Patent Application
20100048415
The present invention is directed to a microarray for the detection of an angiostatic tumor stage/tumor area of colorectal carcinoma in a patient, wherein the microarray comprises gene probes capable of specifically hybridizing to predefined nucleic acids. The invention is further directed to an inhibitor or modulator of one or more of these nucleic acids, as well as to a pharmaceutical composition, comprising those inhibitors or modulators. In a further aspect, the present invention is directed to an ex vivo method for the diagnosis of an angiostatic tumor stage/tumor area in a patient suffering from a colorectal carcinoma. In a further aspect the invention is directed to predict the response of patients with colorectal carcinoma but also other diseases to therapy.
Colorectal Cancer is the third most frequently occurring cancer in both sexes worldwide. It ranks second in developed countries (Hawk and Levin, 2005). The cumulative life time risk of developing colorectal cancer is about 6% (Smith et al., 2002). Despite the advances in the treatment of this disease the 5-year survival is only 62% (Smith et al., 2002).
Three pathways have been described as the basis for malignant transformation within the colon. These are the chromosomal instability pathway, the microsatellite instability pathway (Vogelstein et al., 1988) and the methylation pathway (Jass, 2002).
Malignant transformation of the colorectal epithelium typically occurs as a multistep process that requires cumulative damage to different genes within several cellular generations. Initially cryptal hyperplasia, a proliferation of normal-appearing cells, commonly results from genetic or epigenetic changes in pathways regulating cell cycle progression or apoptosis such as APC or Bcl-2 (Baylin and Herman, 2000). The transition from hyperproliferation to dysplasia is characterized by abnormal nuclear and/or cellular shapes in crypts with larger cells, often characterized by mutations in k-ras (Takayama et al., 2001). Progression from these aberrant crypt foci to adenoma, and subsequently to carcinoma, is typically associated with additional aberrations involving SMAD-2/4, DCC, and p53 (Ilyas et al., 1999). In addition to the genetic changes in the tumor cells two important stroma reactions are associated with colorectal cancer pathogenesis: angiogenesis and inflammation.
Angiogenesis in Colorectal Carcinoma:
Tumor growth beyond the critical two to three millimeter diameter and metastasis require angiogenesis. The important role of angiogenesis in colorectal cancer progression has been convincingly documented. It has been shown that microvessel density increases around primary tumors compared with normal mucosa or adenomas (Bossi et al., 1995), and is a strong independent predictor of poor outcome (Takebayashi et al., 1996). High microvessel density is associated with a greater than 3-fold risk of death from colorectal cancer (Choi et al., 1998). In addition, vascular endothelial growth factor (VEGF) expression is significantly increased in patients with all stages of colorectal carcinoma as compared to controls (Kumar et al., 1998). Intratumor expression of VEGF was found to be associated with a nearly 2-fold increase of death risk from colorectal cancer (Ishigami et al., 1998) and correlated with increasing tumor stage, decreased overall survival, and decreased disease-free survival (Kahlenberg et al., 2003; Kang et al., 1997). Recently, all of these observations were convincingly supported in a clinical study. In this study an anti-VEGF antibody (Bevacizumab, Avastin) was added to flourouracil-based combination chemotherapy. This approach resulted in statistically significant and clinically meaningful improvement in survival among patients with metastatic colorectal cancer (Hurwitz et al., 2004). This was the first report on successful tumor therapy with antiangiogenic treatment strategies, which clearly documented the importance of angiogenesis in colorectal cancer pathogenesis.
Endothelial Cell and Inflammatory Cell Interaction:
As yet, the effect of inflammation on angiogenesis in colorectal carcinoma has not been investigated in detail. Blood vessels can be detected in inflammatory areas of colorectal carcinomas. In addition, angiogenesis is a characteristic feature of inflammatory tissues. Both observations apparently suggest that inflammation may positively contribute to angiogenesis in colorectal carcinoma. However, it is well known that inflammatory cytokines such as interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma are potent inhibitors of endothelial cell proliferation and invasion in vitro (Cozzolino et al., 1990; Frater-Schroder et al., 1987; Friesel et al., 1987; Guenzi et al., 2001; Guenzi et al., 2003; Schweigerer et al., 1987). In addition, inflammatory cytokines have been shown to inhibit angiogenesis in different animal models in vivo (Cozzolino et al., 1990; Fathallah-Shaykh et al., 2000; Norioka et al., 1994; Yilmaz et al., 1998). In contrast, in some other animal models an induction of angiogenesis has been observed in the presence of inflammatory cytokines (Frater-Schroder et al., 1987; Gerol et al., 1998; Mahadevan et al., 1989; Montrucchio et al., 1994; Torisu et al., 2000) and it has been reported that according to their concentrations inflammatory cytokines may act either as pro- or anti-angiogenic molecules in the same model system (Fajardo et al., 1992).
The antiangiogenic effect of inflammatory cytokines may be caused by their direct inhibitory effects on endothelial cell proliferation and invasion (Guenzi et al., 2001; Guenzi et al., 2003; Naschberger et al., 2005). The angiogenic effects of inflammatory cytokines have been attributed to indirect mechanisms, via the recruitment of monocytes into tissues that in turn may release angiogenic factors (Fajardo et al., 1992; Frater-Schroder et al., 1987; Joseph and Isaacs, 1998; Montrucchio et al., 1994) or to the induction of basic fibroblast growth factor (bFGF) or VEGF expression in resident cells (Samaniego et al., 1997; Torisu et al., 2000). Altogether, these results indicate that angiogenesis in colorectal carcionoma may critically depend on the specific micromilieu generated by the interplay of tumor cells, inflammatory cells and endothelial cells. This may significantly vary in different tumor stages but also in different areas of the same tumor. Thus, angiogenesis may be activated in certain tumor areas/stages and inhibited in others.
The relationship of inflammation and cancer has been a matter of debate up to now. Chronic inflammatory diseases such as ulcerative colitis and Crohn's disease predispose patients for colorectal carcinoma with an up to 10-fold increased risk (reviewed in Itzkowitz and Yio, 2004; Clevers, 2004; Farrell and Peppercorn, 2002). It has been demonstrated that chronic inflammation not only triggers the progression of cancer but also the initiation. For example, chronic inflammation is believed to be responsible for the neoplastic transformation of intestinal epithelium (reviewed in Itzkowitz and Yio, 2004). In contrast, acute inflammation of the Th1-type is considered as a host response which antagonizes tumor progression. Efforts have been undertaken to induce acute inflammation in tumor patients by e.g. systemic IL-2 immunotherapy in renal cell carcinoma where but the responses were low (Negrier et al., 1998). The relationship of inflammation, tumor initiation/progression and angiogenesis in the sporadic CRC remains largely unclear.
Recently, a concept determined as “immunoangiostasis” has been introduced by Strieter and colleagues. It was described that under certain pathological conditions in the tissue a micromilieu is established that corresponds to an IFN-γ-dependent (Th-1-like) immune reaction which finally leads to an intrinsic angiostatic reaction. This angiostatic activity has been largely attributed to the induction of the anti-angiogenic chemokines CXCL9 (monokine induced by IFN-γ CXCL10 (IFN-γ inducible protein-10 [IP-10]) and CXCL11 (IFN-inducible T-cell α chemoattractant [I-TAC]) by IFN-γ. These chemokines belong to the CXC chemokine subfamily that all lack a so called “ELR” amino acid motif (Glu-Leu-Arg) (Strieter et al., 2005b). Currently, the anti-angiogenic chemokines consist of five members that are CXCL4 (platelet factor-4 [PF-4]) (Spinetti et al., 2001), CXCL9, CXCL10, CXCL11 and CXCL13 (B-cell chemoattractant-1 [BCA-1]) (Romagnani et al., 2004). All angiostatic chemokines except from CXCL4 are induced by IFN-gamma (Romagnani et al., 2001). CXCL4, CXCL9, CXCL10 and CXCL11 bind to the same receptor, namely CXCR3 that is expressed by CD4 and CD8 lymphocytes, B cells, NK cells and endothelial cells. The CXCR3 receptor exists in two alternatively spliced variants CXCR3-A and CXCR3-B and the latter is responsible for the anti-angiogenic action of the chemokines (Lasagni et al., 2003).
One of the most abundant proteins induced by IFN-γ is the guanylate binding protein-1 (GBP-1) that belongs to the family of large GTPases (Prakash et al., 2000; Cheng et al., 1983; Naschberger et al., 2005).
The inventors demonstrated that GBP-1 is not only induced by IFN-γ, rather by a group of inflammatory cytokines (IFN-α/γ, interleukin [IL]-1α/β and tumor necrosis factor [TNF]-α) (Lubeseder-Martellato et al., 2002; Naschberger et al., 2004). GBP-1 expression was preferentially associated with endothelial cells (EC) in vitro and in viva (Lubeseder-Martellato et al., 2002) and GBP-1 was shown to regulate and mediate the inhibition of proliferation induced by inflammatory cytokines (IC) in endothelial cells as well as their invasive capacity (Guenzi et al., 2001; Guenzi et al., 2003). The protein was established as a histological marker of normal endothelial cells that are activated by IC and display an anti-angiogenic phenotype.
Thus, inflammation and angiogenesis are important stroma reactions of colorectal carcinoma (CRC). Inflammation can exert pro- or antiangiogenic activity. These effects of inflammation may vary in different patients. Pre-therapeutic differentiation of angiogenic and angiostatic inflammation therefore may clearly improve the efficacy of antiangiogenic but also of other forms of therapy of CRC. In addition, this approach may also be adequate to predict therapy response in other diseases.
Therefore, it is an object of the invention to provide a means and method for the detection, prediction and/or diagnosis of an angiostatic tumor stage/tumor area of colorectal carcinoma in a patient. It is a further object of the present invention to provide molecular markers to predict responses to therapy of patients with colorectal carcinoma and also other diseases (e.g. breast carcinoma, lung canarcinoma also). It is a further object of the present invention to provide substances, which are suitable for the treatment of colorectal carcinoma.
These objects are achieved by the subject-matter of the independent claims. Preferred embodiments are set forth in the dependent claims.
The inventors investigated whether guanylate binding protein-1 (GBP-1) may be a marker of angiostatic inflammation in CRC, because it characterizes endothelial cells exposed to inflammatory cytokines and mediates the direct antiangiogenic effects of these factors.
It was found that GBP-1 is strongly expressed in endothelial cells and monocytes in the desmoplactic stroma of some CRC. Transcriptome analysis of GBP-1-positive and -negative CRC (n=24) demonstrated that GBP-1 is highly significant (p<0.001) associated with an interferon-γ (IFN-γ)-dominated micromillieu and high expression of antiangiogenic chemokines (CXCL9, CXCL10, CXCL11). Corresponding conditions have been referred to as immunoangiostasis (IAS) recently. The association of GBP-1 and angiostaxis was confirmed by the detection of an inverse relation of GBP-1 expression and endothelial cell proliferation in the tumor vessels. Moreover, this association was affirmed in an independent disease, namely caseating tuberculosis. This avascular disease is the prototype of highly active IAS and exhibited an extremely robust expression of GBP-1. Most importantly, an immunohistochemical analysis of 388 colonic carcinoma tissues showed that GBP-1 was associated with a highly significant (p<0.001) increased (16.2%) cancer-related 5-year survival of the patients. Moreover, the relative risk of cancer-related death was lowered by 50% in GBP-1-positive colonic carcinoma.
It is shown herein that GBP-1 is a novel marker, among others, and active component of IAS in CRC and it is demonstrated that GBP-1-associated IAS is beneficial for the survival of CRC patients. GBP-1 expression along with the coexpression of several other markers may be a valuable prognostic marker to identify tumors with high intrinsic antiangiogenic activity and GBP-1-positive CRC will differentially respond to antiangiogenic therapy but also to all other forms of therapy as compared to GBP-1-negative CRC. The induction of GBP-1-associated IAS may be a promising approach for the clinical treatment of CRC.
At present an angiostatic stage is not considered to exist in CRC. The inventors have demonstrated that such a stage exists, concommitantly with the availability of means and methods, which allow to detect this stage.
The availability of a method to detect patients with “angiostatic CRC” has three major advantages: (1) It allows at an early stage to apply appropriate treatment strategies to these patients. (2) The specific selection of patients will improve the clinical efficacy of antiangiogenic therapy but likely also to other forms of therapy. (3) Improved selection criteria for therapy responsive patients will significantly reduce the costs for the health system.
Specific forms of therapy which are referred to above include the following but also additional drugs which are used for treatment of colorectal carcinoma but also additional diseases:
(1) Direct and indirect inhibitors of angiogenesis, immunomodulatory molecules and other drugs (clinically approved): monoclonal antibodies (e.g. bevacizumab, cetuximab, ranibizumab, panitumumab), tyrosine kinase inhibitors (e.g. erlotinib, sunitinib/SU11248, sorafenib, temsirolimus), aptamers (e.g. pegaptanib), endogenous angiogenesis inhibitors (e.g. endostatin), thalidomide, paclitaxel, celecoxib, bortezomib, trastuzumab, lenalidomid.
(2) Direct and indirect inhibitors of angiogenesis, immunomodulatory molecules and other drugs (clinically non-approved, in clinical trial): e.g. PTK787, SU5416, ABT-510, CNGRC peptide TNF-alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, LY317615, soy isoflavone, ADH-1, AG-013736, AMG-706, AZD2171, BMS-582664, CHIR-265, pazopanib, PI-88, everolimus, suramin, XL184, ZD6474, ATN-161, cilenigtide.
Altogether, the invention will contribute to predict therapy responses to a variety of different drugs in different diseases. In addition, the invention will contribute an important tool to the development of improved treatment strategies for cancer, which are considering the specific cellular activation phenotype predominating in individual patients to gain optimal therapeutic success.
According to a first aspect, the present invention provides a microarray for the detection of an angiostatic tumor stage/tumor area of colorectal carcinoma in a patient, wherein the microarray comprises gene probes capable of specifically hybridizing to the nucleic acids according to Seq. No. 1-108 or derivatives thereof, wherein the array comprises gene probes hybridizing to a subset of at least 4 of the above nucleic acid sequences, and further, wherein the array comprises gene probes specifically hybridizing to the nucleic acid sequences of Seq. No. 1, 4, 8 and 41.
The term “microarray” as used herein is meant to comprise DNA microarrays as well as protein microarrays.
A DNA microarray in the meaning of the present invention (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip forming an array for the purpose of expression profiling, monitoring expression levels for several genes simultaneously.
The affixed DNA segments are known and termed herein as probes, and many of them can be used in a single DNA microarray. The term gene probe generally means a specific sequence of single-stranded DNA or RNA. The term “probe” generally is here defined as a nucleic acid which can bind to a target nucleic acid via one or more kind of chemical binding, usually via complementary base pairing which usually utilizes hydrogen bonds. A probe thus is designed to bind to, and therefore single out, a particular segment of DNA to which it is complementary. Therefore, it is sufficient for the purposes of the present invention that the gene probe only hybridizes to a small part of the nucleic acid sequences indicated herein.
For performing an analysis, the following approach might be chosen:
At first, RNA is extracted from a patient sample, than the RNA is transcribed into cDNA or cRNA following purification and/or amplification steps. The cDNA or cRNA obtained may be provided with labels, if required. These nucleic acids in the next step are hybridized with the microarray as defined herein, whereby labelled cDNA or cRNA pieces are binding to its complementary counterpart on the array. Following washing away unbound cDNA or cRNA pieces, the signal of the labels in each position of the microarray may be recorded by a suitable device.
As mentioned above and as it can be derived from Table 4, GBP-1 (Seq. No. 41) is a powerful biomarker of an angiostatic immune reaction in colorectal cancer (CRC) and might already serve alone as a valuable tool for detecting an angiostatic tumor stage in a patient suffering from CRC. However, it also turned out that an even more valuable tool can be established, if the expression of at least three additional markers is evaluated, being the genes corresponding to Seq. No. 1, 4, and 8 (CXCL11, CXCL9 and CXCL 10). Interestingly, these three chemokines CXCL9, CXCL10, CXCL11 were among the 15 highest upregulated genes in GBP-1-positive tumors and were also found to be clearly higher expressed in GBP-1-positive as compared to -negative tumors. Thus, they can serve to enhance the sensitivity of detecting an angiostatic stage in an individual patient.
Therefore, it is an essential element of the invention that the microarray is at least comprising gene probes which are capable of hybridizing to the nucleic acid sequences of Seq. No. 1, 4, 8 and 41.
Although it is sufficient that the array contains these probes in order to achieve the object of the present invention, i.e. to detect, whether an angiostatic stage is present in an individual CRC patient or not (in order to subsequently chose the appropriate therapeutical steps), additional gene probes may be included which are capable of hybridizing to further nucleic acids selected from the group of Seq. No. 1-108.
Among these, further subgroups of genes preferably may be selected, specifically those, which are expressed in increased levels in GBP-1-positive CRC and have been shown to play an important role in the regulation of the cellular response to IFN: 1, 4, 8, 14, 25, 26, 41, 54 59, 65, 76, 81, 105, 106, 107, 108 and those whose expression is more than 10fold increased in GBP-1 positive CRC: 1-17. Further subgroups may be identified as Seq. No. 26, 54, 59, 65, 81, 105, 106, 107 and/or 108. It is noted that it is also preferred to additionally use these nucleic acids alone or in combination which each other, for example, and more preferred, subgroups Seq. No. 26, 54, 59, 65, 81 and /or 105, 106, 107, 108.
In a further embodiment, the microarray may additionally contain gene probes capable of specifically hybridizing to at least one of the nucleic acids according to Seq. No. 109-157, being 49 gene probes of genes with increased expression in hGBP-1-negative CRC (see the genes indicated in Table 5. Seq. No.'s correspond to the order of the sequences indicated in the table starting from Seq. No. 109). These additional nucleic acid sequences and the respective gene probes hybridizing to them may be used as “negative” control in order to further enhance the predictive value of the microarray.
Because it has been shown that vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) are major regulators of angiogenesis, the microarray may preferably also contain probes also to these genes. Both genes were not found to be differentially expressed in GBP-1-positive and -negative CRC, because they are generally expressed in increased levels in all CRC as compared to healthy tissues. However, due to their specific activity which antagonizes the effects of GBP-1-associated immunoangiostasis, probes for VEGF (including VEGF-A, VEGF-B, VEGF-C, VEGF-D) and bFGF and all splice variants of the respective genes will be used as a standard to determine basic angiogenic activation. To these goal the probes for VEGF and bFGF will be applied in combination with all gene groups mentioned above: namely 1-108 or 109-157, or 1, 4, 8, 14, 25, 26, 41, 59, 65, 76, 81, 105, 106, 107, 108 or 1-17.
The microarray of the present invention additionally may contain appropriate control gene probes, e.g. actin or GAPDH. Those can be included as control gene probes to determine relative signal intensities.
In a preferred embodiment, the gene probes used in the microarray of the invention are oligonucleotides, cDNA, RNA or PNA molecules.
As mentioned above, the nucleic acids as defined above preferably are labelled in order to allow a better detection of their binding to the corresponding gene probe on the array. Preferably, such a label is selected from the group consisting of a radioactive, fluorescence, biotin, digoxigenin, peroxidase labelling or a labelling detectable by alkaline phosphatase.
In a further embodiment, the gene probes of the array may be bound to a solid phase matrix, e.g. a nylon membrane, glass or plastics.
In a second aspect, the present invention is directed to a protein microarray, capable of detecting at least a subset of four amino acid sequences of a group of amino acid sequences corresponding to the nucleic acid sequences of Seq. No. 1-108, wherein the array is capable of at least detecting the amino acids corresponding to the nucleic acid sequences of Seq. No. 1, 4, 8 and 41.
Or in other words, the protein microarray is capable of detecting all amino acids corresponding to nucleic acid sequences and subgroups as defined hereinabove.
In the protein microarray of the present invention, the array preferably is an antibody microarray or a Western-blot microarray.
An antibody microarray is a specific form of a protein microarray, i.e. a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and a silicon chip for the purpose of detecting antigens.
The term “antibody”, is used herein for intact antibodies as well as antibody fragments, which have a certain ability to selectively bind to an epitope. Such fragments include, without limitations, Fab, F(ab′)2, ScFv and Fv antibody fragment. The term “epitop” means any antigen determinant of an antigen, to which the paratop of an antibody can bind. Epitop determinants usually consist of chemically active surface groups of molecules (e.g. amino acid or sugar residues) and usually display a three-dimensional structure as well as specific physical properties.
The antibodies according to the invention can be produced according to any known procedure. For example the pure complete protein according to the invention or a part of it can be produced and used as immunogen, to immunize an animal and to produce specific antibodies.
The production of polyclonal antibodies is commonly known. Detailed protocols can be found for example in Green et al, Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, editor), pages 1-5 (Humana Press 1992) and Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols In Immunology, section 2.4.1 (1992). In addition, the expert is familiar with several techniques regarding the purification and concentration of polyclonal antibodies, as well as of monoclonal antibodies (Coligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).
The production of monoclonal antibodies is as well commonly known. Examples include the hybridoma method (Kohler and Milstein, 1975, Nature, 256:495-497, Coligan et al., section 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988).), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
In brief, monoclonal antibodies can be attained by injecting a mixture which contains a protein/peptide into mice/rats. The antibody production in the mice/rats is checked via a serum probe. In the case of a sufficient antibody titer, the mouse/rat is sacrificed and the spleen is removed to isolate B-cells. The B cells are fused with myeloma cells resulting in hybridomas. The hybridomas are cloned and the clones are analyzed. Positive clones which contain a monoclonal antibody against the protein are selected and the antibodies are isolated from the hybridoma cultures. There are many well established techniques to isolate and purify monoclonal antibodies. Such techniques include affinity chromatography with protein A sepharose, size-exclusion chromatography and ion exchange chromatography. Also see for example, Coligan et al., section 2.7.1-2.7.12 and section “Immunglobulin G (IgG)”, in Methods In Molecular Biology, volume 10, pages 79-104 (Humana Press 1992).
In a third aspect, the present invention provides an inhibitor or modulator of one or more of the nucleic acids of Seq. No. 1-108, or of the amino acids expressed therefrom. Such substances may be used for the treatment of colorectal carcinoma.
The inhibitor or modulator is preferably selected from the group consisting of an antisense nucleic acid, a ribozyme, double stranded RNA, siRNA, microRNA an antibody, a receptor, a mutated transdominant negative variant of the protein, a peptide and a peptidomimetic.
In a fourth aspect, the invention provides a pharmaceutical composition, which comprises an inhibitor/modulator as defined above and a pharmaceutically acceptable carrier.
The active compounds of the present invention are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease can be treated or at least alleviated. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.
The term “pharmaceutically acceptable” is defined as non-toxic material, which does not interfere with effectiveness of the biological activity of the active compound. The choice of the carrier is dependent on the application.
The pharmaceutical composition can contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve a synergistic effects or to minimize adverse or unwanted effects.
Techniques for the formulation or preparation and application/medication of compounds of the present invention are published in “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latest edition. A therapeutically effective dose relates to the amount of a compound which is sufficient to improve the symptoms, for example a treatment, healing, prevention or improvement of such conditions. An appropriate application can include for example oral, dermal, rectal, transmucosal or intestinal application and parenteral application, including intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal or intranasal injections. The intravenous injection is the preferred treatment of a patient.
A typical composition for an intravenous infusion can be produced such that it contains 250 ml sterile Ringer solution and for example 10 mg protein compound. See also Remington's Pharmaceutical Science (15. edition, Mack Publishing Company, Easton, Pa., 1980).
The active component or mixture of it in the present case can be used for prophylactic and/or therapeutic treatments.
A fifth aspect of the present invention is directed to an ex vivo method for the diagnosis of an angiostatic tumor stage/tumor area in a CRC patient comprising the steps of:
a) providing a sample of the patient;
b) extracting RNA from the sample;
c) optionally transcribing RNA to cDNA or cRNA;
d) detecting, whether at least four nucleic acid sequences selected from the group consisting of Seq. No. 1-108 are present in the sample, and whether the sample contains at least the nucleic acid sequences of Seq. No. 1, 4, 8 and 41;
e) wherein the presence of said nucleic acids is indicative for the presence of an angiostatic tumor stage/tumor area of CRC in said patient.
The sample used in this method preferably is a CRC tissue sample or a cell lysate or a body fluid sample.
The detection preferably is performed by PCR, more preferably by RT-PCR, most preferably multiplex RT-PCR. The PCR method has the advantage that very small amounts of DNA are detectable. Dependent on the to be analyzed material and the equipment used the temperature conditions and number of cycles of the PCR have to be adjusted. The optimal conditions can be experimentally determined according to standard procedures.
Multiplex-PCR conditions for the simultaneous detection of GBP-1, CXCL9, CXCL10 and CXCL11 might be set as follows:
Reaction mixture:
cDNA 1 μl (corresponding to 50 ng total-RNA)
dNTP 200 μM
GBP-1, CXCL10 and CXCL11 primer each 0.4 μM, CXCL9 primer 0.8 μM
10× FastStart High Fidelity Reaction Buffer (Fa. Roche) 5 μl
FastStart High Fidelity Enzyme (Fa. Roche) 0,5 μl
Ad 50 μl Millipore-H2O
Program:
95° C. 2 min 1×
95° C. 30 sec 35×
55° C. 30 sec
72° C. 30 sec
72° C. 4 min 1×
4° C. unlimited
⅓ of the PCR-product are applied to a agarose gel.
The during the PCR amplification accrued, characteristic, specific DNA fragments can be detected for example by gel electrophoretic or fluorimetric methods with the DNA labeled accordingly. Alternatively, other appropriate, known to the expert, detection systems can be applied.
The DNA or RNA, especially mRNA, of the to be analyzed probe can be an extract or a complex mixture, in which the DNA or RNA to be analyzed are only a very small fraction of the total biological probe. This probe can be analyzed by PCR, e.g. RT-PCR. The biological probe can be serum, blood or cells, either isolated or for example as mixture in a tissue.
The detection is—as already outlined above—preferably performed by means of complementary gene probes. Those gene probes preferably are cDNA or oligonucleotide probes. Furthermore, these gene probes preferably are capable of hybridizing to at least a portion of the nucleic acid sequences of Seq. No. 1-108, or to RNA sequences or derivatives derived therefrom.
According to the invention, the hybridization to the nucleic acids according to the invention is done at moderate stringent conditions.
Stringent hybridization and wash conditions are in general the reaction conditions for the formation of duplexes between oligonucleotides and the desired target molecules (perfect hybrids) or that only the desired target can be detected. Stringent washing conditions mean 0.2×SSC (0.03 M NaCl, 0.003 M sodium citrate, pH 7)10.1% SDS at 65° C. For shorter fragments, e.g. oligonucleotides up to 30 nucleotides, the hybridization temperature is below 65° C., for example at 50° C., preferably above 55° C., but below 65° C. Stringent hybridization temperatures are dependent on the size or length, respectively of the nucleic acid and their nucleic acid composition and will be experimentally determined by the skilled artisan. Moderate stringent hybridization temperatures are for example 42° C. and washing conditions with 0.2×SSC/0.1% SDS at 42° C.
The expert can according to the state of the art adapt the chosen procedure, to reach actually moderate stringent conditions and to enable a specific detection method. Appropriate stringent conditions can be determined for example on the basis of reference hybridization. An appropriate nucleic acid or oligonucleotide concentration needs to be used. The hybridization has to occur at an appropriate temperature (the higher the temperature the lower the binding).
In a preferred embodiment, the microarray as defined above is used for the detection.
A sixth aspect of the present invention provides an ex vivo method for the diagnosis of an angiostatic tumor stage/tumor area in a CRC patient comprising the steps of:
a) providing a sample from the patient;
b) detecting, whether at least four amino acid sequences corresponding to the nucleic acid sequences selected from the group of Seq. No. 1-108 are present in the sample, and whether the sample contains at least the amino acids corresponding to the nucleic acid sequences of Seq. No. 1, 4, 8 and 41;
c) wherein the presence of said proteins is indicative for the presence of an angiostatic tumor stage/tumor area of CRC in said patient.
In a preferred embodiment, the detection is performed by contacting the sample with antibodies, which specifically recognize an amino acid expressed from a nucleic acid sequence of one of Seq. No. 1-108.
Preferably, the sample is a CRC tissue sample, a cell lysate or a body fluid. The amino acid sequences are preferably detected by means of multiplex Western blot or ELISA.
The present invention will be further described with reference to the following figures and examples; however, it is to be understood that the present invention is not limited to such figures and examples.
Robust expression of GBP-1 was detected in the desmoplastic stroma of colorectal carcinomas obtained from two different patients by immunohistochemistry (
To characterize the GBP-1-associated micromilieu, 12 GBP-1-positive and 12 GBP-1-negative CRC of patients with closely matched clinical parameters (Table 1, lower panel) were identified by immunohistochemistry and subjected to a transcriptome analysis (HG-U133A, Affymetrix, 22,215 probe sets). Signals were normalized and listed according to their probability to reflect differential expression (p<0.05), significant signal intensity (>300 RLUs) and robust upregulation of expression (>4-fold) in GBP-1-positive tumors. 104 genes fulfilled these criteria (Table 4). Most of these genes were either well-known IFN-induced genes, and/or encoded chemokines or immune reaction-associated genes (Table 4). Interestingly, the three major angiostatic chemokines (CXCL9, CXCL10, CXCL11: table 4, shaded) (Strieter et al., 2005b; Romagnani et al., 2004) were among the eight most strongly upregulated genes in GBP-1-positive tumors. Expression of angiogenic growth factors such as VEGF and basic fibroblast growth factor (bFGF) was not increased in GBP-1-positive CRC.
High reproducibility of the microarray analyses is demonstrated by the fact that within the groups of GBP-1-positive and −negative tumors highly reproducible results were obtained for each gene as shown exemplarily for GBP-1, CXCL9 and CXCL11 (
An IFN-γ-dominated micromilieu characterized by the presence of the angiostatic chemokines has recently been described to regulate an intrinsic angiostatic immune reaction (IAR) (Stricter et al., 2005a; Stricter et al., 2006; Stricter et al., 2004; Strieter et al., 2005b). The antiangiogenic chemokines CXCL9-11 inhibit angiogenesis via the chemokine receptor CXCR3-B (Lasagni et al., 2003; Ehlert et al., 2004), RT-PCR showed that this receptor is constitutively expressed in both, GBP-1-positive and −negative CRC (
In addition, 49 genes were identified, which were significantly increased in GBP-1-negative tumors (Table 5).
GBP-1 expression in UICC stage II-IV colonic carcinoma (n=388) was investigated by immunohistochemical tissue array technology (Tables 1 and 2). Nine different areas of each tumor were analyzed. Numbers of GBP-1-positive cells and expression levels were quantitatively determined (
Interestingly, patients with GBP-1-positive colonic carcinoma had a highly significant (p<0.001) increased cancer-related 5-year survival rate of 16.2% in univariate analysis (Table 3, upper panel;
Material and Methods
Clinical Samples
Affymetrix Array: After informed consent was obtained, 24 patients who underwent surgery for the first manifestation of CRC were included in the study. The investigation was carried out in accordance with the Helsinki declaration. Patients who underwent preoperative radiation or chemotherapy did not participate in the study (Table 1). Patients with familial CRC (familial adenomatous polyposis, hereditary nonpolyposis CRC) were excluded. Stage (UICC 2002), sex ratio, patient age, T-, N-, M-stage, histopathological grading and tumor site were used as conventional clinicopathological parameters (Table 1, lower panel).
Tissue Array: This study was based on the prospectively collected data of the Erlangen Registry of Colo-Rectal Carcinomas (ERCRC) from 1991 to 2001. 388 patients with the following inclusion criteria were selected: Solitary invasive colon carcinoma (invasion at least of the submucosa), localisation >16 cm from the anal verge, no appendix carcinoma; no other previous or synchronous malignant tumor, except squamous and basal cell carcinoma of the skin and carcinoma in situ of the cervix uteri; carcinoma not arisen in familial adenomatous polyposis, ulcerative colitis or Crohn's disease; treatment by colon resection with formal regional lymph node dissection at the Surgical Department of the University of Erlangen; residual tumor classification RO (no residual tumor, clinical and pathohistological examination); UICC stage II-IV 2002 (UICC (2002) TNM classification of malignant tumors. 6th ed (Sobin L H, Wittekind Ch, eds). John Wiley & Sons, New York) (Table 1, upper panel). Patients who died postoperatively and patients with unknown tumor status (with respect to local and distant recurrence) at the end of the study (Jan. 1, 2006) were excluded. A total of nine punches from each of the 388 patients originating from tumor center (three punches), invasive front (three punches) and desmoplastic stroma in/adjacent to the tumor (three punches) were applied to the tissue array analysis. Median follow-up was 83 months (range 1-177). At the end of the study 88 patients (22.7%) had died of their colon carcinoma. Patient and tumor characteristics of the ERCRC patients are shown in Table 1, upper panel. Curatively resected distant metastases were located in the liver (n=29), distant lymph nodes (n=3), peritoneum (n=3), and others (n=3). The carcinomas were graded in accordance with the recommendations of the WHO using the categories low and high grade (Jass and Sobin 1989). With regard to venous invasion we distinguished between no or only intramural venous invasion (EVI negative [−]) and extramural venous invasion (EVI positive [+]). Emergency presentation was defined as the need for urgent surgery within 48 hours of admission (Soreide et al. 1997).
Caseating tuberculosis: Tissue sections of lung biopsies from six patients with the confirmed diagnosis caseating tuberculosis were obtained by the local pathology and areas including caseating granulomas were stained immunohistochemically.
Immunohistochemical Staining
Staining for GBP-1, CD31, CD68 and Ki-67 was performed as previously described (Lubeseder-Martellato et al., 2002; Guenzi et al., 2001; Guenzi et al., 2003). The latter three antibodies were purchased from DAKO (Hamburg, Germany) and diluted as follows: CD31 (1:50), CD68 (1:200) and Ki-67 (1:300). Stained sections were evaluated by two independent persons. Differing results were evaluated by a third person and discussed until consensus was obtained.
In Situ Hybridization
Biopsy specimens were processed as previously described (Stürzl et al., 1999; Stürzl et al., 1992). As a template for transcription of 35S-labeled RNA sense/antisense hybridization probes full length GBP-1-encoding cDNA (M55542) was inserted into the pcDNA3.1 expression vector in sense/antisense orientation. T7 polymerase was used for in vitro transcription. After autoradiography sections were stained with haematoxylin and eosin and analyzed in the bright field (expression signals are black silver grains) and dark field (light scattering by silver grains produces white signals) with a Leica aristoplan microscope.
RT-PCR Analysis
RT-PCR analysis was carried out by using the PCR primers (forward/reverse, 5′-3′ orientation) for both, RT-PCR and multiplex RT-PCR: GBP-1 (M55542): ATGGCATCAGAGATCCACAT, GCTTATGGTACATGCCTTTC; CXCL10 (NM—001565.1): AAGGATGGACCACACAGAGG, TGGAAGATGGGAAAGGTGAG; CXCL9 (NM—002416.1): TCATCTTGCTGGTTCTGATTG, ACGAGAACGTTGAGATTTTCG; CXCL11 (AF030514.1): GCTATAGCCTTGGCTGTGATAT, GCCTTGCTTGCTTCGATTTGGG; IDO (M34455): GCAAATGCAAGAACGGGACACT, TCAGGGAGACCAGAGCTTTCACAC; MCP-2 (NM—005623): ATTTATMCCCCAACCTCC, ACAATGACAMTGCCGTGA; M×1 (NM—002462.2): TACAGCTGGCTCCTGAAGGA, CGGCTAACGGATAAGCAGAG; OAS2 (NM—002535): TTAAATGATAATCCCAGCCC, AAGATTACTGGCCTCGCTGA; Granzyme A (NM—006144.2): ACCCTACATGGTCCTACTTAG, AAGTGACCCCTCGGAAAACA; CXCR3-B (AF469635): AGTTCCTGCCAGGCCTTTAC, CAGCAGAAAGAGGAGGCTGT; GAPDH: AGCCACATCGCTCAGAACAC, GAGGCATTGCTGATGATCTTG.
Affymetrix GeneChip Analysis
Affymetrix GeneChip analysis was carried out as described previously (Croner et al., 2005a; Croner et al., 2005b; Croner et al., 2004). The whole microarray experiment design, setup and results are available through ArrayExpress (http://www.eblac.uk/arrayexpress/) using the access number E-MEXP-833.
Statistical Analysis
Tissue array: The Kaplan-Meier method was used to calculate 5-year rates of cancer-related survival. An event was defined as “cancer-related death”, i. e. death with recurrent locoregional or distant cancer. The 95% confidence intervals (95% Cl) were calculated accordingly (Greenwood et al., 1926). Logrank test was used for comparisons of survival. A Cox regression analysis was performed to identify independent prognostic factors. All factors which were found significant in univariate survival analysis were introduced in the multivariate model. 2 patients were excluded because of missing data on extramural venous invasion (n=386). Chi-square test was used to compare frequencies. A p-value of less than 0.05 was considered to be statistically significant. Analyses were performed using SPSS software version 13 (SPSS Inc., Chicago, USA).
Affymetrix array: Raw data derived from GeneChips were normalized by “global scaling” using Affymetrix Microarray Suite, Data Mining Tool. Signals of the 12 GBP-1-positive and 12 GBP-1-negative CRCs, respectively, were averaged and upregulated genes selected according to p≦0.05, overall signal intensity >300 RLU and fold change >4.
Tables
Homo sapiens interferon stimulated T-cell alpha
Homo sapiens immunoglobulin lambda gene locus DNA
Homo sapiens putative alpha chemokine (H174)
Homo sapiens monokine induced by gamma interferon
Homo sapiens natural killer cell group 7 sequence
Homo sapiens small inducible cytokine subfamily B
Homo sapiens interferon-induced protein 44-like
Homo sapiens rearranged gene for kappa
Homo sapiens immunoglobulin lambda joining 3
Homo sapiens small inducible cytokine B subfamily,
Homo sapiens partial IGKV gene for immunoglobulin
Homo sapiens clone KM36 immunoglobulin light chain
Homo sapiens immunoglobulin kappa-chain VK-1
Homo sapiens partial IGVH3 gene for immunoglobulin
Homo sapiens partial IGVH1 gene for immunoglobulin
Homo sapiens IgH VH gene for immunoglobulin heavy
Homo sapiens isolate donor N clone N88K
Homo sapiens clone bsmneg3-t7 immunoglobulin
Homo sapiens SLAM family member 7 (SLAMF7)
Homo sapiens partial IGVH3 gene for immunoglobulin
Homo sapiens clone ASPBLL54 immunoglobulin
Homo sapiens partial mRNA for human Ig lambda light
Homo sapiens NKG5 gene
Homo sapiens mRNA for alternative activated
Homo sapiens, guanylate binding protein 1,
Homo sapiens immunoglobulin kappa variable 1-13
Homo sapiens chemokine (C-C motif) receptor 5
Homo sapiens bone marrow stromal cell antigen 2
Homo sapiens clone ASMneg1-b3 immunoglobulin
Homo sapiens retinoic acid receptor responder
H. sapiens (T1.1) mRNA for IG lambda light chain.
H. sapiens mRNA for IgG lambda light chain V-J-C
Homo sapiens granulysin (GNLY), transcript variant
Homo sapiens immunoglobulin lambda locus
Homo sapiens mRNA for CC chemokine
Homo sapiens mRNA for immunoglobulin heavy chain
Homo sapiens mRNA for single-chain antibody
Homo sapiens mRNA for single-chain antibody
Homo sapiens granzyme A (granzyme 1, cytotoxic
Homo sapiens partial IGKV gene for immunoglobulin
Homo sapiens interferon-induced protein with
Homo sapiens Fc fragment of IgG, low affinity IIIb,
Homo sapiens isolate donor N clone N8K
Homo sapiens 2′-5′-oligoadenylate synthetase 2
Homo sapiens cDNA clone IMAGE 2009047
Homo sapiens mRNA for VEGF single chain antibody
Homo sapiens T cell receptor beta chain (TCRBV13S1-
Homo sapiens IMAGE clone similar to: chloride
Homo sapiens MHC class II DPw3-alpha-1 chain
Homo sapiens interferon, gamma-inducible protein 30
Homo sapiens XIAP associated factor-1 (BIRC4BP)
Homo sapiens, Similar to signal transducer and activator
Homo sapiens interferon, alpha-inducible protein (clone
Homo sapiens myxovirus (influenza) resistance 1,
Homo sapiens diubiquitin (UBD)
Homo sapiens protein tyrosine phosphatase, receptor
Homo sapiens CD52 antigen (CAMPATH-1 antigen) (CD52)
Homo sapiens CD38 antigen (p45) (CD38)
Homo sapiens lectin, galactoside-binding, soluble, 2
Homo sapiens interferon-alpha inducile (clone IFI-ISK)
Homo sapiens interferon-induced, protein 44 (IFI 44)
Homo sapiens, Similar to kynureninase (L-kynurenine
Homo sapiens homeo box C6 (HOXC6)
Homo sapiens erythrocyte membrane protein band 4.1-
Homo sapiens CD163 antigen (CD163)
Homo sapiens interferon stimulated gene (20 kD) (ISG20)
Homo sapiens major histocompatibility complex, class
Homo sapiens, guanylate binding protein 2,
Homo sapiens, guanylate binding protein 3,
Homo sapiens, guanylate binding protein 4,
Homo sapiens, guanylate binding protein 5,
Homo sapiens proprotein convertase subtilisiakexin type 1 (PCSK1)
Homo sapiens wingless-type MMTV integration site family. member 11
Homo sapiens collagen, type IX, alpha 3 (COL9A3)
Homo sapiens frizzled (Drosophila) homolog 10 (FZD10)
Homo sapiens Wnt inhibitory factor-1 (WIF-1)
Homo sapiens fibroblast growth factor receptor 4. soluble-form splice
Homo sapiens creatine kinase. brain (CKB)
Homo sapiens neurexin 3 (NRXN3)
Homo sapiens sema domain. immunoglobulin domain (Ig), short basic domain.
Homo sapiens type I transmembrane receptor (seizure-related protein)
Homo sapiens meprin A, alpha (PABA peptide hydrolase) (MEP1A)
Homo sapiens Purkinje cell protein 4 (PCP4)
Homo sapiens membrane-bound aminopeptidase P (XNPEP2)
Homo sapiens teratocarcinoma-derived growth factor 1 (TDGF1)
Homo sapiens carboxyl ester lipase-like (bile salt-stimulated
Homo sapiens BTB (POZ) domain containing 2 (BTBD2)
Homo sapiens neural proliferation, differentiation and control.
Homo sapiens defensin, alpha 6, Paneth cell-specific (DEFA6)
Homo sapiens hepatocellular carcinoma-associated antigen 112 (HCA112)
Homo sapiens calcium channel, voltage-dependent, beta 2 subunit
Homo sapiens mucin and cadherin-like (MUCDHL)
Homo sapiens phosphoenolpyruvate carboxykinase 1 (soluble) (PCK1)
Homo sapiens KIAAI305 protein (KIAA1305)
Homo sapiens peptidylarginine deiminase type I (hPAD-colony 10)
Homo sapiens tyrosine protein kinase (CAK) gene
Homo sapiens N-acetyltransferase 2 (arylamine N-acetyltransferase
Homo sapiens RAR-related orphan receptor C (RORC)
Homo sapiens LDL induced EC grotein (LOC51157)
Homo sapiens EWS proteinELA enhancer binding protein chimera
Homo sapiens MUC3B mRNA for intestinal mucin
Homo sapiens RAS protein activator like 1 (GAP1 like) (RASAL1)
Homo sapiens PTK5 protein tyrosine kinase 6 (PTK6)
Homo sapiens coagulation factor X (F10)
Homo sapiens hydroxysteroid (11-beta) dehydrogenase 2 (HSD1182)
Homo sapiens sodium channel. nonvoltage-gated 1 alpha (SCNN1A)
Homo sapiens discoidin domain receptor family, member 1 (DDR1),
Homo sapiens copine I (CPNE1)
Homo sapiens vasoactive intestinal peptide receptor 1 (VIPR1)
Homo sapiens periplakin (PPL)
Homo sapiens Link guanine nucleotide exchange factor II (LOC51195)
Homo sapiens FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1
Sequences:
Gerol, M., L. Curry, L. McCarroll, S. Doctrow, and A. RayChaudhury. 1998. Growth regulation of cultured endothelial cells by inflammatory cytokines: mitogenic, anti-proliferative and cytotoxic effects. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 120:397-404.
Prakash B, Praefcke G J, Renault L, Wittinghofer A and Herrmann C. Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature 2000; 403(6769):567-71.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/062522 | 11/19/2007 | WO | 00 | 5/27/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60861624 | Nov 2006 | US |
