WO2011044902A1

WO2011044902A1 - Tff3 hypomethylation as a novel biomarker for prostate cancer

Info

Publication number: WO2011044902A1
Application number: PCT/DK2010/050253
Authority: WO
Inventors: Karina Dalsgaard SØRENSEN; Else Marie Vestergaard; Torben Falck ØRNTOFT
Original assignee: Aarhus Universitet; Region Midtjylland
Priority date: 2009-10-13
Filing date: 2010-10-05
Publication date: 2011-04-21

Abstract

The present invention relates to a TFF3 gene as a marker of prostate cancer. The present invention thus relates to methods for diagnosing prostate cancer or predicting or prognosticating the disease outcome comprising steps of determining the methylation status of a TFF3 gene and/or the transcriptional and translational expression levels of a TFF3 gene. The present invention further relates to methods for monitoring disease progression in an individual having contracted prostate cancer as well as methods for determining the treatment regime of an individual having contracted prostate cancer comprising steps of determining the methylation status of a TFF3 gene and/or the transcriptional and translational expression levels of a TFF3 gene.

Description

TFF3 hypomethylation as a novel biomarker for prostate cancer

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to a TFF3 gene as a marker of prostate cancer. The present invention thus relates to methods for diagnosing prostate cancer, predicting or prognosticating the disease outcome, for example recurrence following surgery, as well as methods for monitoring disease progression in an individual having contracted prostate cancer. The present invention also pertains to a kit for use in the methods, in addition to methods of treatment.

Background of invention

Prostate adenocarcinoma is a major cause of cancer morbidity and mortality in the

Western world. The gold standard algorithm for diagnosis currently entails digital rectal exam and measurement of serum prostate-specific antigen (PSA) and if either is suspicious it is followed by histopathologal inspection of needle biopsies obtained trans-rectal prostatic recovering <1 % of the prostate volume. However, serum PSA can be elevated in benign conditions and needle biopsy may fail to identify even significant amounts of cancer due to sampling error. The use of PSA testing for prostate cancer detection has increased the incidence of diagnosis as well as shifted detection to earlier and theoretically curable stages. The PSA method, however, is associated with significant false negative rates and does not distinguish well between clinically indolent or aggressive tumors (Nelson WG et al.: Front Biosci 2007;12: 4254-66).

Indicators currently used for outcome prediction following intended curative radical prostatectomy of primary prostate cancer are PSA, Gleason score, pathological stage and surgical margin status (Humphrey PA et al.: Mod Pathol 2004;17: 292-306), but additional markers are needed to improve stratification of low, medium and high risk patients. Therefore, the introduction of additional diagnostic tests is needed to improve the sensitivity of prostate cancer diagnosis.

Prostate cancer development and progression is characterized by the accumulation of genetic and epigenic alterations. Epigenetic changes seem to generally occur at earlier stages of carcinogenesis and may be more common and consistent

(Yegnasubramanian S et al.: Cancer Res 2004;64: 1975-86). Accordingly, mapping of „ epigenetic alterations could be particularly valuable for biomarker discovery. Several types of epigenetic changes have been reported for prostate cancer including DNA hypomethylation, loss of imprinting, and altered histone modification patterns. The best described epigenetic alteration in prostate carcinogenesis, however, is DNA

hypermethylation of specific CpG islands located near gene promoters, as reported for numerous genes including GSTP1, APC, and RASSF1A (for recent reviews see Nelson WG et al.: Front Biosci 2007;12: 4254-66 and Dobosy JR et al.: J Urol

2007;177: 822-31 ). CpG island hypermethylation has been closely linked to gene silencing (Jones PA et al.: Nat Rev Genet 2002;3: 415-28). Only a small number of genes activated through hypomethylation of both CpG island and non-CpG island regions have been identified in PC, e.g. MAGE-1 1 (Karpf AR et al, Mol Cancer Res 2009;7:523-35), LINE-1 (Yegnasubramanian S et al, Cancer Rec 2008;68:8954- 67), WNT5a, CRIP1 and S100P (Wang Q et al, Oncogene 2007 ;26:6560-5).

Methylation of DNA is a mechanism for changing the base sequence of DNA without altering its coding function. DNA methylation is a heritable, reversible and epigenetic change. DNA methylation harbours the potential to alter gene expression which in turn affects developmental and genetic processes. The methylation reaction involves flipping a target cytosine out of an intact double helix thereby allowing the transfer of a methyl group from S-adenosylmethionine in a cleft of the enzyme DNA (cystosine-5)- methyltransferase (Klimasauskas et al., Cell 76:357-369, 1994) to form 5- methylcytosine (5-mCyt). This enzymatic conversion is the only epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic

development (Bird, Cell 70:5-8, 1992; Laird and Jaenisch, Human Mol. Genet. 3:1487- 1495, 1994; and Bestor and Jaenisch, Cell 69:915-926, 1992).

CpG-rich sequences are known as CpG islands. CpG islands are distributed across the human genome and often span the promoter region as well as the first exon of protein coding genes. Methylation of individual promoter region CpG islands usually turns off transcription by recruiting histone deacetylases, which supports the formation of inactive chromatin (Humphrey PA et al.: Mod Pathol 2004;17: 292-306). CpG islands are typically between 0.2 to about 1 kb in length and are located upstream of many housekeeping and tissue-specific genes, but may also extend into gene coding regions. Therefore, it is the methylation of cytosine residues within CpG islands in somatic tissues, which is believed to affect gene function by altering transcription (Cedar, Cell 53:3-4, 1988).

In cancer cells CpG sequences at genes that are normally methylated and silenced in a tissue-specific manner may become aberrantly hypomethylated, causing inappropriate activation of the corresponding gene. Several studies have described a correlation between site-specific hypomethylation and increased expression of genes related with cancer such as MAGE, S100A4, and others. And because DNA hypomethylation has been considered a reversible biological modification, such genes may represent novel targets for cancer therapy.

Abnormal methylation of CpG islands associated with tumor suppressor genes may also cause decreased gene expression. Increased methylation of such regions may lead to progressive reduction of normal gene expression giving abnormal cells a growth advantage (i.e., a malignancy).

Methylation of promoter regions, particularly in tumour suppressor genes, and genes involved in apoptosis and DNA repair, is one of the hallmarks of cancer (Humphrey PA et al.: Mod Pathol 2004;17: 292-306). Changes in the methylation status of these genes are an early event in cancer and continue throughout the different stages of the cancer. Specifically, distinct tumour types often have characteristic patterns of methylation, which can be used as markers for early detection and/or monitoring the progression of carcinogenesis. For therapeutic purposes, the methylation of certain genes, particularly DNA repair genes, can cause sensitivity to specific

chemotherapeutics and methylation of those genes can thereby act as a predictive marker if those chemotherapeutic agents or treatment should be used.

The mammalian trefoil factor family include the breast cancer-associated peptide TFF1 (pS2), TFF2 (spasmolytic polypeptide SP) and TFF3 (intestinal trefoil factor) (7). Trefoil factors are small mucin-associated peptides that are expressed in several tissues of the human body, but most pronouncedly in the gastrointestinal epithelium. Members of the trefoil peptide family are overexpressed at the gene and protein level in a variety of cancers, including breast, intestinal, pancreas, and PCs (8-12), and may contribute to the malignant behaviour of cancer cells through their proposed biological functions as scatter factors, proinvasive, antiapoptotic, and angiogenic agents (13-15). As the PSA method is associated with significant false negative rates and does not distinguish well between clinically indolent or aggressive tumors (Nelson WG et al.: Front Biosci 2007; 12: 4254-66), there is a need for novel markers of prostate cancer that can be used on their own, or in combination with existing markers, or other diagnostic and/or predictive methods, such as histopathological examination of biopsies.

In the present invention, TFF3 is disclosed as a novel marker of prostate cancer which can be used as an independent marker of prostate cancer, or in combination with established markers such as PSA, Gleason score, tumor stage, and surgical margin outcome. TFF3 is furthermore disclosed as a predictor of biochemical recurrence after radical prostatectomy. Summary of invention

The present invention relates to a TFF3 gene as a marker of prostate cancer. The present invention thus relates to methods for diagnosing prostate cancer, predicting or prognosticating the diasease outcome, for example recurrence following surgery, as well as methods for monitoring disease progression in an individual having contracted prostate cancer.

One aspect relates to a method for assisting in diagnosing and/or for diagnosing prostate cancer in an individual comprising the steps of

i) determining the methylation status of a TFF3 gene (SEQ ID NO:1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:1 , or part thereof in a sample from said individual, and/or ii) determining the transcriptional expression level of said TFF3 gene, or nucleotide sequence having at least 90% sequence identity with SEQ ID NO:2, or part thereof in said sample and/or

iii) determining the translational expression level of said TFF3 gene, or amino acid sequence having at least 90 % sequence identity with SEQ ID

NO:3, or part thereof in said sample

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of presence or absence of prostate cancer. A decreased methylation status is indicative of the presence of prostate cancer, and similarly an increased transcriptional and/or translational expression level in the sample is indicative of the presence of prostate cancer.

A second aspect of the present invention pertains to a method for assisting in prognosing and/or for prognosing the disease progression of prostate cancer in an individual having contracted prostate cancer comprising the steps of

iii) determining the translational expression level of said TFF3 gene, or amino acid sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof in said sample

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of presence or absence of prostate cancer.

It is appreciated that a decreased methylation status is indicative of disease

progression of prostate cancer, and similarly an increased transcriptional and/or translational expression level in the sample is indicative of disease progression of prostate cancer. Prognosis is used to predict the outcome or disease progression in the absence of intervention for example in the form of surgery.

Another aspect of the present invention relates to predicting the outcome of prostate cancer in an individual having contracted cancer. Prediction is here used to describe the outcome or disease progression following intervention. Thus, the present invention relates to a method for assisting in predicting and/or for predicting the outcome of prostate cancer in an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the outcome of prostate cancer.

It is appreciated that an increased methylation status is indicative of disease progression of prostate cancer, and similarly a decreased transcriptional and/or translational expression level in the sample is indicative of disease progression of prostate cancer.

A further aspect of the present invention relates to predicting the recurrence risk of prostate cancer in an individual having contracted cancer. Thus, claims are directed against a method for assisting in predicting and/or for predicting the recurrence risk of prostate cancer in an individual having contracted prostate cancer comprising the steps of

i) determining the methylation status of a TFF3 gene (SEQ ID NO:1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:1 , or part thereof in a sample from said individual, and/or ii) determining the transcriptional expression level of said TFF3 gene, or nucleotide sequence having at least 90% sequence identity with SEQ ID

NO:2, or part thereof in said sample and/or

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the recurrence risk of prostate cancer.

It is appreciated that a decreased methylation status is indicative of recurrence risk of prostate cancer, and similarly an increased transcriptional and/or translational expression level in the sample is indicative of recurrence risk of prostate cancer. Such recurrence risk is for example determined following radical prostatectomy, radiation therapy, cryotherapy or brachytherapy. Yet another aspect of the present invention relates to a method for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression in an individual having contracted prostate cancer, comprising the steps of

NO:3, or part thereof in said sample

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of progression of prostate cancer.

It is appreciated that an increased methylation status is indicative of the progression of prostate cancer, and similarly a decreased transcriptional and/or translational expression level in the sample is indicative of the progression of prostate cancer.

A further aspect pertains to methods for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent/indolent to an aggressive prostate cancer in an individual having contracted prostate cancer comprising the steps of i) determining the methylation status of a TFF3 gene (SEQ ID NO:1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:1 , or part thereof in a sample from said individual, and/or ii) determining the transcriptional expression level of said TFF3 gene, or nucleotide sequence having at least 90% sequence identity with SEQ ID NO:2, or part thereof in said sample and/or

iii) determining the translational expression level of said TFF3 gene, or amino acid sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof in said sample _n wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the progression of prostate cancer from a silent/indolent to an aggressive prostate cancer. A silent/indolent prostate cancer is a slow-growing and slow-progressing organ- confined prostate cancer with no or only minor clinical symptoms, whereas an aggressive prostate cancer is which has progressed or will progress relatively fast (i.e. within the remaining life expectancy of a given patient) to non-organ-confined prostate cancer.

Yet a further aspect of the present invention relates to a method for assisting in determining and/or determining the treatment regime of an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the treatment regime to be offered to the individual having contracted prostate cancer. It is appreciated that the methods optionally comprises a step of comparing the methylation status of said TFF3 gene determined in the sample to the methylation status of a control sample, wherein the methylation status of said sample can be determined as increased or decreased. The methylation status of said TFF3 gene in a sample being <50%, is indicative of a decreased methylation level, wherein said sample is a tissue sample and/or an urine sample. In one embodiment, the methylation status of said TFF3 gene in a sample is decreased in comparison with a control sample, for example a fully methylated DNA fragment.

The methods of the present invention may optionally comprise a step of comparing the transcriptional and/or translational expression level of the TFF3 gene determined in the sample to the expression level of a control sample, wherein the expression level in the sample can be determined as increased or decreased.

The samples used in the methods are typically selected from tissue sample, blood, plasma, serum, semen, or urine. In one particular embodiment the sample is a biopsy of the prostate gland or resected prostate tissue following radical prostatectomy.

The methods of the present invention may further comprise a step of measuring the level of prostate specific antigen (PSA) in an individual.

Another aspect of the present invention relates to a pharmaceutical composition for the treatment of prostate cancer comprising

i) at least one transcription inhibitor capable of decreasing the

transcript levels of a TFF3 gene transcript (SEQ ID NO: 2), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:2, or part thereof, and/or

ii) at least one translational inhibitor capable of decreasing the

translational expression levelof the TFF3 gene (SEQ ID NO: 3), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof. The transcriptional inhibitor may be a functional RNA molecule displaying

complementary sequence to a TFF3 gene transcript (SEQ ID NO: 2), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:2, or part thereof. The functional RNA molecule is for example a siRNA molecule, a microRNA molecule, a shRNA molecule and/or an antisense RNA molecule. The translational inhibitor may be an antibody directed against an epitope of the TFF3 protein or part thereof.

Yet a further aspect relates to an assay comprising at least one detection member for a TFF3 gene, transcriptional and/or translational product or part thereof for use in the methods of the present invention. Such a detection member is an antibody directed against an epitope of the TFF3 protein or part thereof, oligonucleotides, primers and/or probes. The assay may further comprise means for providing the level and/or means for providing informations as to whether the level is above or below a cut off value.

A final aspect of the present invention relates to the use of an antibody directed against an epitope of the TFF3 protein or part thereof in the detection of the translational expression level of a TFF3 gene, or part thereof

i) for assisting in the diagnosis and/or for diagnosing of prostate

cancer

ii) for assisting in the prognosis and/or for the prognosis of the disease progression of prostate cancer

iii) for assisting in the prediction and/or the prediction of the progression of prostate cancer

iv) for assisting in predicting and/or for predicting the recurrence risk of prostate cancer

v) for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression

vi) for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cancer vii) for assisting in determining and/or determining the treatment regime The antibody may be monoclonal, polyclonal, or a mixture of at least two monoclonal antibodies. Description of Drawings

Figure 1. Genomic bisulfite sequencing of the TFF promoter associated CpG sites in prostate cell lines with low/absent (BPH1 , PNT1A, DU 145, and PC3) and high

(DUCAP, VCP, and LNCAP) endogenous TFF1 and TFF3 expression. A, schematic representation of the promoter/enhancer region of the TFF1 , TFF2, and TFF3 genes and location of PCR primers (arrows). The positions of CpG dinucleotides are marked by vertical lines and putative binding sites for transcription factors predicted by

Matlnspector 2.2 software are depicted upon the sequence of TFF1 , TFF2, and TFF3 genes. For the investigation of the methylation of the TFF3 promoter we used the revised coding sequence which has the 5' end of exon 1 extended by 170-bp compared to the transcript originally described in (49). For TFF2 and TFF3 nucleotide positions relative to the translation start site (+1 ) are given. For TFF1 the positions are as indicated in (50). B, TFF promoter methylation in DNA from PC cell lines. Average methylation at each analyzed CpG site in the TFF1 , TFF2, and TFF3 promoter is indicated based on bisulfite sequencing of at least 7 individual clones.

Figure 2. Normalized TFF1 (A) and TFF3 (B) RNA expression (grey bars) and protein concentrations (black bars) as measured in cell culture supernatants (pmol/L) in untreated prostate cell lines. Bars denote SD of replicate experiments. 5-aza, normalized TFF expression in cell lines treated with 1 μη"ΐοΙ/Ι_ 5-aza-2-deoxycytidine; 5- aza +PBA, normalized TFF expression in cell lines treated with 1 μη"ΐοΙ/Ι_ 5-aza-2- deoxycytidine in combination with 1 mmol/L 4-phenylbutyric acid. All reactions were run in triplicate. ^*, no induction of TFF1/TFF3 RNA expression; nd, not determined.

Figure 3. TFF1 and TFF3 methylation patterns in nonmalignant (KPC) and PC (PC) tissue samples determined by bisulfite sequencing. Each line represents a single clone. Open and closed squares, unmethylated and methylated CpGs, respectively.

Figure 4. Average TFF3 promoter methylation levels for CpG sites -127, -108, -89, and -80 (·) in a control group (BPH; n=12) and in patients with PC (PC; n=10). Horizontal lines indicate median methylation values and serum TFF3 values, respectively.

Detailed description of the invention

Epigenetic changes, resulting from DNA and histone modifications, may lead to heritable silencing of genes without a change in their coding sequence. These changes are usually established in parental germ cells and inherited post fertilization to the offspring during successive cell divisions.

The most prominent DNA epigenetic modification is by methylation typically on CpG islands. These are sequence regions of more than 500 base pairs in size with a GC content greater than 55%, normally kept free of DNA methylation and are the sites of DNA methylation in various conditions or pathologies. CpG islands are located within the promoter regions of about 40% of mammalian genes and when methylated, cause gene silencing. DNA hypomethylation at specific CpG sites is often associated with activation of affected genes. Epigenetic changes may also occur on chromatin.

Chromatin modifications such as histone acetylation, deacetylation, methylation or demethylation of conserved lysine residues on the amino-terminal tail domains are associated with transcriptional activation or silencing.

The present invention discloses the TFF3 gene as a marker of prostate cancer. The present inventors have found that the TFF3 is a common target for promoter hypomethylation in prostate cancer. Thus, the methylation status of TFF3 can be used as a novel tool for assisting in the diagnosis of prostate cancer presence or absence, as well as for assisting in the prediction and/or prognosis of the disease progression in an individual having contracted cancer. Furthermore, it is disclosed that the

transcriptional and/or translational expression level of the TFF3 gene can further be used for assisting in the diagnosis of prostate cancer presence or absence, as well as for assisting in the prediction and/or prognosis of the disease progression in an individual having contracted cancer. Definitions

The terms 'prostate cancer' is herein used interchangeably with and is equivalent to the term 'prostate adenocarcinoma'. The terms 'diagnosis, diagnostic, diagnosing' are used herein as terms for the act of determining the nature and cause of a disease for example through evaluation of patient history, examination, and review of laboratory data.

The term 'assisting' as used to describe the methods herein is to emphasise that the methods herein are directed solely to the step of gathering data to assist in the diagnosis, prediction, and prognosis of prostate cancer. Accordingly, methods for assisting in such methods are not to be construed as diagnostic methods practised on the human body. The terms 'prognosis, prognostic, prognosing' are used herein as terms for predicting the outcome or disease progression independent of possible intervention for example in the form of surgery, medication etc. The terms 'prediction, predictive, predicting' are used herein as terms for predicting the outcome or disease progression following intervention for example in the form of surgery, medication etc. For example such an intervention may be radical

prostatectomy.

The term "treatment", as used herein comprises any type of therapy, which aims at terminating, preventing, ameliorating and/or reducing the susceptibility to prostate cancer. In a preferred embodiment, the term treatment relates to prophylactic treatment, i.e. a therapy to reduce the susceptibility of prostate cancer.

The term 'individual' and individual in need thereof as used herein refers to a male mammal, preferably a male human being at any age which is suspected of having prostate cancer, is predisposed to develop prostate cancer or has contracted prostate cancer.

The terms 'therapeutically effective amount' means an amount that is sufficient to elicit a desired response.

The term 'gene' as used herein refers to its normal meaning, a nucleic acid sequence with a transcriptional capability, i.e., which can be transcribed into an RNA sequence (an expressed sequence) which in most cases, is translated into an amino acid sequence, along with the regulatory sequences that regulate expression or engage in the expression of expressed sequences.

A double stranded polynucleotide contains two strands that are complementary in sequence and capable of hybridizing to one another.

A nucleotide is herein defined as a monomer of RNA or DNA. A nucleotide is a ribose or a deoxyribose ring attached to both a base and a phosphate group. Both mono-, di-, and tri-phosphate nucleosides are referred to as nucleotides.

The term 'nucleotides' as used herein refers to both natural nucleotides and non- natural nucleotides capable of being incorporated - in a template-directed manner - into an oligonucleotide, preferably by means of an enzyme comprising DNA or RNA dependent DNA or RNA polymerase activity, including variants and functional equivalents of natural or recombinant DNA or RNA polymerases. Corresponding binding partners in the form of coding elements and complementing elements comprising a nucleotide part are capable of interacting with each other by means of hydrogen bonds. The interaction is generally termed "base-pairing". Nucleotides may differ from natural nucleotides by having a different phosphate moiety, sugar moiety and/or base moiety. Nucleotides may accordingly be bound to their respective neighbour(s) in a template or a complementing template by a natural bond in the form of a phosphodiester bond, or in the form of a non-natural bond, such as e.g. a peptide bond as in the case of PNA (peptide nucleic acids). Nucleotides according to the invention includes ribonucleotides comprising a nucleobase selected from the group consisting of adenine (A), uracil (U), guanine (G), and cytosine (C), and

deoxyribonucleotide comprising a nucleobase selected from the group consisting of adenine (A), thymine (T), guanine (G), and cytosine (C). Nucleobases are capable of associating specifically with one or more other nucleobases via hydrogen bonds. Thus it is an important feature of a nucleobase that it can only form stable hydrogen bonds with one or a few other nucleobases, but that it can not form stable hydrogen bonds with most other nucleobases usually including itself. The specific interaction of one nucleobase with another nucleobase is generally termed "base-pairing". The base pairing results in a specific hybridisation between predetermined and complementary nucleotides. Complementary nucleotides according to the present invention are nucleotides that comprise nucleobases that are capable of base-pairing. Of the naturally occurring nucleobases adenine (A) pairs with thymine (T) or uracil (U); and guanine (G) pairs with cytosine (C). Accordingly, e.g. a nucleotide comprising A is complementary to a nucleotide comprising either T or U, and a nucleotide comprising G is complementary to a nucleotide comprising C.

The term 'oligonucleotide' is used herein interchangebly with polynucleotide. As used herein the term "oligonucleotide" refers to a single stranded or double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring bases, sugars and covalent internucleoside linkages (e.g., backbone) as well as oligonucleotides having non-naturally-occurring portions which function similarly to respective naturally-occurring portions (see disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;

5,286,717; 5,321 ,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541 ,306; 5,550,1 1 1 ; 5,563,253; 5,571 ,799;

5,587,361 ; and 5,625,050). The term oligonucleotide thus also refers to any

combination of oligonucleotides of natural and non-natural nucleotides. The natural and/or non-natural nucleotides may be linked by natural phosphodiester bonds or by non-natural bonds. Preferred oligonucleotides comprise only natural nucleotides linked by phosphodiester bonds. Oligonucleotide is used interchangeably with polynucleotide. The oligomer or polymer sequences of the present invention are formed from the chemical or enzymatic addition of monomer subunits. The term "oligonucleotide" as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptide nucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), and the like, capable of specifically binding to a single stranded polynucleotide tag by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units, e.g. 40-60. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'→ 3' order from left to right and the "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Usually oligonucleotides of the invention comprise the four natural nucleotides; however, they may also comprise methylated or non-natural nucleotide analogs. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers (Tetrahedron Lett., 22, 1859-1862, 1981 ), or by the triester method according to Matteucci, et al. (J. Am. Chem. Soc, 103, 3185, 1981 ), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSI PS. TM. technology. When oligonucleotides are referred to as "double-stranded," it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical configuration typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term "double-stranded" as used herein is also meant to refer to those forms which include such structural features as bulges and loops. For example as described in US 5.770.722 for a unimolecular double-stranded DNA. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required. When nucleotides are conjugated together in a string using synthetic procedures, they are always referred to as oligonucleotides. A plurality of individual nucleotides linked together in a single molecule may form a polynucleotide. Polynucleotide covers any derivatized nucleotides such as DNA, RNA, PNA, LNA etc. Any oligonucleotide is also a polynucleotide, but every polynucleotide is not an oligonucleotide. The term "dinucleotide" as used herein refers to two sequential nucleotides. The dinucleotide may be comprised in an oligonucleotide or a polynucleotide. In particular, the dinucleotide CpG, which denotes a cytosine linked to a guanine by a

phosphodiester bond, may be comprised in an oligonucleotide according to the present invention. A CpG dinucleotide is also herein referred to as a CpG site.

Methylation status: the term "methylation status" as used herein, refers to the presence or absence of methylation. The methylation status of a given DNA sample is given as the ratio of methylated versus methylated and non-methylated allelles for either an individual CpG dinucleotide or for a long stretch of DNA sequence comprising at least two CpG dinucleotides, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or more CpG dinucleotides.

The term "hypomethylation" as used herein refers to a decrease in the methylation of CpG dinucleotides.

By the term expression level is meant the level of TFF3 transcripts or part thereof, or TFF3 protein or part thereof in a given sample, for example in a tissue sample. The level of TFF3 protein can be determined by for example immunohistochemistry or other methods using antibodies that specifically recognize TFF3 protein or fragment thereof. The level of TFF3 transcript is determined by for example quantitative RT_PCR with normalisation to at least one relevant other gene.

The term "Test Sensitivity" is the ability of a screening test to identify true disease, also characterised by being a test with high sensitivity and few false negatives. The test sensitivity is calculated as true positive tests per total affected patients tested, expressed as a percentage. The term "Test Specificity" is a screening test which is correctly negative in the absence of disease, has high specificity and few false positives. The test specificity is calculated as true negative tests per unaffected individual s tested, expressed as a percentage. The term "PPV" (Positive Predictive Value) is the percent of patients with positive test having disease, and thus assesses reliability of positive test. Calculation: PPV = (True positive) / (True + False positives)

The term "NPV" (Negative Predictive Value) refers to patients with negative test that do not have disease, and assesses reliability of negative test. Calculation: NPV = (True negative) / (true and false negatives).

Amino acid: Entity comprising an amino terminal part (NH₂) and a carboxy terminal part (COOH) separated by a central part comprising a carbon atom, or a chain of carbon atoms, comprising at least one side chain or functional group. NH₂ refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adopted in 37 C.F.R., section 1.822(b)(2) belong to the group of amino acids listed in Table 2 herein below. Non-natural amino acids are those not listed in Table 2. Examples of non-natural amino acids are those listed e.g. in 37 C.F.R. section 1 .822(b)(4), all of which are incorporated herein by reference. Further examples of non- natural amino acids are listed herein below. Amino acid residues described herein can be in the "D" or or "L" isomeric form.

Symbols Amino acid

1 -Letter 3-Letter

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine s Ser serine

I lie isoleucine

L Leu leucine

T Thr threonine

V Val valine

P Pro proline

K Lys lysine

H His histidine

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

Table 2. Natural amino acids and their respective codes.

Amino acid residue: the term "amino acid residue" is meant to encompass amino acids, either standard amino acids, non-standard amino acids or pseudo-amino acids, which have been reacted with at least one other species, such as 2, for example 3, such as more than 3 other species. In particular amino acid residues may comprise an acyl bond in place of a free carboxyl group and/or an amine-bond and/or amide bond in place of a free amine group. Furthermore, reacted amino acids residues may comprise an ester or thioester bond in place of an amide bond

The term 'stringent conditions' as used herein shall denote stringency as normally applied in connection with Southern blotting and hybridization as described e.g. by Southern E. M., 1975, J. Mol. Biol. 98:503-517. For such purposes it is routine practise to include steps of prehybridization and hybridization. Such steps are normally performed using solutions containing 6x SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 μg ml denaturated salmon testis DNA (incubation for 18 hrs at 42°C), followed by washings with 2x SSC and 0.5% SDS (at room temperature and at 37°C), and a washing with 0.1 x SSC and 0.5% SDS (incubation at 68°C for 30 min), as described by Sambrook et al., 1989, in "Molecular Cloning/A Laboratory Manual", Cold Spring Harbor), which is incorporated herein by reference.

Functional equivalents and variants are used interchangeably herein. In one preferred embodiment of the invention there is also provided variants of TFF3 gene variants of fragments thereof. When being polypeptides, variants are determined on the basis of their degree of identity or their homology with a predetermined amino acid sequence, said predetermined amino acid sequence being of SEQ ID NO: 3, when the variant is a fragment, a fragment of any of the aforementioned amino acid sequences, respectively.

Accordingly, variants preferably have at least 90 % sequence identity, such as at least

91 % sequence identity, for example at least 91 % sequence identity, such as at least

92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least

96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity with the predetermined sequence. Sequence identity is determined in one embodiment by utilising fragments of SEQ ID NO:3 peptides comprising at least 25 contiguous amino acids and having an amino acid sequence which is at least 80%, such as 85%, for example 90%, such as 95%, for example 99% identical to the amino acid sequence of SEQ ID NO: 3, wherein the percent identity is determined with the algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

The following terms are used to describe the sequence relationships between two or more polynucleotides: "predetermined sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity".

A "predetermined sequence" is a defined sequence used as a basis for a sequence comparison; a predetermined sequence may be a subset of a larger sequence, for example, as a segment of a full-length DNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of SEQ ID NO:1 , or may comprise a complete DNA or gene sequence. Generally, a predetermined sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.

Since two polynucleotides may each (1 ) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two

polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a

"comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a predetermined sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the

predetermined sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.

Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described and present a detailed consideration of sequence alignment methods and homology calculations, such as VECTOR NTI. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences will be. The NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at

http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at

http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Homologs of the disclosed polypeptides are typically characterised by possession of at least 94% sequence identity counted over the full length alignment with the disclosed amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Alternatively, one may manually align the sequences and count the number of identical amino acids. This number divided by the total number of amino acids in your sequence multiplied by 100 results in the percent identity.

The term "promoter" refers to the regulatory region located upstream, or 5' to the structural gene. Sequence analysis of the promoter region of TFF3 shows that nearly 13% of the nucleotides are C or G and about 13% are CpG dinucleotides.

Prostate cancer

The present invention discloses a marker for prostate cancer and methods including the use of the marker gene for for example assisting in diagnosing, monitoring, predicting the outcome, prognosticating the outcome of prostate cancer as described elsewhere herein.

Prostate cancer is a disease wherein cancer develops in the prostate gland of the male reproductive system. Thus, prostate cancer is classified as an adenocarcinoma (glandular cancer) in which normal secretorial prostate gland cells mutate into cancer cells. The region of prostate gland where the adenocarcinoma is most common is the peripheral zone. Initially, small clumps of cancer cells remain confined to otherwise normal prostate glands, a condition known as carcinoma in situ or prostatic

intraepithelial neoplasia (PIN). Although PIN has not been proven to be a cancer precursor, it is closely associated with cancer. Over time these cancer cells begin to multiply and spread to the surrounding prostate tissue, known as the stroma, forming a tumor. Eventually, the tumor may grow large enough to invade nearby organs such as the seminal vesicles or the rectum, or the tumor cells may develop the ability to travel in the bloodstream and lymphatic system. Prostate cancer is a malignant neoplasm because it displays the three key malignant properties of cancers: Uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). Prostate cancer most commonly metastasizes to the bones, lymph nodes, rectum, and bladder.

Prostate cancer is evaluated by the stage of the cancer i.e., how much the cancer has spread. As for the marker in the present invention, knowledge of the stage is useful in defining the prognosis of prostate cancer and for deciding on treatment regimes. The four stage TNM system (Tumor/Nodes/Metastases) is widely used for staging prostate cancer. The system makes use of information regarding the size of the tumor, the number of involved lymph nodes, and the presence of any other metastases.

The most important distinction is whether or not the cancer is still confined to the prostate. In the TNM system, clinical T1 and T2 cancers are found only in the prostate, while T3 and T4 cancers have spread elsewhere. Prostate cancer is staged according to the TNM system. The following terms are used to stage a prostate cancer:

T - Primary Tumour

Tx Primary tumour cannot be assessed

TO No evidence of primary tumour

T1 Clinically inapparent tumour neither palpable nor visible by imaging:

1 a Tumour incidental histologic finding in 5% or less of tissue resected

1 b Tumour incidental histologic finding in more than 5% of tissue resected

1 c Tumour identified by needle biopsy (e.g. because of elevated PSA)

T2 Tumour confined within the prostate¹:

2a Tumour involves one-half of one lobe or less

2b Tumour involves more than one-half of one lobe but not both lobes

2c Tumour involves both lobes

T3 Tumour extends through the prostatic capsule²:

3a Extracapsular extension in periprostatic tissue (unilateral or bilateral)

3b Invasion of the seminal vesicle(s)

T4 Tumour is fixed or invades adjacent structures other than the seminal vesicles bladder neck, external sphincter, rectum, levator muscles, or pelvic wall³ ¹Tumour found in one or both lobes by needle biopsy, but not palpable or visible by imaging, is classified as T1 c.

invasion into the prostatic apex but not beyond the prostate is not classified as T3, but as T2.

³lf radical prostatectomy shows that the bladder neck contains microscopic tumour, this must be classified as T3a.

N - Regional lymph nodes

Nx Regional lymph nodes cannot be assessed

NO No regional lymph node metastases

N1 Metastasis in regional lymph nodes

M - Distant metastasis

Mx Distant metastasis cannot be assessed

MO No distant metastasis

M1 Distant metastasis

1 a Non-regional lymph nodes

1 b Bone(s)

1 c Other site(s)

Tissue samples are examined microscopically to determine the features of any cancer identified. The so-called Gleason-score is used to help evaluating the prognosis of individuals having contracted prostate cancer. Upon examination of biopsy samples under a microscope a pathologist assigns grades to the most common tumor pattern of the sample. The two grades are added together to get a Gleason score. The Gleason grade ranges from 1 to 5, with 5 having the worst prognosis. The Gleason score ranges from 2 to 10, with 10 having the worst prognosis.

Gleason scores are associated with the following features: Grade 1 - The cancerous prostate closely resembles normal prostate tissue. The glands are small, well-formed, and closely packed; Grade 2 - The tissue still has well-formed glands, but they are larger and have more tissue between them; Grade 3 - The tissue still has recognizable glands, but the cells are darker. At high magnification, some of these cells have left the glands and are beginning to invade the surrounding tissue; Grade 4 - The tissue has few recognizable glands. Many cells are invading the surrounding tissue; Grade 5 - The tissue does not have recognizable glands. There are often just sheets of cells throughout the surrounding tissue. The present invention wherein the TFF3 gene is disclosed as a marker of prostate cancer relates to all stages, and grades of prostate cancer.

Methods of the present invention

The present invention of the TFF3 gene as a marker of prostate cancer has resulted in a number of methods useful in regard to prostate cancer. For any one of the methods it is appreciated that the methods may be used on their own with TFF3 used as an independent marker of prostate cancer, or in combination with other markers of prostate cancer known. In particular the TFF3 marker may be used in combination with the prostate specific antigen (PSA). PSA is produced by the normal prostate, where small amounts leak into the circulation under normal circumstances. Enlarged prostates and cancerous prostates leak substantial amounts of PSA which can be measured.

One aspect of the present invention relates to a method for assisting in diagnosing and/or for diagnosing prostate cancer in an individual comprising the steps of

According to this method for assisting in diagnosing and/or for diagnosing prostate cancer, a decreased methylation status is indicative of the presence of prostate cancer. Similarly, an increased transcriptional and/or translational expression level in the sample is indicative of the presence of prostate cancer.

Another aspect of the present invention relates to a method for assisting in prognosing and/or for prognosing the disease progression of prostate cancer in an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the progression of prostate cancer.

According to this aspect of wherein a decreased methylation status is indicative of disease progression of prostate cancer. Similarly, an increased transcriptional and/or translational expression level in the sample is indicative disease progression of prostate cancer.

A third aspect of the present invention pertains to a method for assisting in predicting and/or for predicting the outcome of prostate cancer in an individual having contracted prostate cancer comprising the steps of

iii) determining the translational expression level of said TFF3 gene, or amino acid sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof in said sample wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the outcome of prostate cancer. A decreased methylation status is indicative of the disease progression of prostate cancer whereas an increased transcriptional and/or translational expression level in the sample is indicative of the disease progression of prostate cancer.

Method for assisting in predicting and/or for predicting the recurrence risk of prostate cancer

A further aspect of the present invention concerns a method for assisting in predicting and/or for predicting the recurrence risk of prostate cancer in an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the recurrence risk of prostate cancer. It is appreciated that the recurrence risk is the risk of cancer recurring after

intervention, for example following surgery, radical prostatectomy, radiation therapy, cryotherapy or brachytherapy. Recurrence can occur over a wide span in time, for example 2 months after intervention, such as 3 months, 4, months, 5 months, 6 months, 1 year, two years, three years, 4 years, 5, years, 6 years, 7 years, 8 years, 9 years, 10 years, 1 1 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years after intervention or even longer. Thus, the methods of the present invention can be used over a wide span of time to determine whether recurrence has occurred. Tumor recurrence is often measured by increased levels of PSA defined as PSA level greater than or equal to 0.1 ng/ml serum measured at least one month after for example surgery. Individuals having PSA levels above 0.1 ng/ml serum measured less than 1 month after surgery, are not considered to suffer from reoccurrence but rather an indication that the primary tumor was not removed, for example in the event of advanced cancer at the time of surgery. In rare cases metastases are detected without an increase in PSA level. The present invention provides a marker which can be used independently of other markers or in combination with other markers and techniques, for example in combination with PSA measurements.

According to this aspect of the present invention a decreased methylation status is indicative of a risk of recurrence of prostate cancer at any of the indicated times after intervention. Similarly, an increase in the transcriptional and/or translational expression level in the sample is indicative of risk of recurrence of prostate cancer.

In one embodiment the risk of recurrence is determined following radical

prostatectomy.

According to the examples of present invention overall survival is defined as survival, whereas recurrence-free survival corresponds to cancer-specific survival which is survival from prostate cancer where the individual may die from other causes than prostate cancer. The recurrence is recurrence-free survival and overall survival

A further aspect of the present invention pertains to a method for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression in an individual having contracted prostate cancer, comprising the steps of

iii) determining the translational expression level of said TFF3 gene, or amino acid sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof in said sample wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of progression of said prostate cancer.

A decreased methylation status is indicative of a progression of said prostate cancer. Similarly, an increase in the transcriptional and/or translational expression level of the TFF3 gene in the sample is indicative a progression of said prostate cancer.

Assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cancer

Individuals suspected of having contracted, or suspected to be at risk of contracting prostate cancer, or having been diagnosed with prostate cancer may be enrolled in an active surveillance programme. Active surveillance refers to observation and regular monitoring of outbreak without invasive treatment. Thus, active surveillance is often used when an early stage, slow-growing prostate cancer is suspected. For younger men (>10 years life expectancy), a programme of active surveillance may not mean avoiding treatment altogether, but may allow a delay of a few years or more, during which time the quality of life impact of active treatment can be avoided carefully selected men will not miss a window for cure with this approach. Careful selection of individuals for active surveillance ensures that the selected individuals will not be less prone to cure than other individuals.

Programmes for 'watchful waiting' may also be suggested when the risks of surgery, radiation therapy, or hormonal therapy outweigh the possible benefits. Other treatments can be started if symptoms develop, or if there are signs that the cancer growth is accelerating (e.g., rapidly rising PSA, increase in Gleason score on repeat biopsy, etc.).

The TFF3 marker of the present invention can be used for monitoring prostate cancer progression, as an independent marker, but preferably with other markers such as the PSA marker, and characterisation of stage and grade of the prostate cancer in said individual.

The present invention thus in one aspect relates to a method for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cancer in an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the progression of prostate cancer from a silent/indolent to an aggressive prostate cancer.

It is appreciated that an aggressive prostate cancer may require treatment in order for the disease not to progress further, in an attempt to contain the disease.

A decreased methylation status is indicative of a progression of the prostate cancer and an increase in the transcriptional and/or translational expression level of the TFF3 gene in the sample is indicative a progression of said prostate cancer. It is appreciated that a silent/indolent prostate cancer is a slow-growing and slow- progressing organ-confined prostate cancer with no or only minor clinical symptoms, such as obstructive voiding symptoms. Individuals with silent/indolent prostate cancer are more likely to die with prostate cancer than from prostate cancer. The majority of low grade prostate cancers (Gleason score 2-4) and/or low stage (T1 a/b/c-T2a) prostate cancers, typically also having low serum PSA levels (<10 ng/mL) are in the present context comprised within the term silent/indolent prostate cancer.

Silent prostate cancer also include prostate cancers with low risk of recurrence following a given treatment, e.g. radical prostatectomy, as well as prostate cancers that do not cause considerable morbidity or mortality to an individual even if left untreated (watchful waiting/active surveillance). An aggressive prostate cancer according to the present invention is a prostate cancer which has progressed or will progress relatively fast (i.e. within the remaining life expectancy of a given patient) to non-organ-confined prostate cancer (locally advanced prostate cancer and/or dissiminated prostate cancer with distant metastasis and/or castration-resistant prostate cancer) and which will cause severe morbidity and high risk of mortality to the patient. In contrast to patients with indolent/silent prostate cancer, patients with aggressive prostate cancer are likely to die from prostate cancer rather than with prostate cancer. The majority of high grade prostate cancers (Gleason score 8-10) and/or high stage (T3-T4), typically also having high serum PSA levels

(>10 ng/mL) are in the present context comprised within the term aggressive prostate cancer.

The term aggressive prostate cancer also includes prostate cancers with high risk of recurrence after treatment such as radical prostatectomy.

Method for assisting in determining and/or determining the treatment regime of an individual having contracted prostate cancer comprising the steps of

A further aspect of the present invention relates to a method for assisting in

determining and/or determining the treatment regime of an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the treatment regime to be offered to the individual having contracted prostate cancer. According to this aspect a decreased methylation status is indicative of the presence and/or the progression of said prostate cancer. Analogously, an increased

transcriptional and/or translational expression level in the sample is indicative of the presence and/or the progression of said prostate cancer.

Additionally, for all the methods of the present inventions, the methods may further comprise a step of measuring the level of prostate specific antigen (PSA) in an individual. In one embodiment the individual has a normal PSA level of less than 4 ng per ml serum. In another embodiment the individual has a PSA level higher than a normal PSA level, corresponding to a PSA level above 4 ng per ml serum.

Digital rectal examination (DRE) can be used for diagnosing the presence or absence of prostate cancer in an individual, DRE can therefore be used in combination with the method for diagnosing prostate cancer employing TFF3 as a marker. Most CaPs are located in the peripheral zone of the prostate and may be detected by DRE when the volume is about 0.2 mL or larger. The risk of a positive DRE turning out to be cancer is heavily dependent on the PSA value. When PSA is measured at a concentration of 0-1 ng/ml, then the positive predictive value for cancer is 2.8 to 5%. With PSA

concentrations in the range of 1 - 2.5 ng/m! serum, the positive predictive value for cancer is 10.5 to 14%. For PSA concentrations in the range of 2.5 to 4 ng/m! serum, the positive predictive value for cancer is 22 to 30%. For PSA concentrations in the range of 4- 10 ng/ml serum, the positive predictive value for cancer is 41 %, whereas PSA concentrations above 10 ng/ml results in a positive predictive value of 69%. Samples

The sample that is used in the methods of the present invention may be in a form suitable to allow analysis by the skilled artisan. The samples according to the present invention may be selected from a tissue sample, or from body fluids such as blood, plasma, serum, semen, or urine. In a preferred embodiment of the present invention the sample is a urine sample, blood sample, and/or a tissue sample. In case of urine samples a preferred urine sample is a urine sample where the prostate gland has been massaged prior to the sampling in order to transfer as many cells of prostate origin to the urine.

In one particular embodiment of the present invention, the sample is a tissue sample, such as a biopsy of the tissue, or a superficial sample scraped from the tissue. In another embodiment the sample may be prepared by forming a suspension of cells made from the tissue. The sample may, however, also be an extract obtained from the tissue or obtained from a cell suspension made from the tissue. It is appreciated that for the various methods of the present invention different tissue samples are preferred or available. For assisting in the diagnosis/ diagnosing prostate cancer according to the present invention the tissue sample is typically a biopsy of the prostate gland.

However, the sample can also be a urine, sample or blood sample. In a particular embodiment the sample to be used for diagnosing is a urine sample, obtained following the massage of the prostate gland, whereby prostate cells are released into the urine. In a preferred embodiment the sample used in the diagnosis of prostate cancer is a biopsy of the prostate gland,

For assisting in the prognosis and/or for the prognosis of the disease progression of prostate cancer; for assisting in the prediction and/or the prediction of the progression of prostate cancer; for assisting in predicting and/or for predicting the recurrence risk of prostate cancer; for assisting in classifying and/or for classifying prostate cancer; for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression; for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cancer, and/or for assisting in determining and/or determining the treatment regime it is preferred that the sample is from a biopsy of the prostate tissue, from a resected prostate cancer tumour, or from a resected prostate following radical prostatectomy.

Working with tumor material requires biopsies or body fluids suspected to comprise relevant cells. Working with RNA requires freshly frozen or immediately processed biopsies, or chemical pretreatment of the biopsy. Apart from the cancer tissue, biopsies do inevitably contain many different cell types, such as cells present in the blood, connective and muscle tissue, endothelium etc. In the case of DNA studies,

microdissection or laser capture are methods of choice, however the time-dependent degradation of RNA makes it difficult to perform manipulation of the tissue for more than a few minutes. The sample may be fresh or frozen, or treated with chemicals.

Control sample

A control sample is any corresponding sample (tissue sample, blood, plasma, serum, semen, or urine) that is non-malignant, for example a non-malignant prostate biopsy of a healthy individual. Synthetic DNA corresponding to DNA extracted from the above may also be used as control sample.

The control sample should be used as a standard level of the methylation status to be used in evaluation whether the methylation status of a sample to be tested is increased or decreased. For determination of the methylation status of the TFF3 gene, the control sample is a DNA fragment with a sequence corresponding to that of the promoter region or part thereof of the TFF3 gene, wherein the methylation status is known, for example a fully methylated DNA fragment. The DNA fragment is in one embodiment of synthetic origin, however, in another embodiment the DNA fragment is derived from DNA extracted from DNA vector constructs, comprising said promoter region or part thereof.

By the term control sample is also meant a standard transcriptional and/or translational expression level of the TFF3 gene. Such a standard level is determined in samples from non-diseased prostate glands of a statistically significant number of healthy individuals such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more healthy individuals. Such a standard transcriptional and/or translational expression level from non-diseased prostate glands of a statistically significant number of healthy individuals forms the cut off value. If the transcriptional and/or translational expression level from a test sample is above said cut off value the expression level is increased and thus indicative of prostate cancer according to the methods of the present invention.

It is appreciated that for determining the methylation status according to the methods of the present invention the sample is in any of the form described herein above.

Similarly it is appreciated that for determining the transcriptional level according to the methods of the present invention the sample is in any of the form described herein above.

In analogy it is appreciated that for determining the translational level according to the methods of the present invention the sample is in any of the form described herein above.

Determining the methylation status

The methods of the present invention comprises a step of determining the methylation status of a TFF3 gene (SEQ ID NO: 1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO.: NO: 1 , or part thereof in a sample from the individual.

In one embodiment the methylation status is determined in position 1 basepair to 5000 basepairs of SEQ ID NO: 1 (corresponding to SEQ ID NO: 4)

In another embodiment the methylation status is determined in position 5000 basepairs to 10500 basepairs of SEQ ID NO: 1

In another embodiment the methylation status is determined in position 1 basepair to 2000 basepairs of SEQ ID NO: 1

In another embodiment the methylation status is determined in position 2000 basepairs to 4000 basepairs of SEQ ID NO: 1

In another embodiment the methylation status is determined in position 4000 basepairs to 6000 basepairs of SEQ ID NO: 1

In another embodiment the methylation status is determined in position 6000 basepairs to 8000 basepairs of SEQ ID NO: 1

In another embodiment the methylation status is determined in position 8000 basepairs to 9000 basepairs of SEQ ID NO: 1

In a preferred embodiment the methylation status is determined in position 9000 basepairs to 10500 basepairs of SEQ ID NO: 1

In a preferred embodiment the methylation status is determined in position 9664 basepairs to 10080 basepairs of SEQ ID NO: 1

It is appreciated that the the methylation status of at least one CpG dinucleotide is determined, such as two, three, four, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32,33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46,47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 100 CpG dinucleotides.

DNA extraction prior to determining methylation status

Prior to the determination of the methylation status DNA is extracted from the sample. For those embodiments where whole cells, or other tissue samples are being analyzed, it will typically be necessary to extract the nucleic acids from the cells prior to continuing with the various sample preparation operations. Accordingly, following sample collection, nucleic acids may be liberated from the collected cells into a crude extract followed by additional treatments to prepare the sample for subsequent operations, such as denaturation of contaminating (DNA binding) proteins, purification, filtration and desalting.

Liberation of nucleic acids from the sample cells, and denaturation of DNA binding proteins may generally be performed by physical or chemical methods. For example, chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins. Alternatively, physical methods may be used to extract the nucleic acids and denature DNA binding proteins, such as physical protrusions within microchannels or sharp edged particles piercing cell membranes and extract their contents. Combinations of such structures with piezoelectric elements for agitation can provide suitable shear forces for lysis.

More traditional methods of cell extraction may also be used, e.g., employing a channel with restricted cross-sectional dimension which causes cell lysis when the sample is passed through the channel with sufficient flow pressure. Alternatively, cell extraction and denaturing of contaminating proteins may be carried out by applying an alternating electrical current to the sample. More specifically, the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow. Subjecting cells to ultrasonic agitation or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture are also possible extraction methods.

Following extraction, it will often be desirable to separate the nucleic acids from other elements of the crude extract, e.g. denatured proteins, cell membrane particles and salts. Removal of particulate matter is generally accomplished by filtration or flocculation. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step. Desalting of the sample and isolation of the nucleic acid may generally be carried out in a single step, e.g. by binding the nucleic acids to a solid phase and washing away the contaminating salts, or performing gel filtration chromatography on the sample passing salts through dialysis membranes. Suitable solid supports for nucleic acid binding include e.g. diatomaceous earth or silica (i.e., glass wool). Suitable gel exclusion media also well known in the art may be readily incorporated into the devices of the present invention and is

commercially available from, e.g., Pharmacia and Sigma Chemical.

Alternatively, desalting methods may generally take advantage of the high

electrophoretic mobility and negativity of DNA compared to other elements.

Electrophoretic methods may also be utilized in the purification of nucleic acids from other cell contaminants and debris. Upon application of an appropriate electric field, the nucleic acids present in the sample will migrate toward the positive electrode and become trapped on the capture membrane. Sample impurities remaining free of the membrane are then washed away by applying an appropriate fluid flow. Upon reversal of the voltage, the nucleic acids are released from the membrane in a substantially purer form. Further, coarse filters may also be overlaid on the barriers to avoid any fouling of the barriers by particulate matter, proteins or nucleic acids, thereby permitting repeated use.

In a similar aspect, the high electrophoretic mobility of nucleic acids with their negative charges may be utilized to separate nucleic acids from contaminants by utilizing a short column of a gel or other appropriate matrices or gels which will slow or retard the flow of other contaminants while allowing the faster nucleic acids to pass.

This invention provides nucleic acid affinity matrices that bear a large number of different nucleic acid affinity ligands allowing the simultaneous selection and removal of a large number of preselected nucleic acids from the sample. Methods of producing such affinity matrices are also provided. In general the methods involve the steps of a) providing a nucleic acid amplification template array comprising a surface to which are attached at least 50 oligonucleotides having different nucleic acid sequences, and wherein each different oligonucleotide is localized in a predetermined region of said surface, the density of said oligonucleotides is greater than about 60 different oligonucleotides per 1 cm. sup.2, and all of said different oligonucleotides have an identical terminal 3' nucleic acid sequence and an identical terminal 5' nucleic acid sequence, b) amplifying said multiplicity of oligonucleotides to provide a pool of amplified nucleic acids; and c) attaching the pool of nucleic acids to a solid support.

For example, nucleic acid affinity chromatography is based on the tendency of complementary, single-stranded nucleic acids to form a double-stranded or duplex structure through complementary base pairing. A nucleic acid (either DNA or RNA) can easily be attached to a solid substrate (matrix) where it acts as an immobilized ligand that interacts with and forms duplexes with complementary nucleic acids present in a solution contacted to the immobilized ligand. Unbound components can be washed away from the bound complex to either provide a solution lacking the target molecules bound to the affinity column, or to provide the isolated target molecules themselves. The nucleic acids captured in a hybrid duplex can be separated and released from the affinity matrix by denaturation either through heat, adjustment of salt concentration, or the use of a destabilizing agent such as formamide, TWEEN.TM.-20 denaturing agent, or sodium dodecyl sulfate (SDS).

Affinity columns (matrices) are typically used either to isolate a single nucleic acid typically by providing a single species of affinity ligand. Alternatively, affinity columns bearing a single affinity ligand (e.g. oligo dt columns) have been used to isolate a multiplicity of nucleic acids where the nucleic acids all share a common sequence (e.g. a polyA).

The type of affinity matrix used depends on the purpose of the analysis. For example, where it is desired to analyze mRNA expression levels of particular genes in a complex nucleic acid sample (e.g., total mRNA) it is often desirable to eliminate nucleic acids produced by genes that are constitutively overexpressed and thereby tend to mask gene products expressed at characteristically lower levels. Thus, in one embodiment, the affinity matrix can be used to remove a number of preselected gene products (e.g., actin, GAPDH, etc.). This is accomplished by providing an affinity matrix bearing nucleic acid affinity ligands complementary to the gene products (e.g., mRNAs or nucleic acids derived therefrom) or to subsequences thereof. Hybridization of the nucleic acid sample to the affinity matrix will result in duplex formation between the affinity ligands and their target nucleic acids. Upon elution of the sample from the affinity matrix, the matrix will retain the duplexes nucleic acids leaving a sample depleted of the overexpressed target nucleic acids.

The affinity matrix can also be used to identify unknown mRNAs or cDNAs in a sample. Where the affinity matrix contains nucleic acids complementary to every known gene (e.g., in a cDNA library, DNA reverse transcribed from an mRNA, mRNA used directly or amplified, or polymerized from a DNA template) in a sample, capture of the known nucleic acids by the affinity matrix leaves a sample enriched for those nucleic acid sequences that are unknown. In effect, the affinity matrix is used to perform a subtractive hybridization to isolate unknown nucleic acid sequences. The remaining "unknown" sequences can then be purified and sequenced according to standard methods.

The affinity matrix can also be used to capture (isolate) and thereby purify unknown nucleic acid sequences. For example, an affinity matrix can be prepared that contains nucleic acid (affinity ligands) that are complementary to sequences not previously identified, or not previously known to be expressed in a particular nucleic acid sample. The sample is then hybridized to the affinity matrix and those sequences that are retained on the affinity matrix are "unknown" nucleic acids. The retained nucleic acids can be eluted from the matrix (e.g. at increased temperature, increased destabilizing agent concentration, or decreased salt) and the nucleic acids can then be sequenced according to standard methods.

Similarly, the affinity matrix can be used to efficiently capture (isolate) a number of known nucleic acid sequences. Again, the matrix is prepared bearing nucleic acids complementary to those nucleic acids it is desired to isolate. The sample is contacted to the matrix under conditions where the complementary nucleic acid sequences hybridize to the affinity ligands in the matrix. The non-hybridized material is washed off the matrix leaving the desired sequences bound. The hybrid duplexes are then denatured providing a pool of the isolated nucleic acids. The different nucleic acids in the pool can be subsequently separated according to standard methods (e.g. gel electrophoresis).

As indicated above the affinity matrices can be used to selectively remove nucleic acids from virtually any sample containing nucleic acids (e.g. in a cDNA library, DNA reverse transcribed from an mRNA, mRNA used directly or amplified, or polymerized from a DNA template, and so forth). The nucleic acids adhering to the column can be removed by washing with a low salt concentration buffer, a buffer containing a destabilizing agent such as formamide, or by elevating the column temperature.

In one particularly preferred embodiment, the affinity matrix can be used in a method to enrich a sample for unknown RNA sequences (e.g. expressed sequence tags (ESTs)). The method involves first providing an affinity matrix bearing a library of oligonucleotide probes specific to known RNA (e.g., EST) sequences. Then, RNA from undifferentiated and/or unactivated cells and RNA from differentiated or activated or pathological (e.g., transformed) or otherwise having a different metabolic state are separately hybridized against the affinity matrices to provide two pools of RNAs lacking the known RNA sequences.

In a preferred embodiment, the affinity matrix is packed into a columnar casing. The sample is then applied to the affinity matrix (e.g. injected onto a column or applied to a column by a pump such as a sampling pump driven by an autosampler). The affinity matrix (e.g. affinity column) bearing the sample is subjected to conditions under which the nucleic acid probes comprising the affinity matrix hybridize specifically with complementary target nucleic acids. Such conditions are accomplished by maintaining appropriate pH, salt and temperature conditions to facilitate hybridization as discussed above.

Detection of methylation status

A number of current methodologies for methylation studies exist. Sequencing of bisulphite-treated DNA is the gold standard for methylation studies as it reveals directly the status of each CpG dinucleotide. Bisulphate-based methylation genomic sequencing is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. This method utilizes defined procedures that maximize the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.

Methylation specific PCR (MSP) is one of the most widely used assay for the sensitive detection of methylation. US patent 5,786,146 discloses a method of methylation specific PCR (MSP) for identifying DNA methylation patterns in a CpG containing nucleic acid. The method uses agents to modify unmethylated cytosine in the nucleic acid. Prior to amplification, the DNA is treated with sodium bisulphite to convert all unmethylated cytosines to uracils. The bisulphite reaction effectively converts methylation information into sequence difference. CpG specific oligonucleotide primers are used to distinguish between modified methylated and unmethylated nucleic acid. The identification of the methylated nucleic acid is based on the presence or absence of amplification product resulting from the amplification and distinguishing modified methylated and non-methylated nucleic acids. The generated PCR product can for example be visualized on a gel.

A critical parameter for the specificity of methylation-specific PCR is determined by primer design. Since modification of DNA by bisulfite destroys strand complementarity, either strand can serve as the template for subsequent PCR amplification and the methylation pattern of each strand can then be determined. It will be appreciated, though, that amplifying a single strand (e.g., sense) is preferable in practice. Primers are designed to amplify a region that is 80-250 bp in length, which incorporates a sufficient number of cytosines in the original strand to assure that unmodified DNA does not serve as a template for the primers. In addition, the number and position of cytosines within the CpG dinucleotide determines the specificity of the primers for methylated and unmethylated templates. Typically, 1 -3 CpG sites are included in each primer and concentrated in the 3' region of each primer. This provides optimal specificity and minimizes false positives due to mispriming. To facilitate simultaneous analysis of each of the primers of a given gene in the same thermocycler, the length of the primers is adjusted to give nearly equal melting/annealing temperatures.

Real-time fluorescent MSP (MethyLight) is based on real time PCR employing fluorescent probes in conjunction with MSP and allows for a homogeneous reaction which is of higher throughput. If the probe does not contain CpGs, the reaction is essentially a quantitative version of MSP. However, the fluorescent probe is typically designed to anneal to a site containing one or more CpGs, and this third

oligonucleotide increases the specificity of the assay for completely methylated target strands. Because the detection of the amplification occurs in real time, there is no need for a secondary electrophoresis step. Since there is no post PCR manipulation of the sample, the risk of contamination is reduced. The MethyLight probe can be of any format including but not limited to a Taqman probe or a LightCycler hybridization probe pair and if multiple reporter dyes are used, several probes can be performed simultaneously [Eads (1999) Cancer Res. 59:2302-2306; Eads (2000) Nucleic Acids Res. 28:E32; Lo (1999) Cancer Res. 59:3899-390]. Real-time fluorescent MSP is the preferred method for determing the methylation status according to the present invention.

Methods such as a PCR-based high-resolution melting analysis assay may, however, also be used to determine the methylation status. Methylation-sensitive high-resolution melting (MS-HRM) analysis is another PCR-based technology which can be used for determination of TFF3 status and for highly specific and higly sensitive detection of methylated TFF3. This method takes advantage of the fact that methylated DNA and unmethylated DNA acquire different sequences after bisulphite treatment, which results in PCR products with markedly different melting profiles/temperature. PCR is used to amplify both methylated and unmethylated sequences in the same reaction, and this method can be optimised to detect methylation levels as low as 0.1 %. MS-HRM also allows estimation of methylation levels by comparison of melting profiles for a test sample to the melting profiles of PCR products derived from standards with known ratios of methylated:unmethylated alleles. MS-HRM analysis protocols are simple and the method is characterized by high reproducibilty (Wojdacz TK, Dobrovic A, Hansen LL. 2008, Nat Protoc. 2008;3(12): 1903-8).

TFF3 methylation status can also be measured quantitatively by pyrosequencing of bisulfite converted TFF3 DNA following PCR amplification. Pyrosequencing is a sequencing-by-synthesis method that relies on the sequential addition and

incorporation of nucleotides in a primer-directed polymerase extension. Only one of the four nucleotides is present at any time in the reaction vessel, and only if the added nucleotide is complementary to the template DNA will it be incorporated by a DNA polymerase. This event is monitored in real time and hence can be used to quantitate the ratio between methylated and unmethylated CpG dinucleotides. The

pyrosequencing technology makes use of the release of PP, molecules during the iterative incorporation of unmodified nucleotides that are quantitatively converted into a bioluminometric signal (Tost, J. & Gut, I.G. DNA methylation analysis by

pyrosequencing, Nature Protocols 2, - 2265 - 2275 (2007)

TFF3 methylation status may also be determined by targeted resequencing of bisulfite converted DNA (or PCR amplicons hereof) using massively parallel sequencing technologies (aka next-generation sequencing) and possibly future sequencing platforms able to sequence individual DNA molecules. This would allow digital quantification of the ratio between methylated and unmethylated CpG dinucleotide(s). Next-generation sequencing platforms are characterized by the ability to process millions of sequence reads in parallel rather than 96 at a time, as typically seen for capillary-based sequencing. The workflow to produce next-generation sequence-ready libraries is straightforward; DNA fragments are prepared for sequencing by ligating specific adaptor oligos to both ends of each DNA fragment. Importantly, relatively little input DNA (a few micrograms at most) is needed to produce a library. Currently available platforms for next-generation sequencing produce shorter read lengths (35- 250 bp, depending on the platform), but longer reads may be possible in the future. (Mardis, E.R. The impact of next-generation sequencing technology on genetics, Trends Genet. 2008 Mar;24(3):133-41 .

For rapid assessment of CpG methylation density of a DNA region the quantitative methylation density assay may be used as previously described by Galm et al. (2002) Genome Res. 12, 153-7. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with nested primers. PCR products are purified and DNA amount is determined. A predetermined amount of DNA is incubated with .sup.3H-SAM (TRK581 Bioscience, Amersham) and Sssl methyltransferase for methylation quantification. Once reactions are terminated products are purified from the in-vitro methylation mixture. 20% of the eluant volume is counted in .sup.3H counter.

Normalizing radioactivity DNA of each sample is measured again and the count is normalized to the DNA amount.

Restriction analysis of bisulphite modified DNA is a yet another quantitative technique which can be used to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation- dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation levels in original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. This technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

In another embodiment, differential methylation hybridization (DMH) may be used to determine the methylation status of the promoter region of theTFF3 gene. DHM integrates a high-density, microarray-based screening strategy to detect the presence or absence of methylated CpG dinucleotide genomic fragments. Array-based techniques are used when a number (e.g., >3) of methylation sites in a single region are to be analyzed. First, CpG dinucleotide nucleic acid fragments from a genomic library are generated, amplified and affixed on a solid support to create a CpG dinucleotide rich screening array. Amplicons are generated by digesting DNA from a sample with restriction endonucleases which digest the DNA into fragments but leaves the methylated CpG islands intact. These amplicons are used to probe the CpG dinucleotide rich fragments affixed on the screening array to identify methylation patterns in the CpG dinucleotide rich regions of the DNA sample. Unlike other methylation analysis methods such as Southern hybridization, bisulfite DNA

sequencing and methylation-specific PCR which are restricted to analyzing one gene at a time, DMH utilizes numerous CpG dinucleotide rich genomic fragments specifically designed to allow simultaneous analysis of multiple of methylation-associated genes in the genome (for further details see U.S. Pat. No. 6,605,432).

In yet another embodiment, immunoprecipitation of methylated sequences can be used to isolate sequence-specific methylated DNA fragments. Briefly, genomic DNA is sonicated to yield fragments of 200-300 bp. The DNA is then denatured, precleaned with a protein A Fast FlowSepharose) and further incubated with a 5-methylcytidine monoclonal antibody. The complex may be purified using protein A Sepharose and subsequently washed. The immunoprecipitated samples are then analyzed using specific PCR primers.

In the present application the level of methylation may be expressed as percentage of methylation, wherein the percentage is the percentage of cells exhibiting methylation of the promoter as described herein, and/or the percentage of possible methylation sites on the promoter being methylated in a given cell.

Thus, percentage of methylation may either be provided as a percentage of afflicted cells or a percentage or methylation in the cells, or an average percentage in a group of cells, or both percentages may be provided. For both values it is emphasized that the more cells and/or the more methylation sites that are methylated the worse the diagnosis and prognosis, as described above.

The methylation status of TFF3 relates to the indications of prostate cancer of the sample tested, such as diagnosing, predicting, prognosing, monitoring prostate cancer in an individual. The methylation status TFF3 gene may be detected in either a tissue sample as such, or in a body fluid sample, such as blood, serum, plasma, semen and/or urine of the individual.

In one embodiment of the present invention the methylation level of the TFF3- expressing prostate adenocarcinoma cell lines is 0%-45%, in another embodiment the methylation level is 1 %-45%, in another embodiment the methylation level is 5%-45% and in another embodiment the methylation level is 10%-45%. In another embodiment the methylation level of the TFF3-expressing prostate adenocarcinoma cell lines is 0%- 25%, in another embodiment the methylation level is 1 %-20% and in another embodiment the methylation level is 1 %-15%. In another embodiment the methylation level of the TFF3-expressing prostate adenocarcinoma cell lines is 3%-20%, in another embodiment the methylation level is 5%-20%, in another embodiment the methylation level is 5%-15% and in another embodiment the methylation level is 10%-15%. In a preferred embodiment the methylation level of the TFF3-expressing prostate adenocarcinoma cell lines is 7%-14%, for example 7%, 8%, 9%, 10%, 1 1 %, 12%, 13% or 14%. A preferred methylation level of the TFF3-expressing prostate adenocarcinoma cell lines is 10%.

In one embodiment of the present invention the methylation level of the non-malignant prostate cell lines is 50%-100%, in another embodiment the methylation level is 50%- 90%, in another embodiment the methylation level is 50%-80% and in another embodiment the methylation level is 50%-70%. In another embodiment the methylation level of the non-malignant prostate cell lines is 60%-100%, in another embodiment the methylation level is 60%-90% and in another embodiment the methylation level is 60%- 80%. In another embodiment the methylation level of the non-malignant prostate cell lines is 70%-100%, in another embodiment the methylation level is 70%-95%, in another embodiment the methylation level is 70%-85% and in another embodiment the methylation level is 70%-80%. In a preferred embodiment the methylation level of the non-malignant prostate cell lines is 75%-100%, for example 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 80%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A preferred methylation level of the non-malignant prostate cell lines is 88%.

When in the present invention the methylation status of the TFF3 gene in a sample is decreased by 1 % or for example 2%, 3%, 4%, 5%, 6, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or at least 50% and even more, relative to previous measurements on the same individual, or to measurements on non-diseased tissue from the same individual, or relative to a standard level, the methylation status of the TFF3 gene is indicative of the presence of prostate cancer. In a clinical setting, the observation of 1 % methylation, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% methylation or less is indicative of the presence of prostate cancer, when the sample is a tissue sample and/or an urine sample.

In one preferred embodiment the methylation status of the TFF3 gene in a sample being below 50 %, is indicative of a decreased methylation level, wherein the sample is a tissue sample. In another preferred embodiment the methylation status of the TFF3 gene in a sample being below 50 % is indicative of a decreased methylation level, wherein said sample is a urine sample.

A decreased methylation status is indicative of the presence and/or the progression of prostate cancer.

Determining expression levels

Extraction of RNA

RNA or protein can be isolated and assayed from a test sample using any techniques known in the art. They can for example be isolated from a fresh or frozen biopsy, from formalin-fixed tissue, from body fluids, such as blood, plasma, serum, urine or semen.

Methods of isolating total mRNA are well known to those of skill in the art. In one embodiment, the total nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method and polyA.sup. and mRNA is isolated by oligo dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1 -3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-lnterscience, New York (1987)). The sample may be from tissue and/or body fluids, as defined elsewhere herein.

Before analyzing the sample, e.g., on an oligonucleotide array, it will often be desirable to perform one or more sample preparation operations upon the sample. Typically, these sample preparation operations will include such manipulations as extraction of intracellular material, e.g., nucleic acids from whole cell samples, viruses, amplification of nucleic acids, fragmentation, transcription, labeling and/or extension reactions. One or more of these various operations may be readily incorporated into the device of the present invention.

For a number of applications, it may be desirable to extract and separate messenger RNA from cells, cellular debris, and other contaminants. As such, the device of the present invention may, in some cases, include a mRNA purification chamber or channel. In general, such purification takes advantage of the poly-A tails on mRNA. In particular and as noted above, poly- T oligonucleotides may be immobilized within a chamber or channel of the device to serve as affinity ligands for mRNA. Poly-T oligonucleotides may be immobilized upon a solid support incorporated within the chamber or channel, or alternatively, may be immobilized upon the surface(s) of the chamber or channel itself. Immobilization of oligonucleotides on the surface of the chambers or channels may be carried out by methods described herein including, e.g., oxidation and silanation of the surface followed by standard DMT synthesis of the oligonucleotides.

In operation, the lysed sample is introduced to a high salt solution to increase the ionic strength for hybridization, whereupon the mRNA will hybridize to the immobilized poly- T. The mRNA bound to the immobilized poly-T oligonucleotides is then washed free in a low ionic strength buffer. The poy-T oligonucleotides may be immobiliized upon poroussurfaces, e.g., porous silicon, zeolites silica xerogels, scintered particles, or other solid supports.

Following sample preparation, the sample can be subjected to one or more different analysis operations. A variety of analysis operations may generally be performed, including size based analysis using, e.g., microcapillary electrophoresis, and/or sequence based analysis using, e.g., hybridization to an oligonucleotide array.

In the latter case, the nucleic acid sample may be probed using an array of

oligonucleotide probes. Oligonucleotide arrays generally include a substrate having a large number of positionally distinct oligonucleotide probes attached to the substrate. These arrays may be produced using mechanical or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods.

The basic strategy for light directed synthesis of oligonucleotide arrays is as follows. The surface of a solid support, modified with photosensitive protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A selected nucleotide, typically in the form of a 3'-0- phosphoramidite-activated deoxynucleoside (protected at the 5' hydroxyl with a photosensitive protecting group), is then presented to the surface and coupling occurs at the sites that were exposed to light. Following capping and oxidation, the substrate is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second selected nucleotide (e.g., 5'-protected, 3'-0- phosphoramidite-activated deoxynucleoside) is presented to the surface. The selective deprotection and coupling cycles are repeated until the desired set of products is obtained. Since photolithography is used the process can be readily miniaturized to generate high density arrays of oligonucleotide probes. Furthermore, the sequence of the oligonucleotides at each site is known. See Pease et al. Mechanical synthesis methods are similar to the light directed methods except involving mechanical direction of fluids for deprotection and addition in the synthesis steps.

For some embodiments, oligonucleotide arrays may be prepared having all possible probes of a given length. The hybridization pattern of the target sequence on the array may be used to reconstruct the target DNA sequence. Hybridization analysis of large numbers of probes can be used to sequence long stretches of DNA or provide an oligonucleotide array which is specific and complementary to a particular nucleic acid sequence. For example, in particularly preferred aspects, the oligonucleotide array will contain oligonucleotide probes which are complementary to specific target sequences and individual or multiple mutations of these. Such arrays are particularly useful in the diagnosis of specific disorders which are characterized by the presence of a particular nucleic acid sequence.

Following sample collection and nucleic acid extraction, the nucleic acid portion of the sample is typically subjected to one or more preparative reactions. These preparative reactions include in vitro transcription, labelling, fragmentation, amplification and other reactions. Nucleic acid amplification increases the number of copies of the target nucleic acid sequence of interest. A variety of amplification methods are suitable for use in the methods and device of the present invention, including for example, the polymerase chain reaction method or (PCR), the ligase chain reaction (LCR), self sustained sequence replication (3SR), and nucleic acid based sequence amplification (NASBA).

The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of approximately 30 or 100 to 1 , respectively. As a result, where these latter methods are employed, sequence analysis may be carried out using either type of substrate, i.e. complementary to either DNA or RNA.

Frequently, it is desirable to amplify the nucleic acid sample prior to hybridization. One of skill in the art will appreciate that whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids.

Determining transcriptional expression levels

Expression of genes may in general be detected by either detecting mRNA from the cells and/or detecting expression products, such as peptides and proteins. Polymerase Chain reaction (PCR) is a well known and well established technique to determine transcriptional products and therefore also a method that in one embodiment is used to determine the transcriptional expression level of the TFF3 gene, or part thereof.

Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. The high density array may then include probes specific to the internal standard for quantification of the amplified nucleic acid. Thus, in one embodiment, this invention provides for a method of optimizing a probe set for detection of a particular gene. Generally, this method involves providing a high density array containing a multiplicity of probes of one or more particular length(s) that are complementary to subsequences of the mRNA transcribed by the target gene. In one embodiment the high density array may contain every probe of a particular length that is complementary to a particular mRNA. The probes of the high density array are then hybridized with their target nucleic acid alone and then hybridized with a high complexity, high concentration nucleic acid sample that does not contain the targets complementary to the probes. Thus, for example, where the target nucleic acid is an RNA, the probes are first hybridized with their target nucleic acid alone and then hybridized with RNA made from a cDNA library (e.g., reverse transcribed polyA.sup.+ mRNA) where the sense of the hybridized RNA is opposite that of the target nucleic acid (to insure that the high complexity sample does not contain targets for the probes). Those probes that show a strong hybridization signal with their target and little or no cross-hybridization with the high complexity sample are preferred probes for use in the high density arrays of this invention.

PCR amplification generally involves the use of one strand of the target nucleic acid sequence as a template for producing a large number of complements to that sequence. Generally, two primer sequences complementary to different ends of a segment of the complementary strands of the target sequence hybridize with their respective strands of the target sequence, and in the presence of polymerase enzymes and nucleoside triphosphates, the primers are extended along the target sequence. The extensions are melted from the target sequence and the process is repeated, this time with the additional copies of the target sequence synthesized in the preceding steps. PCR amplification typically involves repeated cycles of denaturation,

hybridization and extension reactions to produce sufficient amounts of the target nucleic acid. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.

In PCR methods, strand separation is normally achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase. Typical heat denaturation involves temperatures ranging from about 80. degree C. to 105. degree C. for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity.

In addition to PCR and IVT reactions, the methods of the present invention are also applicable to a number of other reaction types, e.g., reverse transcription, nick translation, and the like.

The nucleic acids in a sample will generally be labelled to facilitate detection in subsequent steps. Labelling may be carried out during the amplification, in vitro transcription or nick translation processes. In particular, amplification, in vitro transcription or nick translation may incorporate a label into the amplified or transcribed sequence, either through the use of labelled primers or the incorporation of labelled dNTPs into the amplified sequence.

Hybridization between the sample nucleic acid and the oligonucleotide probes upon the array is then detected, using, e.g., epifluorescence confocal microscopy. Typically, sample is mixed during hybridization to enhance hybridization of nucleic acids in the sample to nucleic acid probes on the array.

In some cases, hybridized oligonucleotides may be labelled following hybridization. For example, where biotin labelled dNTPs are used in, e.g. amplification or transcription, streptavidin linked reporter groups may be used to label hybridized complexes. Such operations can readily be integrated into the systems of the present invention.

Alternatively, the nucleic acids in the sample may be labelled following amplification. Post amplification labelling typically involves the covalent attachment of a particular detectable group upon the amplified sequences. Suitable labels or detectable groups include a variety of fluorescent or radioactive labelling groups well known in the art. These labels may also be coupled to the sequences using methods that are well known in the art.

Methods for detection of TFF3 transcript or part thereof depend upon the label selected. A fluorescent label is preferred because of its extreme sensitivity and simplicity. Standard labelling procedures are used to determine the positions where interactions between a sequence and a reagent take place. For example, if a target sequence is labelled and exposed to a matrix of different probes, only those locations where probes do interact with the target will exhibit any signal. Alternatively, other methods may be used to scan the matrix to determine where interaction takes place. Of course, the spectrum of interactions may be determined in a temporal manner by repeated scans of interactions which occur at each of a multiplicity of conditions.

However, instead of testing each individual interaction separately, a multiplicity of sequence interactions may be simultaneously determined on a matrix.

Means of detecting labelled target (sample) TFF3 nucleic acids hybridized to the probes of the high density array are known to those of skill in the art. Thus, for example, where a colorimetric label is used, simple visualization of the label is sufficient. Where a radioactive labelled probe is used, detection of the radiation (e.g. with photographic film or a solid state detector) is sufficient.

In a preferred embodiment, however, the target nucleic acids are labelled with a fluorescent label and the localization of the label on the probe array is accomplished with fluorescent microscopy. The hybridized array is excited with a light source at the excitation wavelength of the particular fluorescent label and the resulting fluorescence at the emission wavelength is detected. In a particularly preferred embodiment, the excitation light source is a laser appropriate for the excitation of the fluorescent label.

The target polynucleotide may be labelled by any of a number of convenient detectable markers. A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. Other potential labelling moieties include, radioisotopes, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers, magnetic labels, and linked enzymes. Another method for labelling may bypass any label of the target sequence. The target may be exposed to the probes, and a double strand hybrid is formed at those positions only. Addition of a double strand specific reagent will detect where hybridization takes place. An intercalative dye such as ethidium bromide may be used as long as the probes themselves do not fold back on themselves to a significant extent forming hairpin loops. However, the length of the hairpin loops in short oligonucleotide probes would typically be insufficient to form a stable duplex.

Suitable chromogens will include molecules and compounds which absorb light in a distinctive range of wavelengths so that a color may be observed, or emit light when irradiated with radiation of a particular wave length or wave length range, e.g., fluorescers. Biliproteins, e.g., phycoerythrin, may also serve as labels.

A wide variety of suitable dyes are available, being primarily chosen to provide an intense color with minimal absorption by their surroundings. Illustrative dye types include quinoline dyes, triarylmethane dyes, acridine dyes, alizarine dyes, phthaleins, insect dyes, azo dyes, anthraquinoid dyes, cyanine dyes, phenazathionium dyes, and phenazoxonium dyes. A wide variety of fluorescers may be employed either by themselves or in conjunction with quencher molecules. Fluorescers of interest fall into a variety of categories having certain primary functionalities. These primary functionalities include 1 - and 2- aminonaphthalene, ρ,ρ'-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9- aminoacridines, ρ,ρ'-diaminobenzophenone imines, anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1 ,2-benzophenazin, retinol, bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidzaolylphenylamine, 2-oxo-3-chromen, indole, xanthen, 7- hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins, triarylmethanes and flavin. Individual fluorescent compounds which have functionalities for linking or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N- phenyl 1 -amino-8-sulfonatonaphthalene; N-phenyl 2-amino-6-sulfonatonaphthalene; 4- acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid; pyrene-3-sulfonic acid; 2- toluidinonaphthalene-6-sulfonate; N-phenyl, N-methyl 2-aminoaphthalene-6-sulfonate; ethidium bromide; stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl phosphatidylethanolamine; Ν,Ν'-dioctadecyl oxacarbocyanine; N,N'-dihexyl oxacarbocyanine; merocyanine, 4-(3'pyrenyl)butyrate; d-3-aminodesoxy-equilenin; 12- (9'-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene; 2,2'-(vinylene-p- phenylene)bisbenzoxazole; p-bis>2-(4-methyl-5-phenyl-oxazolyl)!benzene; 6- dimethylamino-1 ,2-benzophenazin; retinol; bis(3'-aminopyridinium) 1 ,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin; chlorotetracycline; N-(7- dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide; N->p-(2-benzimidazolyl)- phenylmaleimide; N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin; 4- chloro-7-nitro-2,1 ,3-benzooxadiazole; merocyanine 540; resorufin; rose bengal; and 2,4-diphenyl-3(2H)-furanone.

Desirably, fluorescers should absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound dye may differ from the unbound dye. Therefore, when referring to the various wavelength ranges and characteristics of the dyes, it is intended to indicate the dyes as employed and not the dye which is unconjugated and characterized in an arbitrary solvent. Fluorescers are generally preferred because by irradiating a fluorescer with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events.

Detectable signal may also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions. One family of compounds is 2,3-dihydro-1 ,-4-phthalazinedione. The most popular compound is luminol, which is the 5-amino compound. Other members of the family include the 5-amino-6,7,8-trimethoxy- and the dimethylaminoca!benz analog. These compounds can be made to luminescence with alkaline hydrogen peroxide or calcium hypochlorite and base. Another family of compounds is the 2,4,5- triphenylimidazoles, with lophine as the common name for the parent product.

Chemiluminescent analogs include para-dimethylamino and -methoxy substituents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence.

Spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like. Exemplary spin labels include nitroxide free radicals.

In addition, amplified sequences may be subjected to other post amplification treatments. For example, in some cases, it may be desirable to fragment the sequence prior to hybridization with an oligonucleotide array, in order to provide segments which are more readily accessible to the probes, which avoid looping and/or hybridization to multiple probes. Fragmentation of the nucleic acids may generally be carried out by physical, chemical or enzymatic methods that are known in the art.

Following the various sample preparation operations, the sample will generally be subjected to one or more analysis operations. Particularly preferred analysis operations include, e.g. sequence based analyses using an oligonucleotide array and/or size based analyses using, e.g. microcapillary array electrophoresis.

In some embodiments it may be desirable to provide additional or alternative means for analyzing the nucleic acids from the sample

Microcapillary array electrophoresis generally involves the use of a thin capillary or channel which may or may not be filled with a particular separation medium.

Electrophoresis of a sample through the capillary provides a size based separation profile for the sample. Microcapillary array electrophoresis generally provides a rapid method for size based sequencing, PCR product analysis and restriction fragment sizing. The high surface to volume ratio of these capillaries allows for the application of higher electric fields across the capillary without substantial thermal variation across the capillary, consequently allowing for more rapid separations. Furthermore, when combined with confocal imaging methods these methods provide sensitivity in the range of attomoles, which is comparable to the sensitivity of radioactive sequencing methods.

In many capillary electrophoresis methods, the capillaries e.g. fused silica capillaries or channels etched, machined or molded into planar substrates, are filled with an appropriate separation/sieving matrix. Typically, a variety of sieving matrices are known in the art may be used in the microcapillary arrays. Examples of such matrices include, e.g. hydroxyethyl cellulose, polyacrylamide and agarose. Gel matrices may be introduced and polymerized within the capillary channel. However, in some cases this may result in entrapment of bubbles within the channels which can interfere with sample separations. Accordingly, it is often desirable to place a preformed separation matrix within the capillary channel(s), prior to mating the planar elements of the capillary portion. Fixing the two parts, e.g. through sonic welding, permanently fixes the matrix within the channel. Polymerization outside of the channels helps to ensure that no bubbles are formed. Further, the pressure of the welding process helps to ensure a void-free system.

In addition to its use in nucleic acid "fingerprinting" and other sized based analyses the capillary arrays may also be used in sequencing applications. In particular, gel based sequencing techniques may be readily adapted for capillary array electrophoresis.

Transcriptional expression products from the TFF3 gene may be detected as indications of prostate cancer of the sample tested, such as diagnosing, predicting, prognosing, monitoring prostate cancer in an individual. The transcriptional expression product of the TFF3 gene may be detected in either a tissue sample as such, or in a body fluid sample, such as blood, serum, plasma, semen and/or urine of the individual.

In one embodiment of the present invention the transcriptional level of the TFF3 gene in a sample is increased by 30%-100% relative to previous measurements on the same individual, or to measurements on non-diseased tissue from the same individual, or relative to a standard level, the transcriptional expression level of the TFF3 gene is indicative of the presence of prostate cancer. In another embodiment the transcriptional level of the TFF3 gene in a sample is increased by 30%-90%, in another embodiment by 30%-80%, in another embodiment by 30%-70% and in another embodiment by 30%-60%. In another embodiment the transcriptional level of the TFF3 gene in a sample is increased by 40%-100%, in another embodiment by 40%-90%, in another embodiment by 40%-80% and in another embodiment by 40%-70%. In a preferred embodiment the transcriptional level of the TFF3 gene in a sample is increased by 45%-55%, for example 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54% or 55%. A preferred increase in transcriptional level of the TFF3 gene in a sample is 50%. When a standard transcriptional expression level has a value of 100, and the test sample has a transcriptional expression level of the TFF3 gene at a value of 150, the expression level is said to be increased by 50%.

In another embodiment a transcriptional level of the TFF3 gene increased by 2 fold or more, such as 3-fold, 4-fold, 5-fold or more is indicative of prostate cancer.

In the comparisons, the control sample is an equivalent RNA sample from one and/or a pool of RNA samples more than one healthy male without (clinically diagnosed) prostate cancer and/or from the same person being tested - but control sample was taken at an earlier time point e.g. in the course of an active surveillance programs.

Methods for determining the translational expression level of the TFF3 gene

The expression products, peptides and proteins, may be detected by any suitable technique known to the person skilled in the art.

In a preferred embodiment the translational expression level of TFF3 or part thereof are detected by means of specific antibodies directed to the TFF3 protein product, such as immunofluorescent and/or immunohistochemical staining of the tissue.

Immunohistochemical localization of expressed TFF3 may be carried out by

immunostaining of tissue sections from for example tissue samples such as a biopsy of the prostate cancer tumor to determine the level of translational expression. For example sections may be cut from paraffin-embedded tissue blocks, mounted, and deparaffinized by incubation at 80 C° for 10 min. followed by immersion in heated oil at 60° C for 10 min. (Estisol 312, Estichem A S, Denmark) and rehydration. Antigen retrieval is achieved in TEG (TrisEDTA-Glycerol) buffer using microwaves at 900 W. The tissue sections may be cooled in the buffer for 15 min before a brief rinse in tap water. Endogenous peroxidase activity is blocked by incubating the sections with 1 % H202 for 20 min. followed by three rinses in tap water, 1 min each. The sections may then be soaked in PBS buffer for 2 min. The next steps can be modified from the descriptions given by Oncogene Science Inc., in the Mouse Immunohistochemistry Detection System, XHC01 (UniTect, Uniondale, NY, USA). Briefly, the tissue sections are incubated overnight at 4° C with primary antibody directed against an epitope of the TFF3 protein, followed by for example three rinses in PBS buffer for 5 min each.

Afterwards, the sections are incubated with biotinylated secondary antibody for 30 min, rinsed three times with PBS buffer and subsequently incubated with ABC (avidin- biotinlylated horseradish peroxidase complex) for 30 min. followed by three rinses in PBS buffer.

Staining may be performed by incubation with AEC (3-amino-ethylcarbazole) for 10 min. The tissue sections are counter stained with Mayers hematoxylin, washed in tap water for 5 min. and mounted with glycerol-gelatin. Positive and negative controls may be included in each staining round with all antibodies.

In yet another embodiment the TFF3 protein or part thereof may be detected by means of conventional enzyme assays, such as ELISA methods. Furthermore, the TFF3 protein or part thereof may be detected by means of

peptide/protein chips capable of specifically binding the peptides and/or proteins assessed. Thereby an expression pattern may be obtained.

Translational expression products from the TFF3 gene may be detected as indications of prostate cancer of the sample tested, such as diagnosing, predicting, prognosing, monitoring prostate cancer in an individual. The translational expression products of the TFF3 gene may be detected in either a tissue sample as such, or in a body fluid sample, such as blood, serum, plasma, semen and/or urine of the individual.

Preferably, the translational level is measured in a tissue sample, for example a biopsy, prostate tumor tissue, or a tissue of a resected prostate.

When in a tissue section following prostatectomy, staining for the presence of TFF3 protein is observed this is considered as an indication that the prediction of disease progression is favourable, for example that the recurrence risk is less severe. In contrast, if no staining can be identified in such a tissue section the prediction of disease progression is unfavourable.

In one embodiment of the present invention the translational level of the TFF3 gene in a sample is increased by 30%-100% relative to previous measurements on the same individual, or to measurements on non-diseased tissue from the same individual, or relative to a standard level, the translational expression level of the TFF3 gene is indicative of the presence of prostate cancer. In another embodiment the translational level of the TFF3 gene in a sample is increased by 30%-90%, in another embodiment by 30%-80%, in another embodiment by 30%-70% and in another embodiment by

30%-60%. In another embodiment the translational level of the TFF3 gene in a sample is increased by 40%-100%, in another embodiment by 40%-90%, in another embodiment by 40%-80% and in another embodiment by 40%-70%. In a preferred embodiment the translational level of the TFF3 gene in a sample is increased by 45%- 55%, for example 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54% or 55%. A preferred increase in translational level of the TFF3 gene in a sample is 50%. When a standard translational expression level has a value of 100, and the test sample has a translational expression level of the TFF3 gene at a value of 150, the expression level is said to be increased by 50%.

In another embodiment a translational expression level of TFF3 is increased by at least 50% is indicative of the presence of prostate cancer. In another embodiment a translational level of the TFF3 gene increased by 2 fold or more, such as 3-fold, 4-fold, 5-fold or more is indicative of prostate cancer. In the comparisons, the control sample is an equivalent sample from one and/or a pool of samples from more than one healthy male without (clinically diagnosed) prostate cancer and/or from the same person being tested - but control sample was taken at an earlier time point e.g. in the course of an active surveillance programs. Methods of treatment and uses

Pharmaceutical composition

The invention also relates to a pharmaceutical composition for treating prostate cancer. Thus, in one aspect of the present invention the pharmaceutical composition for the treatment of prostate cancer comprising i) at least one transcription inhibitor capable of decreasing the transcript levels of a TFF3 gene transcript (SEQ ID NO: 2), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:2, or part thereof, and/or ii) at least one translational inhibitor capable of decreasing the translational expression levelof the TFF3 gene (SEQ ID NO: 3), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof.

In one embodiment of this method the transcription inhibitor is a functional RNA molecule displaying complementary sequence to a TFF3 gene transcript.

The functional RNA molecule is for example a siRNA molecule, a microRNA molecule, a shRNA molecule and/or an antisense RNA molecule.

In one embodiment of this method the translational inhibitor is an antibody directed against an epitope of the TFF3 protein or part thereof.

In the present context the term pharmaceutical composition is used synonymously with the term medicament. The medicament of the invention comprises an effective amount of one or more of the compounds as defined above, or a composition as defined above in combination with pharmaceutically acceptable additives. Such medicament may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration. For most indications a localised or substantially localised application is preferred.

Strategies in formulation development of medicaments and compositions based on the compounds of the present invention generally correspond to formulation strategies for any other protein-based drug product. Potential problems and the guidance required to overcome these problems are dealt with in several textbooks, e.g. "Therapeutic Peptides and Protein Formulation. Processing and Delivery Systems", Ed. A.K. Banga, Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. The preparation may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired , the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or which enhance the effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art. The formu lations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect. Additional formulations which are suitable for other modes of administration include suppositories, and, in some cases, oral formulations. For suppositories, traditional binders and carriers include polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingred ient(s) i n the range of from 0.5% to 1 0% , preferably 1 -2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 1 0-95% of the active ingredient(s), preferably 25-70%.

The preparations are ad m i n istered i n a man ner compati ble with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 1000 μg, such as in the range of from about 1 μg to 300 μg, and especially in the range of from about 10 μg to 50 μg. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosis would be in the interval 30 mg to 70 mg per 70 kg body weight.

Some of the compounds of the present invention are sufficiently active, but for some of th e oth ers , th e effect wi l l be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulation multiple times. Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc.

Kit

According to another aspect of the present invention, it provides kits (assays) useful for detecting prostate cancer and various aspects of prostate cancer, e.g., using the methods disclosed herein.

Thus, the present invention relates to a kit (assay) comprising at least one detection member for a TFF3 gene, transcriptional and/or translational product or part thereof for use in the methods of the present invention: for assisting in the diagnosis and/or for diagnosing of prostate cancer, for assisting in the prognosis and/or for the prognosis of the disease progression of prostate cancer, for assisting in the prediction and/or the prediction of the progression of prostate cancer, for assisting in predicting and/or for predicting the recurrence risk of prostate cancer, for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression, for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cancer, for assisting in determining and/or determining the treatment regime. In one embodiment, the at least one detection member, such as two, three, four, 5, 6, 7, 8, 9, 10 or more detection members is an antibody directed against an epitope of the TFF3 protein or part thereof, oligonucleotides, primers and/or probes that are able to hybridise to the TFF3 gene and/or the TFF3 transcript.

When the at least one detection member is an antibody directed against an epitope of the TFF3 translational product, the antibody is monoclonal, polyclonal, or a mixture of at least two monoclonal antibodies.

In one embodiment the assay further comprises means for providing the level and/or means for providing informations as to whether the level is above or below a cut off value. In the present context the level here refers to methylation status as methylation level, transcriptional expression level and/or translational expression level of the TFF3 gene.

In one embodiment, the present invention provides a kit, e.g., a compartmentalized carrier including a first container containing a pair of primers for amplification of the sample TFF3 gene, a optionally a second container containing a pair of primers for amplification of a region in a reference gene, and a third container containing a first and second oligonucleotide probe specific for the amplification of the TFF3 gene and the region of the reference gene, respectively.

In another embodiment, the kit provided by the present invention further includes a fourth container containing a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid, e.g., uracil. Any suitable modifying agent, such as an agent that modifies unmethylated cytosine nucleotides, can be included in the kit provided by the present invention. For example, the modifying agent can be sodium bisulfite.

The kit may also comprise additional reagents used in the amplifying step of the detection method as disclosed herein. Thus, the kit may further comprise

deoxyribonucleoside triphosphates, DNA polymerase enzyme and/or nucleic acid amplification buffer.

In another embodment, the present invention provides a kit for example a

compartmentalized carrrier including a first container comprising an antibody directed against a TFF3 translation product or part thereof, and a second container containing a reference TFF3 protein.

In yet another embodiment, the present invention provides a kit as for example a compartmentalized carrier including a first container comprising at least one oligo nucleotide or primer pair able to hybridise to the TFF3 transcript, or DNA derived therefrom, and a second container comprising a control sample.

The kit may in preferred embodiments further comprise instructions for the

performance of the detection method of the kit and for the interpretation of the results. The kit involves the method of detecting the methylation status of a CpG-containing nucleic acid, wherein said CpG-containing nucleic acids is modified using an agent which modifies at least one unmethylated cytosine in said methylated CpG-containing nucleic acid and amplifying said CpG- containing nucleic acid by means of at least one methylation-independent oligonucleotide primer. The instructions for performing the method of the kit comprises for example information of particular annealing

temperatures to be used for the at least one methylation-independent primers, as well as for example information on cycling parameters. The kit may further comprise instructions for the interpretation of the results obtained by the method. For example how to interpret the amplified products subsequently analysed by high resolution melting analysis or methods as described elsewhere herein. For example the kit in one embodiment comprises means for providing a methylation level and/or means for providing information as to determine whether the level of methylation is above or below a cut off value. However, the assay in another embodiment comprises means for providing a transcriptional and/or translational expression level, and/or means for providing information as to determine whether the level of expression is above or below a cut off value.

The kit may in preferred embodiments further comprise software comprising an algorithm for calculation of primer annealing temperature and interpretation of results.

In yet another embodiment, the kit provided by the present invention further includes a probe for PSA determination. In still another embodiment, the kit provided by the present invention further includes an instruction insert disclosing normal and/or abnormal methylation ratio ranges for the detection of neoplasia, describing the types of samples suitable or unsuitable for the application of the kit, and/or the specificity or sensitivity provided by the assays utilizing the kit of the present invention.

According to one embodiment of the present invention, the kit provided by the present invention includes a first container containing at least one pair of primers for amplification of a promoter region of TFF3, a second container containing at least one pair of primers for amplification of a region of a reference gene, and a third container containing a first and second oligonucleotide probe specific to the amplification of the promoter region of TFF3 and the region of the reference gene, respectively, provided that one or both primers for amplification of the promoter region of TFF3 or one or more first oligonucleotide probes specific to the amplicon of the promoter region of TFF3 are capable of distinguishing between methylated and unmethylated nucleic acid, either directly or indirectly, e.g., after bisulfite modification. Optionally the kit provided by this embodiment of the present invention can further include an instruction insert, e.g., disclosing the cut off values to be consulted for determining prostate adenocarcinoma or that the kit can be used with a prostate cancer tissue sample, e.g., most suitable to be used with a prostate tissue sample.

The present invention also provides a kit useful for detecting prostate adenocarcinoma, for example in body fluid samples. The kit includes a first container containing at least one pair of primers capable of distinguishing between methylated and unmethylated nucleic acid for amplification of a promoter region of TFF3 and an instruction insert disclosing, among other things, that the kit is useful for detecting prostate cancer in a body fluid sample of an individual and that a methylation level of the promoter region of TFF3 as determined by conventional or non-real-time PCR using the primers provided that is higher than the methylation level of the promoter region of TFF3 in a normal subject is indicative of prostate cancer in the subject.

For example the kit in one embodiment comprises means for providing a methylation level and/or means for providing information as to determine whether the level of methylation is above or below a cut off value, and thus indicative of the presence or absence of prostate cancer, respectively.

The present invention also relates to the use of an antibody directed against an epitope of the TFF3 protein or part thereof in the detection of the translational expression level of a TFF3 gene, or part thereof

viii) for assisting in the diagnosis and/or for diagnosing of prostate

cancer,

ix) for assisting in the prognosis and/or for the prognosis of the disease progression of prostate cancer,

x) for assisting in the prediction and/or the prediction of the progression of prostate cancer,

xi) for assisting in predicting and/or for predicting the recurrence risk of prostate cancer,

xii) for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression,

xiii) for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cance, for assisting in determining and/or determining the treatment regime Examples

Below are non-limiting examples of the present invention in various embodiments. The examples below may thus be regarded as preferred embodiments of the present invention.

Clinical samples

Benign prostate hyperplasia (BPH) (n=12) and prostate adenocarcinoma specimens (n=10) were obtained as transurethral resections or as needle biopsies from patients undergoing radical prostatectomy. Tissue samples were fresh frozen or embedded in Tissue Tek (Bayer Corporation, Pittsburgh, PA) and stored at -80°C. Formalin-fixed and paraffin-embedded (FFPE) tissue samples were also available. Clinicopathological information is provided in Table 1 . Informed consent was obtained from all patients in this study, which was approved by the Scientific Ethics Committee of Aarhus County, Denmark.

Table 1

Clinicopathological data

TFF1 TFF3

TFF1 TFF3 TFF1 TFF3

Sample Methylation Methylation

(pmol/L) (pmol/L) (Ratio) (Ratio)

CpG 1 -13 (%) CpG 5-8 (%)

PC-01 1020 150 1 ,30 4,20 32 19

PC-4 1660 360 2,78 1 ,14 59 47

PC-6 1090 250 3,24 0,89 44 44

PC-07 3980 500 31 ,82 1 ,76 57 79

PC-09 2610 620 20,3 3,21 21 52

PC-29 910 260 3,04 1 ,09 26 20

PC-30 1680 410 2,53 2,07 92 2

PC-41 1870 200 2,95 7,01 2 21

PC-53 930 920 2,97 5,95 65 40

PC-59 1660 700 na na 29 19

KPC-4 510 150 na na 58 48

KPC-16 460 140 na na 49 63

KPC-24 560 140 na na 39 25

KPC-29 310 90 na na 53 53

KPC-31 560 150 na na 78 93

KPC-35 690 200 na na 54 60

KPC-47 750 170 na na 60 79

KPC-50 NA NA na na 66 73

KPC-53 560 190 na na 80 70

KPC-54 540 100 4,48 0,53 65 81

KPC-55 1480 400 12,36 1 ,31 71 83

KPC-57 590 190 2,86 0,37 59 100 ^a Gleason sum and TNM stages refer to the adenocarcinoma in these patient (in italics).

b M stage: 0 (non-metastatic disease); 1 (primary tumor, hormone-naive metastatic prostate cancer); 1 + (hormone- refractory prostate cancer).

na: not applicable

nd: not determined

Cell culture and epigenetic drug treatment

All cell lines were grown in RPMI 1640 with L-glutamine (Gibco, Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. LNCAP, PC3, and DU145 prostate adenocarcinoma cells were purchased from ATCC and BPH1 cells from DSMZ (Braunschweig, Germany). Dr. Kenneth Pienta, University of Michigan, kindly provided VCAP and DUCAP prostate adenocarcinoma cells. PNT1A immortalized prostate epithelial cells are described in (26). All cell lines were treated for 48 hr with 1 μΜ 5-aza-dC (Sigma-Aldrich

Corporation, St. Louis, MO) and allowed 5-days recovery in complete medium prior to RNA and DNA extraction. Four cell lines were given a combination of 1 μΓΤΐοΙ/L 5-aza- dC (48 h treatment + 5 days of recovery) and 1 mmol/L 4-phenylbutyric acid (Sigma) continuously for 7 days. Growth medium was changed daily and cells were harvested on day 7. Untreated cells were grown in parallel. All experiments were performed in duplicate and repeated twice.

For determination of protein content of cell culture supernatants samples were collected at subconfluent densities three days after splitting.

EXAMPLE 1

TFF promoter methylation in prostate cell lines

To investigate possible epigenetic regulation of the trefoil factors in PC, promoter methylation was analyzed for TFF1 -3 by genomic bisulfite sequencing in seven prostate cell lines: Five prostate adenocarcinoma cell lines (DU145, PC3, VCAP, DUCAP and LNCAP), one immortalized non-malignant prostate epithelial cell line (PNT1A), and one benign prostatic hyperplasia cell line (BPH1 ). A total of 13 CpGs in TFF1 , 16 in TFF2, and 8 in TFF3 were examined (Fig. 1 A).

Genomic DNA from prostate cell lines and carefully selected 20-μηι sections of BPH, prostate adenocarcinoma and adjacent non-malignant prostate tissue was isolated with the PUREGENETM DNA Purification Kit (Gentra Systems) and bisulfite-converted using the MethylEasy DNA Bisulfite modification kit (Human Genetic Signatures, Sydney, Australia). TFF1 , TFF2, and TFF3 promoter sequences were amplified from bisulfite-converted DNA by PCR, purified from agarose gels (QIAquick Gel Extraction Kit, Qiagen) and subcloned into the pCR4-TOPO vector (TOPO TA Cloning Kit for Sequencing, Invitrogen). Individual clones were sequenced with M13 forward and reverse primers using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an automated ABI PRISM 3130XL2 Genetic Analyzer (Applied Biosystems). For each sample, at least seven clones were sequenced to identify methylated cytosine residues. Primer sequences were for TFF1 PCR primer sequences (forward and reverse) were for TFF1 (G G AATG GGTTTTATG AGTTTTTTT and CATTACCTCCTCTCTACTCCAAAAA), TFF2

(GGTGTGATTTTGTGTGTGTTTAGTT and AAAACCCTCTCCTTCACTTACAAAA), and TFF3 (AG G AAAG ATAAG GAATTTTTGTGTTTT and

ACATACCTTTATCAAACTCCCAAAC).

For TFFI , the 8 CpGs -181 to +17 were almost completely free of methylation in the TFF1 expressing VCAP, DUCAP, and LNCAP cells (Fig. 1 B and Fig. 2), but hypermethylated in the cell lines with no or low TFF1 expression (BPH1 , PNT1 A, DU145, and PC3). The five CpGs located further upstream (-400 to -301 ) were hypomethylated only in LNCAP cells and showed various degrees of hypermethylation in the remaining six cell lines. No correlation between methylation status of CpGs -400 and -388, overlapping the ERE, and expression of TFF1 was found.

For TFF2 only minor differences in methylation patterns were observed between the different prostate cell lines, which all displayed high levels of hypermethylation of the 10 CpGs located in the 300-bp 5'-flanking region and of the 6 CpGs found after the transcription start site (Fig 1 A, B).

In the TFF3 gene, the four CpGs (-127 to -80) located closest to the ATG start site were almost completely free of methylation in the cell lines VCAP, DUCAP, and LNCAP, but showed high density methylation (>70%) in the remaining cell lines ( Fig. 1 A,B),. The four CpGs located further upstream (-337 to -21 1 ) were hypermethylated in all cell lines.

EXAMPLE 2

Binding sites for transcription factors In silico prediction of transcription factor-binding sites (TFBS) for TFF promoters was carried out using Matlnspector 2.2 software (Genomatix company webpage.

http://www.genomatix.de/products/Matlnspector/) (30). We used the Analyse-it version 1 .69 add-in program package for Microsoft Excel (Analyse-it Software, Ltd., Leeds, United Kingdom) for statistical analysis. We analyzed differences between the median methylation levels of the two groups (PC vs. BPH) by the Mann-Whitney L/ test.

Confidence intervals for sensitivity, specificity, positive predictive value, and negative predictive value were calculated according to (31 ). P < 0.05 was considered statistically significant in a two-sided test.

Several of the hypomethylated 8 CpGs in TFF1 and 4 CpGs in TFF3 correspond with consensus binding sequences for cellular transcription factors, as identified by

Matlnspector software analysis of the TFF promoter regions shown in Fig. 1A. Notably, several of these are putative binding sites for transcription factors with a known role in

PC (CREB, MYB, PAX2, MYC, NFRB, ELK1 ) and for which it has earlier been shown that site-specific CpG methylation of consensus motifs block DNA binding (MYC, NFkB, MYB, CREB, PAX5, and SP1 ) (6;32-39).

Analysis of putative binding sites for transcription factors in the promoter regions of TFF1 and TFF3 revealed several potential binding sites around the CpGs that were hypomethylated in PC cell lines and clinical samples that express trefoil factors.

Functional characterizations of the TFF3 promoter region has previously been based on a genomic annotation with the promoter region extending 170 bp into exon 1 (47). In the present study we showed that the four CpGs closest to the ATG were almost completely free of methylation in TFF3 expressing VCAP, DUCAP, and LNCAP cell lines and corresponding to the proposed epigenetically regulated CpGs we identified putative binding sites for the transcription factors CREB, DMP1 , ELK1 , and SP of which both CREB and SP1 previously have been shown to be methylation sensitive. The influence of the methylated CpG sites on the affinity of transcription factor binding and the correlation with TFF3 gene expression requires further investigation.

EXAMPLE 3

TFF1, TFF2 and TFF3 RNA expression in prostate cell lines

To investigate TFF expression patterns in prostate cells, total RNA was isolated from the seven cell lines. The RNeasy Micro Kit (Qiagen, Valencia, CA) was used to isolate total RNA from cultured cells and carefully selected Tissue Tek-embedded prostate tissue specimens (25-30 twenty-micrometer sections). First-strand cDNA synthesis was performed with Superscript II Reverse Transcriptase (Invitrogen Corporation) using an oligo (dT)24 primer. Trefoil factor expression was measured with TaqMan gene expression assays Hs00170216_m1 (TFF1 ), Hs00193719_m1 (TFF2), and

Hs00173625_m1 (TFF3) and TaqMan Universal PCR Master Mix on a real time ABI

PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). CFL1 (COFILIN-1) was used for normalization, as described in (27). All reactions were run in triplicates. By real-time RT-PCR analysis none or only trace amounts of trefoil factors TFF1 and TFF3 were detected in cell lines with a high degree of methylation (BPH1 and PNT1 A cell lines and in the PC cell lines DU145 and PC3) whereas both genes were significantly expressed in the hypomethylated adenocarcinoma cell lines VCAP, DUCAP, and LNCAP (Fig. 2, grey bars). TFF2 mRNA was expressed in trace amounts in PC3 and VCAP cells, but absent in all other cell lines (data not shown). This is consistent with the observed TFF2 promoter hypermethylation in all prostate cell lines investigated (Fig. 1 B).

Significant expression of TFF1 and TFF3 was detected in androgen responsive cell lines (VCAP, DUCAP, and LNCAP), but not in the androgen independent cell lines DU145 and PC3.

To examine whether expression of TFF genes in PC cells could be restored by treatment with DNA methylation inhibitors cell lines were treated with 5-aza-dC and then analyzed for expression of TFF1 , TFF2, and TFF3 by RT-PCR. 5-aza treatment increased the basal expression of trace amounts of TFF1 eleven-fold in DU145 and nineteen-fold in PC3 cell lines. These cell lines showed high degree of methylation of the CpGs just prior to the transcription start site. Basal expression in VCAP, DUCAP, and LNCAP with low degree of methylation of the CpGs cell lines was increased by 1 .3, 2.7, and 2.7 times respectively after 5-aza treatment. Significant TFF1 expression was not induced in the two benign prostate cell lines BPH1 and PNT1A. TFF2 expression was induced by 5-azacytidine treatment in all cell lines except DU145 and VCAP (data not shown). Trace amounts of TFF3 expression was detected in highly methylated BPH1 , PNT1A, DU145, and PC3 cell lines after 5-aza treatment. TFF3 induction in the cell lines BPH1 , PNT1A, and DU 145 was accompanied by reduction in CpG methylation of 73% to 31 %, 100% to 33%, and 93% to 78%. As little as 6-8% methylation may account for downregulation of genes and may explain why TFF3 also was induced in VCAP, DUCAP, and LNCAP cell lines by 3.8-, 2.0-, and 1.9-fold, respectively (Fig 2 B).

Treatment of four prostate cell lines with 5-aza plus histone deacetylase inhibitor 4- phenylbutyric acid only induced marked TFF1 and TFF3 expression in BPH1 cells. The combined drug treatment did not effectively induce changes in DU 145 (TFF1 and TFF3) and PC3 (TFF1 ) cell lines and did not induce any expression of TFF1 in LNCAP cells and TFF3 in PC3 and LNCAP cells (Fig 2). TFF2 expression was not tested after 4-phenylbutyric acid treatment.

The findings support the notion that DNA methylation is involved in the transcriptional regulation of trefoil factor genes. However, more CpG-sites than the ones examined here may be involved in transcription of TFF genes. Furthermore, indirect cell line specific upregulation of TFF expression may explain the divergent expression patterns after 5-azacytidine and combined treatment with 5-aza and 4-phenylbutyric acid.

EXAMPLE 4

TFF1, TFF2, and TFF3 protein expression in prostate cell lines

Secreted TFF1 , TFF2, and TFF3 protein concentrations in cell-free supernatants from cell cultures were measured by in-house peptide-specific ELISAs as described recently (analytical imprecision, 8% for TFF1 , 7% for TFF2, and 7% for TFF3) (28;29). Samples were subjected to ultrasound and centrifuged at 16,000 xg at room temperature before analysis.

As expected TFF1 was detected in the three hypomethylated cell lines that also expressed mRNA for the peptides. In addition TFF1 was detected in the PC3 cell line (Fig 2A). TFF3 protein was detected in the hypomethylated cell lines DUCAP and LNCAP, whereas no TFF2 protein could be detected in any of the cell culture supernatants (data not shown). Thus, except for the detection of TFF1 protein in PC3 supernatant and the absence of TFF3 protein in VCAP supernatant, all protein measurements correlated well with the corresponding TFF RNA expression levels (Fig 2). These minor discrepancies may be explained by differences in excretion patterns of trefoil peptides by different cell lines and/or low TFF concentrations close to the detection levels of the assays. EXAMPLE 5

TFF1 and TFF3 promoter hypomethylation in PC tissue

To investigate trefoil factor hypomethylation as the possible cause of TFF1 and TFF3 overexpression in primary prostate adenocarcinomas, genomic DNA isolated from twelve BPHs and ten PC tissue samples was analyzed by bisulfite sequencing (Table l and Fig 3). Methylation of the TFF2 promoter in clinical material was not investigated because TFF2 previously showed lower observed accuracy for detection of PC (21 ). Nor did the present in vitro studies show differential methylation of the investigated CpGs in malignant versus non-malignant prostate cell lines.

For TFF1 the individual median methylation of all 13 investigated CpGs were lower in the samples from PC patients and the differences reached statistical significance in the CpGs -370, -354, -56, -13, and -10 (P=0,01 , 0,03, 0,02, 0,05, and 0,04). Overall, for TFF1 the 13 promoter CpGs were hypomethylated (<50% methylated) in 6/10 PC samples and in 2/10 BPH sample. In the TFF3 promoter more marked differences in methylation patterns were observed consistent with the more significant differences previously observed for serum concentrations of TFF3 (21 ). Significant differences in individual CpGs between BPH and PC were observed for the 4 CpGs -127, -108, -89, and -80 (P=0.01 , 0.00, 0.00, and 0.01 ) just prior to the translation start site (Fig 4 and Table 1 ). These four sites were also the most differentially methylated sites in TFF3- expressing versus non-expressing cell lines (Fig. 1 B). TFF1 and TFF3 expression

(peptide and mRNA) were also measured in plasma and prostate samples (Table 1 ). No significant correlations were found between TFF1 and TFF3 methylation status and plasma levels or mRNA expression in prostate samples.

Using a cutoff for the mean methylation of CpGs -127, -108, -89, and -80 in the TFF3 promoter of 50% the sensitivity and specificity of TFF3 promoter methylation for detecting PC was 80 % (44-97%) and 83% (52-98%). Positive and negative predictive values were 80% (44-97%) and 83% (52-98%).

Site-specific hypomethylation of cytosines in CpG dinucleotides located within CpG islands as well as non-CpG islands (i.e. DNA fragments with lower CpG density) at the 5' region of genes has been associated with increased expression of cancer-related genes (45). The trefoil factor genes TFF1 , TFF2, and TFF3 contain CpG dinucleotides at their promoter/5' regions (Fig 1 A), although with lower CpG densities than in classically defined CpG islands (A region of at least 200 bp and with a GC percentage greater than 50% and an observed/expected CpG ratio greater than 60% (46)). The finding that TFF1 and TFF3 are hypomethylated in cancer may reflect a general hypomethylation effect in PC recently proposed to occur later in PC progression (48). In our previous study on plasma levels of trefoil factors in PC we found that patients with advanced PC had significantly higher plasma concentrations.

References

1. Nelson WG, Yegnasubramanian S, Agoston AT, Bastian PJ, Lee BH, Nakayama M, De Marzo AM. Abnormal DNA methylation, epigenetics, and prostate cancer. Front Biosci 2007;12:4254-66.

2. Dobosy JR, Roberts JL, Fu VX, Jarrard DF. The expanding role of epigenetics in the development, diagnosis and treatment of prostate cancer and benign prostatic hyperplasia. J Urol 2007;177:822-31.

3. Jones PA. DNA methylation and cancer. Oncogene 2002;21 :5358-60.

4. Karpf AR, Bai S, James SR, Mohler JL, Wilson EM. Increased expression of androgen receptor coregulator MAGE-1 1 in prostate cancer by DNA hypomethylation and cyclic AMP. Mol Cancer Res 2009;7:523-35.

5. Yegnasubramanian S, Haffner MC, Zhang Y, Gurel B, Cornish TC, Wu Z et al. DNA hypomethylation arises later in prostate cancer progression than CpG island

hypermethylation and contributes to metastatic tumor heterogeneity. Cancer Res 2008;68:8954-67.

6. Wang Q, Williamson M, Bott S, Brookman-Amissah N, Freeman A, Nariculam J et al. Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer. Oncogene

2007;26:6560-5.

7. Thim L. Trefoil peptides: from structure to function. Cell Mol Life Sci 1997;53:888- 903.

8. Garraway IP, Seligson D, Said J, Horvath S, Reiter RE. Trefoil factor 3 is

overexpressed in human prostate cancer. Prostate 2004;61 :209-14.

9. Colombel M, Dante R, Bouvier R, Ribieras S, Pangaud C, Marechal JM, Lasne Y. Differential RNA expression of the pS2 gene in the human benign and malignant prostatic tissue. J Urol 1999;162:927-30.

10. Emami S, Rodrigues S, Rodrigue CM, Le Floch N, Rivat C, Attoub S et al. Trefoil factor family (TFF) peptides and cancer progression. Peptides 2004;25:885-98.

1 1. Taupin D, Ooi K, Yeomans N, Giraud A. Conserved expression of intestinal trefoil factor in the human colonic adenoma-carcinoma sequence. Lab Invest 1996;75:25-32. 12. Terris B, Blaveri E, Crnogorac-Jurcevic T, Jones M, Missiaglia E, Ruszniewski P et al. Characterization of gene expression profiles in intraductal papillary-mucinous tumors of the pancreas. Am J Pathol 2002;160:1745-54.

13. Emami S, Le Floch NN, Bruyneel E, Thim L, May F, Westley B et al. Induction of scattering and cellular invasion by trefoil peptides in src- and RhoA-transformed kidney and colonic epithelial cells. FASEB J 2001 ;15:351-61 .

14. Rodrigues S, Nguyen QD, Faivre S, Bruyneel E, Thim L, Westley B et al. Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways. FASEB J 2001 ;15:1517-28. 15. Rodrigues S, Van Aken E, Van Bocxlaer S, Attoub S, Nguyen QD, Bruyneel E et al. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of

cyclooxygenase-2 and EGF receptor signaling. FASEB J 2003;17:7-16.

16. Welsh JB, Sapinoso LM, Su Al, Kern SG, Wang-Rodriguez J, Moskaluk CA et al. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res 2001 ;61 :5974-8.

17. Dhanasekaran SM, Barrette TR, Ghosh D, Shah R, Varambally S, Kurachi K et al. Delineation of prognostic biomarkers in prostate cancer. Nature 2001 ;412:822-6.

18. Luo J, Duggan DJ, Chen Y, Sauvageot J, Ewing CM, Bittner ML et al. Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling. Cancer Res 2001 ;61 :4683-8.

19. Faith DA, Isaacs WB, Morgan JD, Fedor HL, Hicks JL, Mangold LA et al. Trefoil factor 3 overexpression in prostatic carcinoma: prognostic importance using tissue microarrays. Prostate 2004;61 :215-27.

20. Bonkhoff H, Stein U, Welter C, Remberger K. Differential expression of the pS2 protein in the human prostate and prostate cancer: association with premalignant changes and neuroendocrine differentiation. Hum Pathol 1995;26:824-8.

21. Vestergaard EM, Borre M, Poulsen SS, Nexo E, Torring N. Plasma levels of trefoil factors are increased in patients with advanced prostate cancer. Clin Cancer Res 2006;12:807-12.

22. Tomasetto C, Rockel N, Mattei MG, Fujita R, Rio MC. The gene encoding the human spasmolytic protein (SML1/hSP) is in 21 q 22.3, physically linked to the homologous breast cancer marker gene BCEI/pS2. Genomics 1992;13:1328-30.

23. Chinery R, Williamson J, Poulsom R. The gene encoding human intestinal trefoil factor (TFF3) is located on chromosome 21 q22.3 clustered with other members of the trefoil peptide family. Genomics 1996;32:281 -4. 24. Gott P, Beck S, Machado JC, Carneiro F, Schmitt H, Blin N. Human trefoil peptides: genomic structure in 21 q22.3 and coordinated expression. Eur J Hum Genet

1996;4:308-15.

25. Beck S, Sommer P, Blin N, Gott P. 5'-flanking motifs control cell-specific expression of trefoil factor genes (TFF). Int J Mol Med 1998;2:353-61.

26. Degeorges A, Hoffschir F, Cussenot O, Gauville C, Le DA, Dutrillaux B, Calvo F. Recurrent cytogenetic alterations of prostate carcinoma and amplification of c-myc or epidermal growth factor receptor in subclones of immortalized PNT1 human prostate epithelial cell line. Int J Cancer 1995;62:724-31 .

27. Sorensen KD, Borre M, Orntoft TF, Dyrskjot L, Torring N. Chromosomal deletion, promoter hypermethylation and downregulation of FYN in prostate cancer. Int J Cancer 2008;122:509-19.

28. Vestergaard EM, Poulsen SS, Gronbaek H, Larsen R, Nielsen AM, Ejskjaer K et al. Development and evaluation of an ELISA for human trefoil factor 3. Clin Chem

2002;48:1689-95.

29. Vestergaard EM, Brynskov J, Ejskjaer K, Clausen JT, Thim L, Nexo E, Poulsen SS. Immunoassays of human trefoil factors 1 and 2: measured on serum from patients with inflammatory bowel disease. Scand J Clin Lab Invest 2004;64:146-56.

30. Cartharius K, Freeh K, Grote K, Klocke B, Haltmeier M, Klingenhoff A et al.

Matlnspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 2005;21 :2933-42.

31. Habbema JD., Eijkmanns R, Krijnen P. Analysis of the data on accuracy of diagnostic tests. 2009:177-43.

32. Fox KE, Colton LA, Erickson PF, Friedman JE, Cha HC, Keller P et al. Regulation of cyclin D1 and Wntl Ob gene expression by cAMP-responsive element-binding protein during early adipogenesis involves differential promoter methylation. J Biol Chem 2008;283:35096-105.

33. Garcia GE, Nicole A, Bhaskaran S, Gupta A, Kyprianou N, Kumar AP. Akt-and CREB-mediated prostate cancer cell proliferation inhibition by Nexrutine, a

Phellodendron amurense extract. Neoplasia 2006;8:523-33.

34. Reamon-Buettner SM, Borlak J. Epigenetic silencing of cell adhesion molecule 1 in different cancer progenitor cells of transgenic c-Myc and c-Raf mouse lung tumors. Cancer Res 2008;68:7587-96.

35. Tierney RJ, Kirby HE, Nagra JK, Desmond J, Bell Al, Rickinson AB. Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation. J Virol 2000;74:10468-79.

36. Prendergast GC, Ziff EB. Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. Science 1991 ;251 :186-9.

37. Bednarik DP, Duckett C, Kim SU, Perez VL, Griff is K, Guenthner PC, Folks TM. DNA CpG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs. New Biol 1991 ;3:969-76.

38. Khoubehi B, Kessling AM, Adshead JM, Smith GL, Smith RD, Ogden CW.

Expression of the developmental and oncogenic PAX2 gene in human prostate cancer. J Urol 2001 ;165:21 15-20.

39. Tyagi A, Agarwal R, Agarwal C. Grape seed extract inhibits EGF-induced and constitutively active mitogenic signaling but activates JNK in human prostate carcinoma DU145 cells: possible role in antiproliferation and apoptosis. Oncogene 2003;22:1302- 16.

40. Martin V, Ribieras S, Song-Wang XG, Lasne Y, Frappart L, Rio MC, Dante R. Involvement of DNA methylation in the control of the expression of an estrogen- induced breast-cancer-associated protein (pS2) in human breast cancers. J Cell Biochem 1997;65:95-106.

41 . Sato N, Maitra A, Fukushima N, van Heek NT, Matsubayashi H, lacobuzio- Donahue CA et al. Frequent hypomethylation of multiple genes overexpressed in pancreatic ductal adenocarcinoma. Cancer Res 2003;63:4158-66.

42. Okada H, Kimura MT, Tan D, Fujiwara K, Igarashi J, Makuuchi M et al. Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas. Int J Oncol 2005;26:369-77.

43. Carvalho R, Kayademir T, Soares P, Canedo P, Sousa S, Oliveira C et al. Loss of heterozygosity and promoter methylation, but not mutation, may underlie loss of TFF1 in gastric carcinoma. Lab Invest 2002;82:1319-26.

44. Sorensen KD, Wild PJ, Mortezavi A, Adolf K, Torring N, Heeboll S et al. Genetic and epigenetic SLC18A2 silencing in prostate cancer is an independent adverse predictor of biochemical recurrence after radical prostatectomy. Clin Cancer Res 2009;15:1400-10.

45. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 2002;21 :5400-13.

46. Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol 1987;196:261 -82. 47. Ribieras S, Lefebvre O, Tomasetto C, Rio M. Mouse Trefoil Factor genes: genomic organization, sequences and methylation analyses. Gene 2001 ;266:67-75.

48. Yegnasubramanian S, Haffner MC, Zhang Y, Gurel B, Cornish TC, Wu Z et al. DNA hypomethylation arises later in prostate cancer progression than CpG island hypermethylation and contributes to metastatic tumor heterogeneity. Cancer Res 2008;68:8954-67.

49. Casadei R, Strippoli P, D'Addabbo P, Canaider S, Lenzi L, Vitale L et al. mRNA 5' region sequence incompleteness: a potential source of systematic errors in translation initiation codon assignment in human mRNAs. Gene 2003;321 :185-93.

50. Martin V, Ribieras S, Song-Wang X, Rio MC, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5' region of the pS2 gene and its expression in human breast cancer cell lines. Gene 1995;157:261 -4.

SEQUENCES

SEQ ID NO: 1 TFF3 gene

DNA nucleotide sequence of the entire TFF3 genomic locus including 10000-bp upstream sequence.

The sequence analyzed for methylation according to the present invention is marked in bold and underlined. The ATG start codon is shown in bold and double underlined. >ref|NC_000021 .8|:43732163-43745706 Homo sapiens chromosome 21 , GRCh37 primary reference assembly

GCTGGGTTTGCCCTTTTCTCAGTACAGTGATTCTCCAGCCTGAAGGACCACAGGTTTCCACTGGTGTT TTAGCCGCAACTATACAACACCCACTGTGACCTACCCTCAGGCAAAATCCCATACAGAAATAAATTCG CAGCATTCCCTCTCCTTCTTCCAGGGTCAGCTCCCCTCCAGGATCTCAGACAGCTGTGCTTGGTTTGT TTTTCTCACTTTCCAGTGCCATCAGGCCATTGCTTTGTGCTTTTTCCAGAGTTTTCATTGTTATATCAAG AAGGGCAGGGTCTAGTATGAGCTGGTTGACTATCTCTACTTTTAGTGACTTAAAGTTTTACTTTTCACT TTGGGCCTGTAATTCACTTTGAACACTGTGGGACTTGGACAGAGGGAGGAACAGGCACTCCCCCAGT TTTGGGCACGGAGACACAGACAAGCAATCCCAACACAAATGTGGCATATTGTCATGCTCTGACATACA TGGTCATGCACAGACGGGGAAGGTACATGGATGCATGTGTGTGCATCTGGCCCTGCAATGGGTCAG GAAGCTGAGGAGCCACACAGCATAAACGGGACGGACCGCAGTACTAAGTGTCCTGGCTCAGGGGCA CTGAGCACCCAGGCAGAGCTCGTCCAGCTCCCCAGGAAGCCAGCCAGGGCAGAGAGGAAAACTTGT TAATGGGTTGTTAAATTGTGGTTCTAAATTTACCTTTTTCAATGTAGATAATTAAAAATAGCTTAGCGTC ATTAGGTGGGGATCTGAATTTGTCTTTTTAAAATACAGATAACTGATTGTCATGGTACCATTTATTCAAT TGTCCTGTTTCCCCATTGATATCAAATATATTATCCACATTTTCTTATGTGTATGGATCTATTTCTGACC TTTCTGTTCTGCTCCAGTTGTCTGATTTTATCCCCGCACCCGTACTTCCCTGCTTTAAACACAGTAGTT TTGTAATCAGTCTTAATGTCTGATAGGATAGATATCTTCTGATTCTTCTTTTCCTCTTCCTGGGAACCGT TCTTTAAACTTTTACTAGGTATTTTTGGTGCTTTATCTTTTCATTTAAATCCTGAAGTCAGTTTGTGACAT TTAATTACAATCTTCCTTTGAGACTTTGATAGAAAATAAGTTGAATATATAGTTTATTTTGAGAAAATTAC CACCTTTAGAATCCATGGTCTTCTCATCCTTGAATAGGCACGTCTCTGCTTCCTCAATTCTTCTTTCAG GCCCTTCGGTAAAGTCCATAATTTGTCTGCACATCTAGTCTTCCCTTATACTCCAAAGTCTCTCATGGG AAGGAGCTACACATTTTTTGCCTTTGTTCTCTCTCACCAAAGTCTGTCACAGTTATCTGCCCAGTAGGA GCCCTCAAGAAACACATTTAGACCTCTGTCTTTGGGTTTTATATCTAAATACAGAATCCAATTGTCAGA GGCATATGAACCAGAGCAACTCCATCTTAAATAGGAGCTGGGTAAATGAGGCTGAAACCTACAGAGC TGCATTCCCAGACGGTTAAGACATTCTAAGTCACAGGATGAGATAGGCGGTCAGCACAAAATACAGGT CATAAAGACCTTGCTGATAAAACAGGTTGCAGTGAAGGAGCCGGCCAAAACCCACTGAAACCAAAAT GGCCACGAGAGTGACCTGTGGTCGTCCTCATTTTTACACTCCCACCCGCGCCATGACAGTTTACAAAT GCCATGGCAACGTCAGAAAGTTACCTTAGATGATCTAAAAAGGGGAGGCATGAATAATCCACCCCTTG TTTGCCATATCGTCAAGAATAACCATAAAAATGGGCAACCAGCAGCCCTCAGGGCTGCTCTGTCTATG GAGTAGCCGTTCTTTTATTCCTTTACTTTCTTAATAAACTTGCTTTCTCTTTGCACTGTGAACTCGCCCT GAATTCTTTCTTGCCCAAGATCCAAGAACCCTCTCTTGGGGTTTGGATCAGGACGCCTTTCCCGTAAC ACAATGAATAAATATAGTTGGAACTCTTGGTGTTGTGGGTGTTGGAGAGGAGATTTAGCTGAGACATC AAGAAACAGTAGATGTCATATTTCAAAGTGAGACAATAGACCATGAAACATCAAAAACAATTTGGCTGA GAATTGTTAGATGATTCTTCAGTATTTTGCCTCTACTGATGCTACAAAAGGTTTAAGGGAAGGGAATTT TGTAGAAAATGCTAAGTGTGAGAATTTTTAAGGAGTTGCTGATATGCTGTGAGTCTCTCTCACTGTCAT CTTGCCACAGGGGGTAAGGGTGGGGTGTGCCTGTCTCTAGTCTCAGGTCTTAGGAATGAGGTCTTAG TGGGTCCACCAAGAGAGCGTGGTGCATGTCCTCCATGGTTGGAGGCTACAGGACACCGGTTTCTGAC TCTTGGGGACTCTAATATCTAGTGAGGAACAGGACGAACCGTTAGGATCTGCCTGCATCCCAGAGTG TGTTGGGGTGAAGGAGGCACAAATGTGTCCGGTGAACCACAGGTAAGCCATGGAGCTCAAGAGAACT GTGTCCGTCATCCTAGACTTTGTACAAGAGAGACACTTGAGGTGGGAGCAGACATCATCTCAGAGAA ACCAAAGACAACACCATGTTCCCGGGGATGGGAGGAGAAATTGATGAGCTAGTGGAAAGTGCATCCC CAGTGTCCAAGGATCTGCAGATGAGGAACCCCTGAAGACCCCCCGGAAGCAGCACATGAGATAAGG CGTTTGTTTCAAGCACCTGATGGGCCCAGAGAATGTGGAGCCAACCTGGGAAACATCAGTCCAGTAT AGGAAGAGCTTCCGTTCCCCATTCCCAGTTCCCCTTACTCCCCTCTACCCTGCCCTTGCCCCAACAAC TGTTCCTACCCCTGGAGCAGCAGGCCAGGAGAGGGAGAGGGTTCAATTTATTAATTATGGCAAATTG CAAACATATATTAAAGTAGAGATAATAGTATGATGAACCCCCATCAATGAGTTTCAGCAAGTATCAACT TATGGCCAATCTTGTTTTATCTATACTTACTACCTCTAATTATCCTTCCTGAATTATTTTGAAGACAATTC CACAGATTATACATAATATTTCAGTCTATACCTTGAAGAGATACTGATACATGTAAACGTTTACATCCAT ATGTGTGTCATATACGTAGAGCTACACACTCAAAGATCCACCCCATGCTAAAAATGTCCACTAATTCCT TTATGGCATCAAATGAAAACATCTAATTTTAAGTCAAGTTCAGAATTTTGATTGTCATATGGGACTGGA CATGTCAACTCCTAAATGGCAACTATGCCTAAATGGCAACTATGTTTTGTGACTTTAGAAACCATAGGA TTAACTGACTTTTCTTTCCAAAAGAGAAGGAACCAATCCCCACAGAATGGGAGACAAAGACAAAGACA CCTAGTGCTGTGGTTGGCCCCGTGAGTGCTGTGTGGCTCCTTAGCTCCCCGACCCATTGCAGGGCC AGATGCACACACATGTATCCATGTACCTTCCCCATCTGTGCACGACCACATATGTCAGGGCATGATGC TATGCCACACGTGTGTTGGGATCACCTGACTGTGTCTCCATTTCCAAAGTTGGGGGGGGTGCCTGTT CCTCCCTCTGTCCTAGTTCCACAGTGTTCAAGTCTCTGGAAATGACTGCAGCCCAGTGGGGGGCTTG TGGAATGTCCTCTTGGCAGGGGGTCCTGGTCCCAGTTTGCCAGGCACACTCCCTGGCCACAACTCTT GTCCTGGCATCATTATTCACAGCACCTCCACTCACCTGTCTGAAAAGTGTCCTGGCTTTGACAGCCTC TCACAGGGCTGCCTCTCTGTCCCGCTGTGCCCTGGGCATGAGCTGCCGCGTGCCCTGCCCTCCATC GGGCAGAGCCCAGCCCAAGCCCCACCAGAACGCGAGAGTCCCGGAGCCCTCACCAGCCAGTGTTGT CTTAGCCTCCCCTGAGACAAGGAAATGGGAATGAAAGAGTCCACTGGGGATAATTCTGGTGACAGGG GAGTTTGGGAAGGGATCAAATACATGTCTGGGGATTGATGATAGCCAGGGACTCATGGAGACCAGCA ATCGGATGGGTGCCGGGAAGGACAAAGGCCGAGGCCTGGCGATGTGTGGGGTCAAAGGCATCCAG GCGGCCGAGTGGACCCGTGGTCCTGGCTAGGGGGCACTGAGCACCCGGGAAGAGCTCATCCAGGG CCCCAGGGAGCCAGGCCAGGGCAGGAAGGGAGAGGTCACCATGAGACAAGCCACTGGGATCGTTC AATGTGTCCTTCCCTTCCCTCTTCCCTCACACCAGAAAGCACAGGCTGAAAAGACCCTGCTGCCAGG CGCCCCTTGTGTGTGAGGGTCCTGGGCCATCATAACCAGCACCGCAAGCTGGGTGGTTTAAACAACA GATGTTTATTCTTGTTTATTCTCTCTCAGCTCTGGAGGTCAGAAGTCCAAGGTCAAGGTGTCACAGGG CCTCCTCTCTCTGAGGGCTCCAGGGAAGTGTCCTGCCTGCCTCTTCCGGCTCCTGGGGGTGCTGAA GTCCCCGGCCTCCTTGGCTTGCAGGCATATCACTCGGTCTCTGCCCCAGTTGCCATCGCCATGTGGC CTCCCTGCCCCGTCTGTGCTCACATCTGCACCCCTGTGTGTGTCTCAGTGTTTCTTCTCTTCTTATAAT GACACTAGTCATATTGGATTTAGGGCCCACCCTACTCCATCTAGACCAGGCCAGATGGCGAGCGGAT CCATCTAGACCGAGGGTGTCCAGTCTTCTGGCTTCCCTGGGTCACACCAGAAGAAGAAGAATTGTCT TGGGCCACACGTGAAATACACTAACACTAAAGATAGTTGATGAGCCAAAAAAAAAAATCACAAAAAAAA TCTCATAACATTTTAAGAAAGTTTACGGATTTGTGTAGGGCCGTCTTCAAAGCCATCCTGGGTTGCATG TGGCAGACAGACCTCACATTGAACAAGCTTGATCTAGACCTTATCTTAGTTTAATTACAACCGCAAAAA CTTTATTTCCAAATAAGGTTACATTCCGAGGTTTCAGGAGGGACGTGAATTTTGGGGAGGACACCCTT CAACTCAATGTACTTGGGAATCACTTGGTGATAAAGGCTTGACTCACAGGTATGAGCCAAGTCCGAAG GCCGAGAGTGGCTGCGGCTTCCCAGTCTGGTGGTGGGAGGACCTGGCTCCCCATGGGGTCCCCTGT GCTGTCTGGGGCTGTTGAACTTGGCTGCCTGCACCAATGCATTCAGGGACTGTCTCTGAGAACTGCA TGCCATCAGCCCAGGTCTGGTACAAGAGGGGCTCCCAGCTGTCGGCCCAGGACTGGCTCAGTGCAG GACTGCAGACTGGAGTGCATCACACGGAAGGTGAGGTGACACGGTGCCTCCGGTGCCTGGGTGAGG GCCTTGCACCCTCCTCTCAGGCTCTGCTTCCTGGTAGAGCAGGTCTGGCTGCTGGGGAAGGGCCCA TCTGGGCCCAGCAGGTGGGGCTGCGGGTGAGGTGCTCCTGTCCACCGCGGTCAGCTGCATTATTTC TGGCCGTCAGTCCATGCCTTTCTGGGAACCTTTCCTGTCCCTTGCACGTGGGGCTGATGCAACGGTC CCAAACCCACAGACGCTCTGGGTTTAGAGGGCTCTGTGGCTGCTTCTCAAGACGAGTGGCTGAAACA GCACCAAGTGCTCCTTGAGCTGCACAAGGGAACCCTGGAGCCCATGCCCCCTTCCGAGCCCACTGG GCTGCTCTGCGTTCTGACCTTCCTGTTTTACACGTGGTGGTGGCATTTGCTCTGCTCCTGGTCCCCAC CCCCATTTGTCCTCAGTGGACTATGTGTCAAGACAGAAGCATCCTTGAATGGGTCACTGCGTTTGCCT CCCCAAAGCACCTACATTCAGGTCGTGATGACTCAGGAAGGGTCTTCGAGCCTCTCTCAGGGAGGCA GGAGGAAGTTCTGTGCAGAGTGAGGGGCGGCACCCATGGCAGCTGCAGACCGAGAGACAGCTGGG CAGCAGGGTGCTCCTGGGAGGTGTGCGGCCCTGGGCCACCCACGGGTGCTGATTAGCACAACTTCC TACTTCCTTCCTCCCTCGGACTGTCTGCTGCTTGAGTAATCGAGTGACAATACCAGAAAACTGAGCGG TGATGCAGACCCTATAACCTGCATGTGCCACATCAACTCTGAGTTTTCTGTGGGGCAGTGAGTTAGTG CAAAGTAGGCAGTGTTATGGCCTTACCTGCCCCTCTGCACTTTGGGAGGCTGAGGCAGGCAAATCAC TTGAGGTTAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCTCAGCTCTACTAAAAATACAAAA ATTAGCCGGGCATGGTGGCGTGCACCTGTAATCCTAGCACTTTGGGAGGCCAAGAGAGATGGGTTG CCTGAAGTCAGGAGTTCGAGACCAGCTTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAA AATTAGCCAAGTGTGGTGGCATGCACCTGTAATCCCAGCTACTCGGGAGGCTGAGGGAGGAGGATC GTTTAAGCCTGGGAGGTGTAGGCTGTAGTGAGCTGAGGTTGAACCACTGTACTCCAGCCTGGGTGAT GGGAGAGAAACCCTGTCTCAAAATAAAATAAAATAAAATAAAATAACTTAACATAACATAAAATAAAATA AGATAAGATAAAATAAAATGAAATGTGCCCCTCTGACCTTCACCCCTCCCATCTGGTGAACTTGTACTC ATCCCTCAAGACCTAGTCAGATTTCACCCCACTCTGCCAGCTCTAATCATACTGCACCACAATTCATG CTTGAAGGCTTGTTCCTCCTCACCAGCCTGTGAGCTCTGAGGGCACGGGCTCTGCCTGATGTTCTGG GGCTCCCACATCTGCAGGCCGAGTGCCTGACCTGGGACCCATGCTCAGCTTGTGCTTGTTATATGCA CGTGACAATGACCACGCATCCGGGAGGTGAAAGGTTGCAGCCCCGGCATGGGGGAGCAGACTGTCT TCTTCTTGGATCCATCCAGGAGCCCCTGCACAAGGCCCAGGGTGGGAGGGATTTGCAGGGCTCCTG GGACGGCCCACACCGCCACACCGCACACAGCTGACGAGGCTGCTGCAGGGATGCGGGATGCGGCT CCCAAGGAGGAAAGGGAGAGGCTCCTTTAGAGCCAGTCCTGCTCACCCCCAGGTCTTGGGTTGCCC AGGGGAGCCACCTGTCTGGGTTGGGCCCGATGCCCACTACCGCACGGCCTACAGCTCTGACAACAG CTCTGCTTTTTCTCTGGAACTGGAACTCCCCCAGGGGAATAGGGTGGCTGGGGAAGGCTGTGGGAAT GCAGGCTCTGGAAGGCAGGTTGGGCTCTCCCATGCCATCCTCTGTTCCTGTACAGAGCCCAGGGCT CCTGGGGAGGGGGCTCCTAGAACAGAGAGAGGGGACACACACGGGCCTCCCCATTGACCTCTCCAG AGGTGCAGCCCAAATCCCCCTTCTCTGAGAAGCCTGTGCGTCTCCACCTGACAGGACCCAGCTCTGG CCCCCTTGCCACAGCCTTTGCTCCTACCAGAGCCGGGACCAGGAATCCTCTCCTCTGCCATCATGGT TGATCCAGCACACCCGGCAGGTGGTCAGTGTGTGTCCCAGGCACCTGGCTGGGCTGTGCAGCACCA CTTCATGCTGTCCTGTGGGGTGAGCACCCTGTGACATGTAGGGAAACTGAGGTACAGATGGGACGTG ACTTGCCCAAGGTCAGGCAGCACAGAACCTAGTCTTGGCTTCACCATCTTAACTCTGACATGATCCCG TCTCCCTTTGCGGGGGGACTCTTCAAGGACAAGAATGTGTCACCTTTGTTATTCCTTCCCCAGCCCTA AACCCTGGGTGTAGGCAGTAGGAGCTCAGTCAACATTCACTAGTTAAACGGAAGCAAACCCCAGCAG GGTTCTCGGTGCTGGCCGCAGAGAAGAATCACAGGCGACAGAACCACCTGTCAGGGCGGGGCCCA GG CATTG ATATTCTCCCCACTCCAGG CAATTTTG AGTG CAG CAG GTGG AG ACCACAG CACCTG CAG A AGCTCCTTGCGGGGGTCCTTCCCCAGCAGGTCATGAGGGCCGGAGGGGCAGCAGTCTGTTCCTGGG GTGTTTGCACCCTCCTGGAACTCAGCCCTGCACCACGACCTGGCCTGGCCTCTGGGTACTAGGAGCA GTGGGAAGGCAGGGAGATCTCACCAGATCCCCCAGGGAGATCTCACCAGATCCCCCAGGGATTTCC CCTACAATGACCTTTTTAATAACATGAAAATCCTGCTGTCCATTGATGAACCTGGGTCCGCTGTCTCCC AG AAAAAGTG AGTG CCCCG AAAACAAG G CAGG CAACG CTCTTTCCTTCTTCCTCCTTCG CTGG CCTC AGCGGGAGTGGCACATCAGAGGCCAGGGGCCCTCAAATCCATCCACAAAGGGCGTGCTGAGCGCAA GGGCGGGGCCTGGGAGTCACAGGGCAGGACACATCCTGGTGCTCAGCACGTACGCCATCGAGCTC CCATGGGTCACTCGGGGAAGGGCAGTTTGTGGATGCACCCTTGGAGGCTCAAGGCCGCCCTGCACA CACCACCGGGGACCCCAGCTGCAGGCTAAATCCAAGCAGCCAGGCGCTCCCATTTCTCGGGGTGAT AAACCAAGTCCACCCTTCAGACAGAGGCTCCTGGAAGGGCCAGTGGGGGTCATCAAAATGCCTCCCT GGAAAAGAAACACGGAGAACACAAAATCTAGGGGCCCGTTTTCCTGGAATGAGATTAGTCTCTTGCC CACTACCAGCCAGTCCACTGCAGGAGGTTGGGGGGTTGGGGGATGCTGCGGATGCCGAGGGCGGA GGCTTAGGGTCCTGGGAGTGGGTTAAATACCAGCTATACCAGGGACACTGGAGTAACCTCTGATAGG CACCGGAAATGTTGAGTCTTAGTTTCTTCCTCTGGAAAATGGATAGAATCATCGCTGTCTCATCTGATA TGTTGTTATAAAACAATTCCTGAGATGTAGTCTTTAGTTTTTAAAAGAGCACGCTGGGCAAAGGTGGAG AGTCAGTTCCTGTTTGTGTTAAAAATGTGGCCGAGTGCAGTGGCTCATGCCTGTAATCCTGACACTTT GGGAGGCCAAGGAAGGCAGATCACTTGAGTCCAGGAGTCTGAGACCAGCCTGGCCAACATGGCGAC ACCTGGTCTCTAAAAAAATACAAAATATTAGCTGGACACATGCCTGTGGCCCAGCTACTTAGGAGGCT GAGGTGGGAGGATCACTTGAGCCCCGCGAGGGCGGGGGTCTCAGTGAGTTGAGATTGTGCCACTGC ACTCCAGCCTGGGTAACAGAGCAAGACCCTGTCTCCAAAAAAAAAAAACAAAAAAAAAACAAAAAAAA AACAAAACAAACAAACAAACAAAAACAAACCAAAAAAACGTGCTTAGATCCAGAGAGAAAAATTCTGAA AGGATACCTAAAAAAGTGTTCACAATAGATCAATAGATGCCTCTGGATAGGAAAGGCTCTTTTGTATTA TTTGATTTTAAAAATCAATTATGTGTACCATGTTTTTACTAACATATTTTCAATCAATTCATTTCATCCAC TTATTAAAATGTAGTTACTAATTTTTAGGGGAGAAAGCAAAAAGGAAAGACAAGGAATCTCTGTGTT TCAGGAGTTGTGAGAGAGCCGCAGGGTCCTGACTCACTCAGAGCTGCCTGTCTCCGAGGCCGATC

TGGGATGAAGCAGCCTGGGGCTCTCTTGTCATGGGACCAGGGGTGTTCTGAGGGCTTCTGGCTGG GAGGCTGAGATGGAACGGACACCACACCCTGGTCCTGCCACCCCACATGGCTCCTGCACACTACA CCAGGCCAGGCTAGGAGGGCAATTGACACACATCCGCTCCCCAGTAGAGGACCCGGAACCAGAA CTGGAATCCGCCCTTACCGCTTGCTGCCAAAACAGTGGGGGCTGAACTGACCTCTCCCCTTTGGGA GAGAAAAACTGTCTGGGAGCTTGACAAAGGCATGCAGGAGAGAACAGGAGCAGCCACAGCCAGGA GGGAGAGCCTTCCCCAAGCAAACAATCCAGAGCAGCTGTGCAAACAACGGTGCATAAATGAGGCCTC CTGGACCATGAAGCGAGTCCTGAGCTGCGTCCCGGAGCCCACGGTGGTCATGGCTGCCAGAGCGCT CTGCATGCTGGGGCTGGTCCTGGCCTTGCTGTCCTCCAGCTCTGCTGAGGAGTACGTGGGCCTGTG TGAGTACTGCCCTGACTGCCCCGGTGGCAGGGTGGGCGTGAAGGGAAGGGATCCAGGATAAGGGG GGATTCTGCATTCATTTAATAATGGCCACCTGTCACATATACACTTTTTCCTGCGCTAGCCCTTTGAAG TGGGTCTTTATTGTCCCCATTTCACAGACAAGGAAACCGAGGCTCAGAGAAAGTTAACAACTTATCCA AGGCAGCCCTGCCCAGTCTGTGTTGAAATCAGGGTTTGAGCCTGAGCCCATCCCCTATGACCCCATA GCCATCTTTGCTGGAGATTTCTAAATTACAATATAGGTCTTTATGCATTGTTCCACATTTACAAAGAAAA AGGAAAGATGCAGGAGAAAAACCCTGACTTCAGAACACTGTCAATACCGGCAGGCACAAGGTTCATT TAGCCATTGCATAGCAACCCTGCCATGGGGTGTGGCTGCTCCATTAACCCAAGTTTGAAGGAATGAG GGCATGGCTTTTATCTGGGTGTCTTCTGAGCAGGGTCAAAGGCAGTGGTTCCCGAACTTGCAGCCCA TTAGAATCACCTGGAGAGCTTTAAAAATCCTAATGCTTGGGGCACACCAGTTACATCAGGGCATCTCC AGGCAAGATCCAGGCCTCAGCTGTTTTGTTTTGAGATAGCCTTGCTTTGTCACTCACTGCTGGAGTGC AGTGGCACAATCTCAGCTCACTGCAACCTCCGCCTCCTGGGTTCAAGCAATTCTTGTGCCTCGGCTTC AAGTAGCTGGGATTACAGGCATGCACCACCATGCCCAGCTAATTTTTTGGATTTTTAGTAGAGATGGA GTTTCGCTATGTTGGCCAAGCTGGTCTCAAACTCCTGGCCTCAAGTGATCCTCCTGCCTTGGCCTCCC AAAGTGCTGGAATTACAGGTGTAAGCCACCATGCCCAGCCAACGTCAGTCATTTTTAAAGCTCTGCAG CTGATTCCAGTGTGAGCGAAGTTTGGATGCCAGGAGGATAAGCAATTACGGACTGGGAGCAAGAGAA GGGAATGTAAGACACTGCACGTGATTGCCATTTTCCTAAGGAAATACTCAGTTCGTTAATGAAACGCA GTGAACTTCTGCTGCACATACAGACATAGAGGCTTGCCTGAAACATGAAAATATTGGGGACTGAAGGA TGTCCCGGGAGGGTGGGACATGCTCAACAATTCAGGAAGGGGAGATGCAGAAAAAAGTGAAAAGCA GGCAGCATGCGTTGCAATGATCTCTATGGCGTGTGCCTCTCCTGTCACGGTTTTCATTTAAAACAAAG GGGCAAGGTTTTGTTGGTCAAACAATGAAGGGTAACTTTGTTTCTGGGTTCAAGGGACCCCAGATTCC CCAGGGGTTCCTGCCAGCTGGAAGGTACCCAGGTCCGTATGTGACTTCCCGAGAAGGTGATAAGAG CGTGCCAAGGAGAAAGACACTTAGGCAAATGGCCAGAGTCCCCGAGCTGAGCATTTAACAGACTGCC TCTCTTTAAATATTCACAGGGAAAGTGCATCTTCCTAAGGGCGAGGGTTTCAGCAGTGGTTGAACTCG GCGGGGTGGGGCGGAGCGGGAGGATGCAAACTTGCAAAGTGAAGCAAACACACTCACCGCAGCCC AGCAAGGGCTCTGGCAGCTGACAGGGCTTTGTCTGGGACAGCTGCAAACCAGTGTGCCGTGCCAGC CAAGGACAGGGTGGACTGCGGCTACCCCCATGTCACCCCCAAGGAGTGCAACAACCGGGGCTGCTG CTTTGACTCCAGGATCCCTGGAGTGCCTTGGTGTTTCAAGCCCCTGCAGGAAGCAGGTAAGGCCCCA GTGGCATCGTGGTCTGGGCCCAGCCCCATAAGGCAGGGGGTCTCAGGGCCTCCCTGTCCTTTCTGG GCTGGAGATGGAGGCACAAGGACCCCAGGAAGCCACACACACACACCTGTTCCAAGGCCTCAGAGC AGAGGCTTCACACTTAGGGCAGCCATGGCCAGGGGCTGTCCTCTTCTGTCCCCTTTATGTAAAACATA AAAGCAATTGTTTCAAAAAGGTGTTCAAAATGATGGCATCGCATAGAGGGAACTGATTTAGTAACTATT CTTGAGAGAAGTGGAAACGCATAGGTGTGGAAAGCCGGGCCGACTTTTGGGCTGTTTTTGCAAATCG GCCCCCCAGAGTCTTGTCATTTGTGGCATCCCCTACACAGACGGCAGGCGGTCCCAGCCCTAGACGT CAGGCCTCGGTGCCACACCCCACCTCCCCCACTCTGCCCCCCACAAGGGTCATCTCCTCTCCCTCTC TCTGCCGTGGTGGAGGGCAGGTGCAGGGCAACCACCCTGGGGGTTCCCTCCCCAGGGGCGGAGAG CCTGCGTGCTGTGCGGGTAACAGATGGCCCTGCACACGGGTTTGCCACCCTGGCTCCACCAGGCTT AGCTGCCCCACATCGTGGGTGGGGCGATTGGCTATAAGCCATCTGCCATGTCCAAGTGCCAGCTCAG CCCCCACGAAGGCCGCACCTGCGTGAGGTACCTTCCTGGAACCAGCATCCAGAGGGGCCTCTCTTG CCCTTTGTCCTAGGGTGAAATGCGGGAGGCTGAGTCCTGCTGGCCCCGGCTCCCTGATCAATGATG GGCCCCTGCCCAGGGCCTCCCTTCACCCTCCCCAGCAAGTCCAGGGTAGGGGTGGGGGTGGGGGT CCAGAGAAGGCCAGGAGAGAGAGGGGTCTGGCTACTGTCCACTGCCGGTCCTGTTCCTTCAGCTCC ACTGGAACTACACTCTCCTCTGAGTGCCAGCCATGGCCCTGCCAAGGCCCATCTCGCTTGTTATCTG CCTGATCCCTGGGTCCCACTATCTTGCTTAGCAACCCGAGGTGGGAATCTTGGCTATTCCCCCATGT GGTGGGGACTCAACACTCCCCGGTGACTCTGGGGAGGAGGCAGCACTAGGTGCTGGCCTTGGAGC CTGCCCTGACCTTGGGAAGCTGGGCAGCGTGGGTGGAGAGAGACTGCTCACACAAGCCTTTGCTCT GTTTGCAGAATGCACCTTCTGAGGCACCTCCAGCTGCCCCCGGCCGGGGGATGCGAGGCTCGGAGC ACCCTTGCCCGGCTGTGATTGCTGCCAGGCACTGTTCATCTCAGCTTTTCTGTCCCTTTGCTCCCGGC AAGCGCTTCTGCTGAAAGTTCATATCTGGAGCCTGATGTCTTAACGAATAAAGGTCCCATGCTCCACC CGAGGACAGTTCTTCGTGCCTGA

SEQ ID NO: 2 TFF3 transcript nucleotide sequence

>gi|48928026|ref|NM_003226.2| Homo sapiens trefoil factor 3 (intestinal) (TFF3), mRNA

GCCAAAACAGUGGGGGCUGAACUGACCUCUCCCCUUUGGGAGAGAAAAACUGUCUGGGAGCUUGA CAAAGGCAUGCAGGAGAGAACAGGAGCAGCCACAGCCAGGAGGGAGAGCCUUCCCCAAGCAAACA AUCCAGAGCAGCUGUGCAAACAACGGUGCAUAAAUGAGGCCUCCUGGACCAUGAAGCGAGUCCUG AGCUGCGUCCCGGAGCCCACGGUGGUCAUGGCUGCCAGAGCGCUCUGCAUGCUGGGGCUGGUCC UGGCCUUGCUGUCCUCCAGCUCUGCUGAGGAGUACGUGGGCCUGUCUGCAAACCAGUGUGCCGU GCCAGCCAAGGACAGGGUGGACUGCGGCUACCCCCAUGUCACCCCCAAGGAGUGCAACAACCGGG GCUGCUGCUUUGACUCCAGGAUCCCUGGAGUGCCUUGGUGUUUCAAGCCCCUGCAGGAAGCAGA AUGCACCUUCUGAGGCACCUCCAGCUGCCCCCGGCCGGGGGAUGCGAGGCUCGGAGCACCCUUG CCCGGCUGUGAUUGCUGCCAGGCACUGUUCAUCUCAGCUUUUCUGUCCCUUUGCUCCCGGCAAG CGCUUCUGCUGAAAGUUCAUAUCUGGAGCCUGAUGUCUUAACGAAUAAAGGUCCCAUGCUCCACC CGAGGACAGUUCUUCGUGCCUGAAAAAAAAAAAAAAAAAA

SEQ ID NO: 3 TFF3 amino acid sequence

>gi|585328|sp|Q07654.1 |TFF3_HUMAN RecName: Full=Trefoil factor 3; AltName:

Full=lntestinal trefoil factor; Short=hlTF; AltName: Full=Polypeptide P1 .B; Short=hP1 .B; Flags: Precursor

MAARALCMLGLVLALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCCFDSRIPGVPWC FKPLQEAECTF

Claims

1 . A method for assisting in diagnosing and/or for diagnosing prostate cancer in an individual comprising the steps of

i) determining the methylation status of a TFF3 gene (SEQ ID NO:1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID

NO:1 , or part thereof in a sample from said individual, and/or ii) determining the transcriptional expression level of said TFF3 gene, or nucleotide sequence having at least 90% sequence identity with SEQ ID NO:2, or part thereof in said sample and/or

The method according to claim 1 , wherein a decreased methylation status indicative of the presence of prostate cancer.

The method according to claim 1 , wherein an increased transcriptional and/or translational expression level in the sample is indicative of the presence of prostate cancer.

A method for assisting in prognosing and/or for prognosing the disease progression of prostate cancer in an individual having contracted prostate cancer comprising the steps of

iii) determining the translational expression level of said TFF3 gene, or amino acid sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof in said sample wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of presence or absence of prostate cancer.

The method according to claim 4, wherein an increased methylation status indicative of the disease progression of prostate cancer.

The method according to claim 4, wherein a decreased transcriptional and/or translational expression level in the sample is indicative of the disease progression of prostate cancer.

A method for assisting in predicting and/or for predicting the outcome of prostate cancer in an individual having contracted prostate cancer comprising the steps of

i) determining the methylation status of a TFF3 gene (SEQ ID NO:1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:1 , or part thereof in a sample from said individual, and/or

ii) determining the transcriptional expression level of said TFF3 gene, or nucleotide sequence having at least 90% sequence identity with SEQ ID NO:2, or part thereof in said sample and/or

The method according to claim 7, wherein a decreased methylation status is indicative of the disease progression of prostate cancer.

The method according to claim 7, wherein an increased transcriptional and/or translational expression level in the sample is indicative of the disease progression of prostate cancer. A method for assisting in predicting and/or for predicting the recurrence risk of prostate cancer in an individual having contracted prostate cancer comprising the steps of

The method according to claim 10, wherein a decreased methylation status indicative of the recurrence risk of prostate cancer.

The method according to claim 10, wherein an increased transcriptional and/or translational expression level in the sample is indicative of the recurrence risk of prostate cancer.

The method of any of claims 10 to 12, wherein said risk of recurrence is determined following radical prostatectomy, radiation therapy, cryotherapy or brachytherapy.

The method of any of claims 10 to 12, wherein recurrence is recurrence-free survival and/or overall survival.

A method for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression in an individual having contracted prostate cancer, comprising the steps of i) determining the methylation status of a TFF3 gene (SEQ ID NO:1 ), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:1 , or part thereof in a sample from said individual, and/or

The method according to claim 15, wherein a decreased methylation status is indicative of a progression of said prostate cancer.

The method according to claim 15, wherein an increased transcriptional and/or translational expression level in the sample is indicative a progression of said prostate cancer.

A method for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent/indolent to an aggressive prostate cancer in an individual having contracted prostate cancer comprising the steps of

19. The method according to claim 18, wherein a silent/indolent prostate cancer is a slow-growing and slow-progressing organ-confined prostate cancer with no or only minor clinical symptoms.

20. The method according to claim 18, wherein an aggressive prostate cancer is which has progressed or will progress relatively fast (i.e. within the remaining life expectancy of a given patient) to non-organ-confined prostate cancer. 21 . The method according to claim 18, wherein an aggressive prostatate cancer is a treatment-requiring prostate cancer.

22. The method according to claim 18, wherein a decreased methylation status is indicative of a progression of said prostate cancer.

23. The method according to claim 18, wherein an increased transcriptional and/or translational expression level in the sample is indicative a progression of said prostate cancer.

A method for assisting in determining and/or determining the treatment regime of an individual having contracted prostate cancer comprising the steps of

wherein the methylation status of i) and/or the transcriptional level of ii) and/or the translational expression level of iii) is indicative of the treatment regime to be offered to the individual having contracted prostate cancer. The method according to claim 24, wherein a decreased methylation status indicative of the presence and/or the progression of said prostate cancer.

The method according to claim 24, wherein an increased transcriptional and/or translational expression level in the sample is indicative of the presence and/or the progression of said prostate cancer.

The method of any of the preceding claims, wherein the method optionally comprises a step of comparing the methylation status of said TFF3 gene determined in the sample to the methylation status of a control sample, wherein the methylation status of said sample can be determined as increased or decreased.

The method according to any of the preceding claims, wherein the methylation status of said TFF3 gene in a sample being below 50 %, is indicative of a decreased methylation level, wherein said sample is a tissue sample.

The method according to any of the preceding claims, wherein the methylation status of said TFF3 gene in a sample being below 50 % is indicative of a decreased methylation level, wherein said sample is a urine sample.

30. The method of claim 27, wherein the methylation status of said TFF3 gene in a sample is decreased in comparison with a control sample.

The method of claim 30, wherein said control sample is a fully methylated nucleotide sequence having at least 90 % sequence identity with SEQ ID ON: 1 or part thereof.

The method according to any of the preceding claims, wherein the method optionally comprises a step of comparing the transcriptional and/or translational expression level of the TFF3 gene determined in the sample to the expression level of a control sample, wherein the expression level in the sample can be determined as increased or decreased. The method according to any of the preceding claims, wherein the

transcriptional expression level of the TFF3 gene is determined by PCR.

The method according to any of the preceding claims, wherein the

transcriptional expression level of the TFF3 gene in a sample is increased 50% in comparison with the transcriptional level of a control sample.

The method according to any of the preceding claims, wherein the translational expression level of the TFF3 gene is determined by immunohistochemical analysis.

The method according to any of the preceeding claims, wherein the

translational expression level of the TFF3 gene is increased 50% in comparison with the translational expression level of a control sample.

The method according to any of the preceding claims, wherein the sample is selected from tissue sample, blood, plasma, serum, semen, or urine.

The method according to any of the preceding claims, wherein the sample is a biopsy of the prostate gland or resected prostate tissue following radical prostatectomy.

The methods according to any of the preceding claims, wherein said individual has a normal prostate specific antigen (PSA) level.

The method of claim 39, wherein a normal PSA level is PSA less than 4 ng per ml serum.

The method according to any of the proceeding claims wherein said individual has a PSA level higher than a normal PSA level.

The method claim 41 , wherein the PSA level higher than a normal PSA level is a PSA level above 4 ng per ml serum. 43. A pharmaceutical composition for the treatment of prostate cancer comprising at least one transcription inhibitor capable of decreasing the transcript levels of a TFF3 gene transcript (SEQ ID NO: 2), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:2, or part thereof, and/or

at least one translational inhibitor capable of decreasing the translational expression level of the TFF3 gene (SEQ ID NO: 3), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:3, or part thereof.

The method according to claim 43, wherein said transcription inhibitor is a functional RNA molecule displaying complementary sequence to a TFF3 gene transcript (SEQ ID NO: 2), or nucleotide sequence having at least 90 % sequence identity with SEQ ID NO:2, or part thereof.

The functional RNA molecule according to claim 44, wherein said at least one functional RNA molecule is a siRNA, microRNA, shRNA and/or antisense RNA

The method according to claim 43, wherein said translational inhibitor is an antibody directed against an epitope of the TFF3 protein or part thereof.

Assay comprising at least one detection member for a TFF3 gene,

transcriptional and/or translational product or part thereof for use in the methods of the present invention.

The assay according to claim 47, wherein said at least one detection member is an antibody directed against an epitope of the TFF3 protein or part thereof, oligonucleotides, primers and/or probes.

The assay according to claim 47, further comprising means for providing the level and/or means for providing information as to whether the level is above or below an increase of at least 30%.

50. Use of an antibody directed against an epitope of the TFF3 protein or part thereof in the detection of the translational expression level of a TFF3 gene, or part thereof i) for assisting in the diagnosis and/or for diagnosing of prostate cancer as defined in claim 1

ii) for assisting in the prognosis and/or for the prognosis of the disease progression of prostate cancer as defined in claim 4 iii) for assisting in the prediction and/or the prediction of the progression of prostate cancer as defined in claim 7

iv) for assisting in predicting and/or for predicting the recurrence risk of prostate cancer as defined in claim 10,

v) for assisting in monitoring and/or for monitoring the effect of treatment on prostate cancer progression as defined in claim 15 vi) for assisting in monitoring and/or for monitoring the progression of prostate cancer from a silent to an aggressive prostate cancer as defined in claim 18

vii) for assisting in determining and/or determining the treatment regime as defined in claim 24.

The use according to claim 50, wherein said antibody is monoclonal, polyclonal, or a mixture of at least two monoclonal antibodies.

The use according to claim 50, wherein said antibody is monoclonal.

53. The use according to claim 50, wherein said antibody is polyclonal.