WO2005021783A2

WO2005021783A2 - New use of gain and loss of notch2 function in brain tumors and the new notch2 genetic alterations of the notch2 gene per se

Info

Publication number: WO2005021783A2
Application number: PCT/EP2004/009565
Authority: WO
Inventors: Adrian Merlo; Jean-Louis Boulay
Original assignee: Adrian Merlo; Jean-Louis Boulay
Priority date: 2003-08-30
Filing date: 2004-08-27
Publication date: 2005-03-10
Also published as: WO2005021783A3

Abstract

The invention concerns the new use of gain and loss of Notch2 function by e.g. alterations of Notch2 in brain tumors, especially glioblastomas, astrocytomas and oligodendrogliomas and the new Notch2 genetic alteration of the Notch2 genes per se. In sum, it had been found that, while impaired Notch2 function is a key of oligodendrogliomas, gain of Notch2 function through overexpression is frequently observed in astrocytomas and glioblastomas. By these observations it had been learned that Notch2 may play a dual role in brain tumor development. All these results create opportunities for diagnostic test or new therapeutic useful.

Description

New use of gain and loss of Notch2 function in brain tumors and the new Notch2 genetic alterations of the Notch2 gene per se

The main motivation of studies on the molecular basis of cancer is to develop new therapies for tumors where there is no good treatment, or where existing therapies have significant side effects or problems. Current cancer therapies, mainly surgery, radiation therapy, and chemotherapy, all have drawbacks. Molecular insights into cancer biology have already resulted in some efficacious therapies. The invention concerns the new use of gain and loss of Notch2 function by e.g. genetic alteration of Notch2 in brain tumors, especially glioblastomas, astrocytomas and oligodendrogliomas and the newly found genetic alterations of the Notch2 gene per se for prognostic purposes and predicted response to therapy.

The Drosophila Notch mutation has been discovered by Morgan in 1916. In the 1980s, molecular analysis revealed that the Notch gene encodes a receptor involved in signalling mechanisms that are highly conserved throughout animal evolution, with various biological implications in development, differentiation and oncogenesis (Simpson, Seminars in Cell & Developmental Biology 9:581-582, 1998).

U.S. Patent No. 5,780,300 describes the roles of Notch proteins in differentiation processes. In short, Notch regulates the competence of many different cell types to respond to differentiation/proliferation/apoptosis signals, with the particular cell fates chosen depending upon the developmental history of each cell type and the specific signaling pathways operating within it.

In Drosophila and C. elegans, members of the Notch/lin12 family are required at multiple steps during the differentiation of a variety of tissues when specific cell fates are determined. In Drosophila, Notch has been shown to be required for appropriate cell-fate decisions in numerous tissues, including the nervous System, eye, mesoderm, ovaries and other areas where multi- potent progenitors are making cell-fate decisions. For example, in the neurogenic region the differential expression of Notch appears to mediate lateral Inhibition in which a single cell within a cluster of equivalent cells adopts a neural fate while adjacent cells adopt epidermal fates. Similarly, in embryos with a homozygous null mutation of the Notch gene, all cells in the neurogenic region become neuroblasts and not epidermal precursors (Artavanis-Tsakonas, Science 284:770-776, 1995).

Weinmaster et al. (Development s 16:931-941, 1992) have isolated and sequenced the entire gene designated Notch2 based on its close similarity with Notch2 from other species and its high homology with Notchl . Further characterization showed that, like Notchl , Notch2 protein is subjected to post-translational and ligand-dependent cleavage.

Toron et al. (Nature Genetics 33:208-213, 2003) unravel novel ways by which the Notch pathway can function as a dominant oncogene. In contrast, Nicolas et al. (Nature Genetics 33:416-421 , 2003) draw our attention to the fact that Notch acts as a tumor surpressor in skin carcinogenesis, through interactions with the WNT/wingless and Hedgehog pathways.

In WO 2002040716, methods of diagnosing cancer comprising the identification of neoplastic molecular markers is disclosed. Examples of neoplastic diseases that can be detected by the disclosed methods include e.g. lung cancer, prostate cancer, colon cancer, breast cancer, brain cancer etc. A method of treating a neoplastic disease is also disclosed. The treatment of brain tumors has not been specifically mentioned or exemplified.

The use of gain and loss of Notch2 function in brain tumors and the new Notch2 alterations of the Notch2 genes per se have not been described and exemplified in any citations mentioned before.

In WO 2002/018544, methods and reagents for epithelial barrier formation and following treatment of malignant and benign skin disorders are mentioned. The invention described in above mentioned application provides a method of preventing or retarding the progression of benign or malignant disorders in skin by modulating Notch pathway. In accordance with this embodiment an agonist or antagonist of the Notch pathway is administered to the skin cancer, whereby upon contact with the agonist or antagonist the progression of the skin cancer is retarded.

Novel methods and compositions for inducing differentiation and apoptosis in tumor cells overexpressing Notch proteins, e.g. Notchl or Notch2 have been described in WO 2000/020576.

In a particular embodiment, the target cell is a tumor cell characterised by increased activity or increased expression of a Notch protein, such as a Notchl or Notch2 protein, relative to Notch activity or expression in a same tissue type that is not neoplastic.

The use of gain and loss of Notch2 function in brain tumors and the new Notch2 alterations of the Notch2 genes per se have not been described and exemplified in any citations mentioned before. ln WO 2002/059285 the production of immortalized precursor cell population, useful for preventing or treating hematopoietic disorders, e.g. cancer has been described. Members of the Notch family encode large transmembrane proteins that play central roles in cell-cell interaction and cell-fate decision during early development in a number of invertebrate Systems. (Simpson, Nature 375:376- 377, 1995; Artavanis-Tsakonas et al. Science 268:225-232, 1995). The Notch receptor is part of a highly conserved pathway that enables a variety of cell types to choose between alternative differentiation pathways based on those taken by immediately neighboring cells.

The invention described in WO 99/76383 relates to an ependymal neural CNS stem cell, which cell expresses the surface protein Notchl together with at least one surface protein chosen from the group of Notch2, Notch3, CAR (transmembrane protein binding adenovirus) and CFTR (cystic fibrosis transmembrane conductance regulator), and which cell also comprises at least one cilium, without describing the use of gain and loss of Notch2 function in brain tumors and the new Notch2 alterations of the Notch2 genes per se have not been described and exemplified in any citations mentioned before.

Talora et al. In have reported that the Notch family of cell surface receptors plays a key role in cell-fate determinations and differentiations, functioning in a cell-and context-specific manner (Genes & Development 16:2253-2263, 2002). It has been found that expression of the endogenous Notchl gene is markedly reduced in a panel of cervical carcinoma cells whereas expression of Notch2 remains elevated, and Notchl expression is similarly reduced or absent in invasive cervical cancers. Furthermore it has been observed that expression of activated Notchl causes strong growh Inhibition of HPV-positive, but not HPV-negative, cervical carcinoma cells, but exerts no such on other epithelial tumor cells.

Increased Notchl signaling, but not Notch2 causes a dramatic down-modulation of HPV-driven transcription of E6/E7 viral genes, through surpression of AP-1 activity by up-regulation of the Fra-1 family members and decreased c-Fos expression.

Lu Q.R. et al., PNAS.September 11 „ 2001 , vol.98, no. 19, pages 10851 - 10856 report in'Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors" that human genes are strongly expressed in oligoden-drogliomas, contrasting absent or low expressionin astrocytoma. Notch2 for the treatment of brain tumors has not been mentioned anywhere. Zoblin A. et al in Our Pharm. Biotech. July 2000, vol.1, pages 83 to 106 mention in their arcticle" Toward The Rational Design of Cell Fate Modifiers" Notch signalling as a target for novel biopharmaceuticals as well as possible strategies to design novel Notch-targeting biopharmaceuticals and their possible clinical application Notch2 functions in brain tumors have not been mentioned anywhere.

In WO 03/042246 application (Bodmer et al) an inhibitor of the Notch signalling pathway is used in the manufacture of a medicament for use in the treatment of cancer. The use of Notch2 function in brain tumors has never been mentioned in this application.

The invention described in WO 94/07474 relates to therapeutic and diagnostic methods and compositions based on Notch proteins and other nucleic acids. The invention provides a possibly treatment of disorders of cell fate differentiation by administration of a therapeutic effective compound of the invention without giving any exact experimental prove thereon.

All the observations do not teach the use of gain and loss of Notch2 function in brain tumors and the new Notch2 alterations of the Notch2 genes per se and have not been described and exemplified in any citations mentioned before

Surprisingly, we found that the gain and loss of Notch2 function mutations of Notch2 have an influence of development of brain tumors, especially of glioblastomas, astrocytomas and oligodendrogliomas.

Tumorigenesis is a stochatic process of clonal evolution selecting increasingly more malignant cells, is driven by gain or loss of function mutations in cancer genes (Nowell et al., Science 194:23-28, 1976; Vogelstein et al., Trends in Genetics 9:138-141 , 1993; Mitelman et al., Nature Genetics 15:417-474, 1997; Cahill et al., Trends in Cell Biology 9:M57-60, 1999).

Among brain tumors of the glial lineage, also designated gliomas, clinical histopathological and cytogenetic analysis have revealed that chromosome 1 p loss highly prevalent in oligdendrogliomas correlates with a much better prognosis where mean survival is around 10 years as compared to 10 months in the prevalent glioblastomas, and with significant response to chemotherapy (Shaw et al., J Neurosurgery 76:428-434, 1992 ; Reifenberger et al., American J Pathology 145 :1175-1190, 1994; Caimcross et al., J National Cancer Institute 90 : 1473-1479, 1998; Louis et al., American J Pathology 159 :779-786, 2001). We have shown that the centromeric deletion breakpoint area of chromosome 1p loss targets the Notch2 locus. Conversely, genetic and functional analyses showed gain of Notch2 function in astrocytomas and glioblastomas. Thus, Notch2 can act as an oncogene in glioblastomas and astrocytomas and as a tumor surpressor in oligodendrogliomas.

The invention concerns the new use of these gains and losses of Notch2 function in brain tumors, especially glioblastomas, astrocytomas and oligodendrogliomas and the new Notch2 genetic alterations of the Notch2 genes per se have not been described and exemplified in any citations mentioned before.

The Notch2 mutant genes for targeting and treatment of brain tumors may be administered by every known route and may be selected from the group consisting of the intravenous route, the intraarterial route, the intracutaneus route, the subcutaneous route, the oral route, the buccal route, the intramuscular route, the anal route, the transdermal route, the intradermal route, tbe intrathecal route and the like.

In one preferred embodiment, the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution. In another equally preferred embodiment, the pharmaceutically acceptable composition is a solid and the pharmaceutical composition is in form of a powder or tablet or any other known solid formulation. In a further embodiment, the pharmaceutical carrier is a gel and the pharmaceutical composition is in the form of a suppository or cream. In a further embodiment, the compound may be formulated as part of a pharmaceutically acceptable transdermal patch.

Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agent, thickening agent, colors, viscosity regulators, stabilizer or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water and other common additives.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions can be utilized for intramuscuslar, intrathecal, intratracheal,, epidural, intraperitoneal or subcutaneaus injections. Sterile solutions can also be administered intravenously. The compounds may be prepared as a sterile solid composition which may be dissolved or suspended (diffused) at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. Very much preferred is the local injection of diffusible peptidic vectors according to the invention for locoregional application. The radiopharmaceutical is injected either into a stereotactically implanted ventricular catheter or into a capsule from which a catheter leads to the tumor center or into the resection cavity (also called port-a-cath-device). A number of different, commercially available capsules and catheters can be used for this purpose.

In non-resected main tumor masses, the tip of the catheter has to be centered into the putative midportion of the tumor which can normally be achieved using a 3 dimensional stereotactic planning System. It is imporant to wait at least one day, preferably a few days, between catheter insertion and the first application in order to control the problem of back flow along the outside of the catheter away from the target site into the subarachnoid, subdural, epidural or subcutaneous space. This phenomenon can further be reduced by lowering the elevated intracranial pressure using osmodiuretics and high dose dexamethasone prior to application of the Notch2 mutant genes. In solid non-resected cases, a slow infusion technique is used taking advantage of an infusion pump that allows continuous infusion of a volume of 5-10 ml over a time period of 1-2 hours. This technique is distinct from the so called "convection enhanced delivery" of macromolecules into brain parenchyma that uses even slower infusion velocities to allow some penetration of large molecules that do not diffuse due to their size. Our radiopeptidic vector are virtually small drugs (1-2 kDaltons) and display highly diffusive properties. Diffusion across large areas of a tumor, even across the corpus callosum to the other hemisphere, has been repeatedly observed within 30 minutes following a bolus injection of 2-3 ml of the radiopharmaceutical.

The compounds of the invention can be used alone or in combination with other pharmaceutically active substances. In the case of combinations with other pharmaceutically active compounds, a fixed combination of two or more components (e.g. kit of parts) are prepared as already known to a person of skill in the art, and the compound of the present invention and any other active compound are administered at an interval that allows common, additional or preferably synergistic effect for brain tumor and/or treatment, e.g.targeting and treatment of gliomas.

In any treatment regimen, the compositions containing Notch 2 genetic alterations of the Notch2 genes may be administered, as mentioned above to a patient either singly or in a cocktail containing two or more targeted toxins, other therapeutic agents compositions, or the like, including, but not limited to, immunosuppressive agents, tolerance-inducing agents, potentiators and side-effect relieving agents- Particularly preferred agents useful in suppressing allergic reactions of a host. Preferred immunosuppressive agents include prednisone, DECADRON (Merck, Sharp and Dohme, West Point, Pa.), cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin or their combination. Preferred potentiators include monensin, ammonium Chloride, perhexiline, verapamil amantadine and chloroquine. All of these agents are administered in generally accepted efficacious dose ranges.

Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the subject being treated, including subject age, weight, gender, diet and time of administration, will result in a need to adjust dosages for this special purpose. The administration of the compound may be effected continously or intermittently.

In the treatment, an appropriate dosage level will generally be about 0.001 to 50 mg/kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.005 to about 25 mg/kg per day, more preferably about 0.01 to about 10 mg/kg per day, and even more preferably about 0.05 to about I mg/kg per day.

Figures 1 to 7 illustrates the invention.

Fig. 1 shows chromosome 1p deletions in primary gliomas. Deletion hotspots are framed and have been defined as haplotype 1 (no 1 p loss); haplotype 2 (loss at D1S2845); haplotype 3 (D1 S507); haplotype 4 (D1 S216); haplotype 5 (D1S2845 and D1S507 and D1S216), and finally haplotype 6 (D1 S514/2696).

Fig. 2 shows that chromosome 1p loss in gliomas targets the Notch2 locus.

Fig. 3 demonstrates Notch2 overexpression in astrocytomas/glioblastomas. DNA (grey bars), RNA (black bars) and protein levels (underneath) in glioma cell lines compared to normal human fetal brain (left).

Fig. 4 shows immunohistochemistry of Notch2 on glioma tissue sections.

Fig. 5 shows Notch2 mutations in glioma cell lines.

Fig. 6 demonstrates haplotype analysis of primary brain tumors on chromosome 1 p showing correlation with the Notch2 locus.

Fig. 7 Survival shows an analysis of glioma patients by Notch2 genomic status. Kaplan- Meier curves (cumulative survival rates) confirm the effects when the stratifying according to histological classifications (oligodendrogliomas grade II, oligodendrogliomas grade HI, and glioblastomas) as well as to the novel molecular genetic classifications (LOH for Notch2 (LOH-N2), retention of Notch2 (RET). Differences of OG versus GBM (P < 0.0001 ) and LOH-N2, LOH-1 P, and RET (P < 0.0001 ) are significant.

The following non-limiting examples illustrate the inventor's preferred method for preparing and using the claimed compounds of the invention, which should not be construed as limiting the scope of this invention. The loss and gain of Notch2 function in brain tumors can be demonstrated in the following examples.

Example 1 : Loss of function mutation of Notch2 in oligodendrogliomas.

From an extensive LOH analysis of chromosome I in primary gliomas of all grades and differentiation Status, we observed a deletion pattern highly prevalent in oligodendrogliomas, designated haplotype II. This pattern is consistent with and refines previous comparative genomic hybridization (CGH) and LOH data (Bigner et al., American. J Pathology 155:375-386, 1999; Zhu et al., Genes Chromosomes Cancer 21 :207-21 , 1998). In contrast, among astrocytomas and glioblastomas, only a minority show the same pattern, while other chromosome 1 p deletions are mostly local and sparse, with three clusters of deletion hotspots (haplotypes II-IV). These observations indicate that loss of chromosome 1p extending to the centromeric region may exert a strong selective pressure mostly on oligodendrogliomagenesis (see Figure 1).

Further detailed deletion analysis by LOH and by gene copy dosage have narrowed the centromeric breakpoint area between the sequenced tag site (STS) marker D1S2344, and the region coding for the carboxy terminal end of Notch2, while the closest STS marker 210WF10 was deleted in only one third of tumors. Thus, all breakpoints, located within or in the close vicinity of the Notch2 locus, lead to deletion of all or part of the Notch2 gene (see Figure 2). Interestingly, the cell line Hs.683 characterised as an oligodendroglioma in vivo in a murine model (Branle et al. Cancer 95:641-655, 2002) displayed a deletion breakpoint in the same region.

The Notch2 gene encodes a 2472 amino acid-transmembrane receptor (Weinmaster et al., Development s 16:931 -941 , 1992; Blaumueller et al., Cell 90:281-291 1997) that belongs to a family of four mammalian proteins evolutionary derived from the single Notch initially identified in Drosophila. Notch signaling is a highly conserved process that controls local cell fates in various lineages, mainly during embryonic development (Artavanis-Tsakonas et al., Science 284:770- 776, 1999; Allman et al., Cell 109:S1-S11 , 2002). Members of the Notch family directly trans- activate the expression of target genes upon ligand-dependent activation and subsequent cleavage of their cytosolic domains (Blaumueller et al., Cell 90:281-291 1997; Artavanis- Tsakonas et al., Science 284:770-776, 1999; Jarriault et al., Nature 377:355-358, 1995). For example, mouse Notchl is required for spatial and temporal regulation of oligodendrocyte differentiation in the central nervous system.

Example 2: Gain of function of Notch2 protein in astrocytomas/glioblastomas.

Notch2 expression levels were also evaluated in glioblastoma cell lines (see Figure 3). Strikingly, in agreement with Northern analysis, quantitation of Notch2 complementary DNAs by real-time PCR showed a marked increase in Notch2 transcripts ranging from three- up to forty-fold (Median: 11.1 ) higher than in normal whole fetal brain or in Hs.683 cells, and consistent with Notch2 protein levels. Consistently, Southern analysis together with genomic DNA dosage of STS D1 S2696 located in Notch2 intron 12 by real-time PCR, indicated a two- to three-fold amplification relative to a normal diploid Situation. Fluorescent in situ hybridization of the Notch2 molecular probe on metaphase chromosomes of the U87 cell line favours local duplication of the Notch2 locus, as previously observed for the EGFR gene on 7p 12 in gliomas (Libermann et al., Nature 313:144-147 (1985), rather than a translocation.

Notch2 immunoreactivity was also assessed by immunohistochemistry on glioma biopsies of all grades and differentiation. Notch2 was clearly absent in all oligodendrogliomas grade II and Ml, and astrocytomas and glioblastomas tumor cells mostly showed strong immunoreactivity, while no signal was observed in normal brain tissue (see Figure 4).

Example 3: Existence of Notch2 mutations in human gliomas.

Sections of Notch2 complementary DNA encoding the intracellular domain and the most highly conserved extracellular domains known to be mutation hotspots in Drosophila were sequenced from 11 glioma cell lines. Of note, none of them showed evidence for alternative splicing. In Hs.683 oligodendroglioma cells, leucine 1711 in the RAM23 domain was replaced by a methionine. Leucine 1711 is located within the tryptophane/Ieucine/phenylalanine-proline triplet shown to be the core of the Notch co-activator CBF-l/RBP-Jk/CSL/Su(H) major binding site (Tamura et al., Current Biology 5:1416-1423, 1995). This suggests that this mutation may impair a Notch2 function critical for normal oligodendrocytes, supporting the concept of loss of Notch2 function in oligodendrogliomagenesis. In addition, the glioblastoma line U87 produces transcripts with aspartate 1760 changed to alanine next to the nuclear localisation signal (NLS) I, and arginine 2105 mutated to tryptophane at NLS 2a (see Figure 5). Example 4: Use of Notch2 as a prognostic and as a predictive marker for therapy in human gliomas

By using factor analysis, age and gender of the patients could be excluded as relevant factors contributing to the time of survival following tumor surgery. Multivariate analysis of variance (MANOVA) of the dataset demonstrated highly significant differences for the histological classification (oligodendroglioma (OG) versus glioblastoma (GBM); P < 0.0001). Multiple markers on chromosome 1 were applied to pinpoint the chromosomal region showing the best association with the prognosis of the patient as expressed in survival in months. Using results from six microsatellite markers, six different haplotypes (1-6, see Figure 1) could be constructed from the molecularly genotyped samples. MANOVA demonstrated highly significant difference of haplotype 6 compared to all other haplotypes with respect to the survival time (P_0Veraiι < 0.0001; P_{6 vs}. ι < 0.0001, P₆vs.₂= 0.0001 , P_{6 vs}.₃= 0.0002, P_{6 vs}.4 < 0.0004, P_{6 vs}.5< 0.0017, P_{6 vs}.₇ n.s.; Bonferroni/Dunn), suggesting a particular role of this haplotype in the assessment of the prognosis. Further analysis showed that this haplotype differs from the other haplotypes by the absence of the microsatellite markers D1S514/D1 S2696 adjacent to and located within the Notch2 gene, respectively. MANOVA for the molecular genetic classification (absence or loss of heterozygosity (LOH) for the markers closely adjacent to the Notch2 gene (LOH-N2) versus presence or retention (RET) confirmed this result; P > 0.0001). Hence, both, the histological classification (P < 0.0001) as well as the molecular genetic classification considering solely the Notch2 gene associated markers (P < 0.0001) correlated to a similar extend with the survival time of patients with brain tumors.

In addition, receiver operating characteristics (ROC) analyses were performed to quantify the statistical parameters of a possble predictive test. Sensitivity, specificity, positive and negative predictive values for a given classification used as a test to predict a survival time of more than 24 months were computed (i.e. the cut-off based on the ROC analyses for our further calculations was > 24 months survival time). When the histological classification (oligodendrogliomas (OG) versus glioblastomas (GBM)) was used for stratification, i.e. the classification as a oligodendroglioma was used as a positive test result to predict a survival time of > 24 months, the sensitivity was 80.8 %, the specificity was 92.0 %, the positive predictive value was 84.0 %, and the negative predictive value was 90.2 %.

When the molecular genetic classification (loss of heterozygositiy (LOH) at the notch locus (LOH-N2) versus LOH at 1p (LOH-1 P) or retention of notch (RET)) was used for stratification, i.e. the classification as LOH at the notch locus was used as a positive test result to predict a survival time of > 24 months, the sensitivity was 80.0 %, the specificity was 88.2 %, the positive predictive value was 76.9 %, and the negative predictive value was 90.0 %.

To investigate whether the novel molecular genetic classification resulted in an added value when combined with the histological classification, we computed two further possibilities: LOH-N2 + and OG + as a positive tests result, LOH-N2 + or OG + as a positive tests result. When the combination of LOH-N2 + and OG + was used as a positive tests result to predict a survival time of > 24 months, the sensitivity was 85.7 %, the specificity was 95.5 %, the positive predictive value was 90.0 %, and the negative predictive value was 82.4 %. When the combination of LOH-N2 + or OG + was used as a positive tests result to predict a survival time of > 24 months, the sensitivity was 92.0 %, the specificity was 96.1 %, the positive predictive value was 92.0 %, and the negative predictive value was 96.1 %. These findings demonstrated a clear added value of the combined tests compared to each of the single classifications (histological or molecular genetic).

Histological and molecular genetic parameters potentially associated with the survival time (in months) were detemined. Factor analysis (orthotran/varimax transformation method) including the computation of correlation matrices were used to identify highly correlated continuous parameters and to define the relevant factors subjected to the subsequent multivariate analysis of variance (MANOVA). MANOVA was used for direct comparison of the effects of the different histological and molecular genetic factors on survival time. To determine the significance level of the correlation of single markers to survival time, multivariate analyses (MANOVA) including post hoc tests was used. To visualize marker pattern-specific differences of survival and to determine the significance levels of particular marker patterns (haplotypes) regarding survival time, classical survival analysis methods (Kaplan-Meier curves with product limit) were used.

Factor analysis, MANOVA, and non-parametric Kaplan-Meier analyses including significance levels of differences were computed using StatView® 5.0 (Abacus Concepts Inc., Berkeley, CA, USA). Receiver operating characteristic analyses, sensitivity, specificity and predictive values were calculated with ROC, version 1.1 (Diagene GmbH, Reinach / BL, Switzerland). All other calculations were performed using StatView^® 5.0 (Abacus Concepts Inc., Berkeley, CA, USA) and SPSS 9.0 (SPSS Inc., Chicago, IL, USA). Results are presented as means (± SEM). ln diseases such as brain tumors that are often based on a complex pathogenesis possibly involving multiple genes, the study of multiple markers and their combinations is crucial when particular effects, such as the influence of the molecular basis on the prognosis are of interest.

Such markers should be localized at physically well-defined positions of the respective chromosome. By constructing haplotypes, i.e. by constructing combination patterns of gene markers, the effect of the coincidence of the presence and/or absence of particular gene regions of interest can be controlled for. RET or retention means therefore the presence of the gene at least in a heterozygous state, LOH or loss of heterozygosity means complete loss of the gene even in a heterozygous state. In somatic mutation studies, the construction of marker patterns allows the direct comparison of multiple gene regions, similarly to a multipoint linkage analysis in the study of germ line mutations. The goal is to pinpoint the chromosomal region containing a gene that is crucial for the prognosis of the patients while distinguishing the gene effect from the effects of adjacent genes. Using such marker patterns (haplotypes) in the present work revealed.

Claims

Claims:

1. A method of diagnosing brain tumors, characterised by making use of gain and loss of function of Notch2 in brain tumors.

2. A method of diagnosing astrocytomas/glioblastomas according to claim I, characterised by making use of gain of function of Notch2 in astrocytomas/glioblastomas.

3. A method of diagnosing oligodendrogliomas according to claim I, characterised by making use of loss of function of Notch2 in oligodendrogliomas.

4. A method of diagnosing brain tumors according to claim 1 , characterised by making use of loss of function of Notch2 using Notch2 as prognostic marker.

5. A method of diagnosing oligodendrogliomas according to claims 1 and 4, characterised by making use of loss of function of Notch2 using Notch2 as prognostic marker.

6. A method of diagnosing brain tumors according to claim 1 , characterised by making use of loss of function of Notch2 using Notch2 as predictive marker.

7. A method of diagnosing oligodendrogliomas according to claims 1 and 6, by making use of loss of function of Notch2 using Notch2 as predictive marker.

8. A method of drug targeting in brain tumors, characterised by making use of gain and loss of function of Notch2 in brain tumors.

9. A method of drug targeting in astrocytomas/glioblastomas according to claim 8 , characterised by making use of gain of function of Notch2 in astrocytomas/glioblastomas.

10. A method of drug targeting in oligodendrogliomas according to claim 8, characterised by making use of loss of function in oligodendrogliomas.

11. New Notch2 genetic alterations of the natural Notch2 human gene, characterised that the genetic alterations of the Notch2 genes are different from naturally occurring proteins.

12. New Notch2 genetic alterations of the natural Notch2 human gene according to claim 11 , characterised that the alterations of the Notch2 genes are as defined, which consist of a precise recombination breakpoint on chromosone 1p 11 at the Notch2 locus.

13. New Notch2 genetic alterations of the natural Notch2 human gene according to claim 11 , characterised that the alterations of the Notch2 are as defined, which consist either of haploinsufficiency or of absence of Notch2 proteins, e.g. caused by epigenetic alterations such as promoter methylation or histone deacetylation.

14. New Notch2 genetic alterations of the natural Notch2 human gene according to claim 11 , characterised that the alterations of the Notch2 genes are as defined, which consist of gene amplification, which enhances expression of Notch2 at the transcriptional and protein level.

15. Notch2 genetic alterations of the natural Notch2 human gene according to claim 11 , characterised that the alterations of the Notch2 are as defined which consist of sequence alterations of the entire Notch2 gene including its promoter.

16. New Notch2 genetic alterations of the natural Notch2 human gene according to claim 11 , characterised that the alterations of the Notch2 are as defined and the oncogenic function of Notch2 is accompanied by expression levels of Notch2, if the overexpression is blocked, cell death can be significantly elevated above background levels of apoptosis.

17. New Notch2 genetic alterations of the natural Notch2 human gene according to claim 11 , characterised that the alteration of the Notch2 genes are as defined which discloses an inverse relation between the number of Notch2 alleles, which defined a prognostic marker and allows molecular subtyping oligodendrogliomas.

18. Pharmaceutical composition comprising a new Notch2 genetic alteration of the human gene according to claim 11 , together with a pharmaceutically acceptable carrier, excipient or diluent.