of the DNA. In this family, tumors of the central nervous system were diagnosed as primary malignancies in all carriers of the mutation. The index patient developed an anaplastic medulloblastoma with unusual genomic profile exhibiting six distinct high-level genomic amplifications, two of them targeting the MYCN and GLI2 genes, respectively. In an extrarenal rhabdoid tumor from the same patient, we found a novel high-level amplification of the MYC oncogene. The father of this patient was diagnosed with myxopapillary ependymoma (WHO degrees I), whereas a brother died from an early relapse of a choroid plexus carcinoma. The analysis of this LFS familiy thus revealed novel oncogene amplifications as different second hits that are likely to also play a role in the pathogenesis of their sporadic counterparts.

Abstract: BACKGROUND: To date, little is known about self-help activities including the acquisition and distribution of information among brain tumour patients and their relatives. The aim of our study was to elucidate patient characteristics, methods of networking and the impact on further treatment. METHODS: A German questionnaire was distributed at nationwide patient meetings and via internet forums. It was returned electronically or by regular mail. RESULTS: Mean age of the 129 patients was 43.2 years. Mean age of the 140 relatives (94% family members) answering the questionnaire was 42.6 years. 51% of the patients and 60% of the relatives had a university degree. 61% of the patients suffered from high-grade tumours, and 80% of the relatives were caring for high-grade tumour patients. The higher the grade of the tumour, the earlier self-help was begun after diagnosis. The majority of the patients (36%) and their relatives (54%) spent between 1-4 h per week on self-help activities. More than 80% used the internet, but more than 85% used print products for the acquisition of information. More than 50% felt that they were not given enough information by their treating physician. Motives for self-help were the acquisition of "independent" information and psychological relief from an exchange with other tumour patients. The vast majority was satisfied with the results obtained, and more than four out of five who responded to the questionnaire exchanged information with other patients. The current therapy was influenced by self-help in more than 50% of cases. CONCLUSION: Physicians treating patients with brain tumours face a subgroup of well-educated people aiming to independently verify and possibly supplement and/or modify their prescribed care. With the steadily increasing use of internet resources, this approach can be expected to expand. Physicians should be prepared to deal appropriately with this subgroup of patients and their relatives to ensure that self-help activities support but do not endanger optimal care.

Abstract: So far, little attention has been paid to the biomechanical aspects of decompressive craniectomy. The brain tissue deformation occurring in these patients is difficult to quantify. Twenty-six patients suffering from a large bone defect after craniectomy were examined in supine position. The third ventricle's axial diameter was measured by transcranial ultrasound. Subsequently, the patient was brought into a sitting position. After 5 minutes, another measurement was taken. This procedure was repeated about 7 days after cranioplasty. The patients were grouped according to "early cranioplasty" (cranioplasty within 40 days after craniectomy, median 30 days) and "late cranioplasty", (cranioplasty more than 40 days, median 80 days). Data of 13 healthy volunteers were used as a reference standard. In the healthy volunteers, the third ventricle was enlarging after reaching the sitting position. The median diameter was 2.35 mm in the lying and 2.9 mm in the sitting position (p > 0.05). In the patients before early cranioplasty, a decrease of the diameter after reaching the sitting position was observed. The mean diameter was 7.0 mm in the lying and 5.9 mm in the sitting position (p > 0.01). This difference was not significant in patients before late cranioplasty (9.7 vs. 9.4 mm). After cranioplasty, the mean diameter was 6.6 and 6.2 mm in the early cranioplasty group and 9.2 mm and 9.4 mm in the late cranioplasty group (lying and sitting position, respectively). This data demonstrate for the first time that unphysiological orthostatic brain tissue deformation occurs in patients after craniectomy.

Abstract: The epidermal growth factor receptor (EGFR) is dysregulated in various tumour types such as glioblastoma multiforme (GBM), breast cancer, ovarian carcinoma, non-small cell lung cancer and other cancers. As the intracellular tyrosine kinase of the EGFR activates signalling cascades leading to cell proliferation, angiogenesis and inhibition of apoptosis, the EGFR represents an attractive target in cancer therapy. In GBM which is the most common primary central nervous system tumour in adults, the EGFR is overexpressed in about 40 to 50% of cases, and almost half of these co-express the mutant receptor subtype EGFRvIII. This EGFR variant is constitutively activated, and thereby may contribute to the aggressive and refractory course of GBM which is associated with a median survival of only 40 to 60 weeks from diagnosis. Various trials are ongoing focusing on EGFR and EGFRvIII as new therapeutic targets in GBM. Anti-EGFR monoclonal antibodies (MAbs), e.g. cetuximab, and tyrosine kinase inhibitors (TKIs), e.g. erlotinib and gefitinib, are the most advanced in clinical development. Several trials are investigating MAbs or TKIs in combination with other agents such as inhibitors of the mammalian target of rapamycin. Other still preliminary approaches targeting the EGFR are small interfering RNA, antisense RNA and ribozymes, which lead to degradation of EGFR mRNA. Further studies are needed to define their clinical potential, to identify biological predictors of response and thus to characterize subgroups of patients who will benefit from treatment with these new agents.

Abstract: High-grade gliomas account for the majority of intra-axial brain tumors. Despite abundant therapeutic efforts, clinical outcome is still poor. Thus, new therapeutic approaches are intensely being investigated. Overexpression of the epidermal growth factor receptor (HER1/EGFR) is found in various epithelial tumors and represents one of the most common molecular abnormalities seen in high-grade gliomas. Dysregulated HER1/EGFR is found in 40% to 50% of glioblastoma, the most malignant subtype of glioma. Several agents such as tyrosine kinase (TK) inhibitors, antibodies, radio-immuno conjugates, ligand-toxin conjugates, or RNA-based agents have been developed to target HER1/EGFR or its mutant form, EGFRvIII. To date, most agents are in various stages of clinical development. Clinical data are sparse but most advanced for TK inhibitors. Although data from experimental studies seem promising, proof of a significant clinical benefit is still missing. Among the problems that have to be further addressed is the prediction of the individual patient's response to HER1/EGFR-targeted therapeutics based on molecular determinants. It is quite possible that blocking HER1/EGFR alone will not sufficiently translate into a clinical benefit. Therefore, a multiple target approach concomitantly aimed at different molecular sites might be a favorable concept. This review focuses on current HER1/EGFR-targeted therapeutics and their development for high-grade gliomas.

Abstract: BACKGROUND: The antiproliferative effects of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, on human glioblastoma multiforme (GBM) cell lines in vitro and in vivo are widely variable and independent of EGFR baseline expression levels, indicating that more complex genetic signatures may form the molecular basis of GBM response to erlotinib. This study sought to determine which genes within two common genetic pathways of GBM pathogenesis, i.e., the primary and secondary pathways, may be involved in mediating the cellular response of human GBM towards erlotinib. MATERIALS AND METHODS: Complementary (c)RNAs from cell lines selected to represent the sensitive, intermediately responsive and resistant phenotypes, respectively, were hybridized to CodeLink Human Whole Genome Bioarrays. RESULTS: Expression analysis of prospectively selected 104 genes pertaining to the primary and secondary pathways of GBM pathogenesis identified two genes (IGF1, PIK3C2B) the expression of which significantly correlated with cellular resistance towards erlotinib. CONCLUSION: Among the genes constituting two common pathways of GBM pathogenesis, two candidate genes may confer GBM resistance towards erlotinib, suggesting that resistance towards this compound may be acquired during the natural evolution of GBM.

Abstract: BACKGROUND: Intracranial dermoid cysts are uncommon, and their clinical features as well as surgical management differ from patient to patient. Dermoids are generally benign lesions, but may cause spontaneous complications such as meningitis and/or hydrocephalus due to rupture and epileptic seizures depending on their location. Little has been reported about characteristic imaging findings with resulting therapeutic considerations, and only a few reports exist about associated hydrocephalus. Imaging modalities have changed and can facilitate differential diagnosis and follow-up if applied correctly. In this paper, we attempt to contribute our clinical experience with the management of dermoid cysts. PATIENTS AND METHODS: The charts of five men and two women with intracranial dermoid cysts were retrospectively reviewed. The patients were treated between September 1993 and September 2006. Selected patients are presented in detail. RESULTS: Tumour location, size and radiographic characteristics varied in each patient. Clinical presentations comprised focal neurological deficits as well as epileptic seizures, persistent headache, mental changes and psycho-organic syndromes. One patient underwent delayed ventriculo-peritoneal shunting after ruptured fatty particles caused obstructive hydrocephalus. Despite dermoid rupture into the subarachnoid space, three patients never developed hydrocephalus. Diffuse vascular supra-tentorial lesions were seen in one patient as a result of aseptic meningitis. Diffusion-weighted imaging (DWI) hyperintensity in dermoids is related to decrease of water proton diffusion and should be used for both the diagnosis and follow-up of this lesion. CONCLUSION: Although dermoid cysts are known to be benign entities per se, their rupture can cause a wide range of symptoms including aseptic meningitis and/or hydrocephalus. This may be due to intraventricular obstruction and/or paraventricular compression. While rupture does not necessarily bring about hydrocephalus, radical removal of the tumour and close monitoring of ventricular size is required. Although not widely recognised as such, DWI is considered to be a useful imaging modality in the diagnosis and follow-up of dermoids.

Abstract: BACKGROUND: Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, exerts highly variable antiproliferative effects on human glioblastoma multiforme (GBM) cells in vitro and in vivo. As these effects are independent of EGFR baseline expression levels, more complex genetic signatures may form the molecular basis of the erlotinib-sensitive and erlotinib-resistant GBM phenotypes. The aim of the current study was to determine which genes within the EGFR signaling pathway are candidates for mediating the cellular response of human GBM towards erlotinib. MATERIALS AND METHODS: Complementary (c)RNAs from cell lines selected to represent the sensitive, intermediately responsive and resistant phenotypes, respectively, were hybridized to CodeLink Human Whole Genome Bioarrays. RESULTS: Expression analysis of the prospectively selected 244 genes whose products constitute the EGFR signaling pathway identified five genes the expression of which significantly correlated with phenotype. Functional annotation analysis revealed one (STATI) and two (FKBP14, RAC1) genes conclusively associated with sensitivity and resistance to erlotinib, respectively. Moreover, two additional genes (PTGER4, MYC) were unexpectedly found to be associated with sensitivity. The gene expressions were confirmed by quantitative polymerase chain reaction. CONCLUSION: Five genes within the EGFR signaling pathway may modulate GBM response to erlotinib, which further emphasizes the importance of this pathway for the biology of GBM.

Abstract: We present two cases of lumbar subdural haematoma, both occurring after an uneventful temporomesial focus resection in patients suffering from medically intractable epilepsy. Initial symptom was back pain and sciatica 4 days and 13 days postoperatively, but no neurological deficits were observed. The diagnosis was confirmed by MRI. No risk factor could be identified. The pain responded well to conservative treatment and the haematomas resolved without neurological sequelae. A review of the literature reveals that the characteristics of spinal subdural haematoma following craniotomy are very similar. Six out of 12 reported cases occurred in temporal epilepsy surgery. All had a benign course and did not require an operative procedure. Back pain after epilepsy surgery may be caused by an intraspinal haematoma and should be investigated by MRI.

Abstract: OBJECTIVE: This paper aims to demonstrate the feasibility and efficacy of a minimal invasive approach by using a muscle dilator system for the treatment of lateral lumbar disc herniations. METHODS: A retrospective analysis of 38 cases with lateral lumbar disc herniations that were treated the METRx-System was performed. Patients were assessed preoperatively and postoperatively with the visual analogue scale (VAS) for leg pain. Follow up ranged from 6 to 24 months. RESULTS: No intraoperative complications were observed. Operation time and intraoperative blood loss were minimized. In 37 cases VAS scores were significantly lower after the procedure. A single patient had to be reoperated due to insufficient removal of the herniated disc material. CONCLUSION: The reported minimal invasive technique is a safe and efficient alternative to conventional methods for the treatment of lateral lumbar disc herniations.

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Abstract: Gliomas are the most common primary central nervous system tumours and about 55% are glioblastoma multiforme (GBM). Between 40% and 50% of GBM have dysregulated epidermal growth factor receptor (HER1/EGFR), and almost half of these co-express the mutant receptor subtype EGFRvIII, which may contribute to the aggressive and refractory course of GBM. Limited therapeutic options exist for GBM, and recurrence is common. Standard therapy is surgical resection, where possible, and radiotherapy. Adjuvant chemotherapy provides a modest survival benefit. New therapies are essential, and HER1/EGFR-targeted agents may provide a viable strategy. The HER1/EGFR tyrosine kinase inhibitors erlotinib and gefitinib are in advanced clinical development for glioma, and a number of trials are in progress, or have recently been completed. Preliminary results with gefitinib show no objective responses, but do provide evidence of disease control. In contrast, preliminary data with erlotinib appear more encouraging. Erlotinib inhibits wild-type HER1/EGFR and EGFRvIII, which may underlie its promising clinical activity. Other HER1/EGFR-targeted agents are also being investigated for glioma, including monoclonal antibodies, radio-immuno conjugates, ligand-toxin conjugates, antisense oligonucleotides and ribozymes. Further studies will define their clinical potential and hopefully provide new, effective treatments for GBM and other malignant brain tumours.

Abstract: BACKGROUND: Overexpression and deletion mutation of the epidermal growth factor receptor (EGFR) gene, as well as murine double minute 2 (MDM2) overexpression have been linked to the absence of p53 gene mutations in human glioblastoma multiforme (GBM). MATERIALS AND METHODS: EGFR and MDM2 messenger (m)RNA expression profiles and p53 status were examined by reverse transcription-polymerase chain rection (RT-PCR) and gene sequencing, respectively, in a set of human wild-type (wt) p53 GBM cell lines (U-87MG, U-87MG.wtEGFR and U-87MG.deltaEGFR) that exclusively differ in EGFR expression (endogenous wt EGFR expression, exogenous wt EGFR overexpression and exogenous 801-bp deletion-mutant [delta] EGFR overexpression, respectively), as well as in two human mutant p53 GBM cell lines that differ approximately two-fold in endogenous wt EGFR mRNA expression. RESULTS: Regardless of the underlying heterogeneity in EGFR mRNA expression and p53 status, MDM2 was similarly overexpressed among the cell lines. CONCLUSION: These data suggest that in human GBM (i) overexpression of wt or deltaEGFR and of MDM2 may constitute independent genetic events, (ii) overexpression of wt EGFR and mutation of p53 in GBM, although considered mutually exclusive in vivo, are not reciprocally prohibitive per se, and (iii) p53 mutations do not necessarily preclude MDM2 overexpression. In addition, this set of human GBM cell lines may constitute a suitable model for evaluating MDM2-targeted therapies in the context of various accompanying genetic alterations.

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Abstract: Intrinsic brainstem tumours in adults have a poor prognosis and surgical resection is rarely performed. Encouraged by successful operations on children performed in our department, we began a more aggressive strategy of open operations. Between 1986 and 1997, we operated upon 16 consecutive patients over 16 years of age (five female, 11 male, mean age 36.9 years) who were suffering from intrinsic tumours located in the pons and/or medulla oblongata. The extent of first open resection was 80 - 100% in two of the cases and more than 50% in nine cases. The mean survival time after the first occurrence of symptoms was 88.1 (median 34.5) months, and 39.9 (median 11) months after the first open operation. The rate of 5-year survival from the first occurrence of symptoms was 37.5% (25% after the first open surgical procedure). Thirteen out of 16 patients died within the follow-up period of at least 6.3 years, two of them within the immediate postoperative period. Eleven patients experienced a postoperative deterioration of symptoms from which only four recovered. Eight patients had from WHO grade II astrocytoma and a similar course as patients with higher-grade gliomas (n = 4). Our results indicate that open microneurosurgery for intrinsic brainstem tumours is of questionable benefit for the patient. Although surgery offers the advantages of reliable confirmation of histopathology and may be associated with prolonged survival, neurological deterioration was common and, unlike in paediatric patients, often irreversible.

Abstract: OBJECT: Quantitative and qualitative alterations in the epidermal growth factor receptor (EGFR) commonly occur in many cancers in humans, including malignant gliomas. The aim of the current study was to evaluate molecular and cellular effects of OSI-774, a novel EGFR tyrosine kinase inhibitor, on nine glioblastoma multiforme (GBM) cell lines. METHODS: The effects of OSI-774 on expression of EGFR messenger (m)RNA and protein, proliferation, anchorage-independent growth, and apoptosis were examined using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemical analysis, Coulter counting, soft agar cloning, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling/fluorescence-activated cell sorting, respectively. All p53 genes were completely and bidirectionally sequenced. Suppression of anchorage-independent growth by OSI-774 was inversely correlated to the induction of EGFR mRNA during relative serum starvation (r = -0.74) and was unrelated to p53 status. Overall, suppression of anchorage-independent growth was a considerably stronger effect of OSI-774 than inhibition of proliferation. The extent of OSI-774-induced apoptosis positively correlated with both proliferation and anchorage-independent growth of GBM cell lines (r = 0.75 and 0.79, respectively). In a single cell line derived from a secondary GBM, exposure to concentrations of greater than or equal to 1 micromol/L resulted in a substantial net cell loss during proliferation studies. CONCLUSIONS: The induction of EGFR mRNA may constitute a cellular mechanism to counteract the inhibitory effect of OSI-774 on the anchorage-independent growth of GBM cells. In contrast, no considerable correlation could be established between baseline expression levels of EGFR (both mRNA and protein) in GBM cell lines and their biological response to OSI-774. The OSI-774 induced greater (p53-independent) apoptosis in more malignant GBM phenotypes and may be a promising therapeutic agent against secondary GBM.

Abstract: New drugs and combination modalities for otherwise non-responsive brain tumors are urgently required. The anti-malarial artesunate (ART) and the EGFR tyrosine kinase inhibitor OSI-774 reveal profound cytotoxic activity. The effectiveness of a combination treatment and the underlying molecular determinants of cellular response are unknown. In the present investigation, we studied ART and OSI-774 in glioblastoma multiforme (GBM) cell lines. Supra-additive inhibition of cell growth was observed in U-87MG.DeltaEGFR cells transduced with a deletion-mutant constitutively active EGFR gene, while additive effects were present in cells transduced with wild-type EGFR (U-87MG.WT-2N), kinase-deficient EGFR (U-87MG.DK-2N), mock vector controls (U-87MG.LUX), or non-transduced parental U-87MG cells. Among nine other non-transduced GBM cell lines, supra-additive effects were found in two cell lines (G-210GM, G-599GM), while ART and OSI-774 acted in an additive manner in the other seven cell lines (G-211GM, G-750GM, G-1163GM, G-1187GM, G-1265GM, G-1301GM, and G-1408GM). Sub-additive or antagonistic effects were not observed. Genomic gains and losses of genetic material in the non-transduced cell lines as assessed by comparative genomic hybridization were correlated with the IC(50) values for ART and OSI-774 and subsequently subjected to hierarchical cluster analysis and cluster image mapping. A genomic profile of imbalances was detected that predicted cellular response to ART and OSI-774. The genes located at the genomic imbalances of interest may serve as candidate resistance genes of GBM cells towards ART and OSI-774. In conclusion, the combination treatment of ART and OSI-774 resulted in an increased growth inhibition of GBM cell lines as compared to each drug alone.

Abstract: Eight cell lines newly established from glioblastoma multiforme were examined by comparative genomic hybridization for their patterns of genomic imbalance. The total number of DNA copy number alterations (CNAs) found in the eight cell lines varied between 15 and 24. This characterized the examined cell lines (or the tumors they were derived from) as distinctly progressed in karyotypic evolution. The most frequent CNAs were gains of the entire chromosome 6 or, at least, parts of it, and of 7p22, which were found in all eight cell lines. Other changes present in seven of the eight cell lines were gains of 3q26qter and the entire chromosome 7 and losses of segments on chromosome 4q (e.g., 4q34q35) and of the short arm of chromosome 10. Enh(3q21q25), dim(4q22q33) and dim(4qter), dim(13q22), enh(15q14), and enh(18q22q23) were found in six of the eight cell lines. Several other CNAs [e.g., dim(9p21)] were found in common in five or less of the eight lines. Using a hierarchical cluster analysis, the specific patterns of genomic imbalance allowed various groupings of the examined cell lines. Although a close relation could be confirmed among all examined lines on the basis of shared CNAs, two main groups could be roughly differentiated. Among those there were also more or less closely related subgroups. However, also alterations which were restricted to one single cell line each were found, e.g., dim(1q41qter), dim(2q22qter), enh(4p), dim(5p), dim(4p13pter), dim(8q21qter), enh(9p), dim(9q), dim(11p14pter), enh(12q15q23), enh(13q21), dim(14q21qter), dim(15q21qter), dim(19q), and enh(22q). The comparison of the obtained data on gains and losses of DNA copy numbers in specific chromosomal segments with the data on localization of genes possibly associated with the biology of glioblastoma multiforme additionally shows high conformity but also disparity of the examined cell lines among each other, as well as compared to primary glioblastoma multiforme. Eventually, each of the cell lines could be characterized by its specific pattern of genomic imbalance.

Abstract: Homoharringtonine (HHT) is an ester of cephalotaxine (CET), both of which derive from the Chinese coniferous tree Cephalotaxus hainanensis. HHT inhibited tumor cell growth at molar ranges comparable to established cytostatic drugs, whereas CET was 3-4 orders of magnitude less active. Inhibition concentration 50% (IC50) values of CET and HHT were significantly correlated to doxorubicin, vincristine, methotrexate, cisplatin, or camptothecin in 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, Bethesda, Md., USA). We tested both drugs for resistance of cell lines which selectively overexpress the multidrug resistance (MDR)-conferring genes P-glycoprotein/ MDR1 (CEM/ADR5000), MDR-related protein 1 MRP1 (HL60/AR), and breast cancer resistance protein BCRP (MDA-MB-231-BCRP). A threefold and ninefold resistance to HHT and CET, respectively, was found in CEM/ADR5000 cells, while the other MDR cell lines did not show cross-resistance compared to their drug-sensitive counterparts. As the tumor suppressor p53 is another important factor of chemoresistance, we also analyzed the possibility that p53 affects the response of tumor cells to CET and HHT. Comparing the p53 mutational status of the 55 NCI cell lines (http://dtp.nci.nih.gov) with the IC50 values showed a significant correlation. Thus, CET and HHT were more active in cell lines without p53 mutation. We correlated the IC50 values of CET and HHT with the cell doubling times of the 55 NCI cell lines as proliferation parameter and observed that rapidly growing cells were more susceptible than slowly growing cell lines. We conducted a search mining the NCI's database for the mRNA expression of 465 genes in 55 cell lines and correlated the data with the IC50 values for CET and HHT. Of these genes 61 (=13%) correlated with the IC50 values for CET and 122 (=26%) with the IC50 values for HHT indicating the multifactorial mode of action of these drugs in cancer cells. We have chosen one example from these genes to test a causative role for drug response. U-87MG.DeltaEGFR cells transfected with an epidermal growth factor receptor ( EGFR) gene truncated in its extracellular domain through a deletion of exons 2-7 (Delta EGFR) were 14-fold more resistant to HHT than control cells transfected with mock expression vector or non-transfected cells. The present investigation presents a starting point to dissect the genes and molecular pathways involved in the tumor cells' response to CET and HHT in greater detail.

Abstract: A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.

Abstract: BACKGROUND: The aim of the current study was to investigate a putative relationship between (i) growth characteristics (proliferation and tumorigenicity) of nine glioblastoma multiforme (GBM) cell lines under different growth-stimulating conditions in vitro and (ii) their basal expression of a panel of growth factor receptors/autocrine cytokines. MATERIALS AND METHODS: Basal expressions of the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-beta (PDGFR-beta), platelet-derived growth factor-AA (PDGF-AA) and PDGF-BB, tumor growth factor-alpha (TGF-alpha) and TGF-beta as well as tumor necrosis factor-alpha (TNF-alpha) were determined by immunocytochemistry at standard cell culture conditions (10% fetal calf serum [FCS]). Proliferation and tumorigenicity at 10% FCS and relative serum starvation (0.5% FCS) were assessed by using Coulter counting and soft agar cloning, respectively. RESULTS: The ratio between cell multiplications at 10% and 0.5% FCS over a 10-day period was defined as a measure of growth factor dependence of cellular proliferation. Expression of EGFR (but not of PDGFR-beta) strongly correlated to this ratio (Spearman rank correlation coefficient R = 0.87). No considerable correlations were present among other appropriate pairs of variables with biologically founded putative relationships. CONCLUSION: Greater expression of EGFR is associated with increased growth factor dependence of cellular proliferation. Our results strengthen the role of EGFR as a rational molecular target of therapeutic intervention in GBM.

Abstract: A distinct 801-bp deletion mutation of the epidermal growth factor receptor (EGFR) gene is frequently present in primary glioblastoma multiforme (GBM), confers enhanced tumorigenicity in vivo and is prognostic of a shorter interval to clinical relapse. This study sought to investigate whether overexpression of deletion-mutant (delta) EGFR affects genotoxic stress-provoked mRNA inductions of p53 and murine double minute 2 (MDM2), two other genes strongly involved in the pathogenesis of GBM. In a set of human wild-type (wt) p53 GBM cell lines (U-87MG and U-87MG.delta EGFR) that exclusively differ in EGFR expression (endogenous wt EGFR expression and exogenous delta EGFR overexpression, respectively), ultraviolet (UV) light irradiation-mediated EGFR, p53 and MDM2 genotoxic stress-provoked mRNA inductions were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and densitometry of electrophoretically separated and stained RT-PCR products. Although baseline (at 0 J/m2) p53 mRNA expression in U-87MG.delta EGFR was 42-fold reduced, maximum p53 induction (at 8 J/m2) amounted to 130% compared to U-87MG. Thus, ultimate UV light-mediated p53 mRNA induction was 131.5-fold in U-87MG.delta EGFR and 2.8-fold in U-87MG. In contrast, neither wt/delta EGFR nor MDM2 mRNA expressions were significantly inducible, and MDM2 mRNA profiles were essentially the same among U-87MG and U-87MG.delta EGFR. These data suggest that in human GBM overexpression of delta EGFR is associated with differential genotoxic stress-provoked p53 mRNA induction whereas MDM2 mRNA expression is apparently not directly affected by EGFR status.

Abstract: OBJECT: The goal of this study was to evaluate the activity of certain hairpin ribozymes against deletion-mutant epidermal growth factor receptor (deltaEGFR) messenger (m)RNA in glioblastomas multiforme (GBMs). A distinct 801-bp deletion mutation associated with amplification of the EGFR gene is present in a large subgroup of primary GBMs and confers enhanced tumorigenicity in vivo. As a result of the deletion mutation, the fusion junction of the gene is created directly upstream of a GTA triplet, which is subsequently transcribed into a ribozyme target codon (GUA). METHODS: In attempts to intercept deltaEGFR gene expression at the mRNA level, the authors designed three different hairpin ribozymes derived from the negative strands of satellite RNAs in tobacco ringspot virus, chicory yellow mottle virus (sCYMV1), and arabis mosaic virus against this target and evaluated their efficiency and specificity in a cell-free system. The sCYMV1, identified as the most active anti-deltaEGFR hairpin ribozyme motif, was cloned into the retroviral plasmid N2A+tRNAi(met). High-titer recombinant retrovirus-containing supernatants (> 10(5) colony-forming units/ml) derived from an amphotropic GP+envAM 12 packaging cell line transfected with the N2A+tRNAi(met)-anti-deltaEGFR-sCYMV1 construct were used to introduce the sCYMV1 hairpin ribozyme into U-87MG.deltaEGFR glioblastoma cells, which overexpress exogenous deltaEGFR. Using a virus/target cell ratio of 40:1 in the absence of drug selection, the ribozyme transfer resulted in a greater than 90% reduction of deltaEGFR mRNA levels, a 69% inhibition of deltaEGFR-mediated proliferation advantage, and a greater than 95% decrease of colony formation in soft agar under relative serum starvation conditions in vitro; transfer of a control mutant ribozyme that was rendered incapable of cleaving its target yielded none of these effects. CONCLUSIONS: These findings indicate that the anti-deltaEGFR-sCYMV1 hairpin ribozyme is capable of specifically inhibiting the expression of deltaEGFR and reversing the deltaEGFR-associated malignant phenotype of GBM cells. This strategy may constitute a promising gene therapy approach for a molecularly defined subgroup of GBMs.

Abstract: Delayed traumatic intracerebral haemorrhage (DTICH) constitutes a serious complication of head injury, and several studies have set out to identify predisposing clinical variables and appropriate management strategies. Here we report a distinct and particularly malignant course of DTICH associated with oral anticoagulant therapy.

Abstract: In colorectal carcinoma, c-Ki-ras-2 mutations predominantly occur in codon 12 and, to a considerably lesser extent, in codon 13. To our knowledge, involvement of codon 61 in c-Ki-ras-2 has been reported only once among the large number of colon cancers investigated altogether. In this study, five human primary colorectal carcinomas were analyzed for the presence of activating c-Ki-ras-2 point mutations in codon 12, 13, and 61. Tumor DNAs were amplified by PCR and subsequently hybridized to a panel of synthetic oligonucleotides representing the complete spectrum of possible mutations. In two of the five tumors, mutations involving codons 13 and 61, respectively, were detected. These data extend previous findings that point mutation of codon 61 may be an improbable yet possible event leading to activation of c-Ki-ras-2 in colorectal carcinoma.

Abstract: We report a case of moyamoya disease (MMD) associated with arteriovenous malformation (AVM). The 30-year-old female patient presented with left-hemispheric transient ischaemic attacks (TIAs) involving dysphasia and right-sided hemiparesis. CT-scan and lumbar puncture showed no evidence of intracranial haemorrhage. Cerebral angiography revealed typical moyamoya vessels and occlusion of multiple cerebral arteries with consecutive collateral blood supply. Moreover, a left-parietal AVM with a diameter of approximately 2 cm was detected. An extra-intracranial arterial bypass (EIAB) connecting the left superficial temporal artery (STA) with a cortical branch of the left middle cerebral artery (MCA) was performed (STA-MCA anastomosis) and yielded subsequent resolution of the neurological deficit. Nine months post-operatively neurological deficits similar to those of the initial presentation recurred. Repeated angiography suggested comparatively increased AVM blood flow, and successful extirpation of the AVM gradually re-established almost full functional ability. However, deterioration of the neurological condition developed again. We herewith present the first European case of moyamoya disease associated with arteriovenous malformation and report the clinical course under an alternative neurosurgical treatment consisting of STA-MCA anastomosis and delayed extirpation of the AVM.

Abstract: The expression of alpha6-integrin receptors (VLA-alpha6) and of mRNA encoding the putative 37 kDa laminin receptor precursor (37 LRP) was determined in ductal pancreatic adenocarcinoma and normal pancreatic tissue from the same patient. VLA-alpha6 expression was enhanced and redistributed in pancreatic carcinoma, and 37 LRP mRNA levels were elevated in carcinomatous pancreatic tissue as well as in five pancreatic tumor cell lines. The molecular weight of the major RNA species detected was higher in carcinoma tissue (1.9 kb) as opposed to cell lines (1.2 kb), possibly reflecting alternative splicing of 37 LRP mRNA in the primary tumor.

Abstract: When MOLT-3 human acute leukemia cells were exposed sequentially to trimetrexate (TMQ) and then to methotrexate (MTX), the cells became resistant to antifolate. We designated this subline MOLT-3/TMQ800-MTX10,000. This cell line was found to contain two point mutations in the dihydrofolate reductase (DHFR) gene: a T-->C transition at nucleotide 95 in codon 31, and a T-->A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we synthesized a ribozyme which perfectly base-paired with the double-mutated DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleaved the wild-type RNA substrate. This observation suggests proceeding with caution when using a ribozyme against a mutated mRNA of an essential enzyme as a specific means of treatment.

Abstract: The development of colon carcinomas is associated with allelic deletions on chromosomes 5q, 17p, and 18q. The DCC gene located on chromosome 18q21.3 codes for a potential tumor suppressor gene related to cellular adhesion receptors. We investigated the expression of this gene in several pancreatic carcinoma cell lines and in patients with ductal adenocarcinomas of the pancreas. In 8 of 11 cell lines and in 4 of 8 primary tumors a complete extinction of DCC gene expression was observed, whereas the c-Ki-ras gene was mutated at codon 12 in 7 of 8 tumors. A highly reduced or absent expression of DCC was found in all low or undifferentiated pancreatic tumor cell lines, whereas in the more differentiated ones DCC expression was conserved. These data suggest that loss of DCC gene expression is an important factor in the development or progress of pancreatic adenocarcinoma and may be linked to the differentiated phenotype of the pancreatic tumor cell.