WO2007137873A1 - Method and nucleic acids for the improved treatment of breast cancers - Google Patents

Abstract

The present invention relates to methods for prognosis and/or predicted outcome of treatment of breast cell proliferative disorder patients, in particular breast carcinoma. This is achieved by determining the expression level of STMN1 of the genomic DNA associated with the gene STMN1.

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, morphology (i.e. grade) 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. Current guidelines recommend adjuvant chemotherapy followed by endocrine therapy for most of the women with node-negative, steroid hormone receptor-positive breast cancer. This recommendation is based on a significant reduction of the risk of disease recurrence by chemotherapy in this population, independent of the risk reduction by endocrine therapy. Yet, these patients have a rather good prognosis and in general derive significant benefit from endocrine treatment. Thus, after endocrine treatment, the majority will never experience a recurrence and would be adequately treated by tamoxifen alone. Unfortunately, traditional prognostic factors are not suited to identify patients at low risk in order to avoid over-treatment by chemotherapy in the majority of hormone receptor-positive, node-negative patients. Moreover, in postmenopausal hormone receptor-positive patients, aromatase inhibitors have become a valid option. However, it is still unclear which patients will be sufficiently treated by routine adjuvant treatments such as tamoxifen and who will benefit from alternative therapies aromatase inhibitors- an important question given the lack of information on long-term side effects and the increased costs for aromatase inhibitors. Accordingly there is a long felt need in the art for improved methods of providing a prognosis of breast cancer patients to prevent over treatment and accurately select patients who will benefit from specific treatments.

The gene STMN1 is a known marker for prognosis, both with, or without Tamoxifen treatment. PCT/EP03/10881 discloses that methylation of the gene STMN1 is a marker for response to breast cancer treatments targeting the estrogen pathway(s) (e.g. Tamoxifen), furthermore PCT/EP2004/014170 discloses that methylation of the gene STMN1 is also a general prognostic marker of breast cancer. In both cases methylation of CpG positions within or associated with the gene STMN 1 were determined to be a characteristic of poor outcome. CpG methylation of genes is a common feature of eukaryotic organisms, and enables the control of gene expression. The gene STMN1 is located on chromosome 1 and multiple mRNA transcript variants of the gene are known. The gene is alternatively known as Stathmin; Phosphoprotein p19; pp19; Oncoprotein 18; Op18; Leukemia-associated phosphoprotein p18; pp17; Prosolin; Metablastin and Protein Pr22. The gene stathmin (NM_005563) codes for an oncoprotein 18 (also commonly referred to as stathmin), a conserved cytosolic phosphoprotein that regulates microtubule dynamics. The protein is highly expressed in a variety of human malignancies. In human breast cancers the stathmin gene has shown to be up-regulated in a subset of the tumours. Brief description of Figures Figures 1 to 4 provide an annotation of the 5 currently known transcript variants of the gene STMN1 as taken from Ensembl version 35. Exons are shown in alternating bold and plain type, and the translation of the codons is shown below the transcript sequence. Non-coding regions are highlighted.

Figure 1 (A-B) provides an annotated overview of the transcript variant of SEQ ID NO: 2 (ENST00000355080).

Figure 2 (A-B) provides an annotated overview of the transcript variant of SEQ ID NO: 3 herein referred to as variant B (ENST00000296492).

Figure 3 provides an annotated overview of the transcript variant of SEQ ID NO: 4 herein referred to as variant C (ENST00000306732). Figure 4 provides an annotated overview of the transcript variant of SEQ ID NO: 5 herein referred to as variant C (ENST00000306732).

Figure 5 provides an annotated overview of the transcript variant of SEQ ID NO: 6 is currently uncharacterised but will be herein referred to as variant D (ENST00000354925).

Figure 6 provides an annotated figure of the gene STMN1 (SEQ ID NO: 1) showing alternative transcripts of the gene.

DESCRIPTION

The present invention provides a novel method for analysis of expression of the gene STMN 1 that has utility for the improved treatment of patients with cell proliferative disorders of the breast tissues. The present invention provides a prognostic marker for breast cancer. According to the present invention overexpression of the gene STMN1 is associated with negative outcome in patients with breast cancer. STMN 1 overexpression, is associated with poor outcome in patients treated with therapies targeting the estrogen pathways, furthermore, STMN 1 overexpression is associated with poor prognosis in patients who have not been treated with said treatment. Accordingly it is herein disclosed that STMN 1 overexpression is an indicator of poor prognosis that is of utility in determining treatment strategy of breast cancer patients. It is particularly preferred that when determining suitable adjuvant treatment strategies patients with a poor prognosis are recommended for chemotherapeutic treatment, whereas patients with a good prognosis may be treated solely by adjuvant treatment targeting the estrogen pathways or spared treatment.

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 rate of progression of tumor or metastases, wherein a decrease in any of said factors (with the exception of increased tumor-related complications rate of progression) as relative to a pre-determined level, is a 'negative' outcome and increase thereof is a 'positive' outcome. A decrease in tumor-related complications and/or rate of progression of tumor or metastases as relative to a pre-determined level, is considered a 'positive' outcome and increase thereof is a 'negative' 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.

In one aspect the prognostic marker according to the present invention is 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 after 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.

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.

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.

The herein described markers have further utility in predicting outcome of a patient after treatment with a therapy comprising one or more treatments which target the estrogen receptor pathway or are involved in estrogen metabolism, production or secretion. This will hereinafter also be referred to as a 'predictive' marker. 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. Over expression of the gene STMN1 , in particular of the B variant, is associated with negative outcome of patients treated accordingly. Patients with predicted positive outcome (i.e. under expression) after said treatment will accordingly have a decreased absolute reduction of risk of recurrence and death after treatment with chemotherapy. Patients with predicted negative outcome (i.e. hypermethylation or under expression) after said treatment will accordingly have a relatively larger absolute reduction of risk of recurrence and death after treatment with chemotherapy. Accordingly patients with a negative outcome after said treatment will be considered more suitable candidates for chemotherapeutic treatment than patients with a positive outcome. Patients with a positive outcome may accordingly be prevented from over prescription of chemotherapeutic treatment. It is particularly preferred that said patients are estrogen receptor positive.

The method according to the invention may be used for the 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.

The present invention discloses a method for the use of the gene STMN1 as a prognostic and/or predictive marker for breast cancer. The sequence of said gene is disclosed in SEQ ID NO: 1 , it is preferred that any transcript thereof or polypeptide transcribed therefrom is analysed and a prognostic and/or predictive outcome of breast cancer in a subject is determined. Particularly preferred is the analysis of an mRNA transcript as disclosed in SEQ ID NO: 2 to SEQ ID NO: 6.

Said method may be enabled by means of any analysis of the expression of a RNA transcribed therefrom or polypeptide or protein translated from said RNA, preferably by means of mRNA expression analysis or polypeptide expression analysis. Particularly preferred is the analysis of an mRNA transcript as disclosed in SEQ ID NO: 2 to SEQ ID NO: 6. Accordingly the present invention also provides prognostic assays and methods, both quantitative and qualitative for detecting the expression of the gene STMN 1 in a subject with a breast cell proliferative disorder and determining therefrom upon the prognosis and/or prediction of treatment outcome in said subject.

Aberrant expression of mRNA transcribed from the gene STMN1 are associated with prognosis and/or prediction of treatment outcome of breast carcinoma. Over expression is associated with poor prognosis and/or prediction of treatment outcome, under expression is associated with good prognosis and/or prediction of treatment outcome.

To detect the presence of mRNA encoding a gene or genomic sequence, a sample is obtained from a patient. The sample may be any suitable sample comprising cellular matter of the tumor, most preferably the primary tumor. Suitable sample types include tumors cells or cell lines, histological slides, paraffin embedded tissues, biopsies, tissue embedded in paraffin, bodily fluids (such as but not limited to nipple aspirate and blood) or another suitable biological sample and all possible combinations thereof.

In a particularly preferred embodiment of the method said source is primary tumor tissue. The sample may be treated to extract the RNA contained therein. The resulting RNA 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 oligo 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. For the present invention particularly preferred is the analysis of an mRNA transcript as disclosed in SEQ ID NO: 2 to SEQ ID NO: 6.

In Northern blot analysis total or poly(A)+ mRNA is run on a denaturing agarose gel and detected by hybridization 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, massa 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 labeling, which generate 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 hybridized in solution. Following hybridization, 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 hybridization of the fluorescently labelled cDNA (comprising the sequences to be interrogated) to the known genes immobilized on the glass slide. After hybridization, arrays are scanned using a fluorescent microarray scanner. Analyzing 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. 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 genes of interest (in this case STMN 1) and tend to be shorter sequences in the range of 25-70 nucleotides. 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.

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 labeling of the cDNA can be accomplished by either direct labeling or indirect labeling methods. During direct labeling, fluorescently modified nucleotides (e.g., Cy^®3- or Cy^®5-dCTP) are incorporated directly into the cDNA during the reverse transcription. Alternatively, indirect labeling 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 labeled 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-hybridization using a fluorescent microarray scanner. The fluorescent intensity of each spot indicates the level of expression for that gene; bright spots correspond to strongly expressed genes, while dim spots indicate weak expression.. Once the images are obtained, the raw data must be analyzed. First, the background fluorescence must be subtracted from the fluorescence of each spot. The data is then normalized to a control sequence, such as an exogenously added RNA, or a housekeeping gene panel to account for any nonspecific hybridization, array imperfections or variability in the array setup, cDNA labeling, hybridization or washing. Data normalization allows the results of multiple arrays to be compared. 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 encoded by the gene STMN1 are associated with breast cell proliferative disorder prognosis and/or treatment outcome.

Accordingly over or under expression of said polypeptides are associable with the prognosis and to treatment outcome of breast cancers. Over expression is associated with poor prognosis and under expression is associated with good prognosis.

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 the STMN1 gene.

Such antibodies are useful for breast cancer prognostic and/or predictive applications. In certain embodiments production of monoclonal or polyclonal antibodies can be induced by the use of the coded polypeptide as an antigene. Such antibodies may in turn be used to detect expressed polypeptides as markers for breast cell proliferative disorder prognosis. 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 standar 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 generic protein (e.g. milk protein) to bind remaining sticky places on the membrane. 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 colored 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). 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.

In the final step of the method the prognosis of the patient is determined, whereby overexpression is indicative of negative prognosis. The term overexpression shall be taken to mean expression at a detected level greater than a pre-determined cut off which may be selected from the group consisting of the mean, median or an optimised threshold value. Another aspect of the invention provides a kit for use in providing a prognosis of a subject with a breast cell proliferative disorder, comprising: a means for detecting STMN1 polypeptides. 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 for use in determining treatment strategy for a patient with a breast cell proliferative disorder, comprises: (a) a means for detecting STMN1 polypeptides; (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. Another aspect of the invention relates to a kit for use in providing a prognosis of a subject with a breast cell proliferative disorder, said kit comprising: a means for measuring the level of transcription of the gene STMN 1. 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 STMN 1 particularly preferred oligonucleotides or polynucleotides which are as disclosed in SEQ ID NO: 2 to SEQ ID NO: 6.. 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 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 for use in determining treatment strategy for a patient with a breast cell proliferative disorder comprises (a) a plurality of oligonucleotides or polynucleotides able to hybridise under stringent or moderately stringent conditions to the transcription products of the gene STMN1; (b) a container suitable for containing the oligonucleotides or polynucleotides and a biological sample of the patient comprising the transcription products wherein the oligonucleotides or polynucleotide 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. The kit may also contain other components such as hybridization 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 optimized for primer extension mediated by the polymerase, such as PCR. Most preferably a kit according to the embodiments of the present invention is used for the determination of expression step of the methods according to other aspects of the invention.

In a further aspect, the invention provides a further method for providing a prognosis of a subject with a breast cell proliferative disorder comprising the following steps. In the first step of the method a breast tumor sample is obtained from the subject. 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 oligo 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 hybridization 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, massa 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 labeling, 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 hybridized in solution. Following hybridization, 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 hybridization of the fluorescently labelled cDNA (comprising the sequences to be interrogated) to the known genes immobilized on the glass slide. After hybridization, arrays are scanned using a fluorescent microarray scanner. Analyzing 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. 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 genes of interest (in this case STMN1) and tend to be shorter sequences in the range of 25-70 nucleotides. 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.

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 labeling of the cDNA can be accomplished by either direct labeling or indirect labeling methods. During direct labeling, fluorescently modified nucleotides (e.g., Cy^®3- or Cy^®5-dCTP) are incorporated directly into the cDNA during the reverse transcription. Alternatively, indirect labeling 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 labeled 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-hybridization using a fluorescent microarray scanner. The fluorescent intensity of each spot indicates the level of expression for that gene; bright spots correspond to strongly expressed genes, while dim spots indicate weak expression. Once the images are obtained, the raw data must be analyzed. First, the background fluorescence must be subtracted from the fluorescence of each spot. The data is then normalized to a control sequence, such as an exogenously added RNA, or a housekeeping gene panel to account for any nonspecific hybridization, array imperfections or variability in the array setup, cDNA labeling, hybridization or washing. Data normalization allows the results of multiple arrays to be compared. 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 encoded by the gene STMN 1 are associated with breast cell proliferative disorder prognosis and/or treatment outcome.

Accordingly over or under expression of said polypeptides are associable with the prognosis and to treatment outcome of breast cancers. Over expression is associated with poor prognosis and under expression is associated with good prognosis.

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 the STMN 1 gene.

Such antibodies are useful for breast cancer prognostic and/or predictive applications. In certain embodiments production of monoclonal or polyclonal antibodies can be induced by the use of the coded polypeptide as an antigene. Such antibodies may in turn be used to detect expressed polypeptides as markers for breast cell proliferative disorder prognosis. 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)₁ 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 generic protein (e.g. milk protein) to bind remaining sticky places on the membrane. 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 colored 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). 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.

In a preferred embodiment said method is achieved by contacting the nucleic acid of the gene STMN1 and/or its regulatory regions, or sequences thereof according to SEQ ID NO: 1 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: Preferably, said 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 (such as but not limited to urine, nipple aspirate and blood) and all possible combinations thereof. In a particularly preferred embodiment of the method said source is blood. 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 30⁰C and 70°C, whereby the temperature is increased to over 85⁰C 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/EP2004/011715 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 of SEQ ID NO: 7 to SEQ ID NO: 10 and sequences complementary thereto.

In an alternate embodiment of the method, the methylation status of preselected CpG positions within SEQ ID NO: 1 , may be detected by use of methylation-specific primer oligonucleotides. 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 primers 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 'T¹ 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: 7 to SEQ ID NO: 10 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 TpG' 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 dehvatized 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: 7 to SEQ ID NO: 10, 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 , and the equivalent positions within SEQ ID NO: 7 TO SEQ ID NO: 10. 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-labeled 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 imbodiments, is designed to hybridize to a GpC-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. In a further preferred embodiment of the method, the fifth step of the method 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 acid according to SEQ ID NO: 1 , 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 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: 7 to SEQ ID NO: 10, 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 ID NO: 1 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: 7 to SEQ ID NO: 10 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 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 is 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 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: 7 to SEQ ID NO: 10 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 , and the complement thererof) without the need for pretreatment.

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.

In the final step of the method the prognosis of the patient is determined. Hypermethylation and over expression of the gene STMN1 and/or genomic sequences thereof according to SEQ ID NO: 1 are associated with negative prognosis and outcome of patients treated by means of therapies targeting the estrogen pathways. Patients with predicted positive outcome (i.e. hypomethylation or under expression) after said treatment, will accordingly have a decreased absolute reduction of risk of recurrence and death after treatment with chemotherapy. Patients with predicted negative outcome (i.e. hypermethylation or over expression) after said treatment will accordingly have a relatively larger absolute reduction of risk of recurrence and death after treatment with chemotherapy. Accordingly patients with a negative outcome after said treatment will be considered more suitable candidates for chemotherapeutic candidate than patients with a positive outcome. Patients with a positive outcome may accordingly be prevented from over prescription of chemotherapeutic treatment.

Nucleic acids

In order to enable this method, the invention further provides the modified sequences of the genomic sequence of SEQ ID NO: 1.

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 prediction of outcome of 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 SEQ ID NO: 1 and sequences complementary thereto.

The disclosed invention provides treated nucleic acids, derived from genomic SEQ ID NO: 1 , 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: 7 to SEQ ID NO: 10, wherein said sequence comprises at least one CpG, TpA or CpA dinucleotide and sequences complementary thereto. The sequences of SEQ ID NO: 7 TO SEQ ID NO: 10 provide non-naturally occurring modified versions of the nucleic acid according to SEQ ID NO: 1 , 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" (i.e., 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. antisense strand), wherein "C" is converted to "T," but "CpG" remains "CpG" (Ae., 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 correspond to SEQ ID NO: 7 AND SEQ ID NO: 8. 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 unmethylated); a final chemically converted version of each sequence, discloses the complement of the disclosed genomic DNA sequence (i.e. antisense 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 (antisense strand) of each genomic sequence, all "C" residues of CpG dinucleotide sequences are unmethylated). The 'downmethylated' converted sequences of SEQ ID NO: 1 correspond to SEQ ID NO: 9 AND SEQ ID NO: 10. 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: 7 to SEQ ID NO: 10. 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: 7 to SEQ ID NO: 10 is provided in the sequence listing.

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: 7 to SEQ ID NO: 10, 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: 7 to SEQ ID NO: 10, or to the complements thereof.

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 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*ⁿ - 9*ⁿ 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*ⁿ - 6*ⁿ 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 (SEQ ID NO: 1) or treated versions of said sequence (SEQ ID NO: 7 to SEQ ID NO: 10). 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.

The sequence 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 prediction of 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 predictive 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 detecting STMN1 polypeptides and a means for determining STMN1 methylation. 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. The means for determining STMN1 methylation comprise preferably a bisulfite-containing reagent; a set of primer oligonucleotides containing at least two oligonucleotides whose sequences in each case correspond, are complementary, or hybridize under stringent or highly stringent conditions to a 16- base long segment of the sequences SEQ ID NO: 1 or more preferably SEQ ID NO: 7 TO SEQ ID NO: 10; oligonucleotides and/or PNA-oligomers; as well as instructions for carrying out and evaluating the described method. In a further preferred 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.

Particularly preferred is a kit comprising a bisulfite (= disulfite, hydrogen sulfite) reagent as well as oligonucleotides and/or PNA-oligomers having a length of at least 16 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 7 TO SEQ ID NO: 10 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG, CpA or TpG dinucleotide.

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 and determining the methylation of the gene STMN1 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 for use in determining treatment strategy for a patient with a breast cell proliferative disorder, comprises: (a) a means for detecting STMN 1 polypeptides; (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); (d) a means for detecting STMN1 polypeptides, preferably oligonucleotides and/or PNA-oligomers having a length of at least 16 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 7 TO SEQ ID NO: 10 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG, CpA or TpG dinucleotide 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. Another aspect of the invention relates to a kit for use in providing a prognosis of a subject with a breast cell proliferative disorder, said kit comprising: a means for measuring the level of transcription of the gene STMN1 and a means for determining STMN1 methylation. 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 STMN1. 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. The means for determining STMN1 methylation comprise preferably a bisulfite-containing reagent; a set of primer oligonucleotides containing at least two oligonucleotides whose sequences in each case correspond, are complementary, or hybridize under stringent or highly stringent conditions to a 16-base long segment of the sequences SEQ ID NO: 1 or more preferably SEQ ID NO: 7 TO SEQ ID NO: 10; oligonucleotides and/or PNA-oligomers; as well as instructions for carrying out and evaluating the described method. In a further preferred 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.

Particularly preferred is a kit comprising a bisulfite (= disulfite, hydrogen sulfite) reagent as well as oligonucleotides and/or PNA-oligomers having a length of at least 16 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 7 TO SEQ ID NO: 10 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG, CpA or TpG dinucleotide.

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 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 for use in determining treatment strategy for a patient with a breast cell proliferative disorder comprises (a) a plurality of oligonucleotides or polynucleotides able to hybridise under stringent or moderately stringent conditions to the transcription products of the gene STMN1 ; (b) a container suitable for containing the oligonucleotides or polynucleotides and a biological sample of the patient comprising the transcription products wherein the oligonucleotides or polynucleotide can hybridise under stringent or moderately stringent conditions to the transcription products, (c) means to detect the hybridisation of (b); (d) a means for detecting STMN1 polypeptides, preferably oligonucleotides and/or PNA-oligomers having a length of at least 16 nucleotides which hybridizes to a pretreated nucleic acid sequence according to one of SEQ ID NO: 7 TO SEQ ID NO: 10 and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG, CpA or TpG dinucleotide and optionally, (e) instructions for use and interpretation of the kit results. The kit may also contain other components such as hybridization 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 optimized for primer extension mediated by the polymerase, such as PCR.

Most preferably a kit according to the embodiments of the present invention is used for the determination of expression step of the methods according to other aspects of the invention. The described invention further provides a composition of matter useful for determining the prognosis of a patient with breast cancer. Said composition comprising at least one nucleic acid 18 base pairs in length of a segment of a nucleic acid sequence selected from the group consisting SEQ ID NO: 7 to SEQ ID NO: 10 , and one or more substances taken from the group comprising : magnesium chloride, dNTP, taq polymerase, bovine serum albumen. 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.

Example 1

The first aim of this study was to evaluate commercially available antibodies against the marker Stathmin on tissue microarrays (TMA) of paraffin-embedded tumors in patients with operable breast carcinoma treated with adjuvant Tamoxifen. The second aim was to analyze the prognostic potential of said protein for disease recurrence and thus validate their clinical importance.

Material and methods

Patient characteristics

The study cohort consisted of 215 operable female breast cancer patients who underwent radical local therapy at the Institute of Oncology, Ljubljana, Slovenia, in the period between 1994 and 1999. All presented with ER positive invasive carcinomas with or without lymph node metastases and were treated with adjuvant Tamoxifen monotherapy. The criterion for selection was based on the histopathological diagnosis. Patients with bilateral breast cancer were excluded from the study. None of the patients had received any treatment before the biopsy procedure. All conventional clinico- pathological data were evaluated. The median age at the time of diagnosis was 67.9 years (range, 36- 83 years). All patients were females and were mostly postmenopausal (96.3%). The median follow-up time was 84 months (range, 5-133 months); among the patients 54 (25.1 %) experienced recurrence and 59 (27.4%) died.

Tumor characteristics

Important tumor characteristics are presented in Table 1

Table 1 : Selected tumor characteristics (n = 215)

Tumor grade was established by Bloom-Richardson-Elston score. Hormonal receptors were determined by IHC.

Generation of tissue microarrays (TMA)

The original H and E slides and paraffin-embedded tumor tissue were retrieved from the archives of Department of Pathology, Institute of Oncology, Ljubljana. H and E stained slides of tumor tissue were reviewed to identify representative tumor regions without necrosis or carcinoma in situ. As previously described, three tissue cylinders with a diameter of 0.6mm were obtained for each tumor from corresponding paraffin tumor block and arrayed into a recipient new paraffin block using the tissue chip microarrayer (Beecher Instruments, Silver Spring, MD). Four recipient tissue blocks were constructed. They were subsequently cut into 2-3 micrometer sections and fixed on silanized glass slides (Knittel Glaeser, Germany) to support adhesion of the tissue samples for subsequent immunohistochemical staining.

lmmunohistochemistry

We performed immunohistochemical staining manually for Stathmin-1 antibodies. For visualization, we applied a standard DAB Envision (DAKO) technique for all three antibodies. The manufacturers and staining information are listed in Table 2. Control slides of tonsil and breast tissue were reviewed and deemed adequate in all cases.

Table 2: Antibodies used in present study

pAb=polyclonal, WB=water bath

Scoring of immunohistochemical assays

Tumor heterogeneity in expression was assessed using semi quantitative histo-score method (H- score). The staining intensity (I) was graded as 0-no staining, 1-weak, 2-moderate, and 3-strong.The proportion of cells (P) with the observed intensity was recorded as 0, 1 (< 33 %), 2 (33-66 %), and 3 (> 66 %). H-score was determined as the product of I and P, ranging from minimum score 0 to maximum score 9. The maximum score (IxP) of 9 would be obtained if more than 66 % of tumor cells stained (P=3) with maximal intensity (l=3).

Statistical analysis

The dependent variable in this study was disease free survival, calculated from the date of the start of primary therapy to the date of breast cancer recurrence (local, loco-regional or distant), the date of death from any cause, or the date of last follow-up; censored observations correspond to patients alive and without evidence of recurrence at the time of last follow-up.

The distributions of disease free survival were estimated by the Kaplan-Meier method and compared with the log-rank test. Cox multivariate analysis was used to assess the prognostic importance of various independent factors. Hazard ratios and 95% confidence intervals were calculated in the cohorts of patients with various expressions of the markers. The reported p values are all two tailed.

Results Stathmin-1 expression proved to be a prognostically important marker.

Stathmin-1 was also found to be consistently expressed in more than 66% of tumor cells in a large majority of tumors (94%). Therefore, we only grouped our patients according to Stathmin-1 intensity and not according to Stathmin-1 proportion or score. Furthermore, due to a very small number of patients with weak Stathmin-1 intensity, we combined tumors with weak and moderate staining intensity into one "low expression" group (126 cases).

Tumors with strong staining intensity formed a group of "high expression" (89 cases). If Stathmin-1 intensity is reported as such a binary variable, a statistically significant difference (p = 0.0122) in DFS curves for these two groups of patients was observed (Fig. 7). At medium follow-up of 7 years, the DFS of patients with Stathmin-1 low and high expression was 84.1% and 63.2%, respectively. Apart from Stathmin-1 intensity, univariate survival analysis revealed that the other prognostic factors for this group of patients were tumor size, lymph node status, and progesterone receptor status (Tab. 3). Multivariate survival analysis of DFS showed that Stathmin-1 intensity retained its independent prognostic value for DFS with a relative risk of relapse of 2.55 (95% Cl = 1.41-4.61 ) for high Stathmin- 1 intensity levels. Of the traditional prognostic factors, only lymph node status retained its prognostic value with the relative risk of relapse of 4.96 (95% Cl = 2.55-9.63) while tumor size and progesterone receptor status lost their prognostic significance for DFS in a multivariate model (Tab. 3).

Table 3: Univariate and multivariate analysis of disease free survival

The favorable distribution of patients with respect to lymph node status allowed us to investigate an additional interesting question whether Stathmin-1 intensity predicts DFS in both subgroups of patients (positive and negative lymph node status) or perhaps just in one of the subgroups. Figures 8 and 9 show the Kaplan-Meier curves according to Stathmin-1 staining intensity for the lymph node negative and lymph node positive patients, respectively. In both cases low expression of Stathmin-1 was associated with better disease-free survival. For lymph node negative patients this association is borderline significant (p = 0.0651), while for lymph node positive patients the association is very significant (p = 0.0010). At medium follow-up of 7 years, the DFS of patients with Stathmin-1 low and high expression was 94.4 % and 79.5%, in lymph node negative sub-group of patients and 74.0 % and 33.9%, in lymph node positive sub-group of patients, respectively.

Discussion Currently, one of the most important questions in planning the treatment of patients with operable breast carcinoma is for which endocrine responsive patients endocrine therapy with Tamoxifen alone is sufficient and for which patients additional adjuvant systemic therapy with other endocrine agents and chemotherapy is needed. From available data we know that 5 years of Tamoxifen reduces the risk of relapse by 41%. However, the likelihood of recurrence in patients treated with adjuvant Tamoxifen alone after surgery is about 15 percent at 10 years. Therefore additional molecular markers that would enable us to identify different subsets of HR+ patients that may respond different to Tamoxifen therapy are urgently needed. DNA-methylation biomarkers that are easy to implement and to determine in paraffin-embedded tissue are certainly some of them.

The first aim of this study was to evaluate commercially available antibodies against Stathmin-1 on tissue microarrays (TMA) of paraffin embedded tumors in patients with operable breast carcinoma treated with adjuvant Tamoxifen. All commercially available antibodies against three methylation markers proved to work on TMAs of breast carcinoma. Tumor cells expressed Stathmin-1 in different intensities. The second aim in our study was to identify if the marker is associated with a low risk of recurrence after Tamoxifen monotherapy and thus validate their clinical importance. Using Stathmin-1 we were able to separate ER+ operable breast cancer patients treated by Tamoxifen alone in two distinctive groups according disease-free survival probability. The difference in the risk of distant recurrence between patients with Stathmin-1 low and high expression was large and statistically significant. The rate of distant recurrence at 7 years was only 16% in Stathmin-1 low group of patients and 37% in Stathmin-1 high group of patients. The independent prognostic value of Statmin-1 intensity was confirmed in multivariate Cox model in which Stathmin-1 staining intensity was found to be the only independent prognostic factors next to nodal involvement in our collective of patients.

Stathmin-1 expression was a predictor of disease recurrence in both lymph node positive and negative group. For lymph node positive patients this association was very significant ( p = 0.0010) while for lymph node negative the association was borderline significant (p = 0.0651). We believe that Stathmin-1 intensity is an important prognostic factor also for lymph node negative patients. The lower (borderline) significance in these patients is most likely due to a smaller number of events (the consequence of a much better survival prognosis due to negative lymph node status) compared to lymph node positive group of patients. Our results provide substantial evidence that Stathmin-1 staining intensity defined by immunohistochemistry on paraffin blocks of primary tumor could be used to predict outcome in patients with Tamoxifen monotherapy, and to identify a low risk group for which 5 years of Tamoxifen is sufficient. Lymph node negative patients with low Stathmin-1 staining activity seem to have an excellent long term prognosis according to our findings. In this group with disease-free survival of more than 90% at 7 years the absolute benefit of chemotherapy or endocrine therapy with aromatase inhibitors would be small and may not be justified according to our findings that have to be confirmed in a prospective fashion in a larger set of patients.

Claims

I/We claim:

1. A method for providing a prognosis of a subject with a breast cell proliferative disorder comprising the following steps of: a) obtaining a breast tumour sample from a subject b) determining the expression status of the gene STMN1 in said sample c) determining therefrom the prognosis of said subject whereby overexpression is indicative of negative prognosis.

2. The method according to claim 1 , wherein said subject is estrogen receptor positive.

3. The method according to any of claims 1 or 2, further comprising d) determining a suitable treatment for said subject.

4. The method according to any of claims 1 to 3, wherein said breast cell proliferative disorder is selected form the group consisting of 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.

5. The method according to any of claims 1 to 4, where the expression is determined by measuring the level of at least one of mRNA, cDNA or polypeptide.

6. The method according to any of claims 1 to 5, wherein the sample is selected from the group consisting of cell lines, histological slides, paraffin embedded tissues, biopsies, tissue embedded in paraffin, urine, nipple aspirate and blood.

7. A kit for use in providing a prognosis of a subject with a breast cell proliferative disorder, comprising: a means for detecting STMN1 polypeptides.

8. The kit according to claim 7, comprising: (a) a means for detecting STMN1 polypeptides; (b) a container suitable for containing the said means and a 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.

9. A kit for use in providing a prognosis of a subject with a breast cell proliferative disorder, comprising: a means for measuring the level of mRNA transcription of the gene STMN1.

0. The kit according to claim 9, comprising: (a) a means for measuring the level of mRNA transcription of the gene STMN 1 ; (b) a container suitable for containing the said means and a biological sample of the patient comprising mRNA of the gene STMN1 wherein the means are able to hybridize to the transcription products of the gene STMN 1 ; (c) a means for detecting the complexes of (b); and optionally (d) instructions for use and interpretation of the kit results.