WO2007019670A1 - Method and nucleic acids for the improved treatment of breast cancers - Google Patents

Abstract

The present invention provides methods and nucleic acids for determining the presense or absence as well as the prognosis of breast cell proliferative disorders. The methods involve examining the methylation patterns of genomic DNA sequences in a sample from a subject.

Description

Method and nucleic acids for the improved treatment of breast cancers.

Field of the Invention

In American women, breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death. In women aged 40-55, breast cancer is the leading cause of death (Greenlee et al., 2000). In 2002, there were 204,000 new cases of breast cancer in the US (data from the American Society of Clinical Oncology) and a comparable number in Europe.

Breast cancer is defined as the uncontrolled proliferation of cells within breasts tissues. Breasts are comprised of 15 to 20 lobes joined together by ducts. Cancer arises most commonly in the duct, but is also found in the lobes with the rarest type of cancer termed inflammatory breast cancer. It will be appreciated by those skilled in the art that there exists a continuing need to improve methods of early detection, classification and treatment of breast cancers. In contrast to the detection of some other common cancers such as cervical and dermal there are inherent difficulties in classifying and detecting breast cancers.

Due to current screening programs and the accessibility of this cancer to self-examination, breast cancer is diagnosed comparatively early: in about 93% of all newly diagnosed cases, the cancer has not yet metastasized, and in 65% of cases, even the lymph nodes are not yet affected.

The first step of any treatment is the assessment of the patient's condition comparative to defined classifications of the disease. However the value of such a system is inherently dependent upon the quality of the classification. Breast cancers are staged according to their size, location and occurrence of metastasis. Methods of treatment include the use of surgery, radiation therapy, chemotherapy and endocrine therapy, which are also used as adjuvant therapies to surgery. Although the vast majority of early cancers are operable, i.e. the tumor can be completely removed by surgery, about one third of the patients with lymph-node negative diseases and about 50-60% of patients with node-positive disease will develop metastases during follow- up.

Based on this observation, systemic adjuvant treatment has been introduced for both node- positive and node-negative breast cancers. Systemic adjuvant therapy is administered after surgical removal of the tumor, and has been shown to reduce the risk of recurrence significantly (Early Breast Cancer Trialists' Collaborative Group, 1998). Several types of adjuvant treatment are available: endocrine treatment (for hormone receptor positive tumors), different chemotherapy regimens, and novel agents like Herceptin.

The selection of suitable adjuvant systemic therapies is determined according to an assessment of the patient's risk of recurrence. Risk of recurrence is assessed primarily according to node status, histological grade, tumour size, oestrogen receptor (ER) status of the primary tumour and menopausal status. Other factors that may be taken into consideration include cerbB2 expression, ratio of lymph nodes positive vs number of lymph nodes resected, presence of vascular invasion and age.

According to the risk of recurrence appropriate treatments may be selected that provide a reduction in risk of recurrence or death. Chemotherapy is often prescribed as an adjuvant systematic therapy. The proportional reduction of risk of recurrence and death for any given chemotherapy regimen is fairly constant within defined age and hormone receptor categories but the absolute benefit achieved varies as a function of a patient's risk. Accordingly, in order to determine whether a patient will benefit from chemotherapy it is necessary to accurately determine the risk of recurrence or death.

Molecular markers associated with breast cancer prognosis are known, including methylation markers, for the measurement of risk of relapse or survival. PCT/EP2004/014170 discloses a large group of markers the expression, as determined most preferably by methylation status, thereof being indicative of the prognosis of breast cancer patients in terms of overall survival or relapse. The present invention provide novel markers the methylation of which are indicative of the prognosis of a patient with breast cancer in terms of survival and/or relapse. DNA methylation plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. In humans, 5-methylcytosine is mainly found in the context of cytosine-guanine (CpG) dinucleotides. However, 5- methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behaviour as cytosine. Moreover, the epigenetic information carried by 5- methylcytosine is completely lost during bisulfite treatment.

The most frequently used method for analyzing DNA for the presence of 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine whereby, upon subsequent alkaline hydrolysis, cytosine is converted to uracil which corresponds to thymine in its base pairing behavior. Significantly, however, 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using standard, art-recognized molecular biological techniques, for example, by amplification and hybridization, or by sequencing. All of these techniques are based on differential base pairing properties, which can now be fully exploited.

The prior art, in terms of sensitivity, is defined by a method comprising enclosing the DNA to be analyzed in an agarose matrix, thereby preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and replacing all precipitation and purification steps with fast dialysis (Olek A, et al., A modified and improved method for bisulfite based cytosine methylation analysis, Nucleic Acids Res. 24:5064-6, 1996). It is thus possible to analyze individual cells for methylation status, illustrating the utility and sensitivity of the method. An overview of art-recognized methods for detecting 5- methylcytosine is provided by Rein, T., et al., Nucleic Acids Res., 26:2255, 1998.

The bisulfite technique, barring few exceptions (e.g., Zeschnigk M, et al., Eur J Hum Genet. 5:94-98, 1997), is currently only used in research. In all instances, short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment, and either completely sequenced (Olek & Walter, Nat Genet. 1997 17:275-6, 1997), subjected to one or more primer extension reactions (Gonzalgo & Jones, Nucleic Acids Res., 25:2529-31, 1997; WO 95/00669; U.S. Patent No. 6,251,594) to analyze individual cytosine positions, or treated by enzymatic - A -

digestion (Xiong & Laird, Nucleic Acids Res., 25:2532-4, 1997). Detection by hybridization has also been described in the art (Olek et al., WO 99/28498). Additionally, use of the bisulfite technique for methylation detection with respect to individual genes has been described (Grigg & Clark, Bioessays, 16:431-6, 1994; Zeschnigk M, et al., Hum MoI Genet., 6:387-95, 1997; Feil R, et al., Nucleic Acids Res., 22:695-, 1994; Martin V, et al., Gene, 157:261-4, 1995; WO 97/46705 and WO 95/15373).

Methylation Assay Procedures. Various methylation assay procedures are known in the art, and can be used in conjunction with the present invention. These assays allow for determination of the methylation state of one or a plurality of CpG dinucleotides (e.g., CpG islands) within a DNA sequence. Such assays involve, among other techniques, DNA sequencing of bisulfite-treated DNA, PCR (for sequence-specific amplification), Southern blot analysis, and use of methylation-sensitive restriction enzymes.

For example, genomic sequencing has been simplified for analysis of DNA methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al., Proc. Natl. Acad. ScL USA 89:1827-1831, 1992). Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used, e.g., the method described by Sadri & Hornsby (Nucl. Acids Res. 24:5058-5059, 1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).

COBRA. COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific gene loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite- treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. ScL USA 89:1827-1831, 1992). PCR amplification of the bisulfite converted DNA is then performed using primers specific for the CpG islands of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

MethyLight™. The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g. TaqMan™) technology that requires no further manipulations after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed either in an "unbiased" (with primers that do not overlap known CpG methylation sites) PCR reaction, or in a "biased" (with PCR primers that overlap known CpG dinucleotides) reaction. Sequence discrimination can occur either at the level of the amplification process or at the level of the fluorescence detection process, or both.

The MethyLight™ assay may be used as a quantitative test for methylation patterns in the genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not "cover" known methylation sites (a fluorescence- based version of the "MSP" technique), or with oligonucleotides covering potential methylation sites.

Ms-SNuPE. The Ms-SNuPE™ technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single- nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest. An oligonucleotide is hybridized next to or close to the CpG position of interest, the oligonucleotide is then extended and on the basis of said extension the methylation status of CpG position of interest is determined. Small amounts of DNA can be analyzed (e.g., microdissected pathology sections), and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.

MSP. MSP (methylation-specific PCR) allows for assessing the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; US Patent No. 5,786,146). Briefly, DNA is modified by sodium bisulfite converting all unmethylated, but not methylated cytosines to uracil, and subsequently amplified with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1 % methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the general methodology of DMH (differential methylation hybridisation) as utilized in Example 2.

Figures 2 to 49 provide matrices produced from bisulfite sequencing data analysed by the applicant's proprietary software (See WO 2004/000463 for further information)according to Example 3. Each column of the matrices represent the sequencing data for one amplificate. Each row of a matrix represents a single CpG site within the fragment and each column represents an individual DNA sample, cell line sample or whole blood sample. The bar on the left represents a scale of the percent methylation, with the degree of methylation represented by the shade of each position within the column from black representing 100% methylation to light grey representing 0% methylation. White positions represented a measurement for which no data was available. In figures 29 to 49 the non-aggresssive samples group (i.e. non-relapse patients) is marked 'A', the aggresssive samples group (i.e. relapsed patients) is marked 'B', the peripheral blood lymphocytes sample group is marked 'C.

DESCRIPTION

The present invention provides molecular genetic markers, as well as methods utilising said markers and nucleic acids thereof, that have novel utility for the improved treatment of patients with cell proliferative disorders of the breast tissues. The present invention provides diagnostic and prognostic markers for breast cancer. The term 'prognosis' is taken to mean a prediction of outcome of disease progression (wherein the term progression shall be taken to also include recurrence after treatment). Prognosis may be expressed in terms of overall patient survival, disease- or relapse-free survival, increased tumor-related complications and faster progression of tumour or metastases, wherein a decrease in any of said factors (with the exception of increased tumor-related complications) is a 'negative' outcome and increase thereof is a 'positive' outcome.

Hereinafter prognosis may also be referred to in terms of 'aggressiveness' wherein an aggressive cancer is determined to have a high risk of negative outcome and wherein a non- aggressive cancer has a low risk of negative outcome.

It is particularly preferred that the prognostic markers according to the present invention are used to provide an estimate of the risk of negative outcome. Characterisation of a breast cancer in terms of predicted outcome enables the physician to determine the risk of recurrence and/or death. This aids in treatment selection as the absolute reduction of risk of recurrence and death in treatments such as chemotherapy can be determined based on the predicted negative outcome . The absolute reduction in risk attributable to treatment may then be compared to the drawbacks of said treatment (e.g. side effects, cost) in order to determine the suitability of said treatment for the patient.

According to the predicted outcome (i.e. prognosis) of the disease an appropriate treatment or treatments may be selected from the group consisting of chemotherapy, radiotherapy, surgery, biological therapy, immunotherapy, antibody treatments, treatments involving molecularly targeted drugs, estrogen receptor modulator treatments, estrogen receptor down-regulator treatments, aromatase inhibitors treatments, ovarian ablation, treatments providing LHRH analogues or other centrally acting drugs influencing estrogen production.

Wherein a cancer is characterised as aggressive it is particularly preferred that a treatment such as, but not limited to, chemotherapy is provided in addition to or instead of further treatments. Conversely, wherein a cancer is characterised as non-aggressive (i.e. positive outcome with low risk of death and/or recurrence)the patient will derive low absolute benefit from chemotherapeutic treatment and may be appropriately treated by other means. Therein lies a great advantage of the present invention. Chemotherapy is currently prescribed as a routine adjuvant systemic therapy in most cases, by providing a means for determining which patients will not significantly benefit from chemotherapy the present invention thereby prevents the routine over-prescription of chemotherapy. Alternative therapies which may be recommended include but are not limited to treatments which target the estrogen receptor pathway or are involved in estrogen metabolism, production or secretion. Said treatments include, but are not limited to estrogen receptor modulators, estrogen receptor down- regulators, aromatase inhibitors, ovarian ablation, LHRH analogues and other centrally acting drugs influencing estrogen production.

Furthermore the present invention provides markers which are particularly useful in the prediction of prognosis in patients according to the estrogen receptor status thereof.

The herein described markers have further utility in the diagnosis of breast cancer. This will hereinafter also be referred to as a 'diagnostic' marker.

As used herein the term 'predictive marker' shall be taken to mean an indicator of response to therapy, said response is preferably defined according to patient survival. It is preferably used to define patients with high, low and intermediate length of survival or recurrence after treatment, that is the result of the inherent heterogeneity of the disease process.

As defined herein the term predictive marker may in some situations fall within the remit of a herein described 'prognostic marker', for example, wherein a prognostic marker differentiates between patients with different survival outcomes pursuant to a treatment, said marker is also a predictive marker for said treatment. Therefore, unless otherwise stated the two terms shall not be taken to be mutually exclusive.

The method according to the invention may be used for determining the risk of recurrence and/or death of patients with a wide variety of cell proliferative disorders of the breast tissues including, but not limited to, ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, comedocarcinoma, inflammatory carcinoma, mucinous carcinoma, scirrhous carcinoma, colloid carcinoma, tubular carcinoma, medullary carcinoma, metaplastic carcinoma, and papillary carcinoma and papillary carcinoma in situ, undifferentiated or anaplastic carcinoma and Paget' s disease of the breast. In one embodiment the invention discloses a method for the use of one or more genomic sequences selected from the group consisting SEQ ED NO: 1 to SEQ ID NO: 117 as prognostic and/or diagnostic markers for breast cancer.

Said use of the genomic sequences may be enabled by means of any analysis of the expression of an RNA transcribed therefrom or protein translated from said RNA, by means of mRNA expression analysis or protein expression analysis. However, in the most preferred embodiment of the invention, the prognosis and/or diagnosis of said breast cancer subjects is enabled by means of analysis of the methylation status of one or more CpG positions of genomic sequences selected from the group consisting SEQ ID NO: 1 to SEQ ID NO: 117 and their promoter or regulatory elements. Methods for the methylation analysis of genes are described herein.

Aberrant levels of expression of mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO: 117 according to Table 1 are associated with prognosis and/or diagnosis of breast carcinoma.

To detect the presence of mRNA encoding a genomic sequence, a sample is obtained from a patient. The sample may be any suitable sample comprising cellular matter of the lesion. The sample can be a tissue biopsy sample or a sample of blood, plasma, serum or other body fluids. The sample may be treated to extract the RNA contained therein. The resulting nucleic acid from the sample is then analysed. Many techniques are known in the state of the art for determining absolute and relative levels of gene expression, commonly used techniques suitable for use in the present invention include in situ hybridisation (e.g., FISH), Northern analysis, RNase protection assays (RPA), microarrays and PCR-based techniques, such as quantitative PCR and differential display PCR or any other nucleic acid detection method.

Particularly preferred is the use of the reverse transcription/polymerisation chain reaction technique (RT-PCR). The method of RT-PCR is well known in the art (for example, see Watson and Fleming, supra).

The RT-PCR method can be performed as follows. Total cellular RNA is isolated by, for example, the standard guanidium isothiocyanate method and the total RNA is reverse transcribed. The reverse transcription method involves synthesis of DNA on a template of RNA using a reverse transcriptase enzyme and a 3' end oligonucleotide dT primer and/or random hexamer primers. The cDNA thus produced is then amplified by means of PCR. (Belyavsky et al, Nucl Acid Res 17:2919-2932, 1989; Krug and Berger, Methods in Enzymology, Academic Press, N.Y., Vol.152, pp. 316-325, 1987 which are incorporated by reference). Further preferred is the "Real-time" variant of RT- PCR, wherein the PCR product is detected by means of hybridisation probes (e.g. TaqMan, Lightcycler, Molecular Beacons & Scorpion) or SYBR green. The detected signal from the probes or SYBR green is then quantitated either by reference to a standard curve or by comparing the Ct values to that of a calibration standard. Analysis of housekeeping genes is often used to normalize the results.

In Northern blot analysis total or poly(A)+ mRNA is run on a denaturing agarose gel and detected by hybridisation to a labelled probe in the dried gel itself or on a membrane. The resulting signal is proportional to the amount of target RNA in the RNA population.

Comparing the signals from two or more cell populations or tissues reveals relative differences in gene expression levels. Absolute quantitation can be performed by comparing the signal to a standard curve generated using known amounts of an in vitro transcript corresponding to the target RNA. Analysis of housekeeping genes, genes whose expression levels are expected to remain relatively constant regardless of conditions, is often used to normalize the results, eliminating any apparent differences caused by unequal transfer of RNA to the membrane or unequal loading of RNA on the gel.

The first step in Northern analysis is isolating pure, intact RNA from the cells or tissue of interest. Because Northern blots distinguish RNAs by size, sample integrity influences the degree to which a signal is localized in a single band. Partially degraded RNA samples will result in the signal being smeared or distributed over several bands with an overall loss in sensitivity and possibly an erroneous interpretation of the data. In Northern blot analysis, DNA, RNA and oligonucleotide probes can be used and these probes are preferably labelled (e.g., radioactive labels, mass labels or fluorescent labels). The size of the target RNA, not the probe, will determine the size of the detected band, so methods such as random-primed labelling, which generates probes of variable lengths, are suitable for probe synthesis. The specific activity of the probe will determine the level of sensitivity, so it is preferred that probes with high specific activities, are used.

In an RNase protection assay, the RNA target and an RNA probe of a defined length are hybridised in solution. Following hybridisation, the RNA is digested with RNases specific for single-stranded nucleic acids to remove any unhybridized, single-stranded target RNA and probe. The RNases are inactivated, and the RNA is separated e.g. by denaturing polyacrylamide gel electrophoresis. The amount of intact RNA probe is proportional to the amount of target RNA in the RNA population. RPA can be used for relative and absolute quantitation of gene expression and also for mapping RNA structure, such as intron/exon boundaries and transcription start sites. The RNase protection assay is preferable to Northern blot analysis as it generally has a lower limit of detection.

The antisense RNA probes used in RPA are generated by in vitro transcription of a DNA template with a defined endpoint and are typically in the range of 50-600 nucleotides. The use of RNA probes that include additional sequences not homologous to the target RNA allows the protected fragment to be distinguished from the full-length probe. RNA probes are typically used instead of DNA probes due to the ease of generating single-stranded RNA probes and the reproducibility and reliability of RNA:RNA duplex digestion with RNases (Ausubel et al. 2003), particularly preferred are probes with high specific activities.

Particularly preferred is the use of microarrays. The microarray analysis process can be divided into two main parts. First is the immobilization of known gene sequences onto glass slides or other solid support followed by hybridisation of the fluorescently labelled cDNA (comprising the sequences to be interrogated) to the known genes immobilized on the glass slide (or other solid phase). After hybridisation, arrays are scanned using a fluorescent microarray scanner. Analysing the relative fluorescent intensity of different genes provides a measure of the differences in gene expression.

DNA arrays can be generated by immobilizing presynthesized oligonucleotides onto prepared glass slides or other solid surfaces. In this case, representative gene sequences are manufactured and prepared using standard oligonucleotide synthesis and purification methods. These synthesized gene sequences are complementary to the RNA transcript(s) of the genes of interest (in this case the genes or genomic sequences selected from Table 1) and tend to be shorter sequences in the range of 25-70 nucleotides. In a preferred embodiment said oligonucleotides or polynucleotides comprise at least 9, 18 or 25 bases of a sequence complementary to or hybridising to the mRNA transcript and sequences complementary thereto. Alternatively, immobilized oligos can be chemically synthesized in situ on the surface of the slide. In situ oligonucleotide synthesis involves the consecutive addition of the appropriate nucleotides to the spots on the microarray; spots not receiving a nucleotide are protected during each stage of the process using physical or virtual masks. Preferably said synthesized nucleic acids are locked nucleic acids.

In expression profiling microarray experiments, the RNA templates used are representative of the transcription profile of the cells or tissues under study. RNA is first isolated from the cell populations or tissues to be compared. Each RNA sample is then used as a template to generate fluorescently labelled cDNA via a reverse transcription reaction. Fluorescent labelling of the cDNA can be accomplished by either direct labelling or indirect labelling methods. During direct labelling, fluorescently modified nucleotides (e.g., Cy^®3- or Cy^®5- dCTP) are incorporated directly into the cDNA during the reverse transcription. Alternatively, indirect labelling can be achieved by incorporating aminoallyl-modified nucleotides during cDNA synthesis and then conjugating an N-hydroxysuccinimide (NHS)-ester dye to the aminoallyl-modified cDNA after the reverse transcription reaction is complete. Alternatively, the probe may be unlabelled, but may be detectable by specific binding with a ligand which is labelled, either directly or indirectly. Suitable labels and methods for labelling ligands (and probes) are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation or kinasing). Other suitable labels include but are not limited to biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, and the like.

To perform differential gene expression analysis, cDNA generated from different RNA samples are labelled with Cy^®3. The resulting labelled cDNA is purified to remove unincorporated nucleotides, free dye and residual RNA. Following purification, the labelled cDNA samples are hybridised to the microarray. The stringency of hybridisation is determined by a number of factors during hybridisation and during the washing procedure, including temperature, ionic strength, length of time and concentration of formamide. These factors are outlined in, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., 1989). The microarray is scanned post-hybridisation using a fluorescent microarray scanner. The fluorescent intensity of each spot indicates the level of expression of the analysed gene; bright spots correspond to strongly expressed genes, while dim spots indicate weak expression. Once the images are obtained, the raw data must be analysed. First, the background fluorescence must be subtracted from the fluorescence of each spot. The data is then normalized to a control sequence, such as exogenously added nucleic acids (preferably RNA or DNA), or a housekeeping gene panel to account for any non-specific hybridisation, array imperfections or variability in the array set-up, cDNA labelling, hybridisation or washing. Data normalization allows the results of multiple arrays to be compared.

Another aspect of the invention relates to a kit for use in the prognosis and/or diagnosis of breast carcinoma in a subject according to the methods of the present invention, said kit comprising: a means for measuring the level of transcription of genomic sequences selected from Table I. In a preferred embodiment the means for measuring the level of transcription comprise oligonucleotides or polynucleotides able to hybridise under stringent or moderately stringent conditions to the transcription products of a genomic sequence selected from Table 1. Preferably said oligonucleotides or polynucleotides are able to hybridise under stringent or moderately stringent conditions to at least one of the transcription products of a genomic sequence selected from Table 1. In one embodiment said oligonucleotides or polynucleotides comprise at least 9, 18 or 25 bases of a sequence complementary to or hybridising to at least one of said sequence or sequences complementary thereto.

In a most preferred embodiment the level of transcription is determined by techniques selected from the group of Northern Blot analysis, reverse transcriptase PCR, real-time PCR, RNAse protection, and microarray. In another embodiment of the invention the kit further comprises means for obtaining a biological sample of the patient. Preferred is a kit, which further comprises a container which is most preferably suitable for containing the means for measuring the level of transcription and the biological sample of the patient, and most preferably further comprises instructions for use and interpretation of the kit results.

In a preferred embodiment the kit comprises (a) a plurality of oligonucleotides or polynucleotides able to hybridise under stringent or moderately stringent conditions to the transcription products of at least one genomic sequence selected from Table 1 ; (b) a container, preferably suitable for containing the oligonucleotides or polynucleotides and a biological sample of the patient comprising the transcription products wherein the oligonucleotides or polynucleotides can hybridise under stringent or moderately stringent conditions to the transcription products, (c) means to detect the hybridisation of (b); and optionally, (d) instructions for use and interpretation of the kit results. It is further preferred that said oligonucleotides or polynucleotides of (a) comprise in each case at least 9, 18 or 25 bases of a sequence complementary to or hybridising to the transcription products and sequences complementary thereto.

The kit may also contain other components such as hybridisation buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container. Alternatively, where the oligonucleotides are to be used to amplify a target region, the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimised for primer extension mediated by the polymerase, such as PCR. Preferably said polymerase is a reverse transcriptase.lt is further preferred that said kit further contains an Rnase reagent.

The present invention further provides for methods for the detection of the presence of the polypeptide encoded by said gene sequences in a sample obtained from a patient.

Aberrant levels of polypeptide expression of the polypeptides of genea associated with genomic sequences selected from Table 1 are associated with the presence and/or prognosis of breast carcinoma.

Any method known in the art for detecting polypeptides can be used. Such methods include, but are not limited to masss-spectrometry, immunodiffusion, Immunoelectrophoresis, immunochemical methods, binder-ligand assays, immunohistochemical techniques, agglutination and complement assays (e.g., see Basic and Clinical Immunology, Sites and Terr, eds., Appleton & Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference). Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labelled polypeptide or derivative thereof.

Certain embodiments of the present invention comprise the use of antibodies specific to the polypeptide encoded by a gene associated with a genomic sequence selected from Table 1.

Such antibodies are useful for the detection and/or prognosis of breast carcinomas. In certain embodiments production of monoclonal or polyclonal antibodies can be induced by the use of an epitope encoded by a said polypeptide as an antigene. Such antibodies may in turn be used to detect expressed polypeptides as prognostic markers for breast cancer. The levels of such polypeptides present may be quantified by conventional methods. Antibody-polypeptide binding may be detected and quantified by a variety of means known in the art, such as labelling with fluorescent or radioactive ligands. The invention further comprises kits for performing the above-mentioned procedures, wherein such kits contain antibodies specific for the investigated polypeptides.

Numerous competitive and non-competitive polypeptide binding immunoassays are well known in the art. Antibodies employed in such assays may be unlabelled, for example as used in agglutination tests, or labelled for use a wide variety of assay methods. Labels that can be used include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates or co- factors, enzyme inhibitors, particles, dyes and the like. Preferred assays include but are not limited to radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays and the like. Polyclonal or monoclonal antibodies or epitopes thereof can be made for use in immunoassays by any of a number of methods known in the art.

In an alternative embodiment of the method the proteins may be detected by means of western blot analysis. Said analysis is standard in the art, briefly proteins are separated by means of electrophoresis, e.g., SDS-PAGE. The separated proteins are then transferred to a suitable membrane (or paper), e.g., nitrocellulose, retaining the spacial separation achieved by electrophoresis. The membrane is then incubated with a blocking agent to bind remaining sticky places on the membrane, commonly used agents include generic protein (e.g., milk protein). An antibody specific to the protein of interest is then added, said antibody being detectably labelled for example by dyes or enzymatic means (e.g., alkaline phosphatase or horseradish peroxidase). The location of the antibody on the membrane is then detected.

In an alternative embodiment of the method the proteins may be detected by means of immunohistochemistry (the use of antibodies to probe specific antigens in a sample). Said analysis is standard in the art, wherein detection of antigens in tissues is known as immunohistochemistry, while detection in cultured cells is generally termed immunocytochemistry. Briefly, the primary antibody to be detected by binding to its specific antigen. The antibody-antigen complex is then bound by a secondary enzyme conjugated antibody. In the presence of the necessary substrate and chromogen the bound enzyme is detected according to coloured deposits at the antibody-antigen binding sites. There is a wide range of suitable sample types, antigen-antibody affinity, antibody types, and detection enhancement methods. Thus optimal conditions for immunohistochemical or immunocytochemical detection must be determined by the person skilled in the art for each individual case.

One approach for preparing antibodies to a polypeptide is the selection and preparation of an amino acid sequence of all or part of the polypeptide, chemically synthesising the amino acid sequence and injecting it into an appropriate animal, usually a rabbit or a mouse (Milstein and Kohler Nature 256:495-497, 1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone and Banatis eds., Academic Press, 1981 which are incorporated by reference in its entirety). Methods for preparation of the polypeptides or epitopes thereof include, but are not limited to chemical synthesis, recombinant DNA techniques or isolation from biological samples.

Another aspect of the invention provides a kit for use in the prognosis and/or diagnosis of a breast carcinoma in a subject according to the methods of the present invention, comprising: a means for detecting polypeptides encoded by a gene associated with a genomic sequence selected from Table 1. The means for detecting the polypeptides comprise preferably antibodies, antibody derivatives, or antibody fragments. The polypeptides are most preferably detected by means of Western Blotting utilizing a labelled antibody. In another embodiment of the invention the kit further comprising means for obtaining a biological sample of the patient. Preferred is a kit, which further comprises a container suitable for containing the means for detecting the polypeptides in the biological sample of the patient, and most preferably further comprises instructions for use and interpretation of the kit results. In a preferred embodiment the kit comprises: (a) a means for detecting polypeptides encoded by a gene associated with a genomic sequence selected from Table 1 ; (b) a container suitable for containing the said means and the biological sample of the patient comprising the polypeptides wherein the means can form complexes with the polypeptides; (c) a means to detect the complexes of (b); and optionally (d) instructions for use and interpretation of the kit results. The kit may also contain other components such as buffers or solutions suitable for blocking, washing or coating, packaged in a separate container. The aim of the invention is most preferably achieved by means of the analysis of the methylation of one or more CpG positions of one or a combination of sequences taken from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 1 17 (see Table 1) and/or their regulatory regions.

It is preferred that the object of the invention is achieved by analysing the methylation patterns of a plurality of sequences, hereinafter also referred to as a 'gene panel'. It is further preferred that said plurality is between two and four sequences.

The invention is characterised in that the nucleic acid of one or a combination of said genes are contacted with a reagent or series of reagents capable of distinguishing between methylated and unmethylated CpG dinucleotides within the genomic sequence of interest.

In a particularly preferred embodiment the sequences of said genes comprise SEQ ID NO: 1 to SEQ ID NO: 117 and sequences complementary thereto.

Wherein the patient is estrogen receptor positive it is particularly preferred that the sequences of said genes comprise SEQ ID NO: 97 to SEQ ID NO: 117 and sequences complementary thereto.

Wherein the patient is estrogen receptor negative it is particularly preferred that the sequences of said genes comprise SEQ ID NO: 1 to SEQ ID NO: 96 and sequences complementary thereto.

In a preferred embodiment said method is achieved by contacting the nucleic acid sequences SEQ ID NO: 1 to SEQ ID NO: 117 in a biological sample obtained from a subject with at least one reagent or a series of reagents, wherein said reagent or series of reagents, distinguishes between methylated and non methylated CpG dinucleotides within the target nucleic acid.

In a preferred embodiment, the method comprises the following steps. In the first step, a sample of the tissue to be analysed is obtained. The source may be any suitable source, such as cell lines, histological slides, paraffin embedded tissues, biopsies, tissue embedded in paraffin, bodily fluids (including but not limited to urine, blood and nipple aspirate) and all possible combinations thereof. In a particularly preferred embodiment of the method said source is bodily fluids (including but not limited to urine, blood and nipple aspirate). The DNA is then isolated from the sample. Extraction may be by means that are standard to one skilled in the art, including the use of commercially available kits, detergent lysates, sonification and vortexing with glass beads. Briefly, wherein the DNA of interest is encapsulated by a cellular membrane the biological sample must be disrupted and lysed by enzymatic, chemical or mechanical means. The DNA solution may then be cleared of proteins and other contaminants e.g. by digestion with proteinase K. The genomic DNA is then recovered from the solution. This may be carried out by means of a variety of methods including salting out, organic extraction or binding of the DNA to a solid phase support. The choice of method will be affected by several factors including time, expense and required quantity of DNA. Once the nucleic acids have been extracted, the genomic double stranded DNA is used in the analysis.

In the second step of the method, the genomic DNA sample is treated in such a manner that cytosine bases which are unmethylated at the 5 '-position are converted to uracil, thymine, or another base which is dissimilar to cytosine in terms of hybridization behavior. This will be understood as 'pretreatment' herein.

This is preferably achieved by means of treatment with a bisulfite reagent. The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite or combinations thereof, useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences. Methods of said treatment are known in the art (e.g. PCT/EP2004/011715, which is incorporated by reference in its entirety). It is preferred that the bisulfite treatment is conducted in the presence of denaturing solvents such as but not limited to n-alkylenglycol, particulary diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In a preferred embodiment the denaturing solvents are used in concentrations between 1% and 35% (v/v). It is also preferred that the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane 2-carboxylic acid (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite conversion is preferably carried out at a reaction temperature between 30°C and 70⁰C, whereby the temperature is increased to over 85°C for short periods of times during the reaction (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite treated DNA is preferably purified prior to the quantification. This may be conducted by any means known in the art, such as but not limited to ultrafiltration, preferably carried out by means of Microcon^Λ(TM) columns (manufactured by Millipore^Λ(TM)). The purification is carried out according to a modified manufacturer's protocol (see: PCT/EP2004/01 1715 which is incorporated by reference in its entirety).

In the third step of the method, fragments of the pretreated DNA are amplified, using sets of primer oligonucleotides according to the present invention, and an amplification enzyme. The amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Typically, the amplification is carried out using a polymerase chain reaction (PCR). The set of primer oligonucleotides includes at least two oligonucleotides whose sequences are each reverse complementary to, identical to, or hybridize under stringent or highly stringent conditions to an at least 16-base-pair long segment of the base sequences of one or more of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto.

In an alternate embodiment of the method, the methylation status of preselected CpG positions within the nucleic acid sequences comprising one or more of SEQ ID NO: 1 to SEQ ID NO: 117 may be detected by use of methylation-specific primer oligonucleotides. Wherein the patient is estrogen receptor positive it is particularly preferred that said nucleic acid sequences comprise SEQ ID NO: 97 to SEQ ID NO: 117 and sequences complementary thereto. Wherein the patient is estrogen receptor negative it is particularly preferred that said nucleic acid sequences comprise SEQ ID NO: 1 to SEQ ID NO: 96 and sequences complementary thereto. This technique (MSP) has been described in United States Patent No. 6,265,171 to Herman. The use of methylation status specific primers for the amplification of bisulfite treated DNA allows the differentiation between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to a bisulfite treated CpG dinucleotide. Therefore, the sequence of said primers comprises at least one CpG or TpG dinucleotide. MSP primers specific for non-methylated DNA contain a 'T' at the 3' position of the C position in the CpG. Preferably, therefore, the base sequence of said primers is required to comprise a sequence having a length of at least 9 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG dinucleotide. A further preferred embodiment of the method comprises the use of blocker oligonucleotides. The use of such blocker oligonucleotides has been described by Yu et al., BioTechniques 23:714-720, 1997. Blocking probe oligonucleotides are hybridized to the bisulfite treated nucleic acid concurrently with the PCR primers. PCR amplification of the nucleic acid is terminated at the 5' position of the blocking probe, such that amplification of a nucleic acid is suppressed where the complementary sequence to the blocking probe is present. The probes may be designed to hybridize to the bisulfite treated nucleic acid in a methylation status specific manner. For example, for detection of methylated nucleic acids within a population of unmethylated nucleic acids, suppression of the amplification of nucleic acids which are unmethylated at the position in question would be carried out by the use of blocking probes comprising a 'CpA' or 'TpA' at the position in question, as opposed to a 'CpG' if the suppression of amplification of methylated nucleic acids is desired.

For PCR methods using blocker oligonucleotides, efficient disruption of polymerase-mediated amplification requires that blocker oligonucleotides not be elongated by the polymerase. Preferably, this is achieved through the use of blockers that are 3'-deoxyoligonucleotides, or oligonucleotides derivatized at the 3' position with other than a "free" hydroxyl group. For example, 3'-O-acetyl oligonucleotides are representative of a preferred class of blocker molecule.

Additionally, polymerase-mediated decomposition of the blocker oligonucleotides should be precluded. Preferably, such preclusion comprises either use of a polymerase lacking 5 '-3' exonuclease activity, or use of modified blocker oligonucleotides having, for example, thioate bridges at the 5 '-termini thereof that render the blocker molecule nuclease-resistant. Particular applications may not require such 5' modifications of the blocker. For example, if the blocker- and primer-binding sites overlap, thereby precluding binding of the primer (e.g., with excess blocker), degradation of the blocker oligonucleotide will be substantially precluded. This is because the polymerase will not extend the primer toward, and through (in the 5 '-3' direction) the blocker - a process that normally results in degradation of the hybridized blocker oligonucleotide.

A particularly preferred blocker/PCR embodiment, for purposes of the present invention and as implemented herein, comprises the use of peptide nucleic acid (PNA) oligomers as blocking oligonucleotides. Such PNA blocker oligomers are ideally suited, because they are neither decomposed nor extended by the polymerase. Preferably, therefore, the base sequence of said blocking oligonucleotides is required to comprise a sequence having a length of at least 9 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585, and sequences complementary thereto, wherein the base sequence of said oligonucleotides comprises at least one CpG, TpG or CpA dinucleotide.

The fragments obtained by means of the amplification can carry a directly or indirectly detectable label. Preferred are labels in the form of fluorescence labels, radionuclides, or detachable molecule fragments having a typical mass that can be detected in a mass spectrometer. Where said labels are mass labels, it is preferred that the labeled amplificates have a single positive or negative net charge, allowing for better detectability in the mass spectrometer. The detection may be carried out and visualized by means of, e.g., matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).

Matrix Assisted Laser Desorption/ionization Mass Spectrometry (MALDI-TOF) is a very efficient development for the analysis of biomolecules (Karas and Hillenkamp, Anal Chem., 60:2299-301, 1988). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapour phase in an unfragmented manner. The analyte is ionized by collisions with matrix molecules. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones. MALDI-TOF spectrometry is well suited to the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut and Beck, Current Innovations and Future Trends, 1 : 147-57, 1995). The sensitivity with respect to nucleic acid analysis is approximately 100-times less than for peptides, and decreases disproportionally with increasing fragment size. Moreover, for nucleic acids having a multiply negatively charged backbone, the ionization process via the matrix is considerably less efficient. In MALDI-TOF spectrometry, the selection of the matrix plays an eminently important role. For desorption of peptides, several very efficient matrixes have been found which produce a very fine crystallisation. There are now several responsive matrixes for DNA, however, the difference in sensitivity between peptides and nucleic acids has not been reduced. This difference in sensitivity can be reduced, however, by chemically modifying the DNA in such a manner that it becomes more similar to a peptide. For example, phosphorothioate nucleic acids, in which the usual phosphates of the backbone are substituted with thiophosphates, can be converted into a charge-neutral DNA using simple alkylation chemistry (Gut and Beck, Nucleic Acids Res. 23: 1367-73, 1995). The coupling of a charge tag to this modified DNA results in an increase in MALDI-TOF sensitivity to the same level as that found for peptides. A further advantage of charge tagging is the increased stability of the analysis against impurities, which makes the detection of unmodified substrates considerably more difficult.

In the fourth step of the method, the amplificates obtained during the third step of the method are analysed in order to ascertain the methylation status of the CpG dinucleotides prior to the treatment.

In embodiments where the amplificates were obtained by means of MSP amplification, the presence or absence of an amplificate is in itself indicative of the methylation state of the CpG positions covered by the primer, according to the base sequences of said primer.

Amplificates obtained by means of both standard and methylation specific PCR may be further analyzed by means of hybridization-based methods such as, but not limited to, array technology and probe based technologies as well as by means of techniques such as sequencing and template directed extension.

In one embodiment of the method, the amplificates synthesised in step three are subsequently hybridized to an array or a set of oligonucleotides and/or PNA probes. In this context, the hybridization takes place in the following manner: the set of probes used during the hybridization is preferably composed of at least 2 oligonucleotides or PNA-oligomers; in the process, the amplificates serve as probes which hybridize to oligonucleotides previously bonded to a solid phase; the non-hybridized fragments are subsequently removed; said oligonucleotides contain at least one base sequence having a length of at least 9 nucleotides which is reverse complementary or identical to a segment of the base sequences specified in the present Sequence Listing; and the segment comprises at least one CpG , TpG or CpA dinucleotide.

In a preferred embodiment, said dinucleotide is present in the central third of the oligomer. For example, wherein the oligomer comprises one CpG dinucleotide, said dinucleotide is preferably the fifth to ninth nucleotide from the 5'-end of a 13-mer. One oligonucleotide exists for the analysis of each CpG dinucleotide within the sequence according to SEQ ID NO: 1 to SEQ ED NO: 117, and the equivalent positions within SEQ ID NO: 118 to SEQ ID NO: 585 (according to Table 1). Said oligonucleotides may also be present in the form of peptide nucleic acids. The non-hybridized amplificates are then removed.The hybridized amplificates are then detected. In this context, it is preferred that labels attached to the amplificates are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.

In yet a further embodiment of the method, the genomic methylation status of the CpG positions may be ascertained by means of oligonucleotide probes that are hybridised to the bisulfite treated DNA concurrently with the PCR amplification primers (wherein said primers may either be methylation specific or standard).

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; also see United States Patent No. 6,331,393) employing a dual-labelled fluorescent oligonucleotide probe (TaqMan™ PCR, using an ABI Prism 7700 Sequence Detection System, Perkin Elmer Applied Biosystems, Foster City, California). The TaqMan™ PCR reaction employs the use of a nonextendible interrogating oligonucleotide, called a TaqMan™ probe, which, in preferred embodiments, is designed to hybridize to a CpG-rich sequence located between the forward and reverse amplification primers. The TaqMan™ probe further comprises a fluorescent reporter moiety and a quencher moiety covalently bound to linker moieties {e.g., phosphoramidites) attached to the nucleotides of the TaqMan™ oligonucleotide. For analysis of methylation within nucleic acids subsequent to bisulfite treatment, it is required that the probe be methylation specific, as described in United States Patent No. 6,331,393, (hereby incorporated by reference in its entirety) also known as the MethylLight assay. Variations on the TaqMan™ detection methodology that are also suitable for use with the described invention include the use of dual-probe technology (Lightcycler) or fluorescent amplification primers (Sunrise technology). Both these techniques may be adapted in a manner suitable for use with bisulfite treated DNA, and moreover for methylation analysis within CpG dinucleotides.

A further suitable method for the use of probe oligonucleotides for the assessment of methylation by analysis of bisulfite treated nucleic acids comprises the use of template- directed oligonucleotide extension, such as MS-SNuPE as described by Gonzalgo and Jones, Nucleic Acids Res. 25:2529-2531, 1997.

In yet a further embodiment of the method, the fourth step of the method comprises sequencing and subsequent sequence analysis of the amplificate generated in the third step of the method (Sanger F., et al, Proc Natl Acad Sci USA 74:5463-5467, 1977). In one preferred embodiment of the method the nucleic acids according to SEQ ID NO: 1 to SEQ ID NO: 117, are isolated and treated according to the first three steps of the method outlined above, namely: a) obtaining, from a subject, a biological sample having subject genomic DNA; b) extracting or otherwise isolating the genomic DNA; and c) treating the genomic DNA of b), or a fragment thereof, with one or more reagents to convert cytosine bases that are unmethylated in the 5-position thereof to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties; and wherein the subsequent amplification of d) is carried out in a methylation specific manner, namely by use of methylation specific primers or blocking oligonucleotides, and further wherein the detection of the amplificates is carried out by means of a real-time detection probes, as described above.

Wherein the patient is estrogen receptor positive it is particularly preferred that said nucleic acid sequences comprise SEQ ID NO: 97 to SEQ ID NO: 117 and sequences complementary thereto. Wherein the patient is estrogen receptor negative it is particularly preferred that said nucleic acid sequences comprise SEQ ID NO: 1 to SEQ ID NO: 96 and sequences complementary thereto.

Wherein the subsequent amplification of d) is carried out by means of methylation specific primers, as described above, said methylation specific primers comprise a sequence having a length of at least 9 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585, and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG dinucleotide.

Step e) of the method, namely the detection of the specific amplificates indicative of the methylation status of one or more CpG positions according to SEQ BD NO: 1 to SEQ ID NO: 1 17 is carried out by means of real-time detection methods as described above. In an alternative most preferred embodiment of the method the subsequent amplification of d) is carried out in the presence of blocking oligonucleotides, as described above. Said blocking oligonucleotides comprising a sequence having a length of at least 9 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG, TpG or CpA dinucleotide. Step e) of the method, namely the detection of the specific amplificates indicative of the methylation status of one or more CpG positions according to SEQ ID NO: 1 18 to SEQ ID NO: 585 is carried out by means of real-time detection methods as described above.

In a further preferred embodiment of the method the nucleic acids according to SEQ ID NO: 1 to SEQ ID NO: 117 are isolated and treated according to the first three steps of the method outlined above, namely: a) obtaining, from a subject, a biological sample having subject genomic DNA; b) extracting or otherwise isolating the genomic DNA; c) treating the genomic DNA of b), or a fragment thereof, with one or more reagents to convert cytosine bases that are unmethylated in the 5-position thereof to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties; and wherein d) amplifying subsequent to treatment in c) is carried out in a methylation specific manner, namely by use of methylation specific primers or blocking oligonucleotides, and further wherein e) detecting of the amplificates is carried out by means of a real-time detection probes, as described above.

Wherein the patient is estrogen receptor positive it is particularly preferred that said nucleic acid sequences comprise SEQ ID NO: 97 to SEQ ID NO: 117 and sequences complementary thereto. Wherein the patient is estrogen receptor negative it is particularly preferred that said nucleic acid sequences comprise SEQ ID NO: 1 to SEQ ID NO: 96 and sequences complementary thereto.

Wherein the subsequent amplification of c) is carried out by means of methylation specific primers, as described above, said methylation specific primers comprise a sequence having a length of at least 9 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG dinucleotide.

Additional embodiments of the invention provide a method for the analysis of the methylation status of genomic DNA according to the invention (SEQ ID NO: 1 to SEQ ID NO: 1 17, and complements thererof) without the need for pretreatment.

Wherein the patient is estrogen receptor positive it is particularly preferred that said genomic DNA sequences comprise SEQ ID NO: 97 to SEQ ID NO: 117 and sequences complementary thereto. Wherein the patient is estrogen receptor negative it is particularly preferred that said genomic DNA comprise SEQ ID NO: 1 to SEQ ID NO: 96 and sequences complementary thereto.

In the first step of such additional embodiments, the genomic DNA sample is isolated from tissue or cellular sources. Preferably, such sources include cell lines, histological slides, paraffin embedded tissues, body fluids, or tissue embedded in paraffin. In the second step, the genomic DNA is extracted. Extraction may be by means that are standard to one skilled in the art, including but not limited to the use of detergent lysates, sonification and vortexing with glass beads. Once the nucleic acids have been extracted, the genomic double-stranded DNA is used in the analysis.

In a preferred embodiment, the DNA may be cleaved prior to the treatment, and this may be by any means standard in the state of the art, in particular with methylation-sensitive restriction endonucleases.

In the third step, the DNA is then digested with one or more methylation sensitive restriction enzymes. The digestion is carried out such that hydrolysis of the DNA at the restriction site is informative of the methylation status of a specific CpG dinucleotide.

In the fourth step, which is optional but a preferred embodiment, the restriction fragments are amplified. This is preferably carried out using a polymerase chain reaction, and said amplificates may carry suitable detectable labels as discussed above, namely fluorophore labels, radionucleotides and mass labels. In the fifth step the amplificates are detected. The detection may be by any means standard in the art, for example, but not limited to, gel electrophoresis analysis, hybridization analysis, incorporation of detectable tags within the PCR products, DNA array analysis, MALDI or ESI analysis.

The presence, absence or prognosis of breast cancer can be determined by comparing the observed methylation (or expression) profile with that shown in the appropriate figure selected from figures 2 to 49. The person skilled in the art will be aware that mRNA and protein expression correlate inversely with methylation i.e. hypomethylation is equivalent to over-expression, whereas hypermethylation is equivalent to under-expression.

Nucleic acids

In order to enable this method, the invention further provides the modified sequences of one or a combination of genomic sequences taken from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 1 17.

The invention further provides oligonucleotides and/or PNA-oligomers for detecting cytosine methylations within said sequences. The present invention is based on the novel disclosure that the cytosine methylation patterns of said genomic DNAs are particularly suitable for improved treatment and monitoring of breast cancers and enables the person skilled in the art to determine a prognosis and/or diagnosis for a subject with said disorder based thereupon.

This objective according to the present invention is achieved using a nucleic acid containing a sequence of at least 18 bases in length of the treated genomic DNA according to one of SEQ ID NO: 1 18 to SEQ ID NO: 585 and sequences complementary thereto. In further preferred embodiments of the invention said nucleic acid is at least 50, 100, 150, 200, 250 or 500 base pairs in length of a segment of the nucleic acid sequence disclosed in SEQ ID NO: 118 to SEQ ID NO: 585. Particularly preferred is a nucleic acid molecule that is not identical or complementary to all or a portion of the sequences SEQ ID NO: 1 to SEQ ID NO: 117 or other naturally occurring DNA.

The disclosed invention provides treated nucleic acids, derived from genomic SEQ ID NO: 1 to SEQ ED NO: 117, wherein the treatment is suitable to convert at least one unmethylated cytosine base of the genomic DNA sequence to uracil or another base that is detectably dissimilar to cytosine in terms of hybridization. The genomic sequences in question may comprise one, or more, consecutive or random methylated CpG positions. Said treatment preferably comprises use of a reagent selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, and combinations thereof. In a preferred embodiment of the invention, the objective comprises analysis of a non-naturally occurring modified nucleic acid comprising a sequence of at least 16 contiguous nucleotide bases in length of a sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585, wherein said sequence comprises at least one CpG, TpA or CpA dinucleotide and sequences complementary thereto. The sequences of SEQ ID NO: 118 TO SEQ ID NO: 585 provide non-naturally occurring modified versions of the nucleic acid according to SEQ ID NO: 1 TO SEQ ID NO: 1 17, wherein the modification of each genomic sequence results in the synthesis of a nucleic acid having a sequence that is unique and distinct from said genomic sequence as follows. For each sense strand genomic DNA, e.g., SEQ ID NO:1, four converted versions are disclosed. A first version wherein "C" is converted to "T," but "CpG" remains "CpG" (Le., corresponds to case where, for the genomic sequence, all "C" residues of CpG dinucleotide sequences are methylated and are thus not converted); a second version discloses the complement of the disclosed genomic DNA sequence (i.e. αnrisense strand), wherein "C" is converted to "T," but "CpG" remains "CpG" (i.e., corresponds to case where, for all "C" residues of CpG dinucleotide sequences are methylated and are thus not converted). The 'upmethylated' converted sequences of SEQ ID NO: 1 to SEQ ID NO: 117 correspond to SEQ ID NO: 1 18 to SEQ ID NO: 351. A third chemically converted version of each genomic sequences is provided, wherein "C" is converted to "T" for all "C" residues, including those of "CpG" dinucleotide sequences (i.e., corresponds to case where, for the genomic sequences, all "C" residues of CpG dinucleotide sequences are Mnmethylated); a final chemically converted version of each sequence, discloses the complement of the disclosed genomic DNA sequence (i.e. αnf/sense strand), wherein "C" is converted to "T" for all "C" residues, including those of "CpG" dinucleotide sequences (i.e., corresponds to case where, for the complement (αnfϊsense strand) of each genomic sequence, all "C" residues of CpG dinucleotide sequences are wwmethylated). The 'downmethylated' converted sequences of SEQ ID NO: 1 to SEQ ID NO: 117 correspond to SEQ ID NO: 352 to SEQ ID NO: 585.

The modified nucleic acids could heretofore not be connected with the ascertainment of disease relevant genetic and epigenetic parameters. The object of the present invention is further achieved by an oligonucleotide or oligomer for the analysis of pretreated DNA, for detecting the genomic cytosine methylation state, said oligonucleotide containing at least one base sequence having a length of at least 9 nucleotides which hybridizes to or is identical to a pretreated genomic DNA according to SEQ ID NO: 1 18 to SEQ ID NO: 585. Preferably said oligomers comprise at least one T nucleotide wherein the corresponding base position within genomic (i.e. untreated) DNA is a C, said genomic equivalent of SEQ ID NO: 118 to SEQ ID NO: 585 is provided in the sequence listing (see Table 1).

It is particularly preferred that said oligonucleotides hybridise under moderately stringent and/or stringent hybridisation conditions to all or a portion of the sequences SEQ ID NO: 118 to SEQ ED NO: 585 , or to the complements thereof. The hybridising portion of the hybridizing nucleic acids is typically at least 9, 15, 20, 25, 30 or 35 nucleotides in length. However, longer molecules have inventive utility, and are thus within the scope of the present invention.

Preferably, the hybridising portion of the inventive hybridising nucleic acids is at least 95%, or at least 98%, or 100% identical to the sequence, or to a portion thereof of SEQ ID NO: 118 to SEQ ID NO: 585, or to the complements thereof.

Hybridising nucleic acids of the type described herein can be used, for example, as a primer (e.g., a PCR primer), or a diagnostic and/or prognostic probe or primer. Preferably, hybridisation of the oligonucleotide probe to a nucleic acid sample is performed under stringent conditions and the probe is 100% identical to the target sequence. Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions.

For target sequences that are related and substantially identical to the corresponding sequence of SEQ ID NO: 1 to SEQ ID NO: 117 (such as allelic variants and SNPs), rather than identical, it is useful to first establish the lowest temperature at which only homologous hybridisation occurs with a particular concentration of salt (e.g., SSC or SSPE).Then, assuming that 1% mismatching results in a I⁰C decrease in the Tm, the temperature of the final wash in the hybridisation reaction is reduced accordingly (for example, if sequences having > 95% identity with the probe are sought, the final wash temperature is decreased by 5°C).In practice, the change in Tm can be between 0.5⁰C and 1.5°C per 1% mismatch.

Examples of inventive oligonucleotides of length X (in nucleotides), as indicated by polynucleotide positions with reference to, e.g., SEQ ID NO:1, include those corresponding to sets (sense and antisense sets) of consecutively overlapping oligonucleotides of length X, where the oligonucleotides within each consecutively overlapping set (corresponding to a given X value) are defined as the finite set of Z oligonucleotides from nucleotide positions: n to (n + (X-I)); where n=l, 2, 3,...(Y-(X-I)); where Y equals the length (nucleotides or base pairs) of SEQ ID NO: 1(2994); where X equals the common length (in nucleotides) of each oligonucleotide in the set (e.g.,

X=20 for a set of consecutively overlapping 20-mers); and where the number (Z) of consecutively overlapping oligomers of length X for a given SEQ ID

NO of length Y is equal to 2994-(2O- 1). For example Z= 2994-19= 2975 for either sense or antisense sets of SEQ ID NO: 1, where X=20.

The oligomer probes according to the present invention constitute important and effective tools which, for the first time, make it possible to ascertain specific epigenetic parameters associated with prognosis and/or diagnosis of breast cancer patients. Said oligonucleotides thereby allow the improved treatment of breast cancers. The base sequence of the oligomers preferably contains at least one CpG, CpA or TpG dinucleotide.

The probes may 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 is within the middle third of said oligonucleotide e.g. the 5^m - 9"¹ nucleotide from the 5'-end of a 13-mer oligonucleotide; or in the case of PNA-oligomers, it is preferred for the cytosine of the CpG dinucleotide to be the 4^ - a¹" nucleotide from the 5 '-end of the 9-mer.

The oligomers according to the present invention are normally used in so called "sets" which contain a plurality of oligomers. In the case of the sets of oligonucleotides according to the present invention, it is preferred that at least one oligonucleotide is bound to a solid phase. It is further preferred that all the oligonucleotides of one set are bound to a solid phase.

The present invention further relates to a set of at least 5(oligonucleotides and/or PNA- oligomers) used for detecting the cytosine methylation state of genomic DNA, by analysis of said sequence or treated versions of said sequence (of the genes according to Table 1 as detailed in the sequence listing and sequences complementary thereto). These probes enable improved treatment and monitoring of breast cancers.

The set of oligomers may also be used for detecting single nucleotide polymorphisms (SNPs) by analysis of said sequence or treated versions of said sequence (of the sequences according to Table 1 as detailed in the sequence listing).

The sequences that form the basis of the present invention may also be used to form a "gene panel", i.e. a selection of a plurality of nucleic acid sequences comprising the particular genetic sequences of the present invention and/or their respective informative methylation sites. The formation of gene panels allows for a quick and specific analysis of specific aspects of breast cancer treatment. The gene panel(s) as described and employed in this invention can be used with surprisingly high efficiency for the treatment of breast cancers by diagnosis and/or prognosis of the patient.

In addition, the use of multiple CpG sites from a diverse array of genomic sequences allows for a relatively high degree of sensitivity and specificity in comparison to single gene prognostic and/or diagnostic tools.

According to the present invention, it is preferred that an arrangement of different oligonucleotides and/or PNA-oligomers (a so-called "array") made available by the present invention is present in a manner that it is likewise bound to a solid phase. This array of different oligonucleotide- and/or PNA-oligomer sequences can be characterized in that it is arranged on the solid phase in the form of a rectangular or hexagonal lattice. The solid phase surface is preferably composed of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold. However, nitrocellulose as well as plastics such as nylon which can exist in the form of pellets or also as resin matrices are suitable alternatives. Therefore, a further subject matter of the present invention is a method for manufacturing an array fixed to a carrier material for the improved treatment and monitoring of breast cancers. In said method at least one oligomer according to the present invention is coupled to a solid phase. Methods for manufacturing such arrays are known, for example, from US Patent 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.

A further subject matter of the present invention relates to a DNA chip for the improved treatment and monitoring of breast cancers. The DNA chip contains at least one nucleic acid according to the present invention. DNA chips are known, for example, in US Patent

5,837,832.

Kits

Moreover, an additional aspect of the present invention is a kit comprising: a means for determining methylation of at least one genomic sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 117. The means for determining methylation comprise preferably a bisulfite-containing reagent; one or a plurality of oligonucleotides consisting whose sequences in each case are identical, are complementary, or hybridise under stringent or highly stringent conditions to a 9 or more preferably up to 18 base long segment of a sequence selected from SEQ ID NO: 118 to SEQ ID NO: 585; and optionally instructions for carrying out and evaluating the described method of methylation analysis. In one embodiment the base sequence of said oligonucleotides comprises at least one CpG, CpA or TpG dinucleotide.

In a further embodiment, said kit may further comprise standard reagents for performing a CpG position-specific methylation analysis, wherein said analysis comprises one or more of the following techniques: MS-SNuPE, MSP, MethyLight™, HeavyMethyl, COBRA, and nucleic acid sequencing. However, a kit along the lines of the present invention can also contain only part of the aforementioned components.

In a preferred embodiment the kit may comprise additional bisulfite conversion reagents selected from the group consisting: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components. In a further alternative embodiment, the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimised for primer extension mediated by the polymerase, such as PCR. In another embodiment of the invention the kit further comprising means for obtaining a biological sample of the patient. Preferred is a kit, which further comprises a container suitable for containing the means for determining methylation of at least one genomic sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 1 17 in the biological sample of the patient, and most preferably further comprises instructions for use and interpretation of the kit results. In a preferred embodiment the kit comprises: (a) a bisulfite reagent; (b) a container suitable for containing the said bisulfite reagent and the biological sample of the patient; (c) at least one set of primer oligonucleotides containing two oligonucleotides whose sequences in each case are identical, are complementary, or hybridise under stringent or highly stringent conditions to a 9 or more preferably upto 18 base long segment of a sequence selected from SEQ ID NO: 1 18 to SEQ ID NO: 585; and optionally (d) instructions for use and interpretation of the kit results. In an alternative preferred embodiment the kit comprises: (a) a bisulfite reagent; (b) a container suitable for containing the said bisulfite reagent and the biological sample of the patient; (c) at least one oligonucleotides and/or PNA-oligomer having a length of at least 9 or 16 nucleotides which is identical to or hybridises to a pre-treated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto; and optionally (d) instructions for use and interpretation of the kit results.

In an alternative embodiment the kit comprises: (a) a bisulfite reagent; (b) a container suitable for containing the said bisulfite reagent and the biological sample of the patient; (c) at least one set of primer oligonucleotides containing two oligonucleotides whose sequences in each case are identical, are complementary, or hybridise under stringent or highly stringent conditions to a 9 or more preferably 18 base long segment of a sequence selected from SEQ ID NO: 1 18 to SEQ ID NO: 585; (d) at least one oligonucleotides and/or PNA-oligomer having a length of at least 9 or 16 nucleotides which is identical to or hybridises to a pre- treated nucleic acid sequence according to one of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto; and optionally (e) instructions for use and interpretation of the kit results. The kit may also contain other components such as buffers or solutions suitable for blocking, washing or coating, packaged in a separate container.

Typical reagents (e.g., as might be found in a typical COBRA™-based kit) for COBRA™ analysis may include, but are not limited to: PCR primers for at least one sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585; restriction enzyme and appropriate buffer; gene-hybridization oligo; control hybridization oligo; kinase labeling kit for oligo probe; and labeled nucleotides. Typical reagents (e.g., as might be found in a typical MethyLight ™ -based kit) for MethyLight™ analysis may include, but are not limited to: PCR primers for a sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585; bisulfite specific probes (e.g., TaqMan ™ or Lightcycler ^TM); optimized PCR buffers and deoxynucleotides; and Taq polymerase.

Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-based kit) for Ms- SNuPE™ analysis may include, but are not limited to: PCR primers for specific gene (or bisulfite treated DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE™ primers for a sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585; reaction buffer (for the Ms-SNuPE reaction); and labelled nucleotides.

Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for a sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585, optimized PCR buffers and deoxynucleotides, and specific probes.

Moreover, an additional aspect of the present invention is an alternative kit comprising a means for determining methylation of at least one genomic sequence selected from the group consisting of SEQ ID NO: 1 to SEQ DD NO: 117, wherein said means comprise preferably at least one methylation specific restriction enzyme; one or a plurality of primer oligonucleotides (preferably one or a plurality of primer pairs) suitable for the amplification of a sequence comprising at least one CpG dinucleotide of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117; and optionally instructions for carrying out and evaluating the described method of methylation analysis. In one embodiment the base sequence of said oligonucleotides are identical, are complementary, or hybridise under stringent or highly stringent conditions to an at least 18 base long segment of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117.

In a further embodiment said kit may comprise one or a plurality of oligonucleotide probes for the analysis of the digest fragments, preferably said oligonucleotides are identical, are complementary, or hybridise under stringent or highly stringent conditions to an at least 16 base long segment of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117.

In a preferred embodiment the kit may comprise additional reagents selected from the group consisting: buffer (e.g., restriction enzyme, PCR, storage or washing buffers); DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column) and DNA recovery components.

In a further alternative embodiment, the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimised for primer extension mediated by the polymerase, such as PCR. In another embodiment of the invention the kit further comprising means for obtaining a biological sample of the patient. In a preferred embodiment the kit comprises: (a) a methylation sensitive restriction enzyme reagent; (b) a container suitable for containing the said reagent and the biological sample of the patient; (c) at least one set of oligonucleotides one or a plurality of nucleic acids or peptide nucleic acids which are identical, are complementary, or hybridise under stringent or highly stringent conditions to an at least 9 base long segment of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117; and optionally (d) instructions for use and interpretation of the kit results.

In an alternative preferred embodiment the kit comprises: (a) a methylation sensitive restriction enzyme reagent; (b) a container suitable for containing the said reagent and the biological sample of the patient; (c) at least one set of primer oligonucleotides suitable for the amplification of a sequence comprising at least one CpG dinucleotide of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 1 17; and optionally (d) instructions for use and interpretation of the kit results.

In an alternative embodiment the kit comprises: (a) a methylation sensitive restriction enzyme reagent; (b) a container suitable for containing the said reagent and the biological sample of the patient; (c) at least one set of primer oligonucleotides suitable for the amplification of a sequence comprising at least one CpG dinucleotide of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117; (d) at least one set of oligonucleotides one or a plurality of nucleic acids or peptide nucleic acids which are identical , are complementary, or hybridise under stringent or highly stringent conditions to an at least 9 base long segment of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117 and optionally (e) instructions for use and interpretation of the kit results.

The kit may also contain other components such as buffers or solutions suitable for blocking, washing or coating, packaged in a separate container.

The invention further relates to a kit for use in detecting and/or providing a prognosis of breast cancer in a subject by means of methylation-sensitive restriction enzyme analysis. Said kit comprises a container and a DNA microarray component. Said DNA microarray component being a surface upon which a plurality of oligonucleotides are immobilized at designated positions and wherein the oligonucleotide comprises at least one CpG methylation site. At least one of said oligonucleotides is specific for the at least genomic sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 117 and comprises a sequence of at least 15 base pairs in length but no more than 200 bp of a sequence according to one of SEQ ID NO: 1 to SEQ ID NO: 117. Preferably said sequence is at least 15 base pairs in length but no more than 80 bp of a sequence according to one of SEQ ID NO: 1 to SEQ ID NO: 117. It is further preferred that said sequence is at least 20 base pairs in length but no more than 30 bp of a sequence according to one of SEQ ID NO: 1 to SEQ ID NO: 117.

Said test kit preferably further comprises a restriction enzyme component comprising one or a plurality of methylation-sensitive restriction enzymes.

In a further embodiment said test kit is further characterized in that it comprises at least one methylation-specific restriction enzyme, and wherein the oligonucleotides comprise a restriction site of said at least one methylation specific restriction enzymes.

The kit may further comprise one or several of the following components, which are known in the art for DNA enrichment: a protein component, said protein binding selectively to methylated DNA; a triplex-forming nucleic acid component, one or a plurality of linkers, optionally in a suitable solution; substances or solutions for performing a ligation e.g. ligases, buffers; substances or solutions for performing a column chromatography; substances or solutions for performing an immunology based enrichment (e.g. immunoprecipitation); substances or solutions for performing a nucleic acid amplification e.g. PCR; a dye or several dyes, if applicable with a coupling reagent, if applicable in a solution; substances or solutions for performing a hybridization; and/or substances or solutions for performing a washing step.

The described invention further provides a composition of matter useful for the prognosis of breast cancers. Said composition comprising at least one nucleic acid 18 base pairs in length of a segment of the nucleic acid sequence disclosed in SEQ ID NO: 118 to SEQ ID NO: 585, and one or more substances taken from the group comprising : 1-5 mM Magnesium Chloride, 100-500 μM dNTP, 0.5-5 units of taq polymerase, bovine serum albumen, an oligomer in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising in each case at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridizes under moderately stringent or stringent conditions to a pretreated genomic DNA according to one of the SEQ ID NO: 1 18 to SEQ ID NO: 585 and sequences complementary thereto. It is preferred that said composition of matter comprises a buffer solution appropriate for the stabilization of said nucleic acid in an aqueous solution and enabling polymerase based reactions within said solution.. Suitable buffers are known in the art and commercially available.

In further preferred embodiments of the invention said at least one nucleic acid is at least 50, 100, 150, 200, 250 or 500 base pairs in length of a segment of the nucleic acid sequence disclosed in SEQ ID NO: 118 to SEQ ID NO: 585.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the invention within the principles and scope of the broadest interpretations and equivalent configurations thereof. Table 1: Sequences according to the present invention

Example 1

AP-PCR. AP-PCR analysis was performed on twenty estrogen receptor positive samples of genomic DNA as shown in Table 2:

Table 2: Samples as used in AP-PCR experiments

SAMPLE NO. 55 1021 19494 Relapse 2

SAMPLE NO. 54 102119684 Relapse 3

SAMPLE NO. 50 101719518 Relapse 4

SAMPLE NO. 49 102119626 Relapse 5

SAMPLE NO. 45 102119543 Relapse 6

SAMPLE NO. 47 1021 19577 Relapse 7

SAMPLE NO. 05 101735209 Relapse 8

SAMPLE NO. 48 101719526 Relapse 9

SAMPLE NO. 51 102119676 Relapse 10

SAMPLE NO. 33 102119527 No relapse 11

SAMPLE NO. 34 102119569 No relapse 12

SAMPLE NO. 35 102119585 No relapse 13

SAMPLE NO. 44 102119600 No relapse 14

SAMPLE NO. 36 102119634 No relapse 15

SAMPLE NO. 37 102119618 No relapse 16

SAMPLE NO. 27 101735580 No relapse 17

SAMPLE NO. 41 102119501 No relapse 18

SAMPLE NO. 19 101735332 No relapse 19

SAMPLE NO. 16 101735225 No relapse 20

1. DNA isolation; genomic DNA was isolated from sample classes using the commercially available Wizzard™ kit;

2. Restriction enzyme digestion; each DNA sample was digested with 3 different sets of restriction enzymes for 16 hours at 37°C: Rsal (recognition site: GTAC); Rsal (recognition site: GTAC) plus HpaII (recognition site: CCGG; sensitive to methylation); and Rsal (recognition site: GTAC ) plus Mspl (recognition site: CCGG; insensitive to methylation);

3. AP-PCR analysis; each of the restriction digested DNA samples was amplified with the primer sets according to TABLE 3 at a 40⁰C annealing temperature, and with [³²P] -dATP.

4. Polyacrylamide Gel Electrophoresis; 1.6 μl of each AP-PCR sample was loaded on a 5% Polyacrylamide sequencing-size gel, and electrophoresed for 4 hours at 130 Watts, prior to transfer of the gel to chromatography paper, covering the transferred gel with saran wrap, and drying in a gel dryer for a period of about 1-hour;

5. Autoradiographic Film Exposure; film was exposed to dried gels for 20 hours at -8O⁰C, and then developed. Glogos was added to the dried gel and exposure was repeated with new film. The first autorad was retained for records, while the second was used for excising bands; and

6. Bands corresponding to differential methylation were visually identified on the gel. Such bands were excised and the DNA therein was isolated and cloned using the Invitrogen TA Cloning Kit.

7. The differentially methylated positions were then sequenced according to Example 3.

TABLE 3. Primers used according to the AP-PCR Protocol Example 1

Example 2: DMH analysis of breast neoplastic tissue.

Figure 1 illustrates the general methodology of DMH (differential methylation hybridisation). 'Aggressive' and 'non-aggressive' samples are fragmented by means of a non-methylation specific restriction enzymatic digest (Step 1). The fragments are then ligated to adaptors which enable the enzymatic amplification of the fragments (Step 2). The ligated fragments are then digested by means of a methylation specific digest (Step 3). The digests are then enzymatically amplified (Step 4) and are then hybridised to a chip (Step 5). Said chip comprises a library of detection oligonucleotidesdesigned on the basis of an in-silico digest of the human genome. Differences in the hybridisation patterns between the 'aggressive' and 'non-aggressive' samples enable the identification of differentially methylated restriction enzyme sites.

1. DNA isolation

Samples

Tissue samples were obtained from 17 estrogen receptor negative patients as shown in Table

4. All patients (9) with a disease free survival of less than 35 months were designated as

'aggressive', 'negative outcome' or 'poor prognosis' whereas all patients with a disease free survival of 90 months or greater were designated as 'non aggressive', 'positive outcome' or

'good prognosis'. Three peripheral blood lymphocyte samples were also analysed as controls.

Table 4: Samples analysed according to Example 1.*

* n.a. = not applicable

2: Generation of DNA-microarrays with oligonucleotides.

The determination of the sequences of the different oligonucleotides to be immobillised on the microarray was carried out as follows. The Ensembl Human Genome database version NCBI 33 provided all sequence upon which the microarray was designed. It was downloaded from the server (www.ensembl.org) in the fasta format. This file included all available contigs of the human genome. All oligonucleotides were designed by the means of a computer program, i.e. "in silico".

Said program simulates the digestion of the human genome by means of selected restriction enzymes, both methylation specific and non-methylation specific (i.e. where restriction is independent of the methylation status of the restriction site). The program then generates the sequence of all restriction digest fragments, a subset of these fragments are then selected to be used on the microarray. Fragments with less than 100 and more than 1200 base pairs are (virtually) rejected. The remaining fragments are selected on the basis of the presence of the recognition site of at least one of the methylation specific restriction enzymes as used previously, of these selected fragments all fragments comprising less than 20% repeats are further selected.

The remaining fragments may be further selected either using further selection criteria or randomly selected. The finally selected group of fragments is then synthesised on the microarray surface according to conventional means.

3: Restriciton digest of the DNA samples as isolated in Example 1. The genomic DNA was prepared for hybridisation to the microarray.

Step 1 : 2 μg of each of the isolated genomic DNA sampleswas digested using 5 units of the non-methylation specific restriction enzymes Msel, BJ «1 and Csp6 (obtainable from New England Biolab and MBI Fermentas) for 16 hours at 37⁰C according to the manufacturer's instructions. Subsequently, the restriction enzymes were inactivated at 65⁰C for 20 minutes.

Step 2: Thereafter, purification was performed with the QuiaQuick PCR product purification column kit (Quiagen, Hilden, Germany). Fragments shorter than 40 bases were rejected according to the manufacturers information. However it cannot be excluded that larger fragments up to a size of about 100 bp also rejected. Subsequently, the ligation of adapters (or linkers) was performed according to the procedure described by Huang et al., Hum MoI Genet, 8 (3): 459-470 (1999), said protocol adapted as follows. The fragmented DNA was mixed with 500 pmol adapter, 400 units T4 DNA ligase (New England Biolabs), the volume recommended by the manufacturer of the ligase 10x buffer and ATP and then incubated for 16 hours at 16°C. Beforehand the adaptors were generated by denaturation of an equimolar mixture of the oligonucleotides H24 (5'-AGG CAA CTG TGC TAT CCG AGG GAT-3') and H12-M (5'-CGA TCC CTC GGA-3') for 5 minutes at 95°C and a subsequent stepwise cooling down to 25°C. Thereafter, the ligated DNA is purified with the QuiaQuick PCR product purification column kit (Quiagen, Hilden, Germany).

After this, the purified ligated DNA was digested with 10 units each of the methylation sensitive (i.e. methylation specific) restriction enzymes Bst\J\, Hap II, H/ryCH4iV and HmPl (obtainable from New England Biolabs) for 8 hours at 37°C and subsequently for 8 hours at 60⁰C according to the manufacturers instructions. Each of the ligated fragments was then amplified, in duplicate reactions. About 10-100 ng were used for a PCR reaction, which amplifies unrestricted DNA fragments in the range of 50-1000 bp , said PCR products being fluorescently labelled due to the use of Cy5 or Cy3 labelled primers. The PCR reaction mixture included 350 μM dNTPs, 5 units Deep Vent (exo- ) DNA polymerase, 10 μl 10x buffer and 5 % DMSO in a volume of 100 μl.

4: Hybridisation of samples on the DNA microarray

The labelled amplificates generated as above were hybridised to the DNA microarray synthesised as above. The hybridisation and detection was carried out according to the instructions in "Gene Chip Mapping Assay Manual" by Affymetrix Inc., in particular chapter 5 (page 69-70), as well as chapter 6 "Washing, Staining & Scanning" (page 75-92).

Each sample thereby generated an individual hybridisation pattern, from which methylation differences between aggressive and non-aggressive tissue or between PBL and tumor tissue could be deduced by determining DNA fragment sequences which show differential hybridisation signals between samples of the compared tissues. Furthermore, for each identified DNA sequence , a corresponding cDNA may also be identified, the implication being that said cDNA may be differentially expressed between said groups.

The differentially methylated fragments were then sequenced to provide further information concerning the extent of methylation according to Example 3.

Example 3: Sequencing

Sequencing of all differentially methylated sequences as identified using AP-PCR and DMH (as described above) was carried out upon bisulfite treated DNA to confirm methylation. Primers used for said sequencing and pre-amplification are shown in Table 5.

Samples 5, 16, 19, 27, 33, 34, 35, 36, 37, 41, 44, 45, 47, 48, 49, 50, 51, 53, 54, and 55 of Example 1 were analysed. The majority of samples according to Example 2 were analysed, however, due to technical reasons (e.g. insufficient DNA) not all samples were analysed.

Table 5: Sequencing primers, matrices, amplificates and genomic equivalent of amplificates.

Total genomic DNA of all samples was bisulfite treated converting unmethylated cytosines to uracil. Methylated cytosines remained conserved. Bisulfite treatment was performed with minor modifications according to the protocol described in Olek et al. (1996) The bisulfite treated DNA was then PCR amplified and the PCR products were sequenced. Sequence data was obtained using ABI 3700 sequencing technology. The G-rich primer was used for sequencing. Percentage methylation was calculated using the Applicant's proprietary bisulphite sequence sequencing trace analysis program (See WO 2004/000463 for further information).

Amplification

Fragments of interest were amplified using the following conditions

PCR Reaction solution :

Taq 5U/μl 0,2 dNTPs 25mM each 0,2

10x buffer 2,5 water 10,1 primer (6,25μM) 2

DNA (lng/μl) 10

Cycling conditions: 15min 95°C

30s 95°C 30s 58°C l :30min 72°C

40 cycles

Sequencing

ExoSAP-IT Reaction solution: 4μl PCR product + 2μl ExoSAP-IT 45min/37°C and 15min/95°C

Cycle sequencing:

480 μl H2O

+ 960 μl Sanger Buffer

+ 960 μl dNTPs

+ 240 μl Big Dye V3.1

l lμl

+

4 μl Primer (2pmol/μl)

+

5μl ExoSAP-IT product

Cycling

2 min 96°C, 26 cycles a (30 s/96°C, 15s/55°C, 4 min/60°C)

Purification

A 96 well MultiScreen (Millipore) plate was filled with Sephadex G50 (Amersham) using an appropriate admeasure device. 300μl water were added to each well and incubated 3h at 4°C.

Water was removed by spinning for 5minutes at 91Og. Cycle sequencing product was loaded to the plate and purified by spinning for 5min at 91Og. lOμl of formamide was added to each eluate. Results:

All PCRs yielded a product.

Figures 2 to 49 provide matrices produced from bisulfite sequencing data analysed by the applicant's proprietary software (See WO 2004/000463 for further information). Each column of the matrices represent the sequencing data for one amplificate. Each row of a matrix represents a single CpG site within the fragment and each column represents an individual DNA sample, cell line sample or whole blood sample. The bar on the left represents a scale of the percent methylation, with the degree of methylation represented by the shade of each position within the column from black representing 100% methylation to light grey representing 0% methylation. White positions represented a measurement for which no data was available.

In figures 29 to 49 the non-aggresssive samples group (i.e. non-relapse patients) is marked 'A', the aggresssive samples group (i.e. relapsed patients) is marked 'B', the peripheral blood lymphocytes sample group is marked 'C.

See Table 5 to determine the relevant figure for each amplificate.

Claims

Claims

1. A method for determining the presence or absence of a breast cell proliferative disorder and/or providing a prognosis of a subject with a breast cell proliferative disorder comprising the following steps of: a) obtaining a genomic DNA sample from the subject b) determining the expression status one or a combination of genomic sequences taken from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 117 c) determining therefrom the presence or absence of a breast cell proliferative disorder and/or the prognosis thereof in said subject.

2. A method according to claim 1 wherein said subject is estrogen receptor positive and the genomic sequences of b) are selected from the group consisting of SEQ ID NO: 97 to SEQ ID NO: 117.

3. A method according to claim 1 wherein said subject is estrogen receptor negative and the genomic sequences of b) are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 96.

4. A method according to claims 1 to 3 wherein said prognosis is determined in terms of at least one term selected from the group consisting overall patient survival, disease- or relapse-free survival, tumor-related complications and rate of progression of tumour.

5. A method according to claims 1 to 4 wherein said breast cell proliferative disorder is selected form the group consisting ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, comedocarcinoma, inflammatory carcinoma, mucinous carcinoma, scirrhous carcinoma, colloid carcinoma, tubular carcinoma, medullary carcinoma, metaplastic carcinoma, and papillary carcinoma and papillary carcinoma in situ, undifferentiated or anaplastic carcinoma and Paget' s disease of the breast.

6. A method according to claims 1 to 5 wherein said expression is determined by analysis of at least one of mRNA expression, LOH, protein expression.

7. A method according to claims 1 to 5 wherein said expression is determined by determining the methylation status of one or more CpG positions within said genes and/or regulatory regions thereof.

8. A method for determining the presence or absence of a breast cell proliferative disorder and/or providing a prognosis of a subject with a breast cell proliferative disorder comprising the following steps of: a) extracting or otherwise isolating genomic DNA from a biological sample obtained from the subject, b) treating the genomic DNA of a), or a fragment thereof, with one or more reagents to convert cytosine bases that are unmethylated in the 5-position thereof to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties; c) contacting the treated genomic DNA, or the treated fragment thereof, with an amplification enzyme and at least one primer comprising, a contiguous sequence of at least 9 nucleotides that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 1 18 to SEQ ID NO: 585, and complements thereof, wherein the treated genomic DNA or the fragment thereof is either amplified to produce at least one amplificate, or is not amplified; and d) determining, based on the presence or absence of, or on the quantity or on a property of said amplificate, the methylation state of at least one CpG dinucleotide sequence of at least one genomic sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 117, or an average, or a value reflecting an average methylation state of a plurality of CpG dinucleotide sequences of at least one genomic sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 117 and e) determining from said methylation state the presence or absence of a breast cell proliferative disorder and/or the prognosis thereof in said subject.

9. The method of claim 8, wherein treating the genomic DNA, or the fragment thereof in b), comprises use of a reagent selected from the group comprising of bisulfite, hydrogen sulfite, disulfite, and combinations thereof.

10. The method of claim 8, wherein contacting or amplifying in c) comprises use of at least one method selected from the group comprising: use of a heat-resistant DNA polymerase as the amplification enzyme; use of a polymerase lacking 5'-3' exonuclease activity; use of a polymerase chain reaction (PCR); generation of an amplificate nucleic acid molecule carrying a detectable label.

11. The method of any of claim 1 to 10, wherein the biological sample obtained from the subject is selected from the group comprising cell lines, histological slides, paraffin embedded tissues, biopsies, tissue embedded in paraffin, bodily fluids, nipple aspirate and blood and combinations thereof.

12. The method of claim 9, further comprising in step d) the use of at least one nucleic acid molecule or peptide nucleic acid molecule comprising in each case a contiguous sequence at least 9 nucleotides in length that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585, and complements thereof, wherein said nucleic acid molecule or peptide nucleic acid molecule suppresses amplification of the nucleic acid to which it is hybridized.

13. The method of claim 9, wherein determining in d) comprises hybridization of at least one nucleic acid molecule or peptide nucleic acid molecule in each case comprising a contiguous sequence at least 9 nucleotides in length that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585, and complements thereof.

14. The method of claim 13, wherein at least one such hybridizing nucleic acid molecule or peptide nucleic acid molecule is bound to a solid phase.

15. The method of claim 13, further comprising extending at least one such hybridized nucleic acid molecule by at least one nucleotide base.

16. The method of claim 9, wherein determining in d), comprises sequencing of the amplificate.

17. The method of claim 9, wherein contacting or amplifying in c), comprises use of methylation-specific primers.

18. A method for determining the presence or absence of a breast cell proliferative disorder and/or providing a prognosis of a subject with a breast cell proliferative disorder comprising the following steps of: a) extracting or otherwise isolating genomic DNA from a biological sample obtained from the subject b) digesting the genomic DNA of a), or a fragment thereof, with one or more methylation sensitive restriction enzymes; c) contacting the DNA restriction enzyme digest of b), with an amplification enzyme and at least two primers suitable for the amplification of a sequence comprising at least one CpG dinucleotide of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 117, d) determining, based on a presence or absence of an amplificate the methylation state or level of at least one CpG dinucleotide of a sequence selected from the group consisting SEQ ID NO: 1 to SEQ ID NO: 117, whereby at least one of determining the presence or absence of a breast cell proliferative disorder and/or the prognosis thereof in said subject, is at least in part, afforded.

19. The method according to claim 18 wherein the presence or absence of an amplificate is determined by means of hybridization to at least one nucleic acid or peptide nucleic acid which is identical, complementary, or hybridizes under stringent or highly stringent conditions to an at least 16 base long segment of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 1 17.

20. A nucleic acid, comprising at least 18 contiguous nucleotides of a treated genomic DNA sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585, and sequences complementary thereto, wherein the treatment is suitable to convert at least one unmethylated cytosine base of the genomic DNA sequence to uracil or another base that is detectably dissimilar to cytosine in terms of hybridization.

21. A nucleic acid, comprising at least 50 contiguous nucleotides of a DNA sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 585, and sequences complementary thereto.

22. A kit suitable for performing the method according to claim 1 comprising (a) a bisulfite reagent; (b) a container suitable for containing the said bisulfite reagent and the biological sample of the patient; (c) at least one set of oligonucleotides containing two oligonucleotides whose sequences in each case are identical , are complementary, or hybridize under stringent or highly stringent conditions to a 9 or more preferably 18 base long segment of a sequence selected from SEQ ID NO: 118 to SEQ ID NO: 585.

23. A composition comprising: i. a nucleic acid comprising a sequence at least 18 bases in length of a segment of the chemically pretreated genomic DNA according to one of the sequences taken from the group comprising of SEQ ID NO: 118 to SEQ ID NO: 585 and sequences complementary thereto, and ii. a buffer comprising at least one of the following substances: magnesium chloride, dNTP, of taq polymerase, an oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising in each case at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridizes under moderately stringent or stringent conditions to a pre-treated genomic DNA according to one of the of SEQ ED NO: 1 18 to SEQ ID NO: 585 and sequences complementary thereto.

24. The use of a method according to claims 1 to 19, a nucleic acid according to claims 20 or 21, a kit according to claim 22 or a composition of matter according to claim 23 in determining the presence or absence of a breast cell proliferative disorder and/or the prognosis thereof.