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WO2010079118A1 - Use of rna obtained from proteolipid complexes circulating in blood for diagnosis and treatment of tumors - Google Patents

Use of rna obtained from proteolipid complexes circulating in blood for diagnosis and treatment of tumors Download PDF

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WO2010079118A1
WO2010079118A1 PCT/EP2010/000001 EP2010000001W WO2010079118A1 WO 2010079118 A1 WO2010079118 A1 WO 2010079118A1 EP 2010000001 W EP2010000001 W EP 2010000001W WO 2010079118 A1 WO2010079118 A1 WO 2010079118A1
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tumor
rna
crna
plc
blood
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PCT/EP2010/000001
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Michael Roth-Chiarello
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Michael Roth-Chiarello
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the use of RNA isolated from circulating RNA-proteolipid complex (cRNA-PLC) from body liquids of tumor patients, especially tumor patients suffering from lung cancer, especially small cell lung cancer (SCLC), wherein said RNA was obtained by a specific filtration method.
  • cRNA-PLC circulating RNA-proteolipid complex
  • SCLC small cell lung cancer
  • NSCLC Non-small-cell lung cancer
  • RNA-proteolipid complex In 1985 Wieczorek et al. (Proc. Natl. Acad. Sci, 82, 3455) described for the first time the isolation of an RNA-proteolipid complex from serum of tumor patients and healthy controls. It was observed that increased serum levels of this circulating RNA- proteolipid complex (cRNA-PLC) correlated with the severity of the disease . Due to its chemical and molecular nature the cRNA-PLC with later described circulating proteasomes it was assumed that both terms describe an identical cellular unit, which either originates from cell degradation or from over active tumor cells by exocytosis. Tumour cells, in general, are over-active compared to non-malignant cells and produce significantly higher number of proteolipids which often also contain RNA from the tumour cells.
  • cRNA-PLC circulating RNA- proteolipid complex
  • this cRNA-PLC contain tumo From each cRNA-PLC preparation 500 micro-litres will be suspended mixed with an equal volume of total RNA extraction buffer (Qiagen, Germany) and than be centrifuged at 10,000 x g for 10 min at 4 0 C. The supernatant will be applied onto a RNA separation column (Qiagen) and RNA will be bound in the column to glass-beads. The RNA will be then eluted from the column with a specific mRA elution buffer (Qiagen) and will be which was rich in phospholipids and glycosphingolipids (1 ).
  • the concentration of the cRNA-PLC increased 10 months prior to the conventional clinical diagnostic tools used to indicate a relapse, and therefore the cRNA-PLC may be used as an early indicator of tumor relapse (Wieczorek et al., Cancer Res. 1987;47:6407- 12.).
  • Cellular proteasomes are non-lysosomal proteolytic cell units which participate in the regulatory mechanisms controlling cell growth and differentiation. Abnormal high expression of cellular proteasomes has been described in haematopoietic malignancies and solid tumors. The increase of cellular proteasomses was paralleled by an increase of plasma levels of circulating proteasomes, as determined by ELISA in patients with malignant melanoma (stage III and IV) compared to controls and patients at earlier stages (Stoebner et al., Br J Dermatol. 2005;152:948-53). The serum level of plasma or circulating proteasomes was further increased in patients with tumor metastasis. Increased levels of circulating proteasomes were also reported from patients with auto-immune inflammatory or peri-operative cellular damage (Zoeger et al., Clin Chem. 2006;52:2079-86).
  • ELISA based measurement of circulating proteasomes showed increased levels in serum and plasma samples obtained from patients with rheumatoid arthritis, myasthenia gravis, autoimmune myositis, systemic lupus erythematosus, primary Sjogren's syndrome, and autoimmune hepatitis compared to control serum (I.e.). However, when compared to levels of circulating proteasomes in tumor patient serum, those observed in auto-immune diseases were significantly lower. A further association of circulating 2OS proteasomes was found in serum of patients suffering from mixed connective tissue disease and correlated with clinically relevant changes in disease activity (Majetschak et al., Clin Vaccine Immunol. 2008;15:1489-93). These findings indicate that proliferating cells may get rid of unwanted or an over-production of cellular products by exocytosis of proteasomes.
  • proteolytic activity (chymotrypsin-like, trypsin-like, caspase-like) of circulating proteasomes and the extent of their ubiquination is an indicator of chronic lymphocytic leukemia and correlated it with the clinical prognosis (Ma et al., Cancer. 2008;112:1306-12).
  • RNA The most important part of the cRNA-PLC is its content of RNA. As the cRNA-PLC is assumed to originate from tumor cells, the therein contained RNA may provide a much more tumor specific array of markers than any other type of circulating RNA in the blood. - A -
  • RNA-PLC circulating cRNA-PLC
  • malignant melanoma gastric cancer, colon cancer, prostrate cancer, colorectal cancers, breast cancer and renal cell cancer.
  • These RNAs may originate from a wide range of sources and will therefore not represent a specific array of tumor associated factors or markers (Feng et al., Anticancer Res. 2008 Jan-Feb;28(1A):321 -6).
  • micro RNAs were isolated from proteolipid like structures (Mitchell et al., Proc Natl Acad Sci U S A. 2008 JuI 29;105(30):10513-8; Lawrie et al., Br J Haematol. 2008 May;141 (5):672-5). Reflecting on the anti-inflammatory and anti-infectious function of naturally occurring micro RNA in otherwise healthy people the inventor postulates that such circulating micro RNAs in serum of tumor patients may, after being taken up by a non-tumor cell, switch the "host" cell's program to the benefit of the tumor or to generate a tumor like cell type by down-regulating tumor suppressor genes.
  • RNA isolation One major problem of the investigation of all known types of circulating RNAs is the method used for their isolation. It is a well established fact based on the chemical nature and of the function of RNA that any type of RNA is highly sensitive to degradation by RNases.
  • RNases present an essential protective mechanism that rapidly degrades viral RNA and is present in abundance in all human body fluids (Garcia et al., RNA. 2008 Jul;14(7):1424-32; Taylor et al., Gynecol Oncol. 2008 Jul;110(1 ):13-21 ; Cerkovnik et al., lnt J MoI Med. 2007 Sep;20(3):293-300; O'Driscoll et al., Cancer Genomics Proteomics. 2008 Mar-Apr;5(2):94-104). This problem seems to be significantly reduced when isolating circulating RNA from the above described cRNA-PLC.
  • RNA in this complex no attention was paid to the function or disease specific nature of the content of RNA in this complex, and whether this RNA can be used as specific and reliable biomarker for specific tumors, above all lung cancer, preferably NSCLC.
  • APAF1Akt1 Apoptotic protease activating factor 1
  • Bcl-2-like 1 protein BCL2L
  • Mitogen activated protein-3 kinase / p38 MAK MAK
  • MDM2 MDM-2 protein
  • RIPK1 Receptor interacting serine/threonine kinase 1
  • caspases A special case are apoptosis inducing enzymes assigned as caspases which have been reported to be up-regulated in adenocarnicomas, a finding which contradicts that of others claiming that apoptosis is down-regulated in tumors.
  • caspases were reported as increased:
  • caspase-1 caspase-2 (Casp2) caspase-3 (Casp3) caspase-5 (Casp ⁇ ) caspase-9 (Casp9) caspase-10 (CaspiO)
  • AXIN1 up-regulated (AXUD1) AXIN1 up-regulated (AXUD1)
  • BCL2-antagonist/killer 1 (BAK1) Cyclin dependent kinase inhibitor 2D (CDKN2D)
  • CTLA1 Cox TRL activated protein
  • DAPK2 Death associated protein kinase 2
  • MAP3K7 Mitogen activate protein 3 kinase 7
  • Tumor necrosis factor receptor surface factor 1 TNFRSF1
  • Tumor necrosis factor receptor surface factor 6 TNFRSF6
  • RNA proteoliposome complexes contain RNA that can be taken up by other cells, in which the RNA functions in a way that creates an environment specifically supporting tumor spreading, and therefore can be used as a diagnostic tool.
  • RNA isolated from the cRNA-PLC could be used as an unambiguous indicator for: (i) the tumor type,
  • the cRNA-PLC is used and isolated from serum or whole blood of tumor patients using the method described in WO 97/35589 or WO 90/10872.
  • RNA contained in the cRNA-PLC can be isolated according to the invention, for example by the glass-milk procedure as described in the example section, under non- denaturing and RNase-free conditions.
  • the blood/serum sample comprising said cRNA-PLC complex is submitted to a specific filtration step, which does not only purify the sample but also does separate free RNA present in the sample from RNA bound to the proteolipid complex.
  • a specific filtration step which does not only purify the sample but also does separate free RNA present in the sample from RNA bound to the proteolipid complex.
  • the bound RNA is an unambiguous indicator for tumor markers, whereas the free RNA in the body fluid sample may derive also from other non-tumor sources.
  • the bound RNA can be easily released from the proteolipid complex trapped on the filter material.
  • the inventors have developed a new method which allows a simple fast forward isolation of proteolipid bound RNAs using a filter device.
  • filter systems are known for separating RNA from samples the nature of the filter materials necessary for separating blood circulating RNA bound to proteolipid complex was not further described and cannot be reconstructed from the provided information of the prior art.
  • the effectiveness and specificity to selectively isolate mRNA from the cRNA-PLC can be increased by using filter materials that are able to bind proteins and proteolipids or RNA bound to lipids.
  • filter materials based on nitro-cellulose and derivatives thereof can be used according to the invention.
  • the preferable pore size of said filters according to the best mode of this invention is between 0.1 and 0.5 ⁇ m, preferably 1.1 and 1.3 ⁇ m.
  • the filters which preferably are used according to the invention are composed of nitro-cellulose compounds which are known to bind proteins and proteolipids, thus, the particles which are held back by the filter contain cRNA-PLC.
  • the isolated and purified mRNA will than be transcribed into complementary cDNA by reverse transcriptase according to standard techniques.
  • the cDNA will then be amplified in an unspecific polymerase chain reaction and the products will be analyzed by micro-array chip technology using specific chips for the detection of the presence of tumor related gene products (mRNAs) according to per se known methods.
  • mRNAs tumor related gene products
  • the invention is related to the use of RNA (ribonucleic acid) which is contained in a circulating RNA-proteolipid complex (cRNA-PLC), to transcribe this RNA into cDNA and screen this cDNA by the use of DNA micro array chips for: (i) tumor type specific markers, (ii) for tumor stage specific markers, and (iii) for monitoring the expression of tumor markers in patients who undergo any type of anti-cancer therapy in order to control the success of the therapy and/or the progression of the tumor.
  • RNA ribonucleic acid
  • cRNA-PLC circulating RNA-proteolipid complex
  • the RNA contained in the cRNA-PLC obtained preferably by the filtration step as described originates directly from tumor cells, which are either dying, or which are overactive and therefore release "unwanted” proteins, lipids and RNA by exocytosis. Therefore, this RNA is very specific for the tumor, although it was obtained from body fluids, such as blood.
  • cRNA-PLC is found also in blood samples of healthy individuals, it could be shown by this invention that cRNA-PLC is significantly increased in blood samples of tumor patients. Therefore cRNA-PLC is thought to be an indicator for tumor disease, its status, progression or disappearance.
  • RNA contained in the cRNA-PLC is intact and can be transcribed into cDNA. Therefore, it is possible to characterize the gene of origin of this RNA according to in principle known methods (Voortman et al., MoI Cancer. 2007;6:73; Chen et al., MoI Pharmacol. 2007;72: 1269-79; Tabata et al., J Biol Chem. 2001 ;276:8029-36; Godley et al., Curr Opin Support Palliat Care. 2007;1 :23-9).
  • cRNA-PLC The cell type of origin of the cRNA-PLC is unknown. Based on the observation that its concentration increases with the duration and staging of tumors in blood samples of tumor patients and the fact that the cRNA-PLC was also found under cell culture conditions, it can be hypothesized by this invention that cRNA-PLC is formed and released by tumor cells into the tumor feeding blood vessels and thereby enters into the blood stream.
  • cRNA-PLC mainly contains RNA which encodes for tumor specific factors either supportive or repressive to the tumor and other cells. This makes the cRNA-PLC a tool to search and identify tumor specific factors which are concentrated in this distinguishable blood component and can be isolated by adapted routine methods.
  • the isolated RNA can than be used to screen for a tumor type specific array of markers, as well as it can be used to monitor the effect of any applied therapy.
  • RNA extraction / elution buffer such as RNeasy Kit ® (Quiagen).
  • RNA profile by applying the method to different individuals suffering from the same tumor.
  • RNA within the proteolipid complex is at least partially anti-sense RNA (siRNA).
  • An ex-vivo method for determining tumor progression / regression, or the effect of an anti-tumor therapy in a tumor patient comprising applying the steps (i) - (iv) of claim 1 and repeating these steps at different stages of tumor development and / or therapy schemes.
  • cRNA-PLC obtained from blood or serum of a patient suffering from cancer for ex-vivo identification of biomarkers that are tumor specific, preferably the respective use, wherein the cRNA-PLC is free of non-bound circulating RNA and was obtained from blood by one step filtration through nitrocellulose filter and release of RNA from the complex by washing with RNA extraction buffer.
  • RNAs / cDNAs are coding for non-small cell lung cancer (NSCLC) marker proteins.
  • NSCLC non-small cell lung cancer
  • the following biomarker genes / gene products can be identified by the method according to the invention : tumor suppressor protein 53 (p53) tumor promoter protein 21 (ras-p21) tumor suppressor protein 21 (Waf1/C
  • p53 tumor suppressor protein 53
  • ras-p21
  • BMP bone morphogenetic protein
  • EGF epithelial growth factor
  • FGF fibroblast growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TGF tumor necrosis factor
  • VEGF vascular endothelial growth factor vaso-intestinal peptide
  • Another aspect of the invention relates to the use of polynucleotide arrays, which allows to qualitatively and quantitatively study mRNA expression levels, whereby the RNA is derived from cRNA-PLC.
  • Polynucleotide or DNA arrays are principally well known and consist of large numbers of DNA molecules spotted in a systematic order on a solid support or substrate such as a nylon membrane, glass slide, glass beads or a silicon chip.
  • DNA or RNA arrays can be categorized as microarrays (each DNA/RNA spot has a diameter less than 250 microns) and macro-arrays (spot diameter is grater than 300 microns). When the solid substrate used is small in size, arrays are also referred to as DNA chips. Depending on the spotting technique used, the number of spots on a glass microarray can range from hundreds to tens of thousands.
  • a DNA microarray is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • the probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others).
  • the solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip or colloquially Affy chip when an Affymetrix chip is used.
  • DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system.
  • DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs) (see Types of arrays section), in genotyping or in resequencing mutant genomes. Microarrays also differ in fabrication, workings, accuracy, efficiency, and cost (see fabrication section). Additional factors for microarray experiments are the experimental design and the methods of analyzing the data
  • DNA microarrays are used for a variety of purposes, including, gene expression profiling, de novo gene sequencing, gene mutation analysis, gene mapping and genotyping.
  • cDNA microarrays are printed with distinct cDNA clones isolated from cDNA libraries. Therefore, each spot represents an expressed gene, since it is derived from a distinct mRNA.
  • a method of monitoring gene expression involves providing
  • RNA transcripts of target genes or nucleic acids derived from the RNA transcripts (1 ) a pool of sample polynucleotides comprising RNA transcripts of target genes or nucleic acids derived from the RNA transcripts;
  • probes for example, polynucleotides obtained from a polynucleotide library including control probes
  • the label used to label polynucleotide samples is selected from the group consisting of radioactive, colorimetric, enzymatic, molecular amplification, bioluminescent or fluorescent label. Detection can also involve
  • the invention relates also to any polynucleotide library as previously described wherein said polynucleotides are immobilized on a solid support in order to form a polynucleotide array.
  • the support is selected from the group consisting of a nylon membrane, glass slide, glass beads, or a silicon chip.
  • the invention relates to a polynucleotide library useful in the molecular characterization of a interstitial lung disease, the library including a pool of polynucleotide sequences or subsequences thereof wherein the sequences or subsequences are either underexpressed or overexpressed in diseased cells, further wherein the sequences or subsequences correspond substantially to any of the polynucleotide sequences set forth in any of SEQ ID NOS: x-y or the complement thereof.
  • the invention is related to specific tumor markers, preferably biomarkers for lung cancer, especially for NSCLC.
  • RNAs encoding for the following known NSCLC markers were characterized by screening of tumor suppressor genes which are down- regulated compared to non-malignant human lung cells.
  • tumor suppressor protein 53 tumor promoter protein 21 (ras-p21) tumor suppressor protein 21 (Waf1/Cip1) ( p 2i W af1/CiP1 >) tumor suppressor protein 27 (Kip1) (p217 (Kip1) ) tumor suppressor protein 16 (p16) retinoblastoma tumor suppressor protein (Rb) fragile histidine triade (FHIT) ras-association domain family (RASSF1A) class 3 semaphorins (SEMA3B) phosphatase and tensin homologue (PTEN) base excision repair gene (hOGGD small phospholipid binding protein (BAP1)
  • RNAs encoding for the following known NSCLC markers was characterized by screening of tumor suppressor genes which are up- regulated compared to non-malignant human lung cells. If they exist in form of an inhibitory micro RNA in the cRNA-PLC it is expected that they are up-regulated, which would lead to a enhanced proliferation of any cell which will take such a cRNA- PLC up.
  • BMP Bone morphogenetic proetin
  • EGF Epithelial growth factor
  • FGF Fibroblast growth factor
  • Nerve growth factor (NGF)
  • PDGF Platelet-derived growth factor
  • TGF Tumor growth factor
  • TNF Tumor necrosis factor
  • VEGF Vascular endothelial growth factor
  • VIP Vaso-intestinal peptide
  • NSCLC markers A small set of known NSCLC markers was suggested to be used as indicators for the prognosis. Possible clusters of prognostic and predictive markers are:
  • BRCA1 , p53, KRAS, betatubulin, and EGFR indicate a worse prognosis
  • Example 1 Blood sampling and plasma preparation
  • RNA from proteolipids by cRNA-PLC by glass beads will be collected into EDTA-tubes containing a second commercially available RNase inhibitor and will be centrifuged at 160Og for 10 minutes at 4 0 C. The serum will be collected and centrifuged at 1600Og for 10 minutes at 4 0 C and the upper layer will be collected as plasma.
  • Example 2 Separation of the RNA from proteolipids by cRNA-PLC by glass beads:
  • RNA-PLC preparation 500 micro-litres will be suspended mixed with an equal volume of total RNA extraction buffer (Qiagen, Germany) and than be centrifuged at 10,000 x g for 10 min at 4 0 C.
  • the supernatant will be applied onto a RNA separation column (Qiagen) and RNA will be bound in the column to glass- beads.
  • the RNA will be then eluted from the column with a specific mRA elution buffer (Qiagen) and will be precipitated by addition of 3M NaCI-solution (containing RNase inhibitor) in 50% propanol for overnight at -2O 0 C.
  • the RNA will be collected by centrifugation (13'0OO x g, 15 min., 4 0 C), the RNA pellet will be dried and solubilized for further analysis in 50 micro-litres of RNase free water (Pierce, USA).
  • Example 3 Separation of the RNA from proteolipids by cRNA-PLC filtration:
  • RNA extraction buffer RNeasy; Qiagen, USA. This will extract RNAs which are bound to proteolipid trapped on the surface of the filter.
  • the solution will then be mixed with 1.4ml of Trizol reagent (Invitrogen, USA) and 0.3 ml of chloroform will be added.
  • the mixture will be vigorously mixed for 30 seconds and then centrifuged at 1200Og for 15 minutes at 4 0 C.
  • the upper layer will be transferred into new tubes and mixed with the double volume of 70% ethanol.
  • This mixture will be incubated at -2O 0 C for 1 hour and then be applied onto a RNeasy column and the RNA will be purified and isolated as recommended by the distributor (Qiagen) in 15ml of RNase free water, followed by 15 minutes treatment with DNase I (Invitrogen).
  • the RNA can then be store at -8O 0 C until analysis for miRNA or mRNA content.
  • Reverse transcription will be used to create the more stable complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, an unspecific poly-thymidine-thymidine primer, and dNTPs in a ready to use mixture (Pierce).
  • dNTP will be labeled for later detection of specific cDNAs on micro-array chips.
  • RNA will be first incubated with a primer at 7O 0 C to denature secondary structures, then quickly chilled on ice to let the primer anneal to the RNA, 2) the other components of the reverse transcription reaction will be added (dNTPs, RNase inhibitor, reverse transcriptase) in an appropriate buffer (Pierce), 3) the reaction is extended at 42 0 C for 30 minutes, 4) the reaction is stopped at 7O 0 C, and 5) the template RNA by addition of RNase H at 37 0 C for 20 minutes which creates the so-called first strand cDNA strand. All steps will be performed as recommended by the producer of the cDNA kit and ay be adjusted to improve performance.
  • Example 5 Characterization of tumor specific cDNAs:
  • the labeled cDNA samples will be mixed with a propriety hybridization solution containing SDS. SSC. or dextran sulfate for denaturing, a blocking agent (COT1 DNA, salmon sperm DNA, calf thymus DNA, PoIvA or PoIyT), Denhardt's solution and formamine (Pierce).
  • a blocking agent COT1 DNA, salmon sperm DNA, calf thymus DNA, PoIvA or PoIyT
  • Denhardt's solution and formamine (Pierce).
  • This mix will be added to a pin hole in a microarray gene chip and will be sealed.
  • the microarray will be hybridized over night, after which all non specific binding will be washed off.
  • the microarray will be dried and scanned using a special machine where a laser exits the dye and a detector measures its emission.
  • the image will be gridded with a template and the intensities (pixels) will be quantified.
  • the raw data has then to be normalized, by subtracting the background intensity and then divide the intensity of each spot to the total intensity of each gene sample to the intensity of a reference genes and then the t- value for all the intensities is calculated.
  • RMA robust multichip analysis
  • Affymetrix chips single-channel, silicon chip
  • Example 6 Preparation of Labeled cRNA and Hybridization to Microarrays.
  • Double-stranded cDNA will be synthesized out of the isolated patients RNA samples using a cDNA synthesis kit (Superscript; Life Technologies) employing oligo(dT) priming.
  • the resulting cDNA will be used for in vitro transcription (Ambion T7 Megascript system) in the presence of biotin-11 -CTP and biotin-16-UTP (Enzo Diagnostics).
  • a total of 25-50 ⁇ g of the cRNA product in buffer [40 mM Tris acetate (pH 8.1)/100 mM potassium acetate/30 mM magnesium acetate] will be fragmented at 94°C for 35 min. It will then be used as a hybridization probe from each patient for hybridization as recommended (Affymetrix, Santa Clara, CA).
  • Scanned output files will be visually inspected for hybridization artifacts and then analyzed with GENECHIP 3.1 software (Affymetrix).
  • the expression analysis files created by GENECHIP 3.1 software will be transferred to a database (Microsoft Access) and linked to Internet genome databases (e.g., NHLBI, or Swiss Prot).

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Abstract

The invention relates to the use of RNA isolated from circulating RNA-proteolipid complex (cRNA-PLC) from body liquids of tumor patients, especially tumor patients suffering from lung cancer, especially small cell lung cancer (SCLC), wherein said RNA was obtained by a specific filtration method. The invention relates above all to the use of this RNA for detecting specific tumor markers present in blood or serum samples of tumor patients by means of corresponding RNA/DNA microchips in order to predict tumor type, disease status and efficacy of a specific anti-cancer treatment.

Description

USE OF RNA OBTAINED FROM PROTEOLIPID COMPLEXES CIRCULATING IN BLOOD FOR DIAGNOSIS AND TREATMENT OF TUMORS
FIELD OF THE INVENTION: The invention relates to the use of RNA isolated from circulating RNA-proteolipid complex (cRNA-PLC) from body liquids of tumor patients, especially tumor patients suffering from lung cancer, especially small cell lung cancer (SCLC), wherein said RNA was obtained by a specific filtration method. The invention relates above all to the use of this RNA for detecting specific tumor markers present in blood or serum samples of tumor patients by means of corresponding RNA/DNA microchips in order to predict tumor type, disease status and efficacy of a specific anti-cancer treatment.
BACKGROUND OF THE INVENTION
Non-small-cell lung cancer (NSCLC) accounts for most cancer death world wide, less than 15% of patients survive 5 years after diagnosis. Furthermore, most NSCLC patients do not respond well to therapy thus, it is advised to develop selection criteria that allow to match individual patients with the best possible therapeutic strategy.
In 1985 Wieczorek et al. (Proc. Natl. Acad. Sci, 82, 3455) described for the first time the isolation of an RNA-proteolipid complex from serum of tumor patients and healthy controls. It was observed that increased serum levels of this circulating RNA- proteolipid complex (cRNA-PLC) correlated with the severity of the disease . Due to its chemical and molecular nature the cRNA-PLC with later described circulating proteasomes it was assumed that both terms describe an identical cellular unit, which either originates from cell degradation or from over active tumor cells by exocytosis. Tumour cells, in general, are over-active compared to non-malignant cells and produce significantly higher number of proteolipids which often also contain RNA from the tumour cells. Therefore, this cRNA-PLC contain tumo From each cRNA-PLC preparation 500 micro-litres will be suspended mixed with an equal volume of total RNA extraction buffer (Qiagen, Germany) and than be centrifuged at 10,000 x g for 10 min at 40C. The supernatant will be applied onto a RNA separation column (Qiagen) and RNA will be bound in the column to glass-beads. The RNA will be then eluted from the column with a specific mRA elution buffer (Qiagen) and will be which was rich in phospholipids and glycosphingolipids (1 ). In this context it is important to note that the naturally occurring low and high density serum lipoproteins are different from this cRNA-PLC in regard of their density, chemical composition, and immunological reactivity (Wieczorek et al., Proc Natl Acad Sci U S A. 1985;82:3455- 9).
In a second study the correlation of serum levels of the cRNA-PLC with staging and progression of malignant diseases was confirmed in a larger cohort of tumor patients and it was shown that the chemical composition of the cRNA-PLC was independent of the tumor type and of the tumor staging. Importantly, the tumor mass correlated with the amount of cRNA-PLC and after effective treatment the serum level of cRNA-PLC fell significantly, and therefore cRNA-PLC was considered as a valid tumor marker (2). Furthermore, in tumor patients who showed a relapse of the malignancy the concentration of the cRNA-PLC increased 10 months prior to the conventional clinical diagnostic tools used to indicate a relapse, and therefore the cRNA-PLC may be used as an early indicator of tumor relapse (Wieczorek et al., Cancer Res. 1987;47:6407- 12.).
Interestingly the molecular and chemical composition of the protein part of the cRNA- PLC resembles characteristics of a classical cellular 2OS proteasome, which plays a regulatory role in the cytosolic generation of major histocompatibility complex (MHC) class I restricted antigens, which represent an abundant type of antigens in several autoimmune diseases (Egerer et al., J Rheumatol. 2002;29:2045-52). Proteasomes occur in normal human plasma and have been assigned as "circulating proteasomes", however, their biological function in this state is unknown.
Cellular proteasomes are non-lysosomal proteolytic cell units which participate in the regulatory mechanisms controlling cell growth and differentiation. Abnormal high expression of cellular proteasomes has been described in haematopoietic malignancies and solid tumors. The increase of cellular proteasomses was paralleled by an increase of plasma levels of circulating proteasomes, as determined by ELISA in patients with malignant melanoma (stage III and IV) compared to controls and patients at earlier stages (Stoebner et al., Br J Dermatol. 2005;152:948-53). The serum level of plasma or circulating proteasomes was further increased in patients with tumor metastasis. Increased levels of circulating proteasomes were also reported from patients with auto-immune inflammatory or peri-operative cellular damage (Zoeger et al., Clin Chem. 2006;52:2079-86).
ELISA based measurement of circulating proteasomes showed increased levels in serum and plasma samples obtained from patients with rheumatoid arthritis, myasthenia gravis, autoimmune myositis, systemic lupus erythematosus, primary Sjogren's syndrome, and autoimmune hepatitis compared to control serum (I.e.). However, when compared to levels of circulating proteasomes in tumor patient serum, those observed in auto-immune diseases were significantly lower. A further association of circulating 2OS proteasomes was found in serum of patients suffering from mixed connective tissue disease and correlated with clinically relevant changes in disease activity (Majetschak et al., Clin Vaccine Immunol. 2008;15:1489-93). These findings indicate that proliferating cells may get rid of unwanted or an over-production of cellular products by exocytosis of proteasomes.
It was then assumed that the circulating 2OS proteasomes are generated via an intracellular protein degrading pathway in diseased or otherwise damaged cells, mainly from blood cells (Majetschak et al., I.e.). However, this assumption was not confirmed and therefore the origin of the circulating proteasomes remains unknown (Zoeger et al., I.e. ). A more recent study showed that extracellular proteasomes can be found on the outside of cell membranes of tissue forming lung cells such as epithelial cells and from there they are shed off and enter into the alveolar lining fluid and the epidermal fluid; possibly during the acrosome reaction (Sixt et al., Biochim Biophys Acta. 2008 Jun 18. PMID: 18602990 ahead of print.). In addition, it had been demonstrated that the proteolytic activity (chymotrypsin-like, trypsin-like, caspase-like) of circulating proteasomes and the extent of their ubiquination is an indicator of chronic lymphocytic leukemia and correlated it with the clinical prognosis (Ma et al., Cancer. 2008;112:1306-12).
The most important part of the cRNA-PLC is its content of RNA. As the cRNA-PLC is assumed to originate from tumor cells, the therein contained RNA may provide a much more tumor specific array of markers than any other type of circulating RNA in the blood. - A -
The concept that circulating RNA is indicative for tumor development and prognosis is not new and had been suggested by several earlier studies for various tumor types (Ma et al., I.e.; Tsang et al., Pathology. 2007 Apr;39(2):197-207; Swamp et al., FEBS Lett. 2007 Mar 6;581 (5):795-9). However, so far the characterization of such circulating cell free RNA in blood or plasma samples of tumor patients did not select for the RNA contained in the above described cRNA-PLC. Most recent studies (2007 - 2008) analyzed total circulating RNA (Mehra et al., Clin Cancer Res. 2007 Jan 15;13(2 Pt 1):421-6; Rappl et al., Ann N Y Acad Sci. 2001 Sep;945: 189-91 ; Tani et al., Anticancer Res. 2007 Mar-Apr;27(2): 1207-12; Lin et al., Cancer. 2007 Aug 1 ;110(3):534-42.; Gabri et al., lnt J MoI Med. 2008 May;21 (5):555-9.; Ellinger et al., BJU Int. 2008 Aug 5;102(5):628-32.; Wong et al., lnt J Surg Pathol. 2008 Apr;16(2):119-26; Garcfa et al., Cancer Lett. 2008 May 18;263(2):312-20.). The malignancies with increased levels of circulating cRNA-PLC include: malignant melanoma, gastric cancer, colon cancer, prostrate cancer, colorectal cancers, breast cancer and renal cell cancer. These RNAs may originate from a wide range of sources and will therefore not represent a specific array of tumor associated factors or markers (Feng et al., Anticancer Res. 2008 Jan-Feb;28(1A):321 -6).
Most studies that investigated circulating RNA in tumor patients considered that the preence of this extracellular RNA is a tumor marker by itself, or they focused on the over-expression of tumor specific marker RNAs in such a preparation. Thereby the studies ignored the fact that circulating RNA can be taken up by other cells and execute a function in those cells (Zhou et al., Cancer Lett. 2008 Jan 18;259(1):50- 60.). Interestingly, there are recent reports on circulating micro RNAs in tumor patient's serum (Kuligina et al., Ann N Y Acad Sci. 2008 Aug;1137:51 -7; Mitchell et al., Proc Natl Acad Sci U S A. 2008 JuI 29;105(30):10513-8), and again the function of those micro RNAs can only be speculated.
In the context of this invention, it is of interest to note that such micro RNAs were isolated from proteolipid like structures (Mitchell et al., Proc Natl Acad Sci U S A. 2008 JuI 29;105(30):10513-8; Lawrie et al., Br J Haematol. 2008 May;141 (5):672-5). Reflecting on the anti-inflammatory and anti-infectious function of naturally occurring micro RNA in otherwise healthy people the inventor postulates that such circulating micro RNAs in serum of tumor patients may, after being taken up by a non-tumor cell, switch the "host" cell's program to the benefit of the tumor or to generate a tumor like cell type by down-regulating tumor suppressor genes.
One major problem of the investigation of all known types of circulating RNAs is the method used for their isolation. It is a well established fact based on the chemical nature and of the function of RNA that any type of RNA is highly sensitive to degradation by RNases.
RNases present an essential protective mechanism that rapidly degrades viral RNA and is present in abundance in all human body fluids (Garcia et al., RNA. 2008 Jul;14(7):1424-32; Taylor et al., Gynecol Oncol. 2008 Jul;110(1 ):13-21 ; Cerkovnik et al., lnt J MoI Med. 2007 Sep;20(3):293-300; O'Driscoll et al., Cancer Genomics Proteomics. 2008 Mar-Apr;5(2):94-104). This problem seems to be significantly reduced when isolating circulating RNA from the above described cRNA-PLC.
The serum level of circulating proteasomes correlated with tumor stages and its proteolytic activity was recently suggested to be an indicator for the progression of chronic leukemia progression.
However, no attention was paid to the function or disease specific nature of the content of RNA in this complex, and whether this RNA can be used as specific and reliable biomarker for specific tumors, above all lung cancer, preferably NSCLC.
Genes that had been reported in the prior art to be up-regulated in lung cancer and other organ adenocarcinomas are:
Survivin (Birkcδ)
Apoptotic protease activating factor 1 (APAF1Akt1) Bcl-2-like 1 protein (BCL2L)
CASP2 and RIPK1 domain containing adaptor with death domain (Cradd) Cytochrom C (Cycs)
Mitogen activated protein-3 kinase / p38 MAK (MAP3K)(isofoms: DDDDDDDDD D) 53 inhibiting MDM-2 protein (MDM2) (40 splice variants have been described) Receptor interacting serine/threonine kinase 1 (RIPK1) A special case are apoptosis inducing enzymes assigned as caspases which have been reported to be up-regulated in adenocarnicomas, a finding which contradicts that of others claiming that apoptosis is down-regulated in tumors. However, in adneocarcinomas the following caspases were reported as increased:
caspase-1 (CaspD caspase-2 (Casp2) caspase-3 (Casp3) caspase-5 (Caspδ) caspase-9 (Casp9) caspase-10 (CaspiO)
Genes which are known to be down-regulated in adenocarcinomas are:
AXIN1 up-regulated (AXUD1)
BCL2-antagonist/killer 1 (BAK1) Cyclin dependent kinase inhibitor 2D (CDKN2D)
CCAAT/enhancer binding protein-alphaD (C/EBP-alpha)DD
CCAAT/enhancer binding protein -betaD (CVEBP-beta)DD
Cox TRL activated protein (CTLA1)
Death associated protein kinase 2 (DAPK2) Mitogen activate protein 3 kinase 7 (MAP3K7)
Nuclear factor kappaB-1 alpha (NFKB1A)
Perforin-1 (PRF1)
Myeloid related protein (S100A9)
Tumor necrosis factor receptor surface factor 1 (TNFRSF1) Tumor necrosis factor receptor surface factor 6 (TNFRSF6)
In order to screen multiple patient blood /serum samples for the presence of cRNA- PLC and the subsequent analysis of tumour specific mi/mRNA pattern, the methods described earlier (Wieczorek at al., I.e.) is far too labour and time intensive. Summary of the invention:
The underlying idea of this invention is that the circulating RNA proteoliposome complexes (cRNA-PLC) contain RNA that can be taken up by other cells, in which the RNA functions in a way that creates an environment specifically supporting tumor spreading, and therefore can be used as a diagnostic tool.
Therefore, an RNA isolated from the cRNA-PLC could be used as an unambiguous indicator for: (i) the tumor type,
(ii) the tumor progression (staging), (iii) monitoring of patients response to therapy, and (iv) detecting efficacy of different anti-tumor therapies.
In a first aspect of the invention, the cRNA-PLC is used and isolated from serum or whole blood of tumor patients using the method described in WO 97/35589 or WO 90/10872.
The RNA contained in the cRNA-PLC can be isolated according to the invention, for example by the glass-milk procedure as described in the example section, under non- denaturing and RNase-free conditions.
In a second, preferred aspect of the invention, the blood/serum sample comprising said cRNA-PLC complex is submitted to a specific filtration step, which does not only purify the sample but also does separate free RNA present in the sample from RNA bound to the proteolipid complex. According to the finding of this invention only the bound RNA is an unambiguous indicator for tumor markers, whereas the free RNA in the body fluid sample may derive also from other non-tumor sources. The bound RNA can be easily released from the proteolipid complex trapped on the filter material.
Hence, the inventors have developed a new method which allows a simple fast forward isolation of proteolipid bound RNAs using a filter device. Although filter systems are known for separating RNA from samples the nature of the filter materials necessary for separating blood circulating RNA bound to proteolipid complex was not further described and cannot be reconstructed from the provided information of the prior art. According to the finding of this invention the effectiveness and specificity to selectively isolate mRNA from the cRNA-PLC can be increased by using filter materials that are able to bind proteins and proteolipids or RNA bound to lipids. Preferably, filter materials based on nitro-cellulose and derivatives thereof can be used according to the invention. The preferable pore size of said filters according to the best mode of this invention is between 0.1 and 0.5 μm, preferably 1.1 and 1.3 μm.
Based on the earlier publications by Wieczorek et al. (I.e.), the inventors of the present invention assumed that the so called particle bound circulating RNA is the same or similar to the cRNA-PLC. The filters which preferably are used according to the invention are composed of nitro-cellulose compounds which are known to bind proteins and proteolipids, thus, the particles which are held back by the filter contain cRNA-PLC.
The isolated and purified mRNA will than be transcribed into complementary cDNA by reverse transcriptase according to standard techniques.
The cDNA will then be amplified in an unspecific polymerase chain reaction and the products will be analyzed by micro-array chip technology using specific chips for the detection of the presence of tumor related gene products (mRNAs) according to per se known methods.
The invention is related to the use of RNA (ribonucleic acid) which is contained in a circulating RNA-proteolipid complex (cRNA-PLC), to transcribe this RNA into cDNA and screen this cDNA by the use of DNA micro array chips for: (i) tumor type specific markers, (ii) for tumor stage specific markers, and (iii) for monitoring the expression of tumor markers in patients who undergo any type of anti-cancer therapy in order to control the success of the therapy and/or the progression of the tumor.
According to the invention, it was found that the RNA contained in the cRNA-PLC obtained preferably by the filtration step as described originates directly from tumor cells, which are either dying, or which are overactive and therefore release "unwanted" proteins, lipids and RNA by exocytosis. Therefore, this RNA is very specific for the tumor, although it was obtained from body fluids, such as blood.
Although the cRNA-PLC is found also in blood samples of healthy individuals, it could be shown by this invention that cRNA-PLC is significantly increased in blood samples of tumor patients. Therefore cRNA-PLC is thought to be an indicator for tumor disease, its status, progression or disappearance.
The data obtained show that the RNA contained in the cRNA-PLC is intact and can be transcribed into cDNA. Therefore, it is possible to characterize the gene of origin of this RNA according to in principle known methods (Voortman et al., MoI Cancer. 2007;6:73; Chen et al., MoI Pharmacol. 2007;72: 1269-79; Tabata et al., J Biol Chem. 2001 ;276:8029-36; Godley et al., Curr Opin Support Palliat Care. 2007;1 :23-9).
The cell type of origin of the cRNA-PLC is unknown. Based on the observation that its concentration increases with the duration and staging of tumors in blood samples of tumor patients and the fact that the cRNA-PLC was also found under cell culture conditions, it can be hypothesized by this invention that cRNA-PLC is formed and released by tumor cells into the tumor feeding blood vessels and thereby enters into the blood stream.
By this invention it could be shown that cRNA-PLC mainly contains RNA which encodes for tumor specific factors either supportive or repressive to the tumor and other cells. This makes the cRNA-PLC a tool to search and identify tumor specific factors which are concentrated in this distinguishable blood component and can be isolated by adapted routine methods.
Since the level of cRNA-PLC correlated with the progression or regression of various tumors, the isolated RNA can than be used to screen for a tumor type specific array of markers, as well as it can be used to monitor the effect of any applied therapy.
In summary the invention is directed to the following:
• An ex-vivo method for identifying and determining tumor-specific biomarker genes and / or gene products in an individual suffering from a tumor, the method comprising
(i) isolating and purifying DNA free circulating RNA proteolipid complex (cRNA-
PLC) from a blood or serum sample of said individual,
(ii) separating mRNA from said proteolipid complex and transcribing the RNA to cDNA by standard techniques, (iii) applying the cDNA after amplification to a microassay gene chip, (iv) determining by technical means and / or devices the cDNA and its intensity in comparison with RNA / DNA originating from tissue of a healthy individual, and
(v) identifying by technical means and / or devices from the measured differences the tumor type and strength in the diseased individual by the tumor- specific genes which are up- or down-regulated compared to the healthy individual.
• A respective method, wherein said isolated cRNA-PLC is free of RNA present in the sample but originally not bound to protein or lipid complexes.
• A respective method, wherein said cRNA-PLC was obtained from the blood or serum sample by one-step filtration.
• The respective, wherein said filtration step is carried out by a nitro-cellulose filter in a pore size range of 0.1 - 0.5 μm, preferably 0.1 - 0.3 μm.
• A respectcive method, wherein the mRNA was separated from the proteolipid complex by extraction with RNA extraction / elution buffer, such as RNeasy Kit ® (Quiagen).
• A respective method, wherein the comparison DNA / RNA of the healthy individual and the cDNA of the diseased individual obtained by step (iv) are related to the same tissue type.
• A corresponding method, comprising generating a tumor specific and individual
RNA profile by applying the method to different individuals suffering from the same tumor.
• A respective method, wherein the circulating RNA within the proteolipid complex is at least partially anti-sense RNA (siRNA).
• Arespective method, wherein the tumor to be determined with said biomarkers that are identified by said cRNA-PLC, is NSCLC.
• An ex-vivo method for determining tumor progression / regression, or the effect of an anti-tumor therapy in a tumor patient, the method comprising applying the steps (i) - (iv) of claim 1 and repeating these steps at different stages of tumor development and / or therapy schemes.
• The method above, wherein the therapy scheme includes administration of an anti-tumor drug.
• The use of cRNA-PLC obtained from blood or serum of a patient suffering from cancer for ex-vivo identification of biomarkers that are tumor specific, preferably the respective use, wherein the cRNA-PLC is free of non-bound circulating RNA and was obtained from blood by one step filtration through nitrocellulose filter and release of RNA from the complex by washing with RNA extraction buffer.
• An ex-vivo method, wherein the RNAs / cDNAs are coding for non-small cell lung cancer (NSCLC) marker proteins. Preferably, the following biomarker genes / gene products can be identified by the method according to the invention : tumor suppressor protein 53 (p53) tumor promoter protein 21 (ras-p21) tumor suppressor protein 21 (Waf1/C|P1) (P21 Wafi/cpDj tumor suppressor protein 27(Kιp1) (p217(Kιp1)) tumor suppressor protein 16 (p16) retinoblastoma tumor suppressor protein (Rb) fragile histidine triade (FHIT) ras-association domain family (RASSF1A) class 3 semaphorins (SEMA3B) phosphatase and tensin homologue (PTEN) base excision repair gene (hOGGD small phospholipid binding protein (BAP1) These biomarkers are found to be down-regulated in NSCLC patients:
The following biomarkers are up-regulated in NSCLC patients: bone morphogenetic protein (BMP) epithelial growth factor (EGF) fibroblast growth factor (FGF) nerve growth factor (NGF) platelet-derived growth factor (PDGF)-BB tumor growth factor (TGF) tumor necrosis factor (TNF) vascular endothelial growth factor (VEGF) vaso-intestinal peptide (VIP)
As already mentioned, another aspect of the invention relates to the use of polynucleotide arrays, which allows to qualitatively and quantitatively study mRNA expression levels, whereby the RNA is derived from cRNA-PLC.
Polynucleotide or DNA arrays are principally well known and consist of large numbers of DNA molecules spotted in a systematic order on a solid support or substrate such as a nylon membrane, glass slide, glass beads or a silicon chip.
Depending on the size of each DNA spot on the array, DNA or RNA arrays can be categorized as microarrays (each DNA/RNA spot has a diameter less than 250 microns) and macro-arrays (spot diameter is grater than 300 microns). When the solid substrate used is small in size, arrays are also referred to as DNA chips. Depending on the spotting technique used, the number of spots on a glass microarray can range from hundreds to tens of thousands.
A DNA microarray is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
In standard microarrays, the probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip or colloquially Affy chip when an Affymetrix chip is used. Other microarray platforms, such as lllumina, use microscopic beads, instead of the large solid support. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system.
DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs) (see Types of arrays section), in genotyping or in resequencing mutant genomes. Microarrays also differ in fabrication, workings, accuracy, efficiency, and cost (see fabrication section). Additional factors for microarray experiments are the experimental design and the methods of analyzing the data
DNA microarrays are used for a variety of purposes, including, gene expression profiling, de novo gene sequencing, gene mutation analysis, gene mapping and genotyping. cDNA microarrays are printed with distinct cDNA clones isolated from cDNA libraries. Therefore, each spot represents an expressed gene, since it is derived from a distinct mRNA.
Typically, a method of monitoring gene expression involves providing
(1 ) a pool of sample polynucleotides comprising RNA transcripts of target genes or nucleic acids derived from the RNA transcripts;
(2) hybridizing the sample polynucleotides or nucleic acids derived from the RNA transcripts to an array of probes (for example, polynucleotides obtained from a polynucleotide library including control probes, and
(3) detecting the hybridized double- calculating/quantifying a relative expression (transcription) level, stranded polynucleotides/nucleic acids. The label used to label polynucleotide samples is selected from the group consisting of radioactive, colorimetric, enzymatic, molecular amplification, bioluminescent or fluorescent label. Detection can also involve
The invention relates also to any polynucleotide library as previously described wherein said polynucleotides are immobilized on a solid support in order to form a polynucleotide array.
Preferably the support is selected from the group consisting of a nylon membrane, glass slide, glass beads, or a silicon chip. The invention relates to a polynucleotide library useful in the molecular characterization of a interstitial lung disease, the library including a pool of polynucleotide sequences or subsequences thereof wherein the sequences or subsequences are either underexpressed or overexpressed in diseased cells, further wherein the sequences or subsequences correspond substantially to any of the polynucleotide sequences set forth in any of SEQ ID NOS: x-y or the complement thereof.
The invention is related to specific tumor markers, preferably biomarkers for lung cancer, especially for NSCLC.
In a first step the expression profile of RNAs encoding for the following known NSCLC markers (34) was characterized by screening of tumor suppressor genes which are down- regulated compared to non-malignant human lung cells.
It was found that the following gene products were down-regulated in NSCLC tumors according to the invention:
tumor suppressor protein 53 (p53) tumor promoter protein 21 (ras-p21) tumor suppressor protein 21 (Waf1/Cip1) (p2i Waf1/CiP1>) tumor suppressor protein 27(Kip1) (p217(Kip1)) tumor suppressor protein 16 (p16) retinoblastoma tumor suppressor protein (Rb) fragile histidine triade (FHIT) ras-association domain family (RASSF1A) class 3 semaphorins (SEMA3B) phosphatase and tensin homologue (PTEN) base excision repair gene (hOGGD small phospholipid binding protein (BAP1)
In a second step the expression profile of RNAs encoding for the following known NSCLC markers was characterized by screening of tumor suppressor genes which are up- regulated compared to non-malignant human lung cells. If they exist in form of an inhibitory micro RNA in the cRNA-PLC it is expected that they are up-regulated, which would lead to a enhanced proliferation of any cell which will take such a cRNA- PLC up.
It was found that the following gene products were up-reαulated in NSCLC tumors according to the invention:
Bone morphogenetic proetin (BMP)
Epithelial growth factor (EGF)
Fibroblast growth factor (FGF)
Nerve growth factor (NGF)
Platelet-derived growth factor (PDGF)-BB Tumor growth factor (TGF)
Tumor necrosis factor (TNF)
Vascular endothelial growth factor (VEGF)
Vaso-intestinal peptide (VIP)
A small set of known NSCLC markers was suggested to be used as indicators for the prognosis. Possible clusters of prognostic and predictive markers are:
a) ERCC1 and RRM1 in untreated patients indicate a better prognosis.
b) BRCA1 , p53, KRAS, betatubulin, and EGFR indicate a worse prognosis
EXAMPLES:
Example 1 : Blood sampling and plasma preparation
Peripheral blood samples (10 ml) will be collected into EDTA-tubes containing a second commercially available RNase inhibitor and will be centrifuged at 160Og for 10 minutes at 40C. The serum will be collected and centrifuged at 1600Og for 10 minutes at 40C and the upper layer will be collected as plasma. Example 2: Separation of the RNA from proteolipids by cRNA-PLC by glass beads:
From each cRNA-PLC preparation 500 micro-litres will be suspended mixed with an equal volume of total RNA extraction buffer (Qiagen, Germany) and than be centrifuged at 10,000 x g for 10 min at 40C. The supernatant will be applied onto a RNA separation column (Qiagen) and RNA will be bound in the column to glass- beads. The RNA will be then eluted from the column with a specific mRA elution buffer (Qiagen) and will be precipitated by addition of 3M NaCI-solution (containing RNase inhibitor) in 50% propanol for overnight at -2O0C. The RNA will be collected by centrifugation (13'0OO x g, 15 min., 40C), the RNA pellet will be dried and solubilized for further analysis in 50 micro-litres of RNase free water (Pierce, USA).
Example 3: Separation of the RNA from proteolipids by cRNA-PLC filtration:
Immediately after the isolation the plasma samples will be passed through nitrocellulose filters with a pore size of 0.22Fehler! Textmarke nicht definiert.m. The filters will be then washed in the reversed direction with 0.7 ml of RNA extraction buffer (RNeasy; Qiagen, USA). This will extract RNAs which are bound to proteolipid trapped on the surface of the filter.
The solution will then be mixed with 1.4ml of Trizol reagent (Invitrogen, USA) and 0.3 ml of chloroform will be added. The mixture will be vigorously mixed for 30 seconds and then centrifuged at 1200Og for 15 minutes at 40C. The upper layer will be transferred into new tubes and mixed with the double volume of 70% ethanol. This mixture will be incubated at -2O0C for 1 hour and then be applied onto a RNeasy column and the RNA will be purified and isolated as recommended by the distributor (Qiagen) in 15ml of RNase free water, followed by 15 minutes treatment with DNase I (Invitrogen). The RNA can then be store at -8O0C until analysis for miRNA or mRNA content.
Example 4: Reverse transcription - cDNA:
Reverse transcription will be used to create the more stable complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, an unspecific poly-thymidine-thymidine primer, and dNTPs in a ready to use mixture (Pierce). One of the dNTP will be labeled for later detection of specific cDNAs on micro-array chips. The steps for the cDNA generation are: 1) RNA will be first incubated with a primer at 7O0C to denature secondary structures, then quickly chilled on ice to let the primer anneal to the RNA, 2) the other components of the reverse transcription reaction will be added (dNTPs, RNase inhibitor, reverse transcriptase) in an appropriate buffer (Pierce), 3) the reaction is extended at 420C for 30 minutes, 4) the reaction is stopped at 7O0C, and 5) the template RNA by addition of RNase H at 370C for 20 minutes which creates the so-called first strand cDNA strand. All steps will be performed as recommended by the producer of the cDNA kit and ay be adjusted to improve performance. Example 5: Characterization of tumor specific cDNAs:
The labeled cDNA samples will be mixed with a propriety hybridization solution containing SDS. SSC. or dextran sulfate for denaturing, a blocking agent (COT1 DNA, salmon sperm DNA, calf thymus DNA, PoIvA or PoIyT), Denhardt's solution and formamine (Pierce). This mix will be added to a pin hole in a microarray gene chip and will be sealed. The microarray will be hybridized over night, after which all non specific binding will be washed off. The microarray will be dried and scanned using a special machine where a laser exits the dye and a detector measures its emission. The image will be gridded with a template and the intensities (pixels) will be quantified. The raw data has then to be normalized, by subtracting the background intensity and then divide the intensity of each spot to the total intensity of each gene sample to the intensity of a reference genes and then the t- value for all the intensities is calculated.
Either RMA (robust multichip analysis) or Affymetrix chips (single-channel, silicon chip) can be used. The choice of the micro-array chip depends on the selected product and will be determined later.
Example 6: Preparation of Labeled cRNA and Hybridization to Microarrays.
Double-stranded cDNA will be synthesized out of the isolated patients RNA samples using a cDNA synthesis kit (Superscript; Life Technologies) employing oligo(dT) priming. The resulting cDNAwill be used for in vitro transcription (Ambion T7 Megascript system) in the presence of biotin-11 -CTP and biotin-16-UTP (Enzo Diagnostics). A total of 25-50 μg of the cRNA product in buffer [40 mM Tris acetate (pH 8.1)/100 mM potassium acetate/30 mM magnesium acetate] will be fragmented at 94°C for 35 min. It will then be used as a hybridization probe from each patient for hybridization as recommended (Affymetrix, Santa Clara, CA).
Aliquots of the hybridization patients cRNA mixtures (10 μg cRNA in 200 μl hybridization mix) will be hybridized to a Human Genome U133A Array. Each array will be washed and scanned (Hewlett Packard, GeneArray scanner G2500A) according to procedures developed by manufacturer (Affymetrix).
Example 7: Analysis of GENECHIP Data.
Scanned output files will be visually inspected for hybridization artifacts and then analyzed with GENECHIP 3.1 software (Affymetrix).
The expression analysis files created by GENECHIP 3.1 software will be transferred to a database (Microsoft Access) and linked to Internet genome databases (e.g., NHLBI, or Swiss Prot).

Claims

Patent Claims:
1. An ex-vivo method for identifying and determining tumor-specific biomarker genes and / or gene products in an individual suffering from a tumor, the method comprising (i) isolating and purifying DNA free circulating RNA proteolipid complex (cRNA-PLC) from a blood or serum sample of said individual,
(ii) separating mRNA from said proteolipid complex and transcribing the RNA to cDNA by standard techniques,
(iii) applying the cDNA after amplification to a microassay gene chip, (iv) determining by technical means and / or devices the cDNA and its intensity in comparison with RNA / DNA originating from tissue of a healthy individual, and
(v) identifying by technical means and / or devices from the measured differences the tumor type and strength in the diseased individual by the tumor-specific genes which are up- or down-regulated compared to the healthy individual.
2. A method of claim 1 , wherein said isolated cRNA-PLC is free of RNA present in the sample but originally not bound to protein or lipid complexes.
3. A method of claim 1 or 2, wherein said cRNA-PLC was obtained from the blood or serum sample by one-step filtration.
4. The method of claim 3, wherein said filtration step is carried out by a nitrocellulose filter in a pore size range of 0.1 - 0.4 μm.
5. A method of any of the claims 1 - 4, wherein the mRNA was separated from the proteolipid complex by extraction with RNA extraction / elution buffer.
6. An ex-vivo method of any of the claims 1 - 5, wherein the comparison DNA / RNA of the healthy individual and the cDNA of the diseased individual obtained by step (iv) are related to the same tissue type.
7. An ex-vivo method of any of the claims 1 - 6, comprising generating a tumor specific and individual RNA profile by applying the method to different individuals suffering from the same tumor.
8. An ex-vivo method of any of the claims 1 - 7, wherein the circulating RNA within the proteolipid complex is at least partially anti-sense RNA (siRNA).
9. An ex-vivo method of any of the claims 1 - 8, wherein the tumor to be determined with said biomarkers that are identified by said cRNA-PLC, is NSCLC.
10. The method of claim 9, wherein the biomarkers that are down-regulated in NSCLC are selected from the group consisting of: tumor suppressor protein 53 (p53) tumor promoter protein 21 (ras-p21) tumor suppressor protein 21(Waf1/Cιp1) (p2i<Waf1/C|P1>) tumor suppressor protein 27(Kιp1) (p217(Kιp1)) tumor suppressor protein 16 (p16) retinoblastoma tumor suppressor protein (Rb) fragile histidine triade (FHIT) ras-association domain family (RASSF1A) class 3 semaphorins (SEMA3B) phosphatase and tensin homologue (PTEN) base excision repair gene (hOGGD small phospholipid binding protein (BAP1)
11. The method of claim 9, wherein the biomarkers that are up-regulated in NSCLC are selected from the group consisting of: bone morphogenetic proetin (BMP) epithelial growth factor (EGF) fibroblast growth factor (FGF) nerve growth factor (NGF) platelet-derived growth factor (PDGF)-BB tumor growth factor (TGF) tumor necrosis factor (TNF) vascular endothelial growth factor (VEGF) vaso-intestinal peptide (VIP)
12.An ex-vivo method for determining tumor progression / regression, or the effect of an anti-tumor therapy in a tumor patient, the method comprising applying the steps (i) - (iv) of claim 1 and repeating these steps at different stages of tumor development and / or therapy schemes.
13. The method of claim 12, wherein the therapy scheme includes administration of an anti-tumor drug.
14. Use of cRNA-PLC obtained from blood or serum of a patient suffering from cancer for ex-vivo identification of biomarkers that are tumor specific.
15. Use of claim 14, wherein the cRNA-PLC is free of non-bound circulating RNA and was obtained from blood by one step filtration through nitrocellulose filter and release of RNA from the complex by washing with RNA extraction buffer.
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