Ten-year analysis of the prospective multicentre Chemo-N0 trial validates American Society of Clinical Oncology (ASCO)-recommended biomarkers uPA and PAI-1 for therapy decision making in node-negative breast cancer patients.

AIM: Final 10-year analysis of the prospective randomised Chemo-N0 trial is presented. Based on the Chemo-N0 interim results and an European Organisation for Research and Treatment of Cancer (EORTC) pooled analysis (n=8377), American Society of Clinical Oncology (ASCO) and Arbeitsgemeinschaft Gynäkologische Onkologie (AGO) guidelines recommend invasion and metastasis markers urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor-1 (PAI-1) for risk assessment and treatment decision in node-negative (N0) breast cancer (BC). METHODS: The final Chemo-N0 trial analysis (recruitment 1993-1998; n=647; 12 centres) comprises 113 (5-167) months of median follow-up. Patients with low-uPA and PAI-1 tumour tissue levels (n=283) were observed. External quality assurance guaranteed uPA/PAI-1 enzyme-linked immunosorbent assay (ELISA) standardisation. Of 364 high uPA and/or PAI-1 patients, 242 agreed to randomisation for CMF chemotherapy (n=117) versus observation (n=125). RESULTS: Actuarial 10-year recurrence rate (without any adjuvant systemic therapy) for high-uPA/PAI-1 observation group patients (randomised and non-randomised) was 23.0%, in contrast to only 12.9% for low-uPA/PAI-1 patients (plog-rank=0.011). High-risk patients randomised to cyclophosphamide-methotrexate-5-fluorouracil (CMF) therapy had a 26.0% lower estimated probability of disease recurrence than those randomised for observation (intention-to-treat (ITT)-analysis: hazard ratio (HR) 0.74 (0.44-1.27); plog-rank=0.28). Per-protocol analysis demonstrated significant treatment benefit: HR 0.48 (0.26-0.88), p=0.019, disease-free survival (DFS) Cox regression, adjusted for tumour stage and grade. CONCLUSIONS: Chemo-N0 is the first prospective biomarker-based therapy trial in early BC defining patients reaching good long-term DFS without adjuvant systemic therapy. Using a standardised uPA/PAI-1 ELISA, almost half of N0-patients could be spared chemotherapy, while high-risk patients benefit from adjuvant chemotherapy. These 10-year results validate the long-term prognostic impact of uPA/PAI-1 and the benefit from adjuvant chemotherapy in the high-uPA/PAI-1 group at highest level of evidence. They thus support the guideline-based routine use of uPA/PAI-1 for risk-adapted individualised therapy decisions in N0 breast cancer.

Angiogenesis in malignant glioma--a target for antitumor therapy?

The prognosis of malignant gliomas is still dismal despite aggressive treatment attempts. Thus, alternative therapy strategies are needed. Malignant gliomas are upon the best vascularized tumors in humans and their proliferation is hallmarked by a distinct proliferative vascular component. Hence it seems to be a logical consequence to apply anti-angiogenic treatment strategies to malignant gliomas. These treatment strategies have shown promising effects in animal models and some experimental clinical studies. This review gives a short introduction into the molecules involved in angiogenesis of malignant gliomas, it provides an overview of the latest experimental developments of glioma angiogenesis inhibition and discusses the results of clinical anti-angiogenic trials in patients with high grade glioma. Additionally the problem of monitoring the treatment success of an anti-angiogenic therapy is addressed.

Presence of 14Hz spindle oscillations in the human EEG during deep anesthesia.

OBJECTIVE: To report on presence of human EEG spindle oscillations on the cortical level within flat periods of the burst-suppression pattern during propofol-induced anesthesia; to search for corresponding oscillations and possible functional connections. METHODS: Artefact-free epochs of spindle activation were selected from the electroencephalograms of opiate-dependent patients undergoing rapid opiate detoxification. Power spectral analysis and source localization using low-resolution-brain-electromagnetic-tomography (LORETA(Key)) were performed. RESULTS: Sinusoidal rhythms with waxing and waning amplitudes appeared after propofol-induced narcosis but no direct correlations could be determined between individual dosage and characteristic spindle attributes. The power maximum stood midline over the cortical areas, especially around C(z). We calculated a peak frequency of 14(+/-1.2) Hz. Motor fields, particularly in the frontal, parietal, and various cingulate areas, were found to be the primary sources of spindle oscillations in the cortex. CONCLUSIONS: The frequent occurrence of these localized spindle sources demonstrates the preference for motor fields. Spindle oscillations observed during propofol-induced narcosis were similar in frequency and shape to those observed in natural sleep. SIGNIFICANCE: The results lend support to models that postulate a close link between the motor system and the organization of behavior. In addition, spindle rhythms under propofol bore some resemblance to spindle types which occur during sleep.

Predicting chronic lung disease in very low birthweight infants: comparison of 3 scores.

AIM: To evaluate 3 scores in their ability to predict Chronic Lung Disease (CLD) in very low birthweight (VLBW) infants. METHODS: The records of 188 VLBWs admitted to neonatal intensive care within two years were retrospectively reviewed. Two mortality scores -the CRIB (clinical risk index for babies) score and the Berlin admission score- and one morbidity score developed to predict CLD (Sinkin score) were assigned to each infant. Areas (AUC) under receiver operating characteristic (ROC) curves were used for comparison. RESULTS: The Sinkin score and the Berlin admission score had AUCs of 89.0 and 85.8% to predict CLD ("alive in oxygen at 28 days of life"). The AUCs were 87.7 and 81.2%, respectively, using the CLD definition "alive in oxygen at 36 weeks gestational age", the CRIB had an AUC of 77.0%. CONCLUSION: To enroll patients in trials aimed at early interference with the course of CLD, the Berlin admission score or the Sinkin score could be used.

Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination.

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus-thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.

Self-deleting retrovirus vectors for gene therapy.

A new generation of retrovirus vectors for gene therapy has been developed. The vectors have the ability to excise themselves after inserting a gene into the genome, thereby avoiding problems encountered with conventional retrovirus vectors, such as recombination with helper viruses or transcriptional repression of transduced genes. The strategy exploited (i) the natural life cycle of retroviruses, involving duplication of terminal control regions U5 and U3 to generate long terminal repeats (LTRs) and (ii) the ability of the P1 phage site-specific recombinase (Cre) to excise any sequences positioned between two loxP target sequences from the mammalian genome. Thus, an independently expressed selectable marker gene flanked by a loxP target sequence was cloned into the U3 region of a Moloney murine leukemia virus vector. A separate cassette expressing the Cre recombinase was inserted between the LTRs into the body of the virus. LTR-mediated duplication placed vector sequences, including Cre, between loxP sites in the integrated provirus. This enabled Cre to excise from the provirus most of the viral and nonviral sequences unrelated to transcription of the U3 gene.

Enrichment of insertional mutants following retrovirus gene trap selection.

The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.

Selective disruption of genes expressed in totipotent embryonal stem cells.

Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.

The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers.

The neurogenic loci of Drosophila are required for proper partitioning of ectodermal cells into epidermal versus neural lineages. The loci appear to encode components of a developmental pathway involving cellular communication. In an effort to understand the role of the neurogenic locus mastermind in these processes, we have characterized its expression and sequence. The locus produces a number of transcripts that accumulate ubiquitously during early embryogenesis but more specifically in the central nervous system during later stages. Sequence analysis of a major cDNA product predicts an unusual protein containing an abundance of amino acid homopolymers and charge clusters typical of regulatory molecules. Nearly half of the mass of the predicted protein derives from only three amino acids: glutamine, glycine, and asparagine. Immunohistochemical studies of the protein in cell culture and early embryos show that the protein accumulates predominantly in the nucleus.