DE10317955A1 - Predicting responsiveness of a subject with breast cell proliferative disorder, useful for treating or differentiating breast cell proliferative disorders comprises analyzing methylation pattern of a genomic DNA from the subject - Google Patents

Abstract

Predicting the responsiveness of a subject with a cell proliferative disorder of the breast tissues to a therapy comprising analyzing the methylation pattern of a target nucleic acid by contacting at least one of the target nucleic acids in a biological sample obtained from the subject prior to or during treatment, is new. Predicting the responsiveness of a subject with a cell proliferative disorder of the breast tissues to a therapy comprising analyzing the methylation pattern of a target nucleic acid by contacting at least one of the target nucleic acids in a biological sample obtained from the subject prior to or during treatment, is new. The therapy for the cell proliferative disorder of the breast tissues comprises one or more drugs, which target the estrogen receptor pathway or are involved in estrogen metabolism, production, or secretion. The target nucleic acid comprises one or a combination of genes selected from STMN1, SFN, S100A2, TGFBR2, TP53, PTGS2, FGFR1, SYK, PITX2, GRIN2D, PSA, CGA, CYP2D6, MSMB, COX7A2L, VTN, PRKCD, ONECUT2, WBP11, CYP2D6, DAG1, ERBB2, S100A2, TFF1, TP53, TMEFF2, ESR1, SYK, RASSF1, PITX2, PSAT1, CGA or PCAF and/or their regulatory regions. Independent claims are also included for the following: (1) a nucleic acid consisting essentially of a sequence at least 18 bases in length selected from 132, 28, 16, 40 or 64 sequences (all sequences not given in the specification) or its complementary sequences; (2) an oligomer, preferably an oligonucleotide or peptide nucleic acid (PNA)-oligomer consisting essentially of at least one base sequence having at least 10 nucleotides which hybridizes to or is identical to one of the nucleic acid sequences above; (3) a set of oligomers comprising at least two oligomers; (4) manufacturing an arrangement of different oligomers (array) fixed to a carrier material for predicting the responsiveness of a subject with a cell proliferative disorder of the breast tissues to a therapy; (5) an arrangement of different oligomers (array) obtained from the method above; (6) an array of different oligonucleotide- and/or PNA-oligomer sequences, where the oligonucleotides are arranged on a plane solid phase in the form of a rectangular or hexagonal lattice; (7) a DNA- and/or PNA-array for predicting breast cell proliferative disorders' response to therapy comprising one or more drugs which target the estrogen receptor pathway or are involved in estrogen metabolism, production, or secretion by analysis of the methylation state of any of the CpG dinucleotides selected from 23 sequences (not given in the specification) and comprising at least one nucleic acid given above; and (8) a kit comprising a bisulfite (=disulfite, hydrogen sulfite) reagent as well as oligonucleotides and/or PNA-oligomers. ACTIVITY : Cytostatic. No biological data given. MECHANISM OF ACTION : Gene Therapy.

Description

The Levels of observation caused by methodological developments that have been studied in molecular biology in recent years the genes themselves, the translation of these genes into RNA and the resulting ones Proteins. The question of which gene at what time in the course the development of an individual is turned on, and how that Activation and inhibition of specific genes in specific cells and tissues are controlled with scale and character the methylation of the gene or genome can be correlated. In this Respects can themselves pathogenic conditions in a changed Manifest methylation patterns from individual genes or genomes.

The For example, DNA methylation plays a role in regulation transcription, genetic imprinting and tumorigenesis. The identification of 5-methylcytosine as part of genetic information is therefore significant Interest. 5-methylcytosine positions can however, cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. Furthermore in the case of PCR amplification, that carried by 5-methylcytosine complete epigenetic information lost.

A relatively new and the most commonly used method for examining DNA for 5-methylcytosine is based on the specific Reaction of bisulfite with cytosine, which after subsequent alkaline Hydrolysis is converted into uracil, which in its base pairing behavior corresponds to the thymidine. 5-methylcytosine, on the other hand, is among these Conditions not modified. This will make the original DNA converted so that methyl cytosine, which originally not distinguished from cytosine by its hybridization behavior could be, now through "normal" molecular biological Techniques as the only remaining cytosine, for example by Amplification and hybridization or sequencing can be. All of these techniques are based on base pairing, which can now be fully exploited. The state of the art what the Sensitivity is defined by a method which thereby enclosing the DNA to be examined in an agarose matrix the diffusion and renaturation of the DNA (bisulfite only reacts with single DNA) prevented and all precipitation and cleaning steps replaced by rapid dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec 15; 24 (24): 5064-6). With this method you can individual cells are examined, what is the potential of the method illustrated. However, currently only individual regions up to about 3000 base pairs in length examined, a global study of cells for thousands of potential Methylation events are not possible. However, it can also this method does not produce very small fragments from small sample amounts reliable analyze. Despite diffusion protection, these pass through the matrix lost.

An overview of the other known methods for the detection of 5-methylcytosine can the following review article are taken from: Rein, T., DePamphilis, M.L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.

The Bisulfite technology has so far been used with a few exceptions (e.g. Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 Mar-Apr; 5 (2): 94-8) only used in research. But always short, specific pieces of a known gene after a bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. 1997 No. v; 17 (3): 275-6) or individual cytosine positions by a "primer extension reaction" (Gonzalgo ML, Jones PA. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE) Nucleic Acids Res. 1997 Jun 15; 25 (12): 2529-31, WO 95/00669) or by enzymatic digestion (Xiong Z, Laird PW.COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun 15; 25 (12): 2532-4) demonstrated. Detection by hybridization is also described (Olek et al., WO 99/28498).

Other publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioassays. 1994 Jun; 16 (6): 431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi / Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 Mar; 6 (3): 387-95; Feil R, Charlton J, Bird AP, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb 25; 22 (4): 695-6; Martin V, Ribieras S, Song-Wang X, Rio MC, Dante R. Genomic sequencing indicates a correlation between DNA hypo methylation in the 5 'region of the pS2 gene and its expression in human breast cancer cell lines. Genes. 1995 May 19; 157 (1-2): 261-4; WO 97/46705, WO 95/15373, and WO 97/45560.

An overview of the State of the art in oligomer array production can be from a special edition of Nature Genetics published in January 1999 (Nature Genetics Supplement, Volume 21, January 1999) and the one there Take cited literature.

For the scan immobilized DNA assays are often fluorescence-labeled probes used. Particularly suitable for Fluorescence labeling is the simple application of Cy3 and Cy5 Dyes at 5'-OH the respective probe. Detection of the fluorescence of the hybridized For example, probes can a confocal microscope. The dyes Cy3 and Cy5 are among many others, commercially available.

Matrix-assisted lasers Desorption / ionization mass spectrometry (MALDI-TOF) is one very powerful Development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15; 60 (20): 2299-301). An analyte is embedded in a light-absorbing matrix. By A short laser pulse evaporates the matrix and the analyte molecule is thus unfragmented transported into the gas phase. By collisions with matrix molecules ionization of the analyte is achieved. An applied voltage accelerates the ions into a field-free flight tube. Because of their different Masses, the ions are accelerated to different extents. smaller Ions reach the detector earlier than bigger ones.

MALDI-TOF Spectrometry is ideal for the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass spectrometry. Current innovations and future trends. 1995 1; 147-57). For nucleic acids sensitivity about 100 times worse than for peptides and decreases disproportionately with increasing fragment size. For nucleic acids that a often negatively charged backbone have, the ionization process is through the matrix much more inefficient. In MALDI-TOF spectrometry the choice of matrix plays an eminently important role. For desorption some very powerful matrices have been found from peptides, which result in a very fine crystallization. Now there is some attractive matrices for DNA, however, the difference in sensitivity was not reduced. The Difference in sensitivity can be reduced by the DNA is chemically modified to be more similar to a peptide becomes. Phosphorothioate nucleic acids, where the ordinary Backbone phosphates are substituted by thiophosphates, can be replaced by simple Converting alkylation chemistry into a charge-neutral DNA (Gut IG, Beck S. A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 1995 Apr 25; 23 (8): 1367-73). The Coupling a “batch tags "to this modified DNA results in an increase in sensitivity to the same thing Level as it is for Peptides is found. Another advantage of "charge tagging" is the increased stability of the analysis against contamination, the detection of unmodified substrates greatly complicate.

genomic DNA is made from the DNA of cellular, Tissue or other test samples using standard procedures receive. This standard methodology can be found in literature, such as Fritsch and Maniatis eds .; Molecular Clonin: A Laboratory Manual, 1989.

breast cancer is currently the second most common Type of cancer in women. In 2001, over 190,000 new cases of invasive breast cancer and over 47,000 more cases diagnosed by in situ breast cancer. The occurrence and death rate increases with age for the period 1994-1998 was the incidence of breast cancer among women between the ages 20 and 24 only 1.5 per 100,000 inhabitants. However, the risk increases up to 498.7 in the 75-79 age group. The mortality rates took at around 5% over the past decade and factors that affect 5-year survival influence, close age, Phase of cancer, socio-economic Factors and race.

breast cancer is defined as the uncontrolled cell division of cells within the breast tissue. The breasts consist of 15 to 20 lobes, which are connected by channels are. Cancer is the most common in a canal, but is also found in the lobes, the rarest type of cancer inflammatory Is called breast cancer.

It will be apparent to those skilled in the art that there is a constant need to improve the early detection, classification and treatment methods for breast cancer. In contrast to the detection of some other common cancers, such as cervical and skin cancer, there are inherent Difficulty in classifying and detecting breast cancer.

The The first step in any treatment is to determine the condition of the patient Patients compared to defined classifications of the disease. The value of such a system depends however inherent on the quality the classification. Breast cancer is classified according to its size, location, and classified as the occurrence of metastases. Treatment procedure conclude the use of surgery, radiation therapy and hormone therapy one, also called follow-up therapies applied to surgery.

endocrine Therapies have been developed to study the effects of estrogen on cancer cells block or decrease serum estrogen levels. tamoxifen (TAM) is the most widely used anti-estrogen ingredient for breast cancer patients. It works by blocking estrogen stimulation of breast cancer cells, whereby both the translocation and the nuclear binding of the Estrogen receptor is inhibited. TAM is used in follow-up therapy approaches primary Breast cancer patients and in the treatment of metastatic Disease used, and its effectiveness has been various clinical Studies confirmed. Treatment over five years away reduces the annual Disease recurrence by 47% and annual deaths by 26%, and this reduction is similar in different age groups. It also reduces the incidence the development of contralateral breast tumors. TAM is located at the least toxic antineoplastic agent since it has estrogenic properties in some tissues, it increases that 3-fold risk of endometrial cancer. Other endocrine therapies conclude Aromatase inhibitors include an aromatase enzyme in adipose tissue (the main one Block and increase source of estrogen in postmenopausal women) a reduced or no production of estrogens in obesity Lead tissue. New agents have also been developed that block the estrogen receptor or modulate, commonly referred to as SERMs = Selective Estrogen Receptor Modulators summarized were.

Yet another way to measure the amount of estrogen available for the tumor to reduce it is to administer funds that provide the feedback Loop in sex hormone regulation (LH-RH analogues) in one higher Interrupt level.

in the general side effects of endocrine treatment are Harmless compared to chemotherapy. However, since the endocrine treatments Remove or reduce estrogen as a growth factor for cancer, they only work for Tumors that depend on an estrogen as a growth factor. The Growth of cancers that do not have a functional ER signaling pathway, cannot be modulated by endocrine therapies.

The current way to determine whether a tumor is on endocrine Therapies will address is the analysis of the expression of hormone receptors. The hormone receptor status should be tested before starting TAM treatment as only one Group of patients will benefit from this therapy. To this Purpose are the ER (estrogen receptor) and PR (progesterone receptor) status routinely in all patients tested and as predictive Viewed a response marker (a predictive marker or factor is any measurement with an answer or lack of an answer is associated with a particular therapy). The ER status is for the response in connection endocrine Therapy approach even with progressive metastatic disease predictive.

in the Right now ER positive and / or PR positive patients are received TAM and the remaining 10% of patients receive chemotherapy. The problem is that the Treatment only for a subset of hormone receptor positive Is effective and that a small subset of ER negative patients initially seem to respond to TAM. Then this would not be appealing Subgroup not only did not benefit from the TAM treatment, but this would produce opposite effects and this would not have the opportunity to get chemotherapy instead. On the other hand, it would ER negative subset that benefits from tamoxifen treatment could have, get chemotherapy instead. Several different tests are available to measure PR and ER, including biochemical and IHC (Immunohistochemical) analysis, but no standardization of staining methods, and there is inter-laboratory variability (Clin Cancer Res 2000, 6: 616-621).

Various predictive markers are currently under investigation. Because most patients have received tamoxifen as an endocrine treatment, most markers have been shown to be associated with the response or resistance to tamoxifen. However, it is generally believed that there is a large overlap between responders between one and the other therapy. Indeed, ER and PR expression are used to train patients for any endocrine treatment choose. Among the markers associated with TAM Response is bcl-2. High bcl-2 levels show a promising correlation with the TAM therapy response in patients with metastatic disease and prolonged survival and add valuable information to the ER-negative patient subgroup (J Clin Oncology 1997, 15 5: 1916-1922; Endocrine , 2000, 13 (1): 1–10 There are conflicting indications regarding the independent predictive value of c-erbB2 (Her2 / new) for expression in patients with advanced breast cancer who require further evaluation and verification (British J of Cancer 1999, 79 (7/8): 1220-1226; J Natl Cancer Inst, 1998, 90 (21): 1601-1608).

Other predictive Close marker SRC-1 (steroid receptor coactivator-1), CGA gene overexpression, cell kinetics and S-phase fraction tests (Breast Cancer Res and Treat, 1998, 48: 87-92; Oncogene 2001, 20: 6955-6959) on. Recently uPA (urokinase type Plasminogen activator) and PAI-1 (plasminogen activator inhibitor Type 1) together as useful to a subset of patients to define that have a poorer forecast and that of a connection systemic Therapy would benefit (J Clinical Oncology, 2002, 20 n ° 4). All of these markers require further investigation to be performed Studies since none of these have been a validated response marker so far is.

Of A number of genes associated with cancer have been shown to be: inactivated by hypermethylation of CpG islets during breast tumorigenesis become. Decreased expression of the calcium binding protein S100A2 (Accession number NM_005978) has been involved in the development of breast cancers connected. Hypermethylation of the promoter region this gene was observed in neoplastic cells, which is why proves that the S100A2 Repression in tumor cells by site-specific methylation is conveyed.

The SYK genes (accession number NM_003177) codes for a protein tyrosine kinase, Syk (spleen tyrosine kinase), which is strong in hematopoietic cells is expressed. Syk is found in normal breast ductal epithelial cells expressed, but not in a subset of invasive breast cancer. Also, the loss of syk expression seems to be associated with malignancy phenotypes such as increased To be associated with mobility and invasion. The loss of expression occurs at the level of transcription as a result of DNA hypermethylation on how by Yuan Y, Mendez R, Sahin A and Dai JL (Hypermethylation leads to silencing of the SYK gene in human breast cancer. Cancer Res. 2001; 61 (14): 5558-61.) shown.

The TGF-β type 2 receptor (encoded by the TGFBR2 gene, NM_003242) plays one Role in the transmembrane signal pathways, via a complex of serine / threonine kinases. Mutations in the gene have been found in some primary tumors and in different ones Types of tumor-derived cells, including breast (Zucke CD, Philpott A, Metcalfe JC, Thompson AM, Hughes-Davies L, Kemp PR, Hesketh R. Inhibiting mutations in the transforming growth factor beta type 2 receptor in recurrent human breast cancer. Cancer Res. 2001 Jan 15; 61 (2): 482-5) discovered.

The COX7A2L and GRIN2D genes were both developed by Watanabe et. al. (Isolation of estrogen-responsive genes with a CpG island library. Molec. Cell. Biol. 18: 442-449, 1998.) using the CpG-GBS (genomic binding site) method identified as new estrogen-responsive elements. The COX7A2L gene (Accession number NM_004718) encodes a polypeptide 2-like Cytochrome C oxidase subunit VIIA. Northern blot analysis indicated an upregulation of COX7A2L after estrogen treatment of a breast cancer Cell line after.

The The GRIN2D gene (accession number NM_000836) codes for the N-methyl-D-aspartate ionotrophic Subunit 2D glutamate receptor, a subunit of the NMDA receptor channel, which is associated with neuronal signal, expression continued the cDNA was observed in an osteosarcoma cell line.

The Gene VTN (also known as vitronectin, accession number NM_000638) encodes for a 75 kD glycoprotein (also serum distribution factor or complement S protein called), which is the attachment and distribution of animal Supports cells in vitro, inhibits cytolysis by the complement C5b-9 complex, and the Antithrombin III thrombin effect modulated during blood coagulation. Furthermore, the expression of this gene with the one with the progression and invasiveness of cancer cells associated.

The SFN gene (also known as stratifin) encodes a 14-3-3 family polypeptide, 14-3-3 Sigma. The 14-3-3 family of proteins mediate signal transduction by binding to phosphoserine-containing proteins. The expression of the SFN gene is lost in breast carcinomas, which may be due to Hy permethylation during the early phases of neoplastic transformation (see Umbricht CB, Evron E, Gabrielson E, Ferguson A, Marks J, Sukumar S. Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer. Oncogene. 2001 Jun 7; 20 (26): 3348-53).

The Gen PSA (accession number NM 021154), also known as PSA-T, should not with the gene also commonly referred to as PSA (accession number NM 001648) are confused with the prostate-specific antigen encoded, and whose technically correct name is Kal-likrein 3. The PSA-7 gene encodes for the Protein phosphoserine aminotransferase, which is the second step catalyzing enzyme in the serine biosynthetic pathway. changes in gene expression levels were determined by mRNA expression analysis tracked and upregulation of the gene has been found in colon cancer a study with 6 samples identified (Ojala P, Sundstrom J, Gronroos JM, Virtanen E, Talvinen K, Nevalainen TJ. mRNA differential display of gene expression in colonic carcinoma. Electrophoresis. 2002; 23 (11): 1667-76).

The Gene stathimin (NM_005563) encodes an oncoprotein 18, too known as stathimin, a preserved cytosolic phosphoprotein, that regulates microtubule dynamics. The protein is in a Number of human malignancies highly expressed. In Stahimingen was considered to be in one human breast cancer Subset of tumors shown highly regulated.

The PRKCD gene encodes for a member of the family of protein kinase c enzymes and is on the B-cell signal transmission and the regulation of growth, apoptosis and differentiation of a Variety of cell types involved.

Some of these molecules interact in a cascade-like manner. Who opposed STMN1 directing PRKCD activity is modulated by SFN binding and SYK phosphorylation. Together affected this is the tubulin polymerization required for cell division is.

description

The The present invention provides methods and nucleic acids Predicting the response of a patient with a cell proliferative Breast tissue disease available on endocrine treatment. Under the use of the methods and nucleic acids described here can be statistical significant models of patient response to the treatment regimen be developed and used to help patients and treating physicians assist in determining treatment options. The described Procedure is intended to be used to determine the suitability of endocrine Treatment a therapy for Patients suffering from cell proliferative disease of the breast tissue suffering to determine. Therefore, the present invention is referred to as the Reduced problems viewed with the current treatment response predictive procedures are associated.

Under the use of the methods and nucleic acids as described here patient sensitivity before or during endocrine treatment a cell proliferative disease of the breast tissue is evaluated, important information for the patient and the treating doctor in terms of the risks, disadvantages and benefits that come with the associated with endocrine treatment. It will therefore it can be seen that the The methods and nucleic acids shown here as examples can serve the quality of life to improve a patient and inconsistencies in treatment success by giving both the patient and the attending physician a more accurate Identification of the patient's treatment options are made possible.

The The aim of the invention is by means of the analysis of the methylation pattern one or a combination of the genes selected from the group PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2, COX7A2L (see table 1) and / or their regulatory regions resolved. The invention is thereby characterized that the nucleic acid one or a combination of the genes selected from the group PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L with one Reagent or a set of reagents is contacted who are able to switch between methylated and unmethylated CpG dinucleotides within the genomic sequence of interest to distinguish.

The present invention provides a method for determining genetic and / or epigenetic parameters of genomic DNA. The method is used to improve the treatment and monitoring of breast cell proliferative diseases by enabling accurate prediction of a patient's response to endocrine treatment. In a particularly preferred embodiment, the method of the invention enables differentiation between patients on endocrine therapy respond and those who don't.

The Method according to the invention can for the analysis of a large one Variety of breast proliferative diseases used become, including, but not limited to, ductal carcinomas in situ, lobular carcinomas, colloidal carcinoma, tubular Carcinomas, medullary Carcinomas, metaplastic carcinomas, intraductal carcinomas in situ, lobular Carcinomas in situ and papillary Carcinomas in situ.

The method according to the invention is particularly suitable for predicting the response to endocrine treatment in two treatment approaches. In one embodiment, the method is applied to patients who receive endocrine treatment as a secondary treatment to a primary treatment, such as surgery (hereinafter referred to as the connecting approach), as in 1 clarified. Such treatment is often prescribed to patients suffering from phase 1 to 3 breast cancer. In this embodiment, responders are defined as those who do not have a detectable relapse of breast cancer within a specific period of time, not responders are those who relapse within the period.

In a further embodiment the procedure is used in patients who have a relapse of Breast cancer following Treatment by a primary Medium (preferably surgery) followed by disease-free Period, suffering, and being the endocrine treatment in response to a relapse the carcinoma has been prescribed. Such treatment is often Prescribed to patients suffering from later stage carcinomas, in particular where metastases have occurred. Therefore, this clinical approach is intended here hereinafter also referred to as the 'metastatic Approach ' become. In this embodiment Responder those who are in partial or full remission occur, i.e. Subjects whose cancer is on undetectable levels decreases, unlike those whose diseases continue metastasize or remain above detectable levels. This Methodology provides further improvements over the state of the art to disposal, that this Procedure can be applied to any subject, regardless of Estrogen and / or progesterone receptor status. Therefore it is in one preferred embodiment not required by the subject that it is on the estrogen or Progesterone receptor status tested has been.

Farther allows the method the analysis of cytosine methylations and single Nucleotide polymorphisms within genes.

The The object of the invention is achieved by analyzing the methylation pattern of the genes PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L and / or their regulatory regions solved in one particularly preferred embodiment the sequences comprising SEQ ID NOs: 27, 90, 41, 40, 121, 78, 115, 86, 43 and 105 and sequences complementary thereto. In a preferred one embodiment the method is accomplished by contacting the nucleic acid sequences in a biological sample obtained from a subject dissolved with at least one reagent or a series of reagents, wherein the reagent or series of reagents between methylated and unmethylated CpG dinucleotides within each Target nucleic acid (s) different.

In a preferred embodiment, the method comprises the following steps:
In the first step of the method, the genomic DNA sample must be isolated from sources such as cells or cellular components containing DNA, where sources of DNA include, for example, cell lines, histological sections, biopsies, paraffin-embedded tissue, breast tissue , Blood, plasma, lymphatic fluid, lymphoid tissue, duct cells, ductal lava fluid, nipple aspiration fluid, bone marrow and combinations thereof. The extraction can be done by means that are standard for those of ordinary skill in the art, including the use of detergent lysates, sonication and vortexing with glass beads. Once the nucleic acids have been extracted, the double stranded genomic DNA is used in the analysis.

In a preferred embodiment can the DNA before the next Step of the process can be split, this can be done by any means happen, which is standard in the art, in particular, however not limited to, with restriction endonucleases.

in the The second step of the procedure is the genomic DNA sample treats such a way that the cytosine bases that are not methylated at the 5 'position are converted to uracil, thymine or another base, which differ in terms of hybridization behavior from Is cytosine. This is understood here as "pretreatment".

The Treatment of the genomic DNA described above is preferred with bisulfite (sulfite, disulfite) and subsequent alkaline hydrolysis carried out, leading to a conversion of unmethylated cytosine nucleobases leads to Uracil or another base that is related to distinguishes the base pairing behavior of cytosine. If one bisulfite for the Reaction is used, is added to the unmethylated Cytosine bases instead. About that out must denaturing agent or a solvent and a radical scavenger present his. A subsequent one alkaline hydrolysis then enables the conversion of unmethylated cytosine nucleobases to uracil. The converted DNA is then used for used the detection of methylated cytosines.

fragments of the pretreated DNA are made using sets of Primer oligonucleotides and a preferably heat-resistant polymerase is amplified. Statistical and practical considerations are preferred more than ten different fragments that are 100 - 2000 base pairs in length have amplified. The amplification of different DNA segments can be carried out simultaneously in one and the same reaction vessel. usually, the amplification is carried out by means of a polymerase chain reaction (PCR) carried out.

The The structure of such primers can be seen by the person skilled in the art. These should contain at least two oligonucleotides, their sequences each reversely complementary or identical to a segment that is at least 18 base pairs long of the base sequences given in the appendix: SEQ ID NO 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587. The primer oligonucleotides are preferably thereby indicates that they contain no CpG dinucleotides. In a particularly preferred embodiment the sequence of the primer oligonucleotides is constructed in such a way that to selectively target only the breast cell-specific DNA anneal and amplify them, thereby amplifying Background or irrelevant DNA is minimized. In context background DNA of the present invention is intended to mean genomic DNA, that has no relevant tissue-specific methylation pattern, in which case the relevant tissue is breast tissue.

According to the present Invention, it is preferred that during the Amplification of at least one primer oligonucleotide to a fixed one Phase is bound. The different oligonucleotide and / or PNA oligomer sequences can on a solid phase in the form of a rectangular or hexagonal Grid can be arranged, with the solid phase surface preferably made of silicon, glass, polystyrene, aluminum, steel, iron, copper, Nickel, silver or gold is composed, with others Materials such as nitrocellulose or plastic are used as possible can be.

The Fragments obtained by amplification can be a direct or indirect wear detectable markers. Markers in the form of fluorescent markers are preferred, Radionuclides or removable Molecular fragments, which have a typical mass in a mass spectrometer can be demonstrated it is preferred that the Fragments that are produced are a single positive or negative Have a net charge for better detection in the mass spectrometer. The proof can be carried out are visualized using matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (IT I).

in the next Step will be the nucleic acid amplificates analyzed to determine the methylation congestion of the genomic DNA before To determine treatment.

The post-treatment analysis of nucleic acids can be made using alternative procedures performed become. Different procedures for the methylation status specific Analysis of the treated nucleic acids are described below, other alternative methods are considered Average specialist can be seen.

Using various methods known in the art, the analysis can be performed during the amplification step of the method. In such an embodiment, the methylation status of selected CpG positions within the genes PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2, COX7A2L PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L and / or their regulatory regions can be detected by using methylation-specific primer oligonucleotides. This technique ("MSP") was described in US Patent 6,265,171 to Herman. The use of methylation status specific primers for the amplification of bisulfite treated DNA enables the differentiation between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer who is interested in a bi sulfite treated CpG dinucleotide hybridized. Therefore the sequence of the primer comprises at least one CG, TG or CA dinucleotide. MSP primers specific for non-methylated DNA contain a "T" at the 3 'position of the C position in the CpG. According to the present invention, it is therefore preferred that the base sequence of the primers necessarily comprise a sequence that is long of at least 9 nucleotides attached to a pretreated nucleic acid sequence according to SEQ ID NOs .: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618 , 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 and sequences complementary thereto, wherein the base sequence of the oligomers hybridizes at least one CG, TG or CA dinucleotide.

In one embodiment The method can determine the methylation status of the CpG positions by means of hybridization analysis be determined. In this embodiment of the method are the amplificates obtained in the second step to an arrangement or a set of oligonucleotides and / or PNA probes hybridized. In this context, the hybridization to the as described below instead. The set of probes used during the Hybridization is used, preferably consists of at least 4 oligonucleotides or PNA oligomers together. Serve in the process the amplificates as probes that hybridize to oligonucleotides, that were previously bound to a solid phase. The non-hybridized Fragments are then away. The oligonucleotides contain at least one base sequence, the one length of 10 nucleotides that are reverse complementary or identical is to a segment of the base sequences specified in the Appendix are, the segment at least one CpG or a TpG dinucleotide contains. In a further preferred embodiment is the cytosine of the CpG dinucleotide, or in the case of TpG the thymidine, the 5th to 9th nucleotide from the 5 'end of the 10s. An oligonucleotide exists for each CpG or TpG dinucleotide.

The non-hybridized amplificates are then removed. In the last The hybridized amplificates are detected in step of the method. In this context it is preferred that attached to the amplificates Markers can be identified at any position of the solid phase, where an oligonucleotide sequence is located.

In a further embodiment the methylation status of the CpG positions Oligonucleotide probes are identified that are attached to the treated DNA are hybridized together with the PCR amplification primers (where the primers are either methylation specific or standard can).

A particularly preferred embodiment of this method is the use of fluorescence-based real-time quantitative PCR (Heid et al., Genome Res. 6: 986-994, 1996), which uses a dual-labeled fluorescent oligonucleotide probe (TaqMan ^™ PCR, below using an ABI Prism 7700 sequence detection system, Perkin Elmer Applied Biosystems, Foster City, California). The TaqMan ^™ PCR reaction uses the use of a non-extendable assay oligonucleotide called the TaqMan ^™ probe that is designed to hybridize to a CpG-rich sequence that locates between the forward and reverse amplification primers is. The TaqMan ^™ sample further includes a fluorescent "reporter group" and a "quencher group" which are covalently linked to linker groups (e.g. phosphoramidites) attached to the nucleotides of the TaqMan ^™ oligonucleotide. To analyze methylation within nucleic acids following bisulfite treatment, it is necessary that the probe be methylation specific, as described in US 6,331,393 (incorporated herein by reference), which is also known as the "MethylLight Test". Variations on TaqMan ^™ Detection methodology also useful for use with the described invention includes the use of dual probe technology (Lightcycler ^™ ) or fluorescent amplification primers (Sunrise ^™ technology), both of which can be suitably adapted for use with bisulfite-treated DNA and also for methylation analysis within CpG dinucleotides.

Another suitable method for using oligonucleotide probes for the determination of methylation by analysis of bisulfite-treated nucleic acids is the use of blocking oligonucleotides. The use of such oligonucleotides has been described in BioTechniques 23 (4), 1997, 714-720 D. Yu, M. Mukai, Q. Liu, C. Steinman. Blocking oligonucleotide probes are hybridized to the bisulfite-treated nucleic acid together with the PCR primers. PCR amplification of the nucleic acid stops at the 5 'position of the blocking probes, thereby suppressing amplification of a nucleic acid when the complementary sequence to the blocking probe is present. The probes can be designed to hybridize to the bisulfite treated probe in a methylation status specific manner. For example, for the detection of methylated nucleic acids within a population of unmethylated nucleic acids, a suppression of the amplification of nucleic acids that are on the position in question is not methylated, using blocking probes comprising a "CG" at the position in question, as opposed to a "CA".

In a further preferred embodiment The method will demonstrate the methylation status of the CpG positions through the use of template-directed oligonucleotide extensions carried out, such as MS-SNuPE as described by Gonzalgo and Jones (Nucleic Acids Res. 25: 2529-2531) described.

In a further embodiment The method will determine the methylation status of the CpG positions by sequencing and subsequent sequence analysis of the im second step of the method enables generated amplificate (sucker F., et al., 1977 PNAS USA 74: 5463-5467).

A further embodiment The invention is a method for analyzing the methylation status of genomic DNA without the need for pretreatment. In the The first and second steps of the procedure must be the genomic DNA sample obtained and isolated from tissue or cellular sources. Such sources can Cell lines, histological sections, body fluid or tissue embedded in paraffin lock in. The extraction can be done by means that are standard for the skilled person are close this the use of detergent lysates, sonication and vortexing with Glass beads. Once the nucleic acids are extracted, the genomic double-stranded DNA used in the analysis.

In a preferred embodiment the DNA can be cleaved prior to treatment any means that are standard in the art, in particular with restriction endonucleases. In the third step, the DNA then with one or more methylation sensitive restriction enzymes digested. The digestion is carried out so that the hydrolysis of the DNA begins the restriction site for the methylation state of a specific CpG dinucleotide is informative is.

In a preferred embodiment the restriction fragments are amplified. In another preferred embodiment this is done using the polymerase chain reaction.

in the in the last step, the amplificates are detected. The proof can by any means that is standard in the art, done, for example, but not limited to, gel electrophoresis analysis, Hybridization analysis, incorporation of detectable markers within the PCR product, DNA array analysis, MALDI or ESI analysis.

The mentioned above The method is preferably used to determine genetic and / or epigenetic parameters of genomic DNA applied.

Around to further enable this process the invention further provides the modified DNA of one or more Combination of genes from the group of PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2, COX7A2L PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L are available as well Oligonucleotides and / or PNA oligomers for the detection of cytosine methylations within these genes. The present invention is based on the Discovery that genetic and epigenetic parameters and especially the cytosine methylation pattern the genomic DNAs especially for the improved treatment and surveillance of proliferative diseases of breast cells are suitable.

The nucleic acids according to the present Invention can for the analysis of genetic and / or epigenetic parameters of genomic DNA can be used.

This Task according to the present Invention is achieved through the use of a nucleic acid which is a Sequence with a length of at least 18 bases of the pretreated genomic DNA according to one of the SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586, 587, 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 and sequences complementary thereto contains solved.

The modified nucleic acids have so far not been able to identify genetic diseases relevant to disease and epigenetic parameters.

The object of the present invention is further achieved by an oligonucleotide or an oligomer Analysis of pretreated DNA to detect the genomic cytosine methylation state solved, wherein the oligonucleotide contains at least one base sequence that is at least 10 nucleotides in length, which is linked to a pretreated genomic DNA according to SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 hybridized. The oligomer probes according to the present invention are important and effective tools that allow, for the first time, specific genetic and epigenetic parameters during the analysis of biological samples for features related to a patient's response to endocrine treatment. The oligonucleotides enable the improved diagnosis, treatment and monitoring of proliferative diseases of breast cells. The base sequence of the oligomers preferably contains at least one CpG or TpG dinucleotide. The probes can also exist in the form of a PNA (peptide nucleic acid) which has particularly preferred pairing properties. Particularly preferred are oligonucleotides according to the present invention, in which the cytosine of the CpG dinucleotide lies within the middle third of the oligonucleotide, e.g. B. the 5th – 9th Nucleotide from the 5 'end of a 13-mer oligonucleotide; or in the case of a PNA oligomer, it is preferred that the cytosine of the CpG dinucleotide be 4-6. Nucleotide from the 5 'end of the 9-mer.

The Oligomers according to the present Invention are normally used in so-called "sets" containing at least one oligomer for each the CpG dinucleotides within SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 included.

In a particularly preferred embodiment of the method, the oligomers according to SEQ ID NOs: 1816-1833, 1114-1129, 1808-1815, 1762-1769, 1552-1559, 1490-1499, 1138-1147 and 1686-1693 for the prediction a subject's response to endocrine treatment in a metastatic Approach used.

in the Case of sets of oligonucleotides according to the present invention it is preferred that at least an oligonucleotide is bound to a solid phase. It is further preferred that all Oligonucleotides of a set are bound to a solid phase.

The The present invention further relates to a set of at least 10 n (oligonucleotides and / or PNA oligomers), which are used to detect the Cytosine methylation state of genomic DNA can be used by analyzing the sequence or treated versions of the sequences (of the genes PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L as indicated in the sequence listing and in Table 1) and more complementary Sequences). Allow these probes improved treatment and monitoring of proliferative diseases of breast cells.

The Set of oligomers can also be used to single nucleotides Polymorphisms (SNPs) by analyzing the sequence or treated Versions of the sequence (PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L as in the sequence listing and shown in Table 1).

It it will be readily apparent to those skilled in the art that the method according to the invention by including markers and clinical indicators in the State of the art are known and currently considered for the exit of endocrine treatment as predictive can be viewed, improved and supplemented. More preferred conclude these markers the node status, age, menopausal status, degree, Estrogen and progesterone receptors.

The Genes that form the basis of the present invention are preferred used to form a "gene panel", i.e. a collection containing the said genetic sequences of the invention and / or their respective informative methylation sites include. The Formation of such gene panels allows quick and specific Analysis of specific aspects of breast cancer. The one like this Gen panels described and used according to the invention can be surprising high efficiency for used the therapy of proliferative diseases of breast cells by predicting the outcome of a treatment that comprises one or more active substances acting on the estrogen receptor Target signaling pathway or estrogen metabolism, production or the secretion are involved.

The efficiency of the method according to the invention is improved when applied to patients who have not been treated with chemotherapy. Accordingly, it is a particularly preferred embodiment of the invention in which the method is used to identify subjects who do not have chemo have undergone therapy.

In addition, the Use of multiple CpG positions from a diverse arrangement of Genes to a relatively high degree sensitivity and specificity compared to single-gene prognostic tools.

According to the present Invention, it is preferred that a Arrangement of different oligonucleotides and / or PNA oligomers (a so-called "array") by the present Invention available is made, and is present in such a way that it is also a solid phase is bound. This arrangement of different Oligonucleotide and / or PNA oligomer sequences can be characterized by: the solid phase in the form of a rectangular or hexagonal grid is arranged. The solid phase surface is preferably made of silicon, glass, polystyrene, aluminum, steel, Composed of iron, copper, nickel, silver or gold. however can Nitrocellulose, as well as plastic materials such as nylon, which can be in the form of pellets or as resin matrices Be alternatives.

Therefore Another object of the present invention is a method for producing an array attached to a carrier material for the improved diagnosis, treatment and monitoring of proliferative Breast cell diseases, differentiation of different Subclasses of breast cancer and / or evidence of predisposition across from proliferative diseases of breast cells. In this process becomes at least one oligomer according to the present Invention coupled to a fixed phase. Manufacturing process such arrays are known, for example from U.S. Patent 5,744,305 using solid-phase chemistry and photolabile protecting groups.

On the present invention further relates to a DNA chip for the improved treatment and monitoring of proliferative diseases of breast cells. The DNA chip contains at least a nucleic acid according to the present Invention. DNA chips are known, for example, from U.S. Patent 5,837,832.

Furthermore One object of the present invention is a kit, for example from a bisulfite-containing reagent, a set of primer oligonucleotides, the at least two oligonucleotides, the sequences of which in each case an 18 base segment of the base sequences described in SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 are indicated, correspond to or are complementary to and / or PNA oligomers and instructions for performing and Evaluation of the described method is composed.

In a further preferred embodiment the kit can continue to use standard reagents to perform a CpG position-specific methylation analysis include, where the analysis comprises one or more of the present techniques: MS-SNuPE, MSP, methyl light, heavy methyl and nucleic acid sequencing. However, can a kit according to the present Invention also contain only a part of the above components.

The Oligomers according to the present Invention or arrays thereof and a kit according to the present invention are for use in improved treatment and monitoring proliferative diseases of breast cells thought. According to the present Invention, the method is preferably used for the analysis of important genetic and / or epigenetic parameters within genomic DNA used, especially for use in the improved treatment and surveillance of proliferative diseases of breast cells.

The Process according to the present Invention are for example for the improved treatment and monitoring of enabling proliferative diseases of breast cells used by more informed treatment regimens.

The present invention also relates to the diagnosis and / or prognosis of events which are disadvantageous or relevant to the patient or individual in which important genetic and / or epigenetic parameters within genomic DNA, these parameters having been obtained by means of the present invention can be compared to another set of genetic or epigenetic parameters, the differences serving as a basis for the diagnosis and / or prognosis of events that are disadvantageous or relevant for patients or individuals. Close examples of such parameters a, but are not limited to the detection of estrogen and progesterone markers.

Under the term "hybridization" in the sense of the present The invention is intended to bind an oligonucleotide with formation a duplex structure to a completely complementary sequence understood in the sense of the Watson-Crick base pairings in the sample DNA become.

"Genetic parameters" in the sense of the present Invention are mutations and polymorphisms of genomic DNA and sequences necessary for their regulation. In particular Insertions, deletions, point mutations, Inversions and polymorphisms and particularly preferably SNPs (single Nucleotide Polymorphisms).

in the In the context of the present invention, the term "methylation status" is used as the extent of in a nucleic acid considering methylation of interest, where these are expressed in absolute or relative terms can, i.e. as a percentage or other numerical value or by comparison with another tissue that is hypermethylated in it, hypomethylated or as a significantly similar or identical methylation status having been described.

in the The term “regulatory Region "of a gene as significantly understood the nucleotide sequence that the expression of a Gens affected. These regulatory regions can located within, proximal or distal of the gene. The regulatory Close regions one, but are not limited on, constitutive promoters, tissue-specific promoters, development-specific Promoters, inducible promoters and the like. Promoter regulatory elements can also include certain enhancer sequence elements that control the transcriptional or translational efficiency of the gene.

in the Context of the present invention, the term "chemotherapy" is used of active substances of chemical substances for the treatment of cancer understood significantly. This definition excludes radiation treatment (Treatment with radiation or high energy particles), hormone therapy (Treatment with hormones or hormone analogues (synthetic substitutes) and surgical treatment.

in the The context of the present invention relates to "epigenetic parameters", in particular cytosine methylations and further modifications of DNA bases of genomic DNA and for their sequences required for further regulation. More epigenetic Close parameters For example, the acetylation of histones, but with the described method cannot be analyzed directly, but again with DNA methylation correlated.

in the Context of the present invention, the term "follow-up treatment" as a treatment of a cancer patient immediately following a first non-chemotherapy treatment, e.g. Surgery, well understood. In the context of the present invention, we take this follow-up treatment using tamoxifen. Generally it is the purpose of follow-up treatment, a significantly lower risk of relapse compared to without one Terminal treatment, available to put.

in the In connection with the present invention, the expression "estrogen and / or Progesterone receptor positive "as Cells on their surface Express receptors that oppose the binding of estrogens and / or progesterones are sensitive, significantly understood.

in the the invention is intended to follow more precisely on the basis of the sequences, Figures and examples are described without being limited thereto to become.

1 shows a preferred application of the method according to the invention. The X-axis shows the tumor mass, with the line '3' showing the limit of detectability. The Y axis shows the time. Accordingly, the figure shows a simplified model of endocrine treatment of a phase 1-3 breast tumor, the primary treatment being surgery (at point 1), followed by follow-up treatment with tamoxifen. In a first scenario, a responder to the treatment ( 4 ) as remaining below the limit of detectability for the duration of the observation. A non-responder on treatment ( 5 ) has a period of disease-free survival ( 2 ), followed by a relapse when the carcinoma reaches the limit of detectability.

2 shows a further preferred application of the method according to the invention. The X-axis shows the tumor mass, with the line '3' showing the limit of detectability. The Y axis shows the time. Accordingly, the figure shows a simplified model of endocrine treatment of a late-stage breast tumor, the primary treatment being surgery (at point 1), followed by a relapse treated with tamoxifen ( 2 ). In a first scenario, a treatment responder (4) is shown to remain below the limit of detectability for the duration of the observation. A non-responder on treatment ( 5 ) does not recover from the relapse.

3 shows the methylation analysis of CpG islands according to Example 1. The CpG islands per gene were grouped and their correlation with objective response was determined by Hotelling's T ² statistics. Black dots indicate the P value of the specified gene. The 20 most informative genes, weighted from left to right with increasing P value, are shown. The upper dotted line marks the uncorrected significance value (P <0.05). The lower dotted line marks the significance after an incorrect detection rate correction of 25%. All genes with a P value less than or equal to the gene with the largest P value that is below the bottom line (in this case COX7A2L) are considered significant. The chosen FDR correction ensures that the identified genes are true discoveries with a 75% chance.

4 shows the crude CpG dinucleotide methylation data of the 10 genes that correlated significantly with the overall response rate, as in 3 determined (Example 1). Tumors from objective responders (CR + PR) are shown on the left, tumors from patients with progressive disease (PD) are shown on the right. The shades of gray indicate the relative distance of the CpG dinucleotide methylation status from the mean. The CpG dinucleotides are grouped per gene. The genes shown are ordered from top to bottom according to increasing significance. Light gray represents a hypomethylated CpG dinucleotide, while darker gray represents a hypermethylated CpG dinucleotide. The methylation data are averaged to a standard deviation for each individual CpG dinucleotide and normalized.

5 shows progression-free survival curves (PFS) of combination values according to example 1. PFS curves after the start of tamoxifen treatment of patients are shown. They are grouped using a predictive value (χ ² = 14.0; 3 degrees of freedom) based on the conventional predictive markers age, dominant position of relapse, disease-free interval and ER level (A). A DNA methylation-based value (χ ² = 27.5; 3 degrees of freedom) is shown, comprising the statistically independent predictive genes phosphoserine aminotransferase (PSA-T), stathmin (STMN1), S100 protein A2 (S100A2, transforming growth factor β receptor II (TGFBR2) and glutamate receptor 2D (GRIN2D) (B) Survival curves were generated and the significance was calculated as described in Example 1. The calculated predictive values were divided into 4 groups (q1-q4) with 50 patients each and using the 25th, 50th and 75th The numbers below the X axis represent the numbers of risk patients in each group at the specified times of 0, 12, 24 and 36 months. Progression-free survival (in percent) is plotted on the Y axis and time to progression (in months) is plotted on the X axis.

SEQ ID Nos: 1 to 121 digits 5'- and / or regulatory regions and / or regions rich in CpG Genes according to the table 1. These sequences are derived from the gene bank and are considered as including all minor variations of the sequence material, that are currently unpredictable, for example, but not limited to minor deletions and SNPs.

SEQ ID Nos: 136 to 619 show the pretreated sequence of DNA of the genes according to the table 1 was derived. These sequences are considered to be all minor variations including the sequence material viewed that are currently unpredictable, for example, but not limited to minor deletions and SNPs.

SEQ ID NO: 856 to SEQ ID NO: 1843 show the sequences of oligomers, which especially for the analysis of pretreated DNA according to SEQ ID NO: 136 to SEQ ID NO: 619 are useful.

Examples

In the following example, candidate genes were analyzed using high-throughput methylation analysis methods. A set of 200 breast cancer samples from advanced breast cancer patients treated with the anti-estrogen active ingredient tamoxifen were analyzed. The measurable effect of endocrine therapy on the size of the metastatic lesion and on progression-free survival (PFS) was used as the main clinical endpoints of the study. A total of 499 CpGs in the promoter regions of 117 genes (2 to 6 CpGs per gene) were analyzed

Example 1

in the The first step was taking the genomic DNA from the cell samples isolated using the Wizzard kit (Promega).

The isolated genomic DNA from the samples was using a bisulfite solution (Hydrogen sulfite, disulfite) treated. The treatment was such that all unmethylated cytosines within the sample converted to thiamidine , conversely, 5-methylated cytosines remained within the Sample unmodified.

The treated nucleic acids were then amplified using multiplex PCRs, where 8 fragments per reaction with Cy5 fluorescent labeled primers were amplified. The PCR primers used are in the table 2 described. The PCR conditions were as follows.

Reaction solution:

• 10 ng bisulfite treated DNA
• 3.5 mmol / l MgCl ₂
• 400 µmol / l dNTPs
• 2 pmoles of each primer
• 1 U Hot Star Taq (Qiagen)

Fourty Cycles were carried out as follows. Denaturation at 95 ° C for 15 min, followed by annealing at 55 ° C for 45 sec, primer elongation at 65 ° C for 2 min. A final elongation at 65 ° C was for 10 minutes.

All PCR products from each individual sample were then hybridized to glass slides, which is a pair of immobilized oligonucleotides for each analyzing CpG position. Each of these detection oligonucleotides was constructed to match the bisulfite converted sequence by one CpG to bind around, either originally unmethylated (TG) or was methylated (CG). See Table 2 for more details of all used Hybridisierungsoligonukleotide. The hybridization conditions were selected so for the detection of the individual nucleotide differences between the Allow TG and CG variants.

5 ul volume of each Multiplex PCR product were in 10 × Ssarc buffer (10 × Ssarc: 230 ml 20 × SSC, 180 ml sodium lauroyl sarcosinate solution 20%, diluted to 1000 ml with dH ₂ O). The reaction mixture was then hybridized to the detection oligonucleotides as follows. Denaturation at 95 ° C, cooling to 10 ° C, hybridization at 42 ° C overnight, followed by washing with 10x Ssarc and dH ₂ O at 42 ° C.

The Fluorescence signals from each hybridized oligonucleotide below the use of Genepix scanners and software. The ratios for the two signals (from the CG oligonucleotide and the TG oligonucleotide, used to analyze each CpG position) were calculated based on the comparison of the intensity of the fluorescence signals.

data analysis

To determine the ability of each gene promoter to predict the success or failure of the tamoxifen treatment, the individual measured CpGs per gene were combined using Hotelling's T ² statistics. To identify potential markers, we performed our initial analysis on the 123 patients who showed the extreme types of response; either an objective response (CR + PR, 45 patients) or a progressive disease, right from the start of treatment (PD, 78 patients). Ten genes were significant with a response to tamoxifen after correction for multiple comparison with a moderately conservative false detection rate of 25% (see 3 ) correlates. The genes were PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L. Furthermore, we concluded from the DNA methylation data matrix ( 4 ) that for the main part of the genes CpG island hypermethylation correlated with an advantageous response to tamoxifen treatment, while for stathmin 1 / oncoprotein 18 (STMN1) and stratifin / 14-3-3 protein σ (SFN) hypomethylation was associated with remission.

Then were every 200 patients, including those with an intermediate Response type (59 patients with stable disease (SD, none change the disease for more than 6 months) and 18 patients with stable disease for 6 months or less) in a logistic regression analysis. We combined for each of the 10 selected Gene the estimates from the logistic regression for the entire response (= objective Response + SD) of the various measured CpG dinucleotides. In the entire cohort was for every ten genes the response rate to tamoxifen treatment is approximately 15 to 20% higher (where the odd ratios [OR's] from 1.5 to 3.5 was enough) for patients whose tumors are cheap DNA methylation status than those with an unfavorable Status. In accordance the mean PFS was for the 10 genes in the previous group of patients on average 1.8 times longer, than in the latter group (the hazard situation (HR's) of 0.54 range up to 0.77). Therefore, these 10 genes were also in the whole Cohort associated with disease outcome after tamoxifen treatment.

The Prediction of response to tamoxifen treatment in advanced Breast cancer patients are traditionally based on the patient, disease and tumor characteristics (pyrhones S, Ellmen J, Vuorinen J, Gershanovich M, Tominaga T, Kaufmann M, Hayes DF. Meta-analysis of trials comparing toremifene with tamoxifen and factors predicting outcome of antiestrogen therapy in postmenopausal women with breast cancer. Breast Cancer Res Treat. 1999; 56 (2): 133-43) i.e. age, first dominant point of relapse, disease-free Interval and ER status. We compared a predictive value with the multivariate estimates the conventional predicative Factors (conventional Factors-based Value) was calculated with a value derived only from DNA methylation data was derived (DNA methylation-based value). For one For DNA methylation-based value, we only included genes whose DNA methylation status predicted the response independently. To establish this, added we get the DNA methylation status information each of the 10 genes simultaneously into a multivariable logistic Regression model. After backward elimination the non-significant genes (P <0.05), were five Genes, i.e. Phosphoserine aminotransferase (PSA-T), stathmin (STMN1), S100 Protein A2 (S 100A2), transforming growth factor β receptor II (TGFBR2), and glutamate receptor 2D (GRIN2D) were statistically significant and said independently of each other's response ahead. The DNA methylation-based value, who then with the conventional Factor-based value was compared from the multivariable estimates of these five Genes derived.

The Value for any tumor based on either conventional predictive factors or DNA methylation status was used to separate patients into 4 groups by their 25th, 50th and 75th percentile values (Table 3). The DNA methylation-based score showed a much better discriminatory one Strength with the overall response than the value based on the conventional factors (Table 3). The overall response rates in the DNA methylation-based Value of 26% for the patients in the first quarter (q1) (OR set to 1.0), 34% for the second Quarter (q2) (OR = 1.5), 66% in the third for q3 (OR = 5.5) and 82% and last quarter for q4 (OR = 13.0) (P <0.001) off while for the usual Factor-based value the rates only from 36% (OR set to 1.0) for q1 this value, via 46% (OR = 1.5) (q2) to 64% each (OR = 3.2) and 62% (OR = 2.9) (q3) (q4), increased (P = 0.013) (Table 3).

Similarly, the mean PFS in the extreme quarters was 4.2 times for the DNA methylation-based score (from 3.8 to 16.1 months), while for the conventional factor-based score, the difference between the two extreme quarters was only half that of which was (2.1 times; from 4.7 to 9.7 months) (compare curves from 5A With 5B ).

Therefore, we have shown for the first time that an epigenetic profile based on the CpG island DNA methylation status of promoter regions of only five genes can predict the likelihood of treatment response in patients with ER-positive advanced breast cancer treated with tamoxifen therapy , Furthermore, it appeared that this prediction was much more accurate than the prediction based on commonly used conventional predictive factors, suggesting that DNA methylation analysis may be useful for clinical therapy decisions. Epigenetic profiling of tumor tissue DNA can therefore be a competitive and simple alternative to tedious mRNA profiling, which is heavily dependent on well-preserved tumor tissue material. In addition, epigenetic DNA profiles are very stable, localized at specific sites (the promoter region of a gene) and can be easily amplified by PCR. In addition and beyond, DNA methylation status determination can be archived, frozen or easily accessible in paraffin embedded Tent tumor tissue material are carried out, which makes them widely applicable in routine clinical setup.

Table 1: Genes which were amplified according to Example 1.

Table 2: Hybridization oligonucleotides.

Table 3: Predictive value for response ^a .

^a , Logistic regression was used to test the strength of a factor's association with the type of response to tamoxifen treatment; ^b , odds ratio; ^c , 95% confidence interval; ^d , value based on multivariable analysis of the conventional additional factors age (> 70 J, 55–70 J, 41–55 J versus ≤ 40 J), dominant place of relapse (relapse to viscera or bone versus soft tissue), disease-free interval (> 12 months versus ≤ 12 months), and log ER values. The value was divided into two groups (q1-q4) based on the 25th, 50th and 75th percentile values; ^e , value based on the DNA methylation status of the 5 independently predictive genes PSA-T, STMN1, GRIN2D, TGFBR2 and S100A2. The value was divided into two groups, q1-q4, the same as for the conventional factor-based value.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform of the DPMA can be downloaded.

Claims (45)

Procedure for predicting the responsiveness of a Subject with proliferative diseases of breast tissue cells on a therapy comprising one or more active substances which target the estrogen receptor pathway or estrogen metabolism production or secretion are involved in the process comprises analyzing the methylation pattern of a target nucleic acid one or a combination of the genes selected from the group consisting of from PSA, STMN1, S100A2, SFN, PRKCD, SYK, VTN, GRIN2D, TGFBR2 and COX7A2L and / or its regulatory regions, through in contact bringing at least one of the target nucleic acids in the biological sample, that of the subject before or during treatment with the substances mentioned above has been obtained, with the above substances. The method of claim 1, wherein the target nucleic acid or acids consisting essentially of one or more sequences from the group from SEQ ID NOs: 27, 90, 41, 40, 121, 78, 115, 86, 43 and 105 and more complementary to that Sequences. The method of claim 1 or 2, wherein the proliferative Disease of cells of the breast tissue selected is composed of the group from ductal carcinoma in situ, lobular carcinoma, colloidal carcinoma, tubulärem Carcinoma, medullary Carcinoma, metaplastic carcinoma, intraductal carcinoma in situ, lobular Carcinoma in situ and papillary Carcinoma in situ. The method of claim 3, wherein the subjects are estrogen and / or progesterone receptor are positive. A method according to claims 1 to 4, wherein the endocrine Therapy to treat a relapse or metastatic proliferative disease of cells of the breast tissue serves. The method of claim 5, wherein the subjects are one Have received chemotherapy. The method of claim 6, wherein the subjects are none Have received chemotherapy. Nucleic acid molecule that is in the consists essentially of a sequence with a length of at least 18 bases according to one of the sequences selected from the group consisting of SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 and sequences complementary thereto consists. Oligomer, especially oligonucleotide or peptide nucleic acid (PNA) oligomer, the oligomer consisting essentially of at least there is a base sequence that is at least 10 nucleotides in length, which hybridizes to or is identical to one of the nucleic acid sequences according to SEQ ID NO: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587. The oligomer of claim 9, wherein the base sequence contains at least one CpG dinucleotide. Oligomer according to claim 10, characterized in that this CpG dinucleotide cytosine approximately is located in the middle third of the oligomer. Set of oligomers comprising at least two oligomers according to one of the claims 9 to 11. Set of oligomers according to claim 12, comprising oligomers for the detection of the methylation state of all CpG dinucleotides within the group of SEQ ID NO: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 and sequences complementary thereto. Set of at least two oligonucleotides after one of claims 9 to 13, which are used as primer oligonucleotides for the amplification of DNA Sequences of one of SEQ ID NO: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586 and 587 and sequences complementary thereto is used. Set of oligonucleotides according to one of claims 9 to 13, characterized in that at least an oligonucleotide is bound to a solid phase. Use of a set of oligonucleotides comprising at least three of the oligomers according to any one of claims 13 to 19 for the detection of the methylation state and / or single nucleotide polymorphisms (SNPs) within the sequences selected from the group of SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586, 587 and sequences complementary thereto. Process for making an array of different on a carrier material fixed oligomers (array) to predict the response of the proliferative Disease of breast cells of the patent on tamoxifen treatment by analyzing the methylation state of each of the CpG dinucleotides from the group of SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586, 587, with at least one Oligomer according to one of claims 9 to 15 is coupled to a fixed phase. Arrangement of different oligomers (array), available according to claim 16. Arrangement of different oligonucleotides and / or PNA oligomer sequences according to claim 18, characterized in that these on a flat solid phase in the form of a rectangular or hexagonal grid are arranged. Array according to one of claims 18 or 19, characterized in that that the solid phase surface made of silicon, glass, polystyrene, aluminum, steel, iron, copper, Nickel, silver or gold is composed. DNA and / or PNA array to predict the response the proliferative disease of breast cells on treatment, which comprises one or more active substances which act on the estrogen receptor Target signaling pathway or estrogen metabolism, production or secretion are involved by analysis of the methylation state each of the CpG dinucleotides from the group of SEQ ID NOs: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456-459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586, 587 comprising at least one nucleic acid one of the preceding claims. Method according to one of claims 1 to 3, comprising the following steps: a) obtaining a biological sample containing genomic DNA, b) extracting the genomic DNA, c) in the genomic DNA sample, cytosine bases which are unmethylated at the 5 'position are converted by chemical treatment to uracil or another base which differs in terms of the hybridization behavior of cytosine; d) amplifying at least one fragment of the pretreated genomic DNA, the fragments being one or more sequences selected from the group of SEQ ID NO: 188, 189, 430, 431, 314, 315, 542, 543, 214-217, 456 -459, 556, 557, 376, 377, 618, 619, 364, 365, 606, 607, 306, 307, 548, 549, 220, 221, 462, 463, 344, 345, 586, 587 and sequences complementary thereto and e) determining the methylation state of one or more genomic CpG dinucleotides by analysis of the amplified nucleic acids. A method according to claim 22, characterized in that step e) by means of the hybridization of at least one oligonucleotide according to SEQ ID NO: 1816-1833, 1114-1129, 1808-1815, 1762-1769, 1552-1559, 1490-1499, 1138-1147 and 1686-1693 carried out becomes. A method according to claim 22, characterized in that step e) by means of the hybridization of at least one oligonucleotide according to the claims 9 to 15 becomes. A method according to claim 22, characterized in that step e) by means of the hybridization of at least one oligonucleotide according to the claims 9 to 13 and extension the hybridized oligonucleotide (s / e) by means of at least one Nucleotide base performed becomes. A method according to claim 22, characterized in that step e) is carried out by means of sequencing. A method according to claim 22, characterized in that step d) is carried out using methylation-specific primers. A method according to claim 22, characterized in that step e) by means of a combination of at least two of the claims 23 to 27 described method is carried out. A method according to claim 22, characterized in that the chemical treatment using a solution of bisulfite, hydrogen sulfite or disulfite becomes. A method according to any one of claims 1 to 3, comprising the following steps: a) Obtaining a biological sample that contains genomic DNA, b) Extracting the genomic DNA, c) digesting the genomic DNA comprising one or more of the sequences selected from the group consisting of SEQ ID NOs 27, 90, 41, 40, 121, 78, 115, 86, 43 and 105 and complementary sequences with one or several methylation-sensitive restriction enzymes, and d) Detect the DNA fragments generated in the digestion from step c) become. A method according to claim 30, characterized in that the in step c) digested target sequence or sequences one or more the sequences from the group consisting of SEQ ID NO 27, 90, 41, 40, 121, 78, 115, 86, 43 and 105. A method according to any one of claims 30 or 31, wherein the DNA digest is amplified before step d). Method according to one of claims 22 to 29 and 32 thereby characterized that more as ten different fragments with a length of 100-200 base pairs, be amplified. Method according to one of claims 22 to 29, 32 and 33 thereby characterized that the Amplification of several DNA segments is carried out in one reaction vessel. Method according to one of claims 22 to 29 and 32 to 34, characterized in that the Polymerase is a heat-resistant DNA polymerase. Method according to one of claims 22 to 29 and 32 to 35, characterized in that the Amplification by means of polymerase chain reaction (PCR) is carried out. Method according to one of claims 22 to 29 and 32 to 36, characterized in that the Am bear pliable detectable markers. The method of claim 37, wherein the markers are fluorescent markers, Radionuclides and / or removable molecular fragments are that have a typical mass in a mass spectrometer can be demonstrated. Method according to one of claims 22 to 29, characterized in that that the Amplificates or fragments detected in the mass spectrometer become. Method according to one of claims 38 and / or 39, characterized characterized that the fragments produced a single positive or negative charge for better traceability in the mass spectrometer. Method according to one of claims 37 to 40, that the detection carried out and visualized using matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (IT I). Method according to one of claims 22 to 37, characterized in that that the genomic DNA is obtained from cells or cellular components, containing DNA, with sources of DNA including, for example, Cell lines, histological sections, biopsies, paraffin embedded Tissue, breast tissue, blood, plasma, lymphatic fluid, Ductus cells, ductal lava fluid, Nipple discharge liquid, Bone marrow and combinations thereof. Kit comprising a bisulfite (bisulfite, bisulfite) Reagent and oligonucleotides and / or PNA oligomers according to one of claims 9 to 15. The kit of claim 43, further comprising standard reagents to carry out a methylation test from the group consisting of MS-SNuPE, MSP, methyl light, heavy methyl, nucleic acid sequencing and combinations from that. Use of a method according to one of claims 1 to 7, 17, and 20 to 42, a nucleic acid according to claim 8, one Oligonucleotide or PNA oligomers according to one of claims 9 to 15, a kit according to the claims 43 or 44, an array according to any one of claims 19 to 21 or a set of oligonucleotides according to one of claims 12 to 15 for the treatment, Characterization, classification, and / or differentiation of proliferative diseases of breast cells.