WO2006114611A2 - Materials and methods relating to the diagnosis of cancer - Google Patents

Abstract

Methods of diagnosing cancer or pre-cancerous conditions are disclosed involving the detection of CTCF.

Description

Materials and Methods Relating to the Diagnosis of Cancer

Field of the Invention

The present invention relates to the role of CTCF polypeptide or protein in cancer and particularly, but not exclusively, to the detection of an up-regulation of CTCF expression and/or the detection of a cancer-specific form of CTCF as a means of diagnosing a cancer or a pre-cancerous condition.

Background to the Invention

Breast cancer is the most commonly diagnosed cancer among women after non-melanoma skin cancer, and is the second leading cause of cancer deaths after lung cancer. Owing to its biological heterogeneity and variable responsiveness to treatment, breast cancer is a complex disease for which clinical management is difficult.

At the present time, definitive diagnosis of breast disease is based on histological examination and evaluation of tissue samples. In order for the disease state to be detectable by the microscope, the disease will almost inevitably be reasonably advanced. Early stage detection of tumours in general, including breast tumours, is desirable in order that treatment programmes can be commenced at an early stage, thus increasing the likelihood of successful treatment.

CTCF (or CCCTC-binding factor) is a ubiquitous 11-zinc finger (ZF) phosphoprotein with highly versatile functions: in addition to transcriptional silencing or activating in a context-dependent fashion, it organizes epigenetically controlled chromatin insulators that regulate imprinted genes in soma (1) . Previous reports indicate CTCF could be a new tumour suppressor gene (TSG) because (i) it suppresses cell growth (2) ; (ϋ) it is localized at chromosome position 16q22 associated with the chromosomal abnormality loss of heterozygosity (LOH) in breast malignancies (3); and (iii) functionally significant, tumour-specific CTCF ZF mutations in various cancers including breast tumours have been identified and characterized (4) .

CTCF binds through combinatorial use of its 11 ZFs to approximately 50bp target sites of remarkable sequence variation. Formation of different CTCF-DNA complexes results in distinct functions including gene activation, repression, silencing and chromatin insulation.

Sequence information for the human transcriptional repressor CTCF is publicly available and can be accessed at http : //www . ncbi . nlm. nih . gov. The 727 amino acid sequence is available under SwissProt accession number P49711 (GI .-1706179) and the 3780bp mRNA sequence under accession number NM_006565 (GI:5729789) .

It is generally acknowledged that the process of tumour development is associated with step-by-step activation of growth promoting cellular oncogenes and inactivation of tumour- suppressor genes, usually resulting in over-expression of oncogenes and under-expression of tumour-suppressors (5) . Suppression of apoptosis is therefore considered to be one of the critical factors supporting tumour progression (6).

Summary of the Invention

It was therefore surprising for the inventors to discover elevated levels of CTCF in breast cancer cell lines.

The inventors analysed the possible function of the increased levels of CTCF in the support of cell survival by protecting cancer cells from apoptosis to test the hypothesis that elevated levels of CTCF in breast cell lines may represent one mechanism of cell protection from apoptosis. By using different cellular models and independent apoptotic markers the inventors have shown that reduction of CTCF levels in breast cancer cells leads to apoptotic cell death.

Using Western blot analysis, the inventors have demonstrated that in each of 67 paired tumour/normal breast tissues (54 invasive ductal carcinomas, 5 lobular carcinomas, 8 ductal carcinomas in situ and paired adjacent normal breast tissues) that a clear and specific transition occurs between an approximately 18OkDa form of CTCF which is present in normal breast tissues and an approximately 13OkDa form of CTCF found exclusively in the cancer tissues. The 13OkDa and 18OkDa species are detectable with a number of anti-CTCF antibodies. The 13OkDa form of CTCF was also detected in each of a panel of 14 breast cancer cell lines analysed (Figure IA) .

Expression levels of the cancer-specific 13OkDa form of CTCF were found to be significantly elevated compared to CTCF expression in immortalized cells derived from normal breast tissue (Figure 2A and Table 1) .

Immunofluorescent staining of CTCF confirmed the elevated levels of CTCF in breast cancer cell lines and in 50 breast tissue tumour/normal pairs analysed.

The ability to detect the cancer-specific 13OkDa form of CTCF by reproducible, sensitive, specific and standardized methods which can be easily interpreted by the clinician makes CTCF an excellent diagnostic indicator of breast disease.

The inventors have also shown that up-regulation of CTCF can overcome Bax-induced apoptosis and thus is implicated in protecting cancer cells from apoptosis. Induction of CTCF knockout by employing an inducible anti-sense CTCF resulted in massive cell death in the MCF7 breast cancer cell line.

At its most general the present invention provides methods for diagnosing a disease, or for diagnosing an increased risk of disease development, in a patient.

According to a first aspect of the present invention there is provided a method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of: detecting in vitro the presence of CTCF having a molecular weight between 12OkDa and 14OkDa in a tissue sample from said patient.

Preferably the CTCF is the CTCF polypeptide or protein and has a molecular weight of approximately 13OkDa. The step of detection may comprise contacting a CTCF antibody, preferably a CTCF monoclonal antibody, with said sample so as to hybridise (bind) said antibody to said CTCF polypeptide or protein.

The first aspect of the invention relies on the inventors' finding that detection of a CTCF isoform having an apparent molecular weight of approximately 13OkDa may be used as a marker of a cancerous condition, or the susceptibility to development of a cancerous condition.

Based on the same finding, an alternative first aspect of the present invention is provided in which a method of diagnosing a cancer or a pre-cancerous condition in a patient is provided, the method comprising the step of detecting, in a sample taken from the patient, an isoform of CTCF polypeptide or protein that has an apparent molecular weight of between 60% and 80% of the apparent molecular weight of a CTCF isoform detectable in healthy tissue of a corresponding type.

The CTCF isoform being detected in the patient sample may more preferably have an apparent molecular weight of between 65% and 75%, or still more preferably between 70% and 75%, of the apparent molecular weight of the CTCF isoform detectable in the healthy tissue.

Preferably, the CTCF isoform of lower molecular weight is not detectable in the healthy tissue.

According to a second aspect of the present invention there is provided a method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of: detecting both a first and a second molecular weight form of CTCF in a tissue sample from a patient.

Preferably the first CTCF form has a molecular weight of between 170 and 19OkDa, more preferably approximately 18OkDa. Preferably the second CTCF form has a molecular weight of between 120 and 14OkDa and more preferably approximately 13OkDa.

The second CTCF form may have a molecular weight that is between 60% and 80% of the molecular weight of the first CTCF form. More preferably, this may be between 65% and 75%, or still more preferably between 70% and 75%.

The first and second CTCF forms may be post-translationally modified, e.g. by poly (ADP-ribosyl) ation. The two forms may have a different ADP-ribosylation profile. Alternatively, the second CTCF form may be a truncate of the first CTCF form.

The step of detection may comprise contacting an antibody with said tissue sample such that hybridization (binding) of the antibody to one or both forms of CTCF polypeptide or protein may occur. In one preferred arrangement the antibody may bind to both the first and second CTCF forms. In another preferred arrangement the step of detection may comprise hybridising a first antibody which binds specifically to said first CTCF form and a second antibody which binds specifically to said second CTCF form. Said antibodies may be separate monoclonal antibodies binding to said first and second CTCF forms respectively and may specifically recognize the ADP-ribosylation profile of each form.

According to a third aspect of the present invention there is provided a method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the steps of: detecting in vitro the level of expression of CTCF in a tissue sample from a patient; and comparing said level to a standard level of CTCF expression in healthy tissue, wherein an increase in the level of expression of CTCF in said tissue sample relative to said healthy tissue is indicative of the presence of a cancer or a pre-cancerous condition.

CTCF expression preferably refers to expression of the approximately 13OkDa or approximately 18OkDa forms of CTCF polypeptide or protein.

Suitably, the tissue sample and healthy tissue from which the standard reading is derived will be of the same type, e.g. both breast tissue samples.

The detection step may comprise quantitatively determining an amount of CTCF polypeptide or protein in said tissue sample. The standard level of CTCF polypeptide or protein is preferably quantitatively determined by the same method used to quantitatively determine the amount of CTCF polypeptide or protein in said sample. The method of quantitation may comprise immunohistochemical scoring of stained samples or quantitative assessment of band intensity observed in Western blot relative to a loading control.

The CTCF polypeptide or protein being detected in the third aspect is preferably a form of CTCF having a molecular weight between 120 and 14OkDa and more preferably approximately 13OkDa, but may alternatively be a form of CTCF having a preferred molecular weight between 170 and 19OkDa, more preferably approximately 18OkDa.

In a fourth aspect of the present invention an anti-CTCF antibody is provided for use in the diagnosis of a cancer or a precancerous condition.

In a fifth aspect of the present invention the use of an antibody to CTCF polypeptide or protein in the manufacture of a product for the diagnosis of a cancer or a pre-cancerous condition is provided.

In one arrangement of the fourth and fifth aspects said antibody may bind specifically to a form of CTCF polypeptide or protein having a molecular weight between 120 and 14OkDa, more preferably approximately 13OkDa. The antibody may be a CTCF monoclonal antibody.

In a sixth aspect of the present invention there is provided an assay kit comprising an antibody which binds specifically to a form of CTCF having a molecular weight between 120-14OkDa.

Preferably the assay kit comprises an antibody which binds specifically to a form of CTCF polypeptide or protein having a molecular weight of approximately 13OkDa.

According to a seventh aspect of the present invention there is provided a method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of detecting the presence or absence of a poly (ADP-ribosyl) modified form of CTCF.

Preferably, said poly (ADP-ribosyl) modified form of CTCF is detected by hybridization of CTCF with an anti-poly (ADP-ribose) antibody, e.g. by Western blotting. The poly (ADP-ribosyl) modified form of CTCF preferably has an apparent molecular weight between 170 and 19OkDa, more preferably approximately 18OkDa. The poly (ADP-ribosyl) modified form of CTCF may also be detected by hybridization with an anti-CTCF antibody, e.g. by Western blotting.

Detection of the presence or absence of the poly (ADP-ribosyl) modified form of CTCF may comprise detecting a reduction or increase in the amount of said poly (ADP-ribosyl) modified form of CTCF in a patient sample relative to a healthy tissue sample.

In an eighth aspect of the present invention there is provided a method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of detecting a change or difference in the level of poly (ADP-ribose) polymerase (PARP) activity or expression in a sample from said patient relative to a level of PARP activity or expression in a healthy tissue.

Detection of PARP activity may be performed by a suitable assay of cell lysates or extracts. Relative levels of PARP expression may be determined by quantitative gel electrophoresis, e.g. by measuring band intensity - which may be performed using research tools available to the skilled person such as the BioRad™ Quantity One™ Quantitation software and Gel Doc™ system.

In the aspects and embodiments of the invention the molecular weight of CTCF polypeptide or protein is preferably an apparent molecular weight of the CTCF polypeptide or protein. This may be determined by electrophoretic separation of CTCF from components of the tissue sample taken from the patient of interest. The electrophoresis experiment may comprise SDS-PAGE wherein total cell lysates are prepared for loading in sodium dodecyl sulfate (SDS) -containing buffer with brief treatment with DNAseI to reduce viscosity, and electrophoresis on a 10% SDS polyacrylamide gel (PAGE) . Selected marker compounds may be used to standardize the molecular weight determination and are preferably included in the same electrophoresis experiment. Suitable molecular weight standards to be included may comprise one or more of the pre- stained protein markers, broad range (premixed format) , P7708S available from New England BioLabs™ (www.neb.com or www. neb. com/neb/msds/new/P7708.pdf) .

Visualisation of bands in the electrophoresis experiment may be performed by use of stains known to the skilled person e.g. Coomassie blue. To determine and visualize bands corresponding to a form of CTCF polypeptide or protein, Western blot may be employed in which the components of the electrophoretic separation are blotted and probed with one or more anti-CTCF antibodies. For Western blotting, the SDS PAGE separated bands are transferred onto Immobilon™ P membranes (Millipore™, Bedford, Mass., USA) by semi-dry blotting and probed with anti-CTCF antibodies. CTCF antibodies which may be used in the Western blotting may be selected from:

(i) CTCF polyclonal antibodies;

(ii) CTCF antibodies raised against the CTCF C-terminal domain amino acid sequence; (iii) CTCF monoclonal antibodies.

The detection of a cancer may comprise the detection of a neoplasm, being an abnormal growth or tumour. In the case of a tumor, it may be benign or malignant and may be a primary tumour or a secondary tumour, i.e. one that has metastasized from a primary tumour located in a different tissue.

The cancer is preferably a form of breast cancer, or is involved in the development of a breast cancer. Breast cancers within the scope of the invention may be selected from the group consisting of: (i) invasive ductal carcinoma (IDC) ;

(ii) ductal carcinoma in situ (DCIS) ;

(iii) invasive lobular carcinoma (ILC);

(iv) invasive lobular carcinoma in situ (ILCIS); (v) adenocarcinoma (AC) ;

(vi) lobular hyperplasia benign (LBH) ;

(vii) medullary carcinoma (MD) .

A pre-cancerous condition may comprise the early stage formation of a cancer, i.e. where a neoplasm or tumour is not yet physically detectable (e.g. by imaging techniques such as Magnetic Resonance Imaging), but in which tumour precursor cells, e.g. cells exhibiting an abnormal protein expression profile, increased resistance to apoptosis or abnormal immortality, have begun to appear or accumulate in the tissue. The pre-cancerous condition may be the result of early stage primary tumour development in the tissue or the result of metastasizing cells becoming lodged in the tissue and forming the basis of a potential secondary tumour.

The methods of the present invention may therefore be useful: in assessing the disposition or predisposition of a patient to develop a cancer, particularly a breast cancer; in early as well as late stage diagnosis of cancer; and in continued monitoring and prognosis reporting during treatment of the cancer which may indicate the degree of success of the treatment and presence or remission of the cancer.

Accordingly, the diagnosis or prognosis may relate to an existing (previously diagnosed) cancerous condition, which may be benign or malignant, may relate to a suspected cancerous condition or may relate to the screening for cancerous conditions in the patient (which may be previously undiagnosed) .

Other diagnostic tests may be used in conjunction with those described here to enhance the accuracy of diagnosis or prognosis of a cancerous condition or to confirm a result obtained by using the tests described here.

The tissue sample may be a quantity of tissue excised from the tissue of interest in the patient, e.g. a biopsy, alternatively it may comprise or may be derived from cells isolated from the patient; a quantity of blood; or a quantity of serum derived from the individual's blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells .

The method of diagnosis may be an in vitro method performed on the patient sample, or following processing of the patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method of diagnosis to be performed and therefore the method may be one which is not practised on the human or animal body.

The patient to be treated may be any animal or human. The patient may be a non-human mammal, but preferably the patient of interest from which the tissue sample is obtained is a human individual, and is more preferably a female patient.

Detection of CTCF

The 13OkDa and/or the 18OkDa CTCF isoforms may be detected using any appropriate agent capable of binding to the selected isoform(s) .

Preferred agents include antibodies (polyclonal or monclonal) and aptamers. Preferred binding agents may have a binding affinity (K_D) for the CTCF isoform of from 100 μM to 10 pM, or better. Particularly preferred binding agents may have a K_D in the range lOμM to InM. Suitable antibodies and aptamers capable of binding specifically to one or both CTCF isoforms can be prepared by persons of skill in the art.

In further aspects of the present invention, the use of such binding agents in the detection of CTCF isoforms is provided. Such use may be in vivo use, but is preferably in vitro use. For example, the use of an antibody, or aptamer, capable of binding the 13OkDa CTCF isoform in the detection of the 13OkDa CTCF isoform in vitro is provided. The use, in vitro, of an antibody, or aptamer, capable of binding the 13OkDa CTCF isoform in the detection of cancer in a patient sample is also provided.

In some embodiments, CTCF detection may be facilitated by adhering the binding agent (s) to a suitable solid support, e.g. a column or plate.

Aptamers

Aptamers, or nucleic acid ligands, are nucleic acid molecules characterised by the ability to bind to a target molecule with high specificity and high affinity. Aptamers to a given target may be identified by the method of Systematic Evolution of Ligands by Exponential enrichment (SELEX™) . Aptamers and SELEX are described in WO91/19813.

Aptamers may be DNA or RNA molecules and may be single stranded or double stranded. The aptamer may comprise chemically modified nucleic, acids, for example in which the sugar and/or phosphate and/or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose. Aptamers can be thought of as the nucleic acid equivalent of monoclonal antibodies and often have K_d's in the nM or pM range. As with monoclonal antibodies, they may be useful in virtually any situation in which target binding is required, including use in therapeutic and diagnostic applications, in vitro or in vivo. In vitro diagnostic applications may include use in detecting the presence or absence of a target molecule.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Brief Description of the Figures

Figure 1. Expression of the CTCF protein in human breast cell lines and tumours.

(A) Western blot analysis. Cellular extracts were prepared from 5xlO⁶ cells, total nuclear protein concentration was determined for each sample and equal amount (40μg) of total protein loaded onto SDS-PAGE. Samples were electrophoretically separated, blotted and probed with the anti-CTCF antibody. The same membrane was stripped and re-probed with the anti-α-tubulin antibody (loading control) ;

(B) Immunofluorescent staining. For immunofluorescent detection of CTCF cells were prepared as described under "Materials and Methods". In all cell lines CTCF demonstrates nuclear localization, however sub-nuclear distribution of CTCF is different. Cell lines have been placed into Groups I-IV according to a characteristic pattern of CTCF distribution: Group I - homogenous diffuse nucleoplasmic; Group II - homogenous diffuse both in nucleoplasm and nucleoli; Group III - Speckled, Group IV - other than I-III. Bars: lOμm.

Figure 2. CTCF is expressed at higher levels in breast tumour tissues .

(A) Western blot analysis of six paired samples (Numbers 1 through 6) of normal and tumour tissues (a representative Western blot is shown) . 50μg of the total protein was used in this assay. The bands of the CTCF isoforms (18OkDa and 13OkDa) were quantified and their additive values were normalized to the corresponding values of α-tubulin used as loading control (bottom panel) ;

(B) Immunohistochemical staining of CTCF in normal breast tissue (top left) and invasive ductal carcinoma (bottom left) . CTCF expression is almost undetectable in normal breast tissue, whereas CTCF expression is high in tumour tissue. A pair normal/tumour tissue number 3 is shown as an example of a typical staining;

(C) Immunohistochemical staining of cell lines expressing high (MCF7) and low (HBLlOO) levels of CTCF. Top right and bottom right - background staining with haematoxylin plus secondary antibodies .

Figure 3. Transient knock-out of CTCF induces apoptotic cell death in breast cancer cells.

(A) Five breast cancer cell lines with up-regulated CTCF protein levels, one immortalized cell line and a non-breast cell line (293T of kidney origin) were transiently transfected with 2.5μg pCTCF-S, pCTCF-AS or pcDNA3 together with 2.5μg pEGFP as a marker. Apoptotic cell death was assessed by two methods: TUNEL assay and DAPI staining as described in Materials and Methods. Average of three experiments showing the percentages of apoptotic cells (TUNEL positive or assessed by DAPI) among the transfected cells (EGFP positive) were calculated 24, 48 and 72 hrs post transfection. The maximum effect for all cell lines (presented on the graphs) was achieved after 72 hours post-transfection, except 293T where it was 24 hrs;

(B) CTCF over-expression can overcome effects of Bax. 2μg of the pSFFV-Bax (pBax) plasmid was co-transfected with 2μg pcDNA3

(control) or 2μg of pCTCF-S, and 2μg of pEGFP. Average of three experiments showing the percentages of apoptotic cells (TUNEL positive or assessed by DAPI) among the transfected cells (EGFP positive) were calculated 24, 48 and 72 hrs post transfection. The maximum effect for all cell lines (presented on the graphs) was achieved after 72 hours post-transfection. Key: TUNEL - boxes with no filling; DAPI- boxes with black filling;

(C) Two typical images of apoptotic cells transfected with pCTCF- AS after TUNEL staining. Top panel: TUNEL positive cells show significantly lower amount of CTCF, an apoptotic cell is indicated by an arrow; Bottom panel: identification of transfected cells by an EGFP marker.

Figure 4. Conditional knock-out of CTCF in MCFl leads to proliferation block and apoptotic cell death.

(A) Schematic outline of the functional elements of the pΞpiLacCTCFanti, containing CTCF cDNA in the reverse orientation relative to the IPTG-inducible promoter (see Materials and Methods for detail) . The abbreviations stand for: Hyg - hygromycin B resistance gene; tk-P - thymidine kinase basic promoter; MMTV-P - mouse mammary tumour virus basic promoter; poly (A) - poly (A+) signal; OriP - origin for plasmid replication of EB virus; EBNAl - EB virus nuclear antigen 1; Lac -lactose operator sequences; CTCF ORF - CTCF cDNA (open reading frame) ;

(B) Western-blot analysis of MCF7/Lap5/CTCFantisense cells after induction with IPTG. IxIO⁶ MCF7/Lap5/CTCFantisense cells were plated in flasks and induced with IPTG. Cells were collected after 2, 4, 8, 12, 24 and 36 hours, non-stimulated cells were used as control point 0. Cell lysates were prepared, total protein measured, 20μg of total protein was loaded onto SDS-PAGE, proteins resolved, blotted and Western blot assay performed as described in "Materials and Methods" with anti-CTCF antibodies;

(C) Phase contrast images of MCF7/Lap5/CTCFantisense cells cultured in the presence of inducer (IPTG) for the indicated times;

(D) MCF7/Lap5/CTCFantisense cells on Day 2, non-induced (top panel) and after induction with IPTG (bottom panel) . Cells were pulsed with BrdU for 3 hr and stained with anti-BrdU antibodies labelled with Texas red (a) and antibody to cytokeratin 18 fragment labelled with fluorescein isothiocyanate (b) ; panel c - (a) and (b) merge.

Figure 5. CTCF-180 appears in MCF-I cells after treatment with sodium butyrate.

Western blot showing the appearance of a 18OkDa form of CTCF in MCF-7 cells following sodium butyrate treatment. CTCF-180 appears in MCF-7 cells after treatment with sodium butyrate: this isoform co-migrates with CTCF-180 detected in normal breast tissue .

Figure 6. Appearance of a 18OkDa form of CTCF and reduction in the 13OkDa form of CTCF in MCF-7 cells treated with sodium butyra te (NaB) .

Western blots showing the appearance of a 18OkDa form of sodium butyrate in MCF-7 cells after 4 hours incubation with sodium butyrate. Tubulin was used as the loading control.

Figure 7. The 18OkDa form of CTCF is recognized by anti-PAR antibodies .

Western blots showing recognition of a 18OkDa form of CTCF by anti-PAR antibodies in normal breast tissue and MCF-7 cell treated with sodium butyrate (NB) .

Figure 8. Poly (ADP-ribose) polymerase is elevated after 4h of MCF-7 with sodium butyrate.

Western blot showing elevation of poly (ADP-ribose) polymerase in MCF-7 cells following treatment with sodium butyrate.

Figure 9. Analysis of CTCF expression in different phenotypes of breast tumours and breast reduction tissue. 6 different types of mammalian breast tissues were analysed for CTCF expression. This included the following tissues: Normal breast reduction tissue; IDC, invasive ductal carcinoma; DCIS, ductal carcinoma in situ; ILC, invasive lobular carcinoma related; AD, adenocarcinoma and MD, medullary carcinoma. CTCF expression is shown as mean IRS values ± standard error.

**, significantly different where each tumour phenotype is compared to normal breast reduction tissue and p= <0.05 and # where only one sample was available for analysis.

Figure 10. Comparison of CTCF IRS values with Tumour grade in IDC tumours only. Included in Graph are normal (breast reduction tissue) and Paired peripheral (PP) tissue mean CTCF IRS values. ** Refers to a statistically significant results (p = <0.05). Figure 11. Comparison of CTCF and PR expression in 19 IDC breast tumours. The PR IRS values were in the clinical data provided with the breast tumours. Mean PR IRS values were plotted against mean CTCF IRS values categorised into low, moderate and high groups. All parameters are displayed as mean values ± standard error.

Figure 12. Mean CTCF expression of all tumour types in relation to breast tumour size. Each group was generated by categorising the tumours into 3 sizes: small, lmm-15mm; medium, 16mm-30mm; and large, 31mm-55mm. The small sized tumour group consisted of only- one sample, as indicated by ^A. Standard errors were calculated and are depicted by error bars. ** represents significant differences when compared to breast reduction tissue (CTCF expression in normal tissue) and * possibly significant but can not be determined due to low sample number.

Figure 13. Analysis of CTCF expression and age of the patient in IDC tumours. Each CTCF IRS value was categorised into 3 groups, as before: low, moderate and high. The mean age of each patient was correlated for each IRS group and depicted as the above graph. Standard error bars are included.

Figure 14. CTCF expression in IDC breast tumour samples treated with chemotherapy at different operative time periods. Time periods for treatments include no treatment, pre-operative treatment and post-operative treatment. Values are represented as mean CTCF IRS values ± standard error. No statistical analysis was carried out.

Figure 15. Western blot showing detection of the 13OkDa CTCF isoform by binding of an antibody specific for the 13OkDa isoform. Detailed. Description of the Best Mode of the Invention

Specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

Materials and methods

Cell lines and tissues

A panel of cell lines representing various types of normal and transformed human breast cells was obtained from M.0' Hare and B.Gusterson (Division of Cancer Sciences and Molecular Pathology, Western Infirmary, Glasgow, UK) . It included two immortalized cell lines, HBLlOO and HB4A (7) derived from normal breast epithelium, and a "normal" luminal cell line (8) and eight estrogen receptor positive (ER+) cell lines originating from human breast tumours at various stages of tumour progression: HMT-3522 are breast epithelial cells derived from fibrocystic immortalized, non-tumourigenic disease; T47D, MCF-7, BT474, CAMA 1, ZR-75-1, and ZR-75-30 are breast carcinomas, and GI-IOl is a breast tumour xenograft line spontaneously metastasing to the lungs of athymic mice. Ten estrogen receptor negative breast cell lines (ER-) included HBLlOO, adherent adenocarcinomas MDA-MB-157, MDA-MB-175, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, SKBR- 5, SKBR-7 and floating carcinoma DU4475.

The ER-negative cell lines also demonstrate various tumourigenic and metastatic potential, ranging from immortalized HBLlOO, non- tumourigenic (MDA-MB-453) to highly malignant (MDA-MB-231) . All cell lines were maintained in RPMI 1640 medium supplemented with HEPES, GlutaMAX and sodium bicarbonate, 20μg/ml gentamicin, 10% foetal calf serum (all from Life Technologies) . For HB4A cells, 5 μg/ml of insulin (Sigma) and 5 μg/ml of hydrocortisone (Sigma) were included in the medium.

Primary human normal and tumour tissues were obtained from patients treated at Colchester General Hospital (Essex, UK) , with written consent taken before surgery. Fifty four invasive ductal carcinomas, five lobular carcinomas, eight ductal carcinomas in situ and paired adjacent normal breast tissue samples were collected during surgery. The samples were immediately frozen and stored at -80⁰C.

Normal Breast and Breast Tumours were assessed for CTCF Immuno- Reactivity by immunostaining frozen tissue sections with anti-CTCF rabbit polyclonal antibodies. As a reference, normal breast tissue from patients after reductive surgery were used for staining. A scale from 0 (no detectable signal) to +++ (very strong signal) was used to assess staining intensity. The results are shown in Table 2.

Expression vectors and transient transfections .

The vectors for transient transfection were made by insertion of the full CTCF cDNA (9) in two orientations into the pcDNA3 vector (Invitrogen™) . The pSFFV-Bax construct was a kind gift from S. Korsmeyer (Dana-Farber Cancer Institute, One Jimmy Fund Way, Boston, MA 02115, USA) . Transient transfections were performed by using a calcium phosphate transfection protocol (10) .

IPTG-inducible anti-sense CTCF system.

MCF/LAP5 cells expressing an isopropyl-β-D-thiogalactopyranoside (IPTG) -dependent transactivator LAP267 (11) were provided by Lester Lau and Dimitri Pestov (University of Illinois College of Medicine, Chicago, IL) .

Construction of the IPTG-inducible episomal vector pEpiLac3 and pEpiLacCTCF (in both orientations) were described previously (12, 13) . The schematic outline of the anti-sense construct, pEpiLacCTCFanti, is shown in Fig.4A. The anti-sense CTCF construct, and the empty pEpiLac3 were transfected into the MCF/LAP5 cells and then selected in Hygromycin B (Hygro) using the technique of Li and Lau (12) . The hygro-resistant cells were single cell cloned and tested for expression of CTCF by western blotting; one of the clones grown as a cell line called

MCF/LAP5/antiCTCF was utilized in this study. To assay for effects of CTCF "knock-out", MCF/LAP5/antiCTCF cell line and control Hygro-resistant cells MCF/LAP5 were treated with 2mM IPTG for indicated time intervals.

Western blotting analysis, sample preparation, imunofluorescence, apoptosis monitoring and immunohistochemistry. Lysates from cell lines and tumour tissues were prepared according to Klenova et al (14) with modifications. Samples from the frozen tissue sections were homogenized in the lysis buffer (2OmM Tris/Hepes pH 8.0, 2mM EDTA, 0.5M NaCl, 0.5% Na deoxycholate, 0.5% Triton X-100, 0.25M Sucrose, 5OmM β- mercaptoethanol, and protease inhibitor kit (Roche) at approximate ratio of 5mg of tissue/ml buffer, the extract incubated on ice for 20 min, filtered, centrifuged at 14,000 rpm at +4°C and the supernatant collected.

Western blot assay was performed as described previously with anti-CTCF or anti-α-tubulin (Sigma) antibodies (15) . Total cell lysates, prepared for loading in sodium dodecyl sulfate (SDS)- containing buffer with brief treatment with DNAseI to reduce viscosity, were run through SDS-10% polyacrylamide gel electrophoresis (PAGE) gels and transferred onto Immobilon™ P membranes (Millipore™, Bedford, Mass., USA) by semi-dry blotting. Membranes were then probed with the appropriate antibody. The molecular weight markers incorporated in the electrophoresis experiment were the pre-stained protein marker, broad range (premixed format), P7708S New England BioLabs™ (www.neb.com or www. neb. com/neb/msds/new/P7708. pdf) . CTCF antibodies used in the visualisation of bands included: (i) CTCF polyclonal antibodies (product code ablO571,

Abeam™ (www. abeam. com) ) ;

(ii) CTCF antibody (Immunogen™) raised against the CTCF C- terminal domain amino acid sequence:

(57O)MARHADNCAGPDGVEGENGGETKKSKRGRKRKMRSKKEDSSDSENAEPD

LDDNEDEEEPAVEIEPEPEPQPVTPAPPPAKKRRGRPPGRTNQPKQNQPTAIIQ VEDQNTGAIENIIVEVKKEPDAEPAEGEEEEAQPAATDAPNGDLTPEMILSMMD

R (727) (SEQ ID No.l) ; (iϋ) as well as monoclonal antibodies (product catalog no.' s 612148 (old catalog no. 39220-050) and 612149 (old catalog no. C39220-150) , both mouse IgGl monoclonal antibodies, BD Pharmingen™ (www.bdbeurope.com) ) .

Quantification of the bands and CTCF/tubulin ratio was performed by using the BioRad™ Quantity One™ Quantitation software and Gel Doc™ system.

For indirect immunofluorescent staining, the standard protocols were used (16) with an additional modification which included a step of microwave heating after fixation of cells in formaldehyde (17) . The primary N-CTCF affinity-purified antibody and the secondary FITC-conjugated anti-rabbit antibodies were used at dilution 1:40.

To monitor apoptotic cell death, several techniques were used; staining for cytokeratin 18 was performed with M30 monoclonal antibody specific for an epitope of cytokeratin 18 uncovered by caspase cleavage (Roche Molecular Biochemicals™) . BrdU incorporation by cells containing IPTG-inducible anti-sense CTCF mRNA was monitored at selected times during culture by pulse- labeling for 3 hrs and staining with a 1:1 dilution of the BrdU antibody according to the manufacturer's instructions (Amersham Pharmacia™) . The TUNEL method was also employed for detection of apoptosis using the in situ cell detection kit (TMR red) from Roche Applied Science™ according to the manufacturer's instructions. For detection of the characteristic chromatin condensation and nuclear fragmentation associated with apoptosis, staining with 4 ' 6-Diamidino-2-phenylindole dilactate (DAPI)

(Sigma™) at 5μg/ml in PBS for 15 min was conducted as described (18) . Immunofluorescence was visualized using Confocal Laser Scanning microscopy (Biorad™) . Immunohistochemical analysis was performed using sections cut from the frozen tissues by staining with the Vectastain Elite ABC™ standard kit (Vector

Laboratories™) as suggested by the manufacturer. Dilutions of the anti-CTCF antibodies applied to the sections were 1:50.

Results and discussion

In preliminary experiments designed to assess CTCF expression in breast cell lines and tumours, the observed levels of the CTCF mRNA were highly variable in the breast cell lines and tumour samples, with no correlation to the ER+/- status of the cells and levels of c-myc or BRCAl mRNA. Higher expression of CTCF was detected in all types of breast tumours compared to reduction samples (Table 2) .

Analysis of the CTCF protein in breast cancer cell lines, however, revealed that all cell lines contained a 13OkDa band characteristic for CTCF (Figure IA) . In general, there was notable elevation of CTCF protein expression in a panel of the breast cancer cell lines compared to the CTCF expression in immortalized HBLlOO or HB4a cells derived from normal breast epithelium (~80% of cells contained from medium to high levels of CTCF) (Figure IA) .

Immunofluorescent staining of CTCF confirmed the results of the Western blot assay revealing elevated levels of CTCF in most of the cell lines compared to HB4A (Figure IB) . In all cell lines inspected CTCF demonstrated clear and exclusively nuclear localization, which was reconfirmed by staining with DAPI and/or phase contrast imaging of the cells (examples are given in Figure IB) . However the distribution of CTCF inside the nucleus was distinct in different cell types. The patterns were determined arbitrary, using classification previously suggested by Sutherland et al (19) : Group I (homogenous diffuse nucleoplasmic distribution of CTCF) ; Group II (homogenous diffuse both in nucleoplasm and nucleoli) ; Group III (speckled pattern of CTCF) and Group IV (clearly defined) . As our previous observations show, cells with "normal" phenotype, e.g. NIH 3T3 (immortalized mouse fibroblasts) demonstrate homogenous diffuse nucleoplasmic distribution of CTCF ((14); E.Klenova and F.Docquier, unpublished) . In the analysed panel, a cell line HB4A falls into this category, whilst the majority of cancer cell lines show sub- nuclear localization different from the HB4A cells, thus implicating that CTCF function (s) in cancer cells may differ from its function (s) in normal cells and CTCF sub-nuclear localization may be a potential marker for a type of breast tumour.

To confirm our observations on breast cancer cell lines, we next determined if the levels of expression of CTCF in breast tumours would be higher than in normal tissues. Western blot analysis revealed that in cell lines, as well as in tumour samples, CTCF protein migrates as a 130 kDa protein, typically seen in cell lysates (9, 14, 15) . However, in normal tissues CTCF protein has a larger size of 18OkDa (Figure 2A) . The observed shift in the size of the CTCF protein has been detected in all paired samples tested and may be associated with cell immortalization/ transformation. The total levels of both CTCF forms were notably higher in breast tumour tissues than in normal paired samples (Figure 2A, top and bottom panels) .

Immunohistochemical analysis confirmed that in all tumours inspected so far expression of CTCF was upregulated compared to normal breast tissues, where CTCF levels were very low/undetectable. A statistical analysis of CTCF expression in the tumours is shown in Table 1. All normal paired tissues showed very weak (+/-) CTCF immunostaining.

A typical pattern of immunostaining of a tumour specimen with the anti-CTCF antibodies is shown in Figure 2B revealing that CTCF expression is increased in tumour cells (Figure 2B, bottom left panel) compared to normal tissue (top left panel) . The level of CTCF detected in breast tumours is comparable with the levels of CTCF in breast cancer cell lines (Figure 2C) .

CTCF is believed to be a tumour suppressor (2, 3) . Therefore the elevation of CTCF protein in breast cell lines and tumours was unexpected. There are rare examples of increased levels of a protein with tumour suppressive functions in cancer cells. In particular, it has been described for the retinoblastoma protein RbI, which is considerably elevated in colorectal carcinomas (20) and believed to have anti-apoptotic function.

The inventors then investigated whether up-regulation of CTCF may also be linked to suppression of apoptosis. Several breast cancer cell lines showing high levels of CTCF expression were transiently transfected with plasmids carrying CTCF cDNA in the sense (pCTCF-S) and anti-sense orientation (pCTCF-AS) , as well as the empty vector pcDNA3 as control. The pEGFP was used as a marker of transfection, hence the number of TUNEL positive cells was calculated among the EGFP cells (Figure 3C, bottom panel) . Immunofluorescent staining of the TUNEL-positive cells revealed that these cells show significantly lower levels of CTCF (Figure 3C, top panel) . As shown in Figure 3A, in all breast cancer cell lines used for this experiment the appearance of an increased number of apoptotic cells correlates with co-transfection with pCTCF-AS . A very low percentage in the number of apoptotic cells was detected when pCTCF-S and pcDNA3 were used in the experiments. Interestingly, introduction of pCTCF-S leading to CTCF over-expression into the immortalized 'normal' breast cell line HBLlOO containing low levels of CTCF and a non-breast cell line 293T resulted in an increase of apoptotic cells. Apoptotic cell death from CTCF over-expression was reported by Qi et al in immature B cells (13) , thus it may be a characteristic feature of breast cancer cells to undergo apoptosis upon CTCF down- regulation.

The inventors then investigated whether these effects could be mediated through a Bax (the apoptosis-associated, cell death- inducing, membrane-associated Bcl2 protein) -dependent mechanism. In these experiments, the Bax-expressing plasmid pSFFV-Bax was transfected alone or in combination with pCTCF-S into breast cell lines. As shown in Figure 3B, CTCF over-expression can overcome, however not entirely, apoptosis induced by Bax in all three breast cancer cell lines tested.

To provide further evidence that down-regulation of CTCF in breast cancer cells can lead to apoptosis, the inventors employed the MCF7 cell line, which expresses high amounts of CTCF protein, to generate a cell line producing the anti-sense CTCF mRNA in an inducible fashion, as described under "Materials and Methods". The inventors observed that after induction with IPTG, resulting in knock out of CTCF protein (Figure 4B) , massive cell death occurred in the culture at day 3 post-induction (Figure 4C) . To determine if cell death was due to apoptosis, cells at day 2 post-induction were stained for caspase-dependent proteolytic fragment of cytokeratin 18. As shown in Figure 4D, the cytokeratin 18 - positive cells (b) appeared in the culture whereas the number of proliferating, BrdU-positive cells (a) , reduced dramatically. These effects were dependent on CTCF knockout, since there were no indications of apoptosis in the MCF7/anti-sense cells not-treated with IPTG, or in the control MCF7/Lap cells treated with IPTG (data not shown) . Taken together, the results of this work indicate that elevated levels of CTCF in breast cancer cells can have anti-apoptotic function and form a possible molecular mechanism for breast cancer development.

The effect of the apoptosis inducing compound, sodium butyrate, on CTCF expression has also been studied by the inventors. It has been shown that CTCF protein expression is down-regulated in sodium butyrate treated breast cancer cells. In particular, following sodium butyrate administration a transition from the

13OkDa form of CTCF to the 18OkDa form of CTCF has been observed (Figure 5 and 6) . After treatment of MCF-7 cells with sodium butyrate for 4 hours a 18OkDa form of CTCF is observed, with the level of expression of the 13OkDa form of CTCF seen to be reduced suggesting a possible transition from the 13OkDa to the 18OkDa form in response to the sodium butyrate treatment (Figure 6) .

Nature of the 13OkDa and 18OkDa CTCF forms observed The observation of a size shift for the CTCF protein in all paired breast tumour cell lines and tissues tested is itself significant and provides an opportunity for straightforward diagnosis of cancerous tissue.

Whilst not bound by any theory, the inventors believe that this observation is due to a differing CTCF modification pattern in normal and tumour tissue. In unpublished results (Wenqiang et al) it has been shown that the chromatin insulator protein CTCF carries a post-translational modification: poly (ADP-ribosyl) ation (PAR) ; with the conclusion that CTCF is PARlated in vivo and that a genome-wide and context dependent link exists between CTCF, PARlation and chromatin insulator function.

The inventors investigated this theory by taking total CTCF fractions, immunoprecipitated from cell lysates obtained from sodium butyrate treated MCF-7 and normal breast tissues, resolving the immunoprecipitates, western blotting and probing with anti-poly (ADP-ribose) antibody (Rabbit polyclonal antibody recognizing Poly (ADP-ribose) , Calbiochem™, Cat# 528815, Lot# B41469) . The results (Figure 7) confirm that the 18OkDa form of CTCF is recognized by anti-PAR antibodies.

Elevation of the Poly (ADP-ribose) polymerase (PARP) level is also observed in MCF-7 cells after 4 hours treatment with sodium butyrate and coincides with the appearance of the 18OkDa form of CTCF (Figure 8) . A characteristic product of PARP cleavage also appears after 48 hours sodium butyrate treatment (indicated by an arrow in Figure 8). PARP sequence information can be obtained from the NCBI database (www.ncbi . nlm. gov) , for example the amino acid sequence of human PARP can be located under accession number Q9UGN5 (GI.-17380230) .

Without being bound by any theory, the appearance of the approx. 13OkDa CTCF form in breast tumour tissues and cell lines may be the result of a change in the dynamic equilibrium of constitutive PARlation of CTCF. In turn, this may lead to a loss of CTCF- dependent chromatin insulator function.

Conclusions

CTCF is a known multifunctional transcription factor and a candidate tumour suppressor gene. Surprisingly however, the levels of CTCF in breast cell lines and tumours are unexpectedly elevated. This up-regulation of CTCF may be linked to protection of cancer cells from apoptosis. Manipulations of CTCF levels in cells, in transient transfections and in an inducible cellular model, in combination with the TUNΞL assay, confirm that reduction of CTCF in all breast cancer cell lines studied, but not in immortalized or non-breast cell lines, leads to apoptotic cell death. The characteristic pattern of CTCF expression (appearance of the 13OkDa form of CTCF specific for tumours and higher levels of CTCF in tumours, and possibly specific localization in the nucleus) observed provides a readily detectable marker for detection of tumour cells.

Specific Detection of the 13OkDa CTCF Isoform

Whilst the 13OkDa CTCF isoform is detectable using antibodies that recognize both the 13OkDa and 18OkDa isoforms, it may be desirable to detect the 13OkDa isoform using an antibody that is specific for the 13OkDa isoform and does not bind the 18OkDa isoform to any significant extent.

The inventors identified an antibody capable of specifically binding to the 13OkDa CTCF isoform (BD Biosciences Pharmingen, www.bdbiosciences.com, Catalog no.s 612148 (C39220-050) and 612149 (C39220-150) ) .

The inventors initiated studies using this antibody to test tumour samples for the presence of the 13OkDa isoform. 48 paired breast tissue samples (tumour/normal) were tested by Western blot using the 13OkDa specific antibody (Figure 15) . This analysis revealed the 13OkDa isoform to be present in 52% (25/48) of breast tumour samples. None of the normal samples were found to express the 13OkDa isoform.

This data confirms the 13OkDa CTCF isoform as a cancer-specific CTCF isoform and confirms that detection of the 13OkDa CTCF isoform can provide a reliable marker and/or prognostic indicator of cancer, particularly breast cancer.

Clinical Significance of CTCF Expression in Breast Tumours

Investigation of CTCF expression in breast tumours Tables 3 to 6 show the results of comparison between the expression profiles of CTCF and clinical parameters of the breast cancer patients. These parameters include tumour histology, tumour grade, PR expression, size of tumour, the patient's age and pre- or post- operative chemotherapy status. 19 IDC (Invasive Ductal Carcinoma) , 3 DCIS (Ductal Carcinoma in situ) , 4 AD (Adenoma) , 1 MD (Medullary carcinoma) and 4 LC (Lobular carcinoma) related tumours were used in this analysis.

All the clinical and pathological features of patients with different types of breast tumours for which CTCF immunoreactivity was assessed have been summarized in Tables 3 to 6. Analysis revealed that there were differences in the amount of CTCF expression in the IDC tumours. Thus, 3 (16%) of the tumours expressed low levels of CTCF (IRS values between 1 and 4), whilst 9 (47%) and 7 (37%) of the tumours expressed moderate (IRS values between 5 and 8) and high levels (IRS values between 9 and 12), respectively. The mean CTCF IRS value was 7.68 ± 0.18. The IRS of CTCF in DCIS was also assessed and is shown in Table 4. This data showed that 2/3 (66%) of these tumours were expressing moderate amounts of CTCF, with the IRS scores of 6 and 8, while one sample 1/3, (33%) , was grouped into the high expression group, providing an IRS value of 9 and a mean CTCF IRS value of 7.67 ± 0.51. 3/4 (75%) of the lobular related tumours (Table 5) expressed CTCF within the high IRS group, with IRS values between 9 and 12. Only 1/4 (25%) of the samples expressed moderate amounts of CTCF, with the mean CTCF IRS values of 8.5 ± 0.83 for all lobular related tumours. Table 6, shows CTCF IRS values for AD and MD (of which only one sample was obtained) tumours. Of the AD related tumours, 1/4 (25%) were low, 2/4 (50%) were moderate and 1/4 (25%) were high expressers of CTCF. The mean CTCF IRS value for AD tumours was 7.5 ± 0.85. The single MD tumour sample was expressing moderate amounts of CTCF IRS, which resulted in the IRS value of 8. Comparison of the expression of CTCF in breast reduction and breast tumour samples clearly showed that during the progression from normal to tumourigenic tissue CTCF was over-expressed.

Correlation between CTCF expression and phenotype of breast tumours

Using the information presented in Tables 3 to 6, CTCF expression in the different types of breast tumours was analysed according to their histology. This data is summarised in Figure 9. As shown in this Figure, there is an increase in the expression of CTCF from normal breast reduction tissue (normal tissue) , mean 1 ± 0.14, to breast tumour tissue, mean of 7.68 ± 0.18 in IDC, 7.67 ± 0.51 for DCIS, 8.5 ± 0.83 for ILC related tumours, 7.5 + 0.85 for AD and 8 + 0 (only one sample available) for MD tumours.

Unpaired Student's t-test showed that there were statistical differences between normal breast reduction tissue and each phenotype of tumour (p= <0.005) except for the MD phenotype, where only one sample was available and could not be statistically tested. These data suggest that during the transition from normal breast to breast tumour, of the aforementioned phenotypes, CTCF expression was up-regulated.

Investigation of the correlation between CTCF expression and tumour grade

CTCF expression levels in the different IDC grades have been compared to the control samples, which include normal (breast reduction) and paired peripheral tissues.

Using an unpaired Student' s t-test the following results were generated for comparison of CTCF expression in breast reduction tissue with each grade of IDC (Figure 10) : grade 1 tumours could not be statistically analysed because only 2 samples were available. However, there were very significant differences in the expression of CTCF between grade 2 tumours and breast reduction tissue (t = -3.78, degrees of freedom = 12 and p = 0.0026) and in grade 3 tumours and breast reduction tissues (t = -5.93, degrees of freedom = 11 and p = <0.0001) .

Comparison of CTCF and PR expression in IDC tumours

Comparison of CTCF and Progesterone Receptor (PR) expression in IDC breast tumours revealed an interesting feature, which was different to CTCF and ER expression patterns. As shown in Figure 11 there was an increase in PR expression (IRS values) from 2.7 ± 0.84 to 5.9 ± 0.16 with an increase in CTCF expression from low to moderate. As CTCF expression continued to increase, from moderate to high, there was a decrease in the PR IRS values from 5.9 ± 0.16 to 3.4 ± 0.43. From this data we can conclude that there is an increase in PR expression with an increase in CTCF expression to moderate levels.

However, when CTCF expression continues to increase from moderate to high levels we see a reduction in PR expression. These data suggest that there is a critical expression level of CTCF with respect to the expression levels of PR. We carried out a Student' s t-test and compared CTCF expression groups with PR expression. We observed that there was a significant difference in the PR status in the low to moderate CTCF expression groups (t-test = -2.93, degrees of freedom = 9 and p = 0.017) . However, we found that there was no significant relationship between the PR expression of the low to high CTCF expression groups or the moderate to high CTCF expression groups (t-test = -0.319, degrees of freedom = 9 and p = 0.76 and t-test = 1.94, degrees of freedom = 14 and p = 0.75, respectively) .

Comparison between CTCF expression and tumour size Using the clinical data of the size of the tumours, three groups of tumour size were generated: Small, lmm-15mm; Medium, 16mm- 30mm; and Large, 31-55mm. The mean CTCF IRS value in each group was: normal breast reduction, 1 ± 0.05; Small, 4 + 0 (of which there was only one sample to compare); Medium, 7.29 ± 0.21; and Large, 8.89 ± 0.31 (Figure 12). These data suggested that there was an increase in CTCF IRS value with an increase in the size of the tumour. Statistical analysis of CTCF expression between breast reduction tissue and each size group showed that there were very significant differences in the CTCF IRS values (Table 7) . However, it should be noted that it was not possible to carry out statistical analysis between the small sized tumour group and breast reduction tissue, because the sample number for the small sized tumour group was too small (n=l) . This prevented us from carrying out a statistical test but if we compare the difference between the mean CTCF IRS value of the small size tumour, IRS 4, and that of the breast reduction tissue, mean IRS of I₇ a difference in CTCF expression can be observed.

The Student' s t-test results were tabulated and are shown in Table 7. The unpaired Student's t-test was carried out to determine whether there was a relationship between the expression of CTCF in normal breast tissue (no tumour therefore size = 0mm) and the CTCF expression in tumours of different sizes.

Together these results showed that there was a very significant increase in CTCF expression with increase in tumour size.

Evaluation of potential correlation between CTCF expression and age

In this test, each CTCF expression group, low, moderate and high, was used to analyse the possible relationship between age and CTCF expression. The result of this analysis is shown in Figure 13. In the low expression group, CTCF IRS 1-4, the mean age of the patient was 70 ± 2.96 years, with an average CTCF IRS value of 3. There was a slight decrease in the average age of the patient in the moderate expression group, CTCF IRS 5-8, from 70 ± 2.96 years to 66 + 1.37 years producing an average CTCF IRS expression value of 6.36. Finally, in the high expression group there was a further decrease in mean age to 60 ± 1.89 years and an average CTCF IRS value of 11.36. These data indicated that as the patient increased in age there was a decrease in the level of CTCF expression.

The Student's t-test was carried out and generated the following non-significant results: comparison of the age of patients within the low CTCF expression group with the age of patients within the moderate CTCF expression group t-test = 0.473, degrees of freedom = 14 and p = 0.6 and the age of patients in the low CTCF expression group with the age of patients in the high CTCF expression group t-test= 1.14, degrees of freedom = 11 and p = 0.28.

Investigation of a correlation between CTCF expression in breast tumours and the time of chemotherapy treatment

We decided to combine the CTCF expression results from the untreated breast tumours with the post-operative chemotherapy treated patients because the tumour would have been removed from the patient before treatment had commenced. With this in mind, we went on to analyse CTCF expression in three groups only: breast reduction (normal breast) , no treatment/post-operative treatment breast tumours and pre-operative treated breast tumours (Figure 14) .

The mean CTCF IRS in the combined un-treated tumours with the post-operatively treated tumours of the IDC tumours was 7.63 ± 0.23, while the tumours that were treated with chemotherapy before operative surgery (pre-treated) showed a lower CTCF expression of 6.0 ± 0.0 (n=2).

Our data suggest that there was a slight decrease in the expression of CTCF in tumours which had been treated pre- operatively, whilst post-operative treatment had little effect on the expression of CTCF. Comparison of CTCF expression between the different groups could not be tested statistically because the pre-treated sample number was too small (n=2) .

Conclusions

CTCF is expressed at higher levels in breast tumours compared with normal breast tissues

A positive correlation was observed between the expression of CTCF and tumour grade and tumour size.

Table 1

CTCF over-expression in Breast Tumour Tissue as ascertained by scoring of Inununohistochemical staining.

Tumour type refers to diagnosis supplied from a local histologist; IDC: Invasive Ductal Carcinoma; DCIS: Ductal Carcinoma in Situ; ILC: Invasive Lobular Carcinoma; ILCIS: Invasive Lobular Carcinoma in Situ; AC: Adenocarcinoma; LBH: Lobular Hyperplasia Benign and MD: Medullary Carcinoma. Type of staining was identified as either nuclear only, nuclear and cytoplasmic or cytoplasmic only.

All normal paired tissue showed very weak (+/-) CTCF immunostaining . Table 2

10

15

20

25

30

35

Table 2. Normal Breast and Breast Tumours assessed for CTCF Immuno- Reactivity: Higher expression of CTCF is detected in all types of breast tumours compared to reduction samples.

Immunostaining of frozen tissue sections was performed with the anti-CTCF rabbit polyclonal antibodies. As a reference, the normal breast tissue from patients after reductive surgery were used for staining. A scale from 0 (no detectable signal) to +++ (very strong signal) was used to assess staining intensity.

Abbreviations used in table: HMEC; Human Mammary Epithelial Cells (obtained from Cambrex™) ; NEp, Normal Epithelial, NBr, normal breast reduction tissue; IDC, Invasive Ductal Carcinoma; DCIS, Ductal Carcinoma in-situ; ILC, Infiltrating Lobular Carcinoma; LCIS, Lobular Carcinoma in-situ; LHP, Lobular Hyperplasia; AD, Adenocarcinoma; MD, Medullary Carcinoma. T, Tumour; R, Reduction.

Table 3

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Table 3. Clinical data and IRS for CTCF of patients with breast tumours. IRS refers to the Immunoreactive score, which is calculated by multiplying the staining intensity by the percentage of immunoreactive positive cells. Abbreviations used in Table: post, post-operative chemotherapy; pre, pre-operative chemotherapy, N. K., not known; IDC, Invasive Ductal Carcinoma; T, tumour; ER, Estrogen Receptor and PR, Progesterone Receptor; Post- or Pre-operative treatment abbreviations: FEC, combination chemotherapy consisting of 5-Fluorourocil, Epirubicin and Cyclophosphamide; Eryth, Erythromycin and CMF, Cyclophosphamide, Metroleate and 5-Fluorourocil.

Table 4

Table 4. Clinical data and IRS values of CTCF expression in benign Ductal Carcinoma in situ breast tumours. IRS refers to the Immunoreactive score, which is calculated by multiplying the staining intensity by the percentage of immunoreactive positive cells. Abbreviations used in Table: post, post-operative chemotherapy; pre, pre-operative chemotherapy; DCIS, Ductal Carcinoma in situ; T, tumour; ER, Estrogen Receptor and PR, Progesterone Receptor. T168* refers to a second biopsy removed from the same patient (in this instance T155 as an early biopsy, which we did not

10 receive) . 4-» o

15

20

Table 5

Table 5. Clinical data and IRS values for CTCF expression in Lobular related breast tumours. IRS refers to the Immunoreactive score, which is calculated by multiplying the staining intensity by the percentage of immunoreactive positive cells. Abbreviations used in Table: post, post-operative chemotherapy; pre, pre-operative chemotherapy; N. K., not known; ILC, Invasive Lobular Carcinoma; LCIS, Lobular Carcinoma in situ; LHP, Lobular Hyperplasia; T, tumour, ER, Estrogen Receptor and I—»

10 PR, Progesterone Receptor. Post or Pre-operative treatment abbreviations: FEC; combination chemotherapy consisting of 5-Fluorourocil, Epirubicin and Cyclophosphamide and Tax; Taxotere.

15

20

Table 6

Table 6. Clinical data and IRS values of CTCF expression in Adeno and Medullary carcinoma breast tumours. IRS refers to the Immunoreactive score, which is calculated by multiplying the staining intensity by the percentage of immunoreactive positive cells. Abbreviations used in Table: post, post-operative chemotherapy; pre, pre-operative chemotherapy; N.K., not known; AD, Adenoma; MD, Medullary carcinoma; T, tumour, ER, Estrogen Receptor and PR, Progesterone Receptor Post- or Pre¬

10 operative treatment abbreviations: FEC; combination chemotherapy consisting of 5-Fluorourocil, Epirubicin and Cyclophosphamide; Tax, Taxotere; Eryth, Erythromycin and EC, Epirubicin and Cyclophosphamide .

Table 7

10 Table 7. Analysis of the association between expression levels of CTCF (IRS) and the size of the breast tumour. The confidence level was set at p= 0.05 (95%). Abbreviations used are: t, t-test; dof, degrees of freedom; and N. D., not determined.

U)

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Claims

Claims :

1. A method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of: detecting the presence of CTCF having a molecular weight between 12OkDa and 14OkDa in a tissue sample from said patient.

2. The method of claim 1 wherein the CTCF polypeptide or protein has a molecular weight of approximately 13OkDa.

3. The method of claim 1 or 2 wherein the step of detection comprises contacting a CTCF antibody with said sample.

4. The method of claim 3 wherein said antibody is a CTCF monoclonal antibody.

5. A method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of: detecting both a first and a second molecular weight form of CTCF in a tissue sample from a patient.

6. The method of claim 5 wherein the first CTCF form has a molecular weight of between 170 and 19OkDa.

7. The method of claim 5 wherein the first CTCF form has a molecular weight of approximately 18OkDa.

8. The method of any one of claims 5 to 7 wherein the second CTCF form has a molecular weight of between 120 and 14OkDa

9. The method of any one of claims 5 to 7 wherein the second CTCF form has a molecular weight of approximately 13OkDa.

10. The method of any one of claims 5 to 9 wherein the step of detection comprises contacting a CTCF antibody with said sample.

11. The method of claim 10 wherein said antibody binds to both said first and second CTCF forms.

12. The method according to any one of claims 5 to 9 wherein the step of detection comprises contacting said sample with a first antibody which binds specifically to said first CTCF form, and a second antibody which binds specifically to said second CTCF form.

13. The method of claim 12 wherein said first and second antibodies are monoclonal antibodies to said first and second CTCF forms respectively.

14. The method of any one of claims 5 to 13 further comprising the step of contacting said sample with an antibody capable of binding to a poly (ADP-ribose) modified form of CTCF.

15. A method according to any one of the preceding claims wherein the molecular weight of CTCF polypeptide or protein is an apparent molecular weight determined by electrophoretic separation of the components of said tissue sample by 10%SDS PAGE.

16. A method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the steps of: detecting the level of expression of CTCF in a tissue sample from a patient; and comparing said level to a standard level of CTCF expression in healthy tissue, wherein an increase in the level of expression of CTCF in said tissue sample relative to said healthy tissue is indicative of the presence of a cancer or a pre-cancerous condition.

17. The method of claim 16 wherein said detection comprises quantitatively determining the amount of CTCF polypeptide or protein in said sample.

18. The method of claim 16 or 17 wherein the standard level of CTCF polypeptide or protein is quantitatively determined by the same method used to quantitatively determine the amount of CTCF polypeptide or protein in said sample.

19. The method of claim 17 or 18 wherein the method of quantitation comprises immunohistochemical scoring of stained samples .

20. A method according to any one of the preceding claims wherein said cancer is a breast cancer.

21. A CTCF antibody for use in the diagnosis of a cancer or a pre-cancerous condition.

22. The antibody of claim 21 wherein said antibody binds specifically to a form of CTCF polypeptide or protein having a molecular weight between 120 and 14OkDa.

23. The antibody of claim 21 wherein said antibody binds specifically to an approimately 13OkDa form of CTCF polypeptide or protein.

24. The antibody of any one of claims 21 to 23 wherein said antibody is a CTCF monoclonal antibody.

25. The antibody of any one of claims 21 to 24 wherein said cancer is a breast cancer.

26. Use of an antibody which specifically binds to CTCF in the diagnosis of a cancer or a pre-cancerous condition.

27. The use as claimed in claim 26 wherein said antibody binds specifically to a form of CTCF polypeptide or protein having a molecular weight between 120 and 14OkDa.

28. The use as claimed in claim 26 wherein said antibody binds specifically to an approximately 13OkDa form of CTCF polypeptide or protein.

29. The use as claimed in any one of claims 26 to 28 wherein said antibody is a CTCF monoclonal antibody.

30. The use as claimed in any one of claims 26 to 29 wherein said cancer is a breast cancer.

31. An assay kit comprising an antibody which binds specifically to a form of CTCF polypeptide or protein having a molecular weight between 120-14OkDa.

32. An assay kit as claimed in claim 31 wherein said molecular weight is approximately 13OkDa.

33. A method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of detecting the presence or absence of a poly (ADP-ribosyl) modified form of CTCF.

34. The method of claim 33 wherein said detection comprises contacting a tissue sample from said patient with an anti- poly (ADP-ribose) antibody.

35. The method of claim 33 or 34 wherein said detection of the presence or absence of a poly (ADP-ribosyl) modified form of CTCF comprises detecting a reduction or increase in the amount of said poly (ADP-ribosyl) modified form of CTCF in a patient sample relative to a healthy tissue sample.

36. A method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of detecting a change or difference in the level of poly (ADP-ribose) polymerase (PARP) activity or expression in a sample from said patient relative to a level of PARP activity or expression in a healthy tissue.

37. A method of diagnosing a cancer or a pre-cancerous condition in a patient comprising the step of detecting, in a sample taken from the patient, an isoform of CTCF polypeptide or protein that has an apparent molecular weight of between 60% and 80% of the apparent molecular weight of a CTCF isoform detectable in healthy tissue of a corresponding type.

38. The method of claim 37 wherein the CTCF isoform being detected in the patient sample has an apparent molecular weight of between 65% and 75% of the apparent molecular weight of the CTCF isoform detectable in the healthy tissue.

39. The method of claim 37 wherein the CTCF isoform being detected in the patient sample has an apparent molecular weight of between 70% and 75% of the apparent molecular weight of the CTCF isoform detectable in the healthy tissue.

40. The method of any one of claims 37 to 39 wherein the CTCF isoform of lower molecular weight is not detectable in the healthy tissue.