WO2010132958A1 - Methods for predicting responsiveness to treatment - Google Patents

Abstract

The present invention relates to a method for predicting a response to a treatment regime, for determining an optimal treatment regime, or for predicting the likelihood or duration of survival of a subject suffering from or suspected of suffering from cancer. The method of the invention is reliant on the analysis of S100A4 expression and/or activity in a biological sample from the subject.

Description

METHODS FOR PREDICTING RESPONSIVENESS TO TREATMENT

FIELD OF THE INVENTION

The present invention relates to the prediction of whether or not a subject suffering from or suspected of suffering from cancer will have a beneficial response to treatment.

BACKGROUND OF THE INVENTION

Modern clinical research has focused on developing therapeutic regimens that effectively prolong survival in diseased subjects. Stratifying individual patients based on stage and phenotype of disease to guide treatment decisions is a basis of modern clinical oncology practice. This stratification has evolved through clinical observation of the natural history and therapeutic responsiveness of the disease and their relationship to morphological, and in more recent times molecular and genetic characteristics. Defining phenotypes of individual cancers has aided stratification of therapy so that optimal treatment is given without delay and minimizes unnecessary adverse side effects. Furthermore, it has guided and focused research to identify mechanisms amenable to the development of novel therapeutic strategies for phenotypes that are non-responsive to conventional treatments. Finally, patients can be better informed with respect to the likely outcome of their disease and their expected quality of life. Consequently, significant international effort is currently focused on characterizing phenotypic subgroups in particular cancers and understanding the molecular mechanisms that underpin their development. Progress has varied between different cancer types. Breast cancer has several defined phenotypes and specific therapeutic approaches which contribute significantly to the current > 90% 5-year survival rate. However, other cancers, especially pancreatic cancer and colorectal cancer are yet to be stratified into clinically relevant subgroups.

Pancreatic cancer is the fourth leading cause of cancer death in Western societies with an overall 5-year survival rate of less than 5%. Advances in neoadjuvant and adjuvant chemotherapeutic/anti-neoplastic regimens have resulted in some improvement in outcome, but pancreatectomy remains the single most effective treatment modality for pancreatic cancer, and offers the only potential for cure. Only 20% of patients present with localized, non-metastatic disease which is suitable for resection. Those who undergo resection and receive adjuvant therapy have a median survival of 12-22 months and a 5-year survival of 20-25%. Colorectal cancer is currently one of the three most frequent malignancies in Western industrial nations. Although the 5 -year survival rate for patients with early stage and local colorectal cancer approaches nearly 90%, survival is dramatically decreased by local recurrence and the development of distant metastases that primarily affect the liver, which are the predominant cause of colorectal cancer-related mortality. Adjuvant chemotherapy for colorectal cancer is associated with improved outcome, but only benefits a minority of individuals (<10%).

It will be clear to the skilled artisan from the foregoing that there is a need in the art for prognostic markers to aid in the prediction of clinical outcomes to available therapeutic regimens, for example, to improve the likelihood of a beneficial treatment outcome and/or survival rate or time and/or quality of life of a subject.

SUMMARY OF THE INVENTION

In work leading up to the present invention, the inventors sought to identify a molecular/biochemical marker that facilitates prediction of the likelihood that a subject suffering from or suspected of suffering from cancer will have a beneficial response to a treatment regime. The inventors have found that levels and/or activity of expression products of the S100A4 gene vary considerably in cancer patients and that the level of expression/activity is associated with the responsiveness of a subject to treatment, for example, surgical treatment, or treatment with a chemotherapeutic or endocrine agent.

As exemplified herein, the inventors have demonstrated that an aberrant level of S100A4 expression and/or activity is prognostic of a subject's response to a treatment regime.

As further exemplified herein, aberrant expression of S100A4 is predictive of a response of a subject suffering from cancer, for example, pancreatic cancer, to surgery, for example, pancreatectomy.

As further exemplified herein, aberrant expression of S100A4 is predictive of a response of a subject suffering from cancer, for example, pancreatic cancer, colorectal cancer, or ovarian cancer to a chemotherapeutic agent, for example, a nucleoside analog, such as gemcitabine, or a platinum based agent, such as oxaliplatin, or a semisynthetic analogue of the natural alkaloid camptothecin, such as irinotecan, or a taxane agent, such as, paclitaxel or docetaxel.

As further exemplified herein, aberrant expression of S100A4 is predictive of a response of a subject suffering from cancer, for example, breast cancer, to an endocrine agent, for example, an agent that inhibits the production of estrogen, lowers the level of estrogen, and/or blocks the estrogen receptor. Accordingly, the present invention provides a method for predicting a response to a treatment regime, for determining an optimal treatment regime, or for predicting the likelihood or duration of survival of a subject suffering from or suspected of suffering from cancer, the method comprising: performing an assay to detect expression and/or activity of a S100A4 gene product in a biological sample of the subject, using the amount of aberrant levels of S100A4 as a basis for predicting the subject's response to the treatment regime, for determining the optimal treatment regime, or for predicting the likelihood or duration of survival of the patient. In one embodiment of the invention, a subject having expression and/or activity of the S100A4 gene product above a predetermined level is unlikely to have a beneficial response to the treatment regime, or a subject having expression and/or activity of the S100A4 gene product at or below the predetermined level is likely to have a beneficial response to the treatment regime. In a further embodiment, the cancer is pancreatic cancer, colorectal cancer, or breast cancer.

In another embodiment of the invention, a subject having expression and/or activity of the S100A4 gene product at or above a predetermined level is likely to have a beneficial response to the treatment regime, or wherein a subject having expression and/or activity of the S100A4 gene product below the predetermined level is unlikely to have a beneficial response to the treatment regime.

In a further embodiment, the cancer is ovarian cancer.

In one embodiment of the invention, the S 100A4 gene product is mRNA. The mRNA may have, for example, at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

Expression of the S100A4 gene product in a biological sample may be determined using an electronic device or a machine.

Expression of the S100A4 gene product in a biological sample may be determined by contacting the biological sample with a nucleic acid probe under conditions to effect specific hybridization of the probe to the mRNA and detecting the amount of mRNA bound probe.

In another embodiment of the invention, the S100A4 gene product is a S100A4 protein. The S100A4 protein may comprise, for example, an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 3. Expression of the S100A4 gene product in a biological sample may be determined by contacting the biological sample with an antibody or antigen binding fragment thereof, under conditions to effect specific binding of the antibody to the protein to form a complex and detecting the amount of the complex.

In one embodiment, the method additionally comprises isolating or obtaining the biological sample from the subject. In another embodiment, the method of the invention is performed in vitro or ex vivo.

In performing the method of the invention, the biological sample may be obtained from the subject either before or after the subject has been diagnosed with cancer. In one embodiment of the invention, the biological sample is obtained from the pancreas, breast, ovary, colon, rectum or anus of the subject.

In another embodiment of the invention, the expression and/or activity of the S100A4 gene product is elevated relative to a reference sample from a normal or responsive subject.

In a further embodiment, the method comprises: (i) determining the level of expression and/or activity of the S100A4 gene product in a biological sample from the subject; and

(ii) comparing the level of expression and/or activity of the S100A4 gene product at (i) to the level of expression and/or activity of the S100A4 gene product in a reference sample from a normal or responsive subject, wherein an elevated level of expression and/or activity of the S100A4 gene product in the biological sample relative to the reference sample indicates that the subject is unlikely to have a beneficial response to the treatment regime.

In another embodiment of the invention, the expression and/or activity of the S100A4 gene product is reduced relative to a reference sample from a normal or responsive subject.

In a further embodiment, the method comprises:

(i) determining the level of expression and/or activity of the S100A4 gene product in a biological sample from the subject; and

(ii) comparing the level of expression and/or activity of the S100A4 gene product at (i) to the level of expression and/or activity of the S100A4 gene product in a reference sample from a normal or responsive subject, wherein a reduced level of expression and/or activity of the S100A4 gene product in the biological sample relative to the reference sample indicates that the subject is unlikely to have a beneficial response to the treatment regime. In one embodiment of the invention, the method further comprises: performing an assay to detect expression and/or activity of a S100A2 gene product in a biological sample of the subject, the method comprising: performing an assay to detect expression and/or activity of a S100A2 gene product in a biological sample of the subject, using the amount of aberrant levels S100A4 and S100A2 as a basis for predicting the subject's response to a treatment regime, for determining the optimal treatment regime, or for predicting the likelihood or duration of survival of the patient.

In one embodiment of the invention, a subject having expression and/or activity of the S100A2 and S100A4 gene products at or below predetermined levels is likely to have a beneficial response to the treatment regime.

Methods for detecting expression and/or activity of a protein and/or the level thereof are described herein and shall be taken to apply mutatis mutandis to this embodiment of the invention.

In another embodiment, the method of the invention additionally comprises the step of recommending a treatment regime for the cancer.

The present invention also provides a method of treating cancer in a subject, the method comprising: performing a method according to the invention to determine whether or not the subject is likely to have a beneficial response to a treatment regime; and treating the subject according to the treatment regime if they are likely to have a beneficial response to the treatment regime.

The present invention also provides a method of treatment comprising administering a therapeutic agent to a subject suffering from cancer who has been previously determined to be likely to have a beneficial response to treatment with the therapeutic agent by virtue of having aberrant levels of expression and/or activity of a

S100A4 gene product in a biological sample therefrom.

The present invention also provides use of a therapeutic agent to treat a subject suffering from cancer who has been previously determined to be likely to have a beneficial response to treatment with the therapeutic agent by virtue of having aberrant levels of expression and/or activity of a S100A4 gene product in a biological sample therefrom.

The present invention also provides use of a therapeutic agent in the manufacture of a medicament for the treatment of a subject suffering from cancer who has been previously determined to be likely to have a beneficial response to treatment with the therapeutic agent by virtue of having aberrant levels of expression and/or activity of a S100A4 gene product in a biological sample therefrom. In one embodiment of the invention, the treatment regime comprises administration of a therapeutic agent. The therapeutic agent may be a chemotherapeutic and/or anti-neoplastic agent.

In one embodiment of the invention, the chemotherapeutic and/or anti- neoplastic agent is a DNA-intercalating agent, for example, cisplatin, carboplatin, or oxaliplatin.

In another embodiment of the invention, the chemotherapeutic and/or antineoplastic agent is a topoisomerase 1 inhibitor, which prevents DNA from unwinding, for example, irinotecan. In another embodiemnt of the invnetion, the chemotherapeutic and/or antineoplastic agent is a mitogenic inhibitor, for example, paclitaxel or docetaxel.

In another embodiment of the invention, the chemotherapeutic and/or antineoplastic agent is a DNA replication inhibitor, for example an anti-metabolite. The anti-metabolite may be, for example, a pyrimidine analog. The pyrimidine analog may be, for example, a thymidylate synthase inhibitor, a DNA polymerase inhibitor, a ribonucleotide reductase inhibitor, or a hypomethylating agent. In one embodiment of the invention, the thymidylate synthase inhibitor is fluorouracil, capecitabine, tegafur, carmofur, or floxuridine; the DNA polymerase inhibitor is cytarabine; the ribonucleotide reductase inhibitor is gemcitabine or a derivative thereof; and the hypomethylating agent is azacitidine or decitabine.

In an exemplary embodiment of the invention, the chemotherapeutic and/or antineoplastic agent is a nucleoside analog. In one embodiment of the invention, the nucleoside analog is a 2', 2'-difluoronucleoside, such as, gemcitabine or a derivative thereof. In another embodiment of the invention, the therapeutic agent is an endocrine agent, for example, a radiotherapeutic agent for ovarian ablation, an LHRH analogue, an aromatase inhibitor, tamoxifen, or fulvestrant.

The present invention also provides a kit for use in the method of the invention. In one embodiment of the invention, the kit comprises a detector molecule capable of detecting the presence, or absence, or level of a S100A4 gene product and optionally, a detector molecule capable of detecting the presence, or absence, or level of a S100A2 gene product. In one embodiment of the invention, the detector molecule is a nucleic acid probe. In another embodiment of the invention, the detector molecule is an antibody or antigen binding fragment thereof. Optionally, the kit is packaged with instructions for use, for example, in a method described herein according to any embodiment. The present invention also provides a kit for treating a subject with cancer, the kit comprising a therapeutic agent for cancer and instructions to perform a method as described herein, according to any embodiment, for example, prior to commencing treatment, to determine whether or not a subject will have a beneficial response to treatment. Optionally, the kit comprises means for performing the method of the invention.

Suitable reagents for inclusion in a kit of the invention are described herein and shall be taken to apply mutatis mutandis to the present embodiment of the invention.

In one embodiment of the invention, the subject is a mammal, for example, a human.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. Expression of Sl 00A4 predicts survival after pancreatectomy

Figure 2. Expression of S100A4 predicts response to adjuvant chemotherapy in pancreatic cancer

Figure 3. Expression of S100A4 predicts survival after resection for colorectal cancer

Figure 4. Expression of S100A4 predicts response to adjuvant chemotherapy in colorectal cancer Figure 5. S100A4 expression predicts response to adjuvant endocrine therapy after curative resection for breast cancer

Figure 6. S100A4 expression predicts response to adjuvant chemotherapy after surgery for ovarian cancer

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 - Nucleotide sequence of S100A4 transcript variant 1 SEQ ID NO: 2 - Nucleotide sequence of S100A4 transcript variant 2 SEQ ID NO: 3 - Amino acid sequence of S100A4 SEQ ID NO: 4 - Nucleotide sequence of S100A2 SEQ ID NO: 5 - Amino acid sequence of S100A2

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

GENERAL

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be considered an admission that the prior art is in any way relevant to the present invention.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Each embodiment described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise.

S100A4

The present inventors have discovered that aberrant expression of a S100A4 gene product in a subject is predictive of responsiveness or non-responsiveness to treatment with, for example, a therapeutic or endocrine agent or with surgery.

Accordingly, the method of the invention comprises detecting S100A4 expression and/or activity in a biological sample of the subject. Optionally, the method of the invention comprises detecting S100A2 expression and/or activity in a biological sample of the subject.

As used herein, the term "S100A4 gene" refers to a human gene at chromosome position Iq21 which encodes SlOO calcium-binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog, mts 1, p9Ka, CAPL, pEL98). The

S100A4 gene comprises, for example, a sequence as shown in SEQ ID NO: 1 or SEQ

ID NO: 2.

As used herein, the term "S100A2 gene" refers to a human gene at chromosome position Iq21 which encodes SlOO calcium-binding protein A2 (CAN19, SlOOL). The S100A2 gene comprises, for example, a sequence as shown in SEQ ID NO: 4.

As used herein, the term "gene" includes the deoxyribonucleotide sequences comprising the protein coding region of a structural gene. The term "gene" also includes 5' and 3' untranslated regions involved in expression of the gene. The term

"gene" encompasses both complementary DNA (cDNA) and genomic forms or clones of the gene. A genomic form or clone of the gene comprises the protein coding region which may be interrupted with non-coding sequences termed "introns" (also referred to as "intervening regions" or "intervening sequences"). Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA) and may contain regulatory elements, such as enhancers. Introns are removed or "spliced out" from the hnRNA or primary RNA transcript and as such, are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes a synthetic or fusion molecule encoding all or part of the protein of the invention and a complementary nucleotide sequence to any one of the above.

As used herein, the term "S100A4 gene product" or "S100A2 gene product" refers to any transcription product of the S100A4 or S100A2 gene, respectively, such as, unprocessed or processed mRNA, including a splice variant, or any translation product encoded by the S100A4 or S100A2 gene, such as a precursor S100A4 or S100A2 polypeptide or a processed S100A4 or S100A2 polypeptide. S100A4 and S100A2 are members of a family of sixteen SlOO calcium binding proteins, that all have in common a functional EF-hand domain that mediates their activity.

By "mRNA" it is meant mRNA encoding a S100A4 or S100A2 polypeptide that has, for example, at least about 80% identity to SEQ ID NO: 3 or SEQ ID NO: 5, respectively, or mRNA comprising a nucleotide sequence that has at least about 80% identity, or at least about 90%, or at least about 95%, or at least about 99% identity to the nucleotide sequence set forth in SEQ ID NO: 1 (S100A4) or SEQ ID NO: 2 (S100A4), or SEQ ID NO: 3 (S100A2). The percentage identity of a polynucleotide can be determined using the GAP program of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et ah, 1984), which utilizes the algorithm of Needleman and Wunsch, 1970, or alternatively, the CLUSTAL W algorithm of Thompson et ah, 1994, for multiple alignments, to maximize the number of identical polynucleotides and to minimize the number and/or length of sequence gaps in the alignment.

By "S100A4 polypeptide or S100A4 protein" or "S100A2 polypeptide or S100A2 protein" it is meant a polypeptide that comprises an amino acid sequence of a S100A4 or S100A2 protein having for example, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% identity to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, respectively. In determining whether or not two amino acid sequences fall within these defined percentage identity limits, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison of amino acid sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical amino acid residues, depending upon the algorithm used to perform the alignment. In the present context, references to percentage identity between two or more amino acid sequences refers to the number of identities between said sequences as determined using any standard algorithm known to those skilled in the art. In particular, amino acid identities are calculated using the GAP program of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et ah, 1984), which utilizes the algorithm of Needleman and Wunsch, 1970, or alternatively, the CLUSTAL W algorithm of Thompson et ah, 1994, for multiple alignments, to maximize the number of identical amino acids and to minimize the number and/or length of sequence gaps in the alignment.

THERAPEUTIC TREATMENT

The methods of the invention can be used to screen subjects in need of treatment, for example, cancer patients, prior to treating said subjects with a therapeutic agent or with surgery. Thus, the inventive methods can be used to screen subjects to enable a care provider to determine whether or not treatment of said subject with a particular therapeutic agent or with surgery will be effective. A subject who is predicted to respond to the treatment based on methods of the invention is a candidate for treatment with the therapeutic agent or with surgery. A subject who is predicted to be unlikely to respond to treatment with a therapeutic agent based on methods of the invention, can be a candidate, inter alia for surgery and/or treatment with a therapeutic agent in conjunction with an inhibitor of expression or activity of the S100A4 gene product, or another treatment method.

As used herein, the term "treatment" includes abrogating, inhibiting, slowing, or reversing the progression of a disease or condition, or ameliorating or preventing a clinical symptom of the disease or condition.

As used herein, "surgery" refers to the incision or curettage of tissue or an organ, particularly surgical resection. The term "resection^" refers io the surgical removal of part or all of a tissue, structure or organ. Exemplary resections include pancreatectomy, partial colectomy, breastectomy,and oophorectomy.

THERAPEUTIC AGENTS

As used herein, the term "therapeutic agent" includes compounds or compositions that provide a desired biological or pharmacological effect when administered to the subject. In one embodiment of the invention, the therapeutic agent is a chemotherapeutic and/or anti-neoplastic agent. The chemotherapeutic and/or anti- neoplastic agent may be a DNA intercalating agent, for example, cisplatin, a 2', T- difluoronucleoside, for example, gemcitabine or a derivative thereof, a topoisomerase 1 inhibitor, for example, irinotecan, or a mitogenic inhibitor, for example, paclitaxel or docetaxel. In another embodiment, the therapeutic agent is an endocrine agent.

DNA intercalating agents

Intercalating agents typically wedge between bases along the DNA, affecting the structure of the DNA and preventing polymerase and other DNA binding proteins from functioning properly. Intercalating agents act to prevent DNA synthesis, inhibit transcription and/or induce mutations. Examples include, platimun based chemotherapeutic drugs (also known as platinum analogues) such as, cisplatin, carboplatin, nedaplatin (trade name: Aqupla), oxaliplatin (trade name: Eloxatin), satraplatin, triplatin, and tetranitrate.

2', 2'-difluoronucleosides

2', 2'-difluoronucleosides useful in the methods of the present invention can be represented by formula I:

wherein:

Ri is hydrogen or

o

-C-R⁵

R-₂ is a base defined by one of the formulae:

X is N or C-R⁴;

R³ is hydrogen, C₁-C₄ alkyl or R⁴ is hydrogen, Ci-C₄ alkyl, amino, bromo, fluoro, chloro or iodo; each R⁵ independently is hydrogen or Ci-C₄ alkyl; and the pharmaceutically-acceptable salts thereof.

2', 2'-difluoronucleosides useful in the methods of the present invention can also be represented by formula II:

wherein:

R⁶ is hydrogen or Ci-C₄ alkyl; R⁷ is a base of one of the formulae:

X is N or C-R⁴ ;

R⁸ is hydrogen or C₁-C₄ alkyl;

R⁴ is hydrogen, Ci-C₄ alkyl, amino, bromo, fluoro, chloro and iodo; and the pharmaceutically-acceptable salts thereof; with the proviso that R⁶ and R⁸ may both be hydrogen only when X is N.

2', 2'-difluoronucleosides useful in the methods of the present invention can also be represented by formula III:

wherein:

R is hydrogen or Ci-C₄ alkyl; R9 is

and the pharmaceutically-acceptable salts thereof.

The 2'2'-dinucelosides useful in the methods of the present invention can be prepared as described in US 5,464,826.

Gemcitabine and its Derivatives

One 2', 2'-difluoronucleoside useful in the methods of the present invention is 2', 2'-difluoro-deoxycytidine (gemcitabine; cytindine, 2'-deoxy-2', 2'-difluoro-; 2'-deoxy-2' 2'-difluorocytidine; CCRIS 8984; DDFC; DFdC; DFdCyd; HSDB 7567; LY 188011; NSC 613327; UNII-B76N6SBZ8R). The term "gemcitabine" as used herein means gemcitabine hydrochloride or gemcitabine free base. Gemcitabine has formula IV:

Gemcitabine is a nucleoside analog of deoxycytidine used in chemotherapy. It is marketed as Gemzar® (gemcitabine hydrochloride) by Eli Lilly and Company. Similar to fluorouracil and other analogs of pyrimidines, the drug replaces one of the building blocks of nucleic acids, in this case cytidine, during DNA replication. The process arrests tumor growth, as new nucleosides cannot be attached to the "faulty" nucleoside, resulting in apoptosis.

Pharmacology

Gemcitabine is an anti-neoplastic anti-metabolite. Anti-metabolites masquerade as purine or pyrimidine - which become the building blocks of DNA. They prevent purine or pyrimidines becoming incorporated into DNA during the "S" phase (or DNA synthesis phase of the cell cycle), stopping normal development and division. Gemcitabine blocks an enzyme which converts the cytosine nucleotide into the deoxy derivative. In addition, DNA synthesis is further inhibited because gemcitabine blocks the incorporation of the thymidine nucleotide into the DNA strand. Mechanism of Action

Gemcitabine inhibits thymidylate synthetase, leading to inhibition of DNA synthesis and cell death. Gemcitabine is a prodrug so activity occurs as a result of intracellular conversion to two active metabolites, gemcitabine diphosphate and gemcitabine triphosphate by deoxycitidine kinase. Gemcitabine diphosphate inhibits ribonucleotide reductase, the enzyme responsible for catalyzing synthesis of deoxynucleoside triphosphates required for DNA synthesis. Gemcitabine triphosphate (diflurorodeoxycytidine triphosphate) competes with endogenous deoxynucleoside triphosphates for incorporation into DNA.

Indication

Gemcitabine has proven activity in pancreatic, non-small cell lung carcinoma, breast, ovarian, bladder, ovarian, and small-cell lung cancer. It is being investigated for use in esophageal cancer, and is used experimentally in lymphomas and various other tumor types.

Derivatives

Gemcitabine has three derivatizable functions, namely the 5'- and 3'-hydroxyl groups and the N^4" amino group. Gemcitabine derivatives include any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound which upon administration to a subject, is capable of providing (directly or indirectly) a compound of formula IV or an active metabolite or residue thereof.

Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

The amine group may be quarternized with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. They may also form amides with groups including lower alkyl carboxylic acids, such as acetic acid and 2, 2- dimethylpropionic acid, or sulfonated with groups including alkyl sulfonic acids, such as methyl sulfonic acid, to form sulfonamides. The hydroxyl groups may be esterified with groups including lower alkyl carboxylic acids, such as acetic acid and 2, 2-dimethylpropionic acid, or sulfonated with groups including alkyl sulfonic acids, such as methyl sulfonic acid.

This invention also encompasses pharmaceutical compositions containing prodrugs of compounds of formula IV.

Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined to the free amino and hydroxy groups of compounds of formula IV. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxy lysine, demosine, isodemosine, 3-methylhistidine, norvlin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include phosphate derivatives of compounds of formula IV (such as acids, salts of acids, or esters) joined through a phosphorus-oxygen bond to a free hydroxyl of compounds of formula rv.

Irinotecan

Irinotecan is a topoisomerase 1 inhibitor, which prevents DNA from unwinding.

Chemically, it is a semisynthetic analogue of the natural alkaloid camptothecin. Its main use is in colon cancer, particularly in combination with other chemotherapy agents. This includes the regimen FOLFIRI which consists of infusional 5-fluorouracil, leucovorin, and irinotecan.

Taxanes Taxanes are diterpenes produced by the plants of the genus Taxus (yews).

Taxanes were first derived from natural sources, but some have been synthesized artificially. Taxanes include, for example, paclitaxel and docetaxel.

Taxanes dirsupt microtubule function which is essential to cell division and as such, taxanes are said to function as mitotic inhibitors.

Endocrine Agent

According to the present invention, the term "endocrine agent" means any agent that disrupts the signal generated by hormone, for example, estrogen or progesterone, binding to its receptor. Nonlimiting examples include agents which inhibit the production of hormone, such as, a radiotherapeutic agent for ovarian ablation, lower the hormone level, such as, LHRH analogues and aromatase inhibitors, or block the hormone receptor, such as, the drugs tamoxifen and fulvestrant.

LHRH analogues AN used herein, the term "LHRH analogue" is intended to encompass peptidic compounds that mimic the structure of luteinizing hormone releasing hormone. An LHRH analogue may be an LHRH agonist or an LHRH antagonist.

As used herein, an "LHRH agonist" is intended to refer \o a compound which stimulates the luteinizing hormone releasing hormone receptor (LHRH-R) such that release of luteinizing hormone is stimulated. Examples of LHRH agonists include leuprolide αraJe name: Lupron). goserelin (trade name: Zoladex), buserelin, triptorelin (also known as Decapeptyl, D-Trp-6-LHRH), nafarelin (trade name: Synarel), lutrelin, cyptoreiin, gonadorelin and histrelin.

As used herein, the term "LHRH antagonist" is intended to refer to a compound that inhibits the LHRH-R such that release of luteinizing hormone is inhibited. Examples of LHRH antagonists include Antide, Cetrorelix, compounds described in US 5,470,947, WO 89/01944, US 5,413,990, US 5,300,492, US 5,371,070, US 5,296,468, US 5,171,835, US 5,003,011, US 4,431,635, US 4,992,421, US 4,851,385 US, 4,801,577, US 4,689,396, US 08/480,494, and PCT/US96/09852. Studies have shown that adjuvant treatment with LHRH analogue for 2-3 years

(with or without tamoxifen for 5 years) produces a treatment benefit similar to (or even greater than) adjuvant cytotoxic chemotherapy with cyclophosphamide, methtrexate, florouracil (CMF) among pre-menopausal women with hormone-receptor-positive breast cancer.

Aromatase Inhibitors

As used herein, the term "aromatase inhibitor" relates to a compound which inhibits the estrogen production, that is, the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to steroids, especially atamestane, exemestane (trade name: Aromasin) and formestane (trade name: Lentaron) and, in particular, non-steroids, especially aminoglutethimide (trade name: Orimeten), roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadrozole (trade name: Afema), anastrozole (trade name: Arimidex) and letrozole (trade names: Femara or Femar).

Adjuvant therapy with the selective, non-steroidal aromatase inhibitor anastrozole has been shown to result in improved recurrence free survival compared to tamoxifen at short term follow up.

Tamoxifen

Tamoxifen (trade names: Nolvadex, Istubal, and Valodex) is an antineoplastic nonsteroidal selective estrogen receptor modulator (SERM). Tamoxifen competitively inhibits the binding of estradiol to estrogen receptors, thereby preventing the receptor from binding to the estrogen-response element on DNA. The result is a reduction in DNA synthesis and cellular response to estrogen. In addition, tamoxifen up-regulates the production of transforming growth factor B (TGFb), a factor that inhibits tumor cell growth, and down-regulates insulin-like growth factor 1 (IGF-I), a factor that stimulates breast cancer cell growth.

Five years of tamoxifen is the standard adjuvant treatment for patients with hormone -receptor-positive early breast cancer irrespective of age, menopausal status, or tumor stage.

Fulvestrant

Fulvestrant (trade name: Faslodex) is a parenteral 7-alpha-alkylsulphinyl analog of estradiol. Fulvestrant is an estrogen receptor antagonist that binds to the estrogen receptor in a competitive manner with affinity comparable to that of estradiol.

Fulvestrant down regulates the estrogen receptor protein in human breast cancer cells. The transcription of estrogen-regulated genes is inhibited as a consequence of the down-regulation of the estrogen receptor by fulvestrant.

CANCER

The method of the present invention is applicable to a wide range of cancers including, but not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. In one embodiment of the invention, the cancer is a carcinoma. In one embodiment of the invention, the cancer is pancreatic cancer, small cell lung cancer, non-small-cell lung cancer, breast cancer, bladder cancer, colorectal cancer, or ovarian cancer.

In another embodiment of the invention, the cancer is a gemcitabine-responsive cancer.

As used herein, the term "pancreatic cancer" includes exocrine and endocrine pancreatic cancers. Exocrine pancreatic cancers include adenocarcinomas (M8140/3), adenosquamous carcinomas, squamous cell carcinomas, and giant cell carcinomas.

Endocrine pancreatic cancers include islet cell carcinomas. The term "small cell lung cancer" includes squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. The term "bladder cancer" refers to any of several types of malignant growths of the urinary bladder. The term "breast cancer" includes ductal and lobular carcinomas. The term "colorectal cancer" includes cancer of the colon, rectum, and/or anus, particuarly, adenocarcinomas, and may also include carcinomas (e.g., squamous cloacogenic carcinomas), melanomas, lymphomas, and sarcomas. Epidermoid

(nonkeratinizing squamous cell or basaloid) carcinomas are also included. The term

"ovarian cancer" includes cancers of epithelial origin, such as, for example, serous, mucinous, endometrioid, clear cell, papillary serous, Brenner cell or undifferentiated adenocarcinoma. In one embodiment of the invention, the cancer is pancreatic cancer.

In another embodiment of the invention, the cancer is colorectal cancer. In another embodiment of the invention, the cancer is breast cancer. In another embodiment of the invention, the cancer is ovarian cancer. In one embodiment of the invention, the subject has undergone or will undergo surgery, for example, a pancreatectomy, a partial colectomy, a breastectomy, or a oophorectomy. DETECTION METHODS AND ANAL YSIS

As used herein "expression and/or activity" means expression and/or activity of a S100A4 or S1000A2 gene product.

mRNA Expression

In the practice of the methods of the invention, S100A4 or S100A2 gene expression may be detected by measuring the expression of corresponding mRNA in a biological sample. mRNA expression may be measured by any suitable method, including but not limited to, those described below. In one embodiment, in situ hybridization (ISH) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. ISH is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ).

In one embodiment, RNA (or isolated mRNA) is detected by Northern blot analysis. Northern blot analysis involves the separation of the RNA by gel electrophoresis and hybridization of a complementary labeled probe.

In another embodiment, RNA (or isolated mRNA) expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; see U.S. Patent Nos. 5,846,717; 6,090,543; 6,001,567; 5,985,557; and 5,994,069). The INVADER assay detects specific nucleic acid sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes.

In a further embodiment, RNA (or isolated mRNA or cDNA) is detected by hybridization to an oligonucleotide probe). A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, a TaqMan assay (US 5,962,233 and US 5,538,848) may be utilized. The TaqMan assay is performed during a PCR reaction. The TaqMan assay exploits the 5 '-3' exonuclease activity of the AMPLITAQ GOLD™ DNA polymerase. A probe consisting of an oligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye) and a 3'-quencher dye is included in the PCR reaction. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of the AMPLITAQ GOLD™ polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter. In yet another embodiment, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA (or isolated mRNA) is enzymatically converted to cDNA using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. Conditions for specifically hybridizing a nucleic acid probe to a target nucleic acid sequence, and conditions for washing to remove non-specific hybridizing nucleic acid are well understood by those skilled in the art. For the purposes of further clarification only, reference to the parameters affecting hybridization between nucleic acid molecules is found in Ausubel et ah, 1992. Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5⁰C to 10⁰C below Tm; intermediate stringency at about 10⁰C to 20⁰C below Tm; and low stringency at about 20⁰C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization is used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization are used to identify or detect similar or related polynucleotide sequences.

In a preferred embodiment, the present invention encompasses the use of nucleotide sequences that can hybridize to a target nucleotide sequence under stringent conditions (e.g. 65°C and 0. IxSSC { IxSSC = 0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0}).

mRNA Analysis

Analysis of S100A4 mRNA expression levels can be carried out by, for example, assessment of tissue staining (in situ hybridization) using a scoring system. Alternatively quantitative RNA analysis can be used to measure the concentration (or copy numbers) of specific RNA sequences. For gene expression analysis, it is important to obtain a homogeneous sample containing only the desired cell type for RNA preparation. Two techniques, laser capture microdissection and flow cytometry can be used to obtain homogenous cells from complex tissue samples. Since cells can respond to environmental changes by changing their expression profiles, it is important that minimum disturbance is inflicted during cell purification and RNA preparation. RNase inhibition reagents (such as RNAlater from Ambion, Austin, USA) are often added prior to cell lysis. Many commercial kits are now available for total RNA and mRNA preparations. For reverse transcription, various engineered AMV reverse transcriptase, engineered M-MLV reverse transcriptase or rTth reverse transcriptase/DNA polymerase can be used with either random hexamer, Oligo(dT) or gene specific primers.

Quantifying gene expression can be carried out by, for example, detection of mRNA on a Northern blot, or PCR products on a gel or Southern blot, real-time PCR and real competitive PCR.

Real-Time PCR

Real-time PCR, also called quantitative real time polymerase chain reaction (Q- PCR/qPCR) or kinetic polymerase chain reaction, can be used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.

The procedure follows the general principle of PCR; however the amplified DNA is detected as the reaction progresses in real time as opposed to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

Frequently, real-time PCR is combined with reverse transcription to quantify mRNA and non-coding RNA in cells or tissues.

Competitive PCR In Competitive PCR, total RNA is reverse transcribed with random hexamers.

Then, a competing DNA oligonucleotide (typically 80 bases long) with one base difference from the gene of interest is added prior to PCR. A base extension reaction at the mutation site is carried out by adding a ThermoSequenase and 3 ddNTPs and 1 dNTP to produce two oligonucleotide products with different molecular weight. These two products are subsequently detected and quantified by MALDI-TOF MS with Allelotyping software (SEQUENOM, Inc.).

Protein Expression

Alternatively, S100A4 or S100A2 gene expression may be detected by measuring the expression of the corresponding S100A4 protein or polypeptide in a biological sample. Protein expression may be detected by any suitable method. In one embodiment, the S100A4 protein is detected by immunohistochemistry, the specific binding of antibodies or antigen binding fragments thereof (hereinafter, collectively referred to as "antibodies") to antigens in biological tissues. An antibody- antigen interaction can be visualized by techniques know in the art; for example, through use of an antibody conjugated to an enzyme, for example, peroxidase (commonly referred to as immunoperoxidase staining), or alternatively, use of an antibody conjugated to a fluorophore, for example, FITC (commonly referred to as immunofluorescence).

In another embodiment, the S100A4 protein is detected by immunoassay, the specific binding of antibodies to antigens in biological liquids. Antibody binding is detected by techniques known in the art, including radioimmunoassay (e.g., enzyme- linked immunosorbant assays (ELISA)), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using for example, colloidal gold, enzyme or radioisotope labels), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays.

As used herein, the term "specific binding" shall be taken to mean an antibody that reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or antigens or cell expressing same than it does with alternative antigens or cells. For example, an antibody that specifically binds to an antigen, or an epitope, or immunogenic fragment thereof, binds that antigen, or the epitope, or the immunogenic fragment with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens, or, epitopes, or immunogenic fragments thereof. It is also understood that, for example, an antibody that specifically binds to a first target may or may not specifically bind to a second target. As such, "specific binding" does not necessarily require exclusive binding or non-detectable binding of another molecule, this is meant by the term "selective binding". Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

In one embodiment, antibody binding is detected by a label on the primary antibody. In another embodiment, the primary antibody is detected by binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in US 5,885,530; 4,981,785; 6,159,750 and 5,358,691. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to prognostic markers is utilized.

The use of monoclonal antibodies for immunohistochemistry and immunoassays is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art (Douillard and Hoffman, 1981; Kohler and Milstein, 1976).

Anti-S100A4 antibodies and anti-S100A2 antibodies can be commercially sourced from, for example, NeoMarkers, Sigma-Aldrich, Novus Bio.

In a further embodiment of the invention, predicting whether a subject will respond to treatment, for example, with a therapeutic agent or with surgery can be determined by assaying an activity of the protein product.

Protein Analysis

Analysis of S100A4 expression levels can be carried out by, for example, assessment of tissue staining (immunohistochemistry) using a scoring system, or by flow cytometry. Alternatively S100A4 expression can be quantitatively measured by, for example, Western blot, ELIZA.

Immunohistochemistry Scoring

Cytoplasmic, nuclear and/or stromal immunoreactivity for S100A4 can be assessed by immunohistochemistry and described in terms of the intensity (0 = negative, 1+ = weak, 2+ = moderate and 3+ = strong) and percentage of immunopositive cells. From these indices a modified 'H score' (i.e., intensity x percentage of positive cells) can be calculated for each core and a final score can be assigned for each tumor. Non limiting examples of how tumors may be assessed are given below:

Pancreatic tumors may be assessed as positive for S100A4 expression if, for example, there is any positive nuclear or cytoplasmic staining at any intensity in > 1% of cells. Colorectal tumors may be assessed as positive for S100A4 expression if, for example, they have a combined (nuclear and cytoplasmic) H-score of > 200.

Breast tumors may be assessed as positive for S100A4 expression if, for example, they have a stromal intensity of 3 and are assessed as low or negative for S100A4 expression if, for example, they have a stromal intensity of 0 to 2.

Ovarian tumors may be assessed as positive for S100A4 expression if, for example, they have a stain intensity of 3 and are assessed as low or negative for S100A4 expression if, for example, they have a stain intensity of 0 to 2.

BIOLOGICAL SAMPLES

As used herein, the term "biological sample" includes a cell, tissue or bodily fluid from a subject and any extract or derivative thereof. A "biological sample" can be, or can comprise, for example, biopsy material (i.e., a tissue specimen); bone marrow; blood, blood plasma, serum or cellular fraction thereof; urine; saliva; tears; semen; ascites fluid; a cell derived from a biological source; nucleic acid derived from a cell; or nucleic acid produced using nucleic acid derived from a cell.

In one embodiment of the invention, the biological sample comprises a cell from the subject's pancreas, lung, breast, bladder, or ovary. In another embodiment of the invention, the biological sample comprises a neoplastic cell. In a further embodiment of the invention, the biological sample is a tumor sample.

The biological sample can be prepared on a solid matrix for histological analysis, or alternatively, in a suitable solution such as, for example, an extraction buffer or suspension buffer.

PREDETERMINED LEVEL AND REFERENCE SAMPLES

According to the methods of the invention, the aberrant level of expression and/or activity of a S100A4 gene product in a biological sample from a subject is determined and compared with the level of expression and/or activity of the gene product in a biological sample from a normal or responsive subject (reference sample) or normal tissue or to a predetermined level.

In one embodiment of the invention, the biological sample is closely matched to the reference sample, for example, they are from the same tissue type. In one example, the biological sample is compared to normal epithelium of a corresponding or closely matched tissue (i.e., tissue from the same organ as the biopsy material) from a normal subject. In another embodiment, the biological sample comprises (or is suspected of comprising) a neoplastic cell and is compared to normal epithelium obtained from the same organ as the biological sample and from the same subject.

As used herein, the term "normal subject" shall be taken to mean an individual (or group of individuals) who is known not to suffer from a given cancer, such knowledge being derived from clinical data on the individual(s). It is preferred that said "normal subject" does not have a large number of risk factors associated with developing cancer, for example familial history.

As used herein, "normal tissue" means tissue with no visible manifestations of disease as determined by, for example, histology.

As used herein, a subject is "responsive" to a treatment if said treatment had, or is having the effect of abrogating, inhibiting, slowing, or reversing the progression of the disease or condition, or ameliorating or preventing a clinical symptom of the disease or condition. In one embodiment, the "responsive subject" had a complete response to treatment as defined by standard accepted clinical definitions. For example, a complete response of a subject to a treatment is indicated by absence of clinical and/or radiological and/or tumor marker evidence of the cancer.

As used herein, the term "predetermined level" refers to a level of a gene product expression and/or activity below which it has been found that the subject is likely to respond to treatment and/or above which the subject is unlikely to respond to the treatment, or vice versa. Such a predetermined level is determined, for example, based on data from a population of subjects comprising a plurality of subjects that are, or were responsive to treatment and a plurality of subjects that are not, or were not responsive to treatment. Those skilled in the art will appreciate that the "predetermined level" against which the expression or activity of the S100A4 gene product is compared may vary from cancer to cancer.

RECOMMENDED THERAPIES

The methods of the present invention may also comprise the step of recommending a treatment regime for the disease or condition. The treatment regime may comprise surgery and/or administering a therapeutic agent alone or in combination with an inhibitor that reduces expression and/or activity of S100A4 in a cell.

In one embodiment, the inhibitor administered comprises nucleic acid. The nucleic acid may be an antagonist of S100A4 expression, such as, for example, an antisense nucleic acid, peptide nucleic acid (PNA), ribozyme, interfering RNA, siRNA or shRNA which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize with the target molecule, in particular, S100A4-encoding RNA. When introduced into a cell using suitable methods, such a nucleic acid inhibits the expression of the S100A4 gene encoded by the sense strand. Antisense nucleic acid, ribozymes (US 4,987,071; US 5, 116,742; Bartel and Szostak, 1993), nucleic acid capable of forming a triple helix (Helene, 1991), PNAs (Hyrup et al, 1996; Perry-O'Keefe et al, 1996), interfering RNAs (Elbashir et al, 2001; Sharp, 2001; Lipardi et al, 2001; Nishikura, 2001), small interfering RNAs (siRNA) or short haipin RNA (shRNA) may be produced by standard techniques known to the skilled artisan, based upon the sequences disclosed herein. In one embodiment of the invention, the antisense nucleic acid, ribozyme, PNA, interfering RNA, siRNA or shRNA comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence having at least about 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2 (i.e., it is complementary to S100A4-encoding mRNA) and can hybridize thereto. For example, such antagonistic nucleic acid can be complementary to a target nucleic acid having the sequence of SEQ ID No: 1, or SEQ ID NO: 2, or a portion thereof, sufficient to allow hybridization. Longer molecules, comprising a sequence that is complementary to at least about 20, or, 25, or 30, or 35, or 40, or 45, or 50 contiguous nucleotides of S 100A4-encoding mRNA are also encompassed by the present invention. In one embodiment of the invention, interfering RNA, for example, siRNA is used to down-regulate S100A4 expression in a cell. This down regulation may cause cells to be more sensitive to therapeutic treatment and subsequent cell death. Such interfering RNAs generally comprise an RNA molecule having a region of self- complementarity capable of forming a double stranded RNA. In one embodiment of the invention, a construct comprising an antisense nucleic acid, ribozyme, PNA, interfering RNA, siRNA or shRNA, can be introduced into one or more suitable cells of the subject to inhibit S100A4 expression and/or activity therein. The antisense nucleic acid, ribozyme, PNA, interfering RNA, siRNA or shRNA, inhibits S100A4 expression and the subsequent formation of deleterious protein-protein complexes involving a S100A4 protein. Accordingly, non- responsiveness to therapeutic treatment that is mediated by S100A4 in the cell containing the construct is inhibited.

The use of antibodies or small molecule inhibitors that can inhibit one or more functions characteristic of a S100A4 protein, such as a binding activity, a signaling activity, and/or stimulation of a cellular hyperproliferative response, is also encompassed by the present invention. In one embodiment, antibodies or small molecule inhibitors of the present invention can inhibit binding of an interacting protein (i.e., one or more interacting proteins) to a S100A4 protein and/or can inhibit one or more functions mediated by a S100A4 protein in response to binding of interacting proteins. In another embodiment of the invention, the method involves recommending that a subject does not receive treatment with an agent and/or that they receive treatment with a different agent and/or surgery.

ADMINISTRA TION The therapeutic agent can be administered to the subject by an appropriate route, either alone or in combination with another drug or an inhibitor that reduces expression and/or activity of S100A4 in a cell. An effective amount of the therapeutic agent is administered. An "effective amount" is an amount sufficient to achieve the desired therapeutic effect, under the conditions of administration. A variety of routes of administration are possible including, but not necessarily limited to oral, dietary, topical, parenteral (e.g., intravenous, intra-arterial, intramuscular, subcutaneous injection), and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration.

Formulation of a therapeutic agent to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule). An appropriate composition comprising the agent to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers include various additives, preservatives, or fluid, nutrient or electrolyte replenishers and the like (See, generally, Remington's Pharmaceutical Sciences, 1985). For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser). Where the therapeutic agent is a protein or peptide, the agent can be administered via in vivo expression of the recombinant protein. In vivo expression can be accomplished via somatic cell expression according to suitable methods (US 5,399,346). In this embodiment, nucleic acid encoding the protein can be incorporated into a retroviral, adenoviral or other suitable vector (preferably, a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the protein for delivery. In the latter embodiment, the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the protein in a therapeutically effective amount.

Administration of Gemcitabine for the Treatment of Cancer The recommended dose for gemcitabine therapy is about 1 g/m² of body surface area. Gemcitabine can be administered intravenously, which requires the active substance be in the form of a solution.

Gemcitabine preparations for parenteral administration can be in lyophilized form (e.g., Gemzar) and reconstituted before administration to the subject. In order to reduce the solution volume to be freeze-dried for the lyophilizate, the active substance is typically dissolved in water at a pH range of 2.7 to 3.3.

Ready-to-use gemcitabine solutions, wherein the solution has a gemcitabine concentration of about 16 mg/ml to about 110 mg/ml and a pH of about 3.5 to about 10 are described in US 20060089329.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

The invention is hereinafter described by way of the following non-limiting

Examples and with reference to the accompanying figures.

EXAMPLES

Example 1 : Materials and Methods

Patients and Tissue Specimen - Pancreatic Cancer

Detailed clinico-pathologic and outcome data for a cohort of 372 consecutive patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent pancreatic resection were obtained from teaching hospitals associated with the New South Wales Pancreatic Cancer Network (NSWPCN; www.pancreaticcancer.net.au) Sydney, Australia (Table 1). This cohort was the combination of a training cohort of 76 patients and an independent validation cohort of 296 patients. The training set was accrued from 4 hospitals. The training set included patients with pancreatic cancer, who were treated with surgery alone prior to 1998, when adjuvant chemotherapy for pancreatic cancer was not used in Australia. Patients that comprised the validation set were accrued from patients with pancreatic cancer from 8 hospitals within the NSWPCN that were treated between 1996 and 2007, with 29% of patients having received adjuvant chemotherapy (gemcitabine and/or 5-fluorouracil). Both cohorts displayed clinical and pathological features that are consistent with the expected clinical behavior of pancreatic cancer and are similar to published pancreatic cancer cohorts worldwide. Ethical approval for the acquisition of data and biological material was obtained from the Human Research Ethics Committee at each participating institution. The diagnosis and all pathological features were reviewed centrally by at least one specialist pancreatic histopathologist, and the date and cause of death was obtained from the NSW Cancer Registry and treating clinicians.

Training Cohort The cohort of 76 patients consisted of 31 women and 45 men. The mean age at diagnosis was 62 years and range of 34 to 82 years. The median follow-up for all patients was 12.2 months (range, 0.3 to 124 months). Three patients (4%) were alive at the census date. The 30-day mortality rate was 2.6%. Sixty-nine patients (90.9%) died from pancreatic cancer, two patients (2.6%) died of other causes, and no patients were lost to follow-up. The median disease-specific survival was 12.2 months, with 3- and 5 -year survival rates of 15.8% and 5.8% respectively. The majority of tumors were moderately differentiated (58%), followed by poor differentiation (33%), and only 9% of tumors were well differentiated. Most tumors were located in the head of the pancreas (82%) and were more than 20 mm in maximal diameter (80%). Forty-two out of 76 patients (55%) had resections with clear surgical margin using RO = 0 mm definition. Lymph node metastases were present in 40 (53%) of 75 patients (one patient data missing), perineural invasion was present in 46 patients (61%), and vascular space invasion was present in 30 patients (40%).

Factors associated with a significantly better survival on univariate analysis including well and moderate differentiation (median survival 14.8 Vs 10.1 months; P 0.0480) compared to poor differentiation, tumors of the pancreatic head (median survival 15.6 Vs 7.9 months; P = 0.0002) compared with those of the body/tail, absence of margin involvement (19.7 Vs 9.5 months; P < 0.0001), absence of lymph node metastases (19.7 Vs 10.1 months; P 0.0007) and absence of vascular space invasion (14.8 Vs 9.2 months; P = 0.0270). Multivariate models using Cox proportional hazard analysis demonstrated that tumor location, margin involvement and vascular space invasion were independent prognostic factors (Table 2, Supplementary Table 1).

Validation Cohort The cohort of 296 patients consisted of 148 women and 148 men. The mean age at diagnosis was 67 years and range of 28 to 87 years. The median follow-up for all patients was 16.1 months (range, 0 to 168 months). Fifty-two patients (17.6%) were alive at the census date. The 30-day mortality rate was 4.4%. Two hundred and twenty-one patients (74.6%) died from pancreatic cancer, 10 patients (3.4%) died of other causes, and no patients were lost to follow-up. The median disease-specific survival was 18.1 months, with 3- and 5 -year survival rates of 25.8% and 13.4% respectively. The majority of tumors were moderately differentiated (67%), followed by poor differentiation (25%), and only 8% of tumors were well differentiated. Most tumors were located in the head of the pancreas (81%) and were more than 20 mm in maximal diameter (77%). One hundred and eight-four out of 296 patients (62%) had resections with clear surgical margins using RO = 0 mm definition. Lymph node metastases were present in 186 (63%) of 296 patients, perineural invasion was present in 215 patients (73%), and vascular space invasion was present in 134 patients (45%).

Factors associated with a significantly better survival on univariate analysis including tumors of the pancreatic head (median survival 19.6 Vs 13.9 months; P = 0.0303) compared with those of the body/tail, tumor size < 20 mm (31.0 Vs 16.2 months; P < 0.0001), absence of margin involvement (23.3 Vs 13.2 months; P < 0.0001), absence of lymph node metastases (23.3 Vs 16.3 months; P 0.0055), absence of perineural invasion (26.5 Vs 16.9 months; P = 0.0044), absence of vascular space invasion (14.8 Vs 9.2 months; P = 0.0046) and administration of > 3 cycles of adjuvant chemotherapy (27.5 Vs 16.5 months; P = 0.0118). Multivariate models using Cox proportional hazard analysis demonstrated that tumor size, margin involvement, lymph node metastases and adjuvant chemotherapy (> 3 cycles) were independent prognostic factors (Table 2, Supplementary Table 2).

Patients and Tissue Specimens - Colorectal Cancer

Cohort description and characteristics as per Table 3.

Patients and Tissue Specimens - Breast Cancer

Cohort description and characteristics as per Table 4.

Tissue Microarrays and Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue blocks for each patient were obtained from anatomical pathology departments. Tissue microarrays were constructed with each resected specimen represented by a minimum of 3 x 1 mm tissue cores. Immunohistochemistry was performed on 4 μm serial sections of paraffin-embedded, formalin-fixed tissue mounted on SuperFrost slides (Menzel-Glaser, Germany).

S100A4 Detection Protocol

Antigen was retrieved using DAKO S 1699 solution in 100⁰C water bath for 15 minutes. Immunostaining was performed using the DAKO Auto-stainer. The microarrays were treated with 3% Peroxidase Block (DAKO, K4011) for 5 minutes then Protein Block (DAKO, X0909) for 10 minutes. Primary Anti-S100A4 rabbit polyclonal antibody (NeoMarkers, Cat. #RB-1804, Fremont, CA, USA) with a dilution of 1 : 100 was used and incubated for 60 minutes. EnVision+ System anti-rabbit was used as secondary antibody (DAKO, K4003), then 3, 3'-diaminobenzidine (DAKO,

K3468) was used as a substrate. The microarrays were then counterstained with

Mayer's haematoxyline. Spleen was used as positive control and anti-rabbit IgG of the same concentration was used as negative control.

BNIP3 Detection Protocol

Immunohistochemistry for BNIP3 was performed on formalin fixed paraffin embedded tissue sectioned at 4μm. The primary antibody used was BNIP3 (Sigma- Aldrich, Cat. No. B7931, Clone ANa40, St Louis, MO, USA) at a dilution of 1 in 300. All slides were stained manually. Heat retrieval was performed using Universal Decloaker Solution (Biocare Medical, Walnut Creek, CA 94597, Cat. No. UDlOOOM) for 7 minutes at 85 C° using the Decloaking Chamber. A biotin-free detection system was employed, MACH4 (Biocare Medical MH4U534L) was used essentially according to the manufacturers protocol.

Immunohistochemistry Scoring Immunostaining was assessed by two independent observers. Standardization of scoring was achieved by comparison of scores between observers, and by conferencing, where any discrepancies were resolved by consensus. Scores were given as a percentage of cells with positive staining within the representative area of the tissue microarray core and the absolute intensity of staining on a scale of 0 to 3. For example, in the case of tissues from subjects with pancreatic cancer, 0 represents no staining, 1 represents heterogenous nuclear staining, 2 represents homogenous nuclear staining and 3 represents intense homogenous nuclear staining.

Pancreatic cancer: positive S100A4 expression was defined as either nuclear or cytoplasmic staining of any intensity in > 1% of cells. For BNIP3, it was observed that if there were the presence of cytoplasmic staining, all the pancreatic cancer cells within the core had cytoplasmic staining. Positive BNIP3 expression was defined as cytoplasmic staining intensity of greater than 1.

Colorectal cancer: positive S100A4 expression was defined as combined (nuclear and cytoplasmic) H-score of > 200 (H-score = intensity x %, intensity = 0 - 3 and % = 0 - 100).

Breast cancer: positive S100A4 expression was defined as stromal intensity of 3 and low or negative S100A4 expression was defined as stromal intensity of 0 to 2.

Ovarian cancer: positive S100A4 expression was defined as stain intensity of 3 and low or negative S100A4 expression was defined as stain intensity of 0 to 2.

Statistical A nalysis

Median survival was estimated using the Kaplan-Meier method and the difference was tested using the log-rank Test. P-values of less than 0.05 were considered statistically significant. Clinico-pathological variables analyzed with a P value of less than 0.25 on log-rank test were entered into Cox Proportional Hazard multivariate analysis. Statistical analysis was performed using StatView 5.0 Software (Abacus Systems, Berkeley, CA, USA). Disease-specific survival was used as the primary endpoint. Table 1: Cohort Characteristics of Patients with Pancreatic Cancer

a Analyzed as a continuous variable. b Stage I tumors Vs Stage Il for survival analysis based on UICC TNM Staging System 6^th Edition, 2002. c Well and Moderately differentiated tumors grouped together for survival analysis. d Patients with tumors located in the head of the pancreas underwent Whipple pancreaticoduodenectomies, and those with tumors of the body/tail had left sided pancreatectomies. e Tumor size was prognostic as a continuous variable (p = 0.0021), > 30 mm (p = 0.0012), and > 40 mm (p =

0.0008) in the validation set. f Gemcitabine or 5-FU: Adjuvant (n = 85), Nil chemotherapy at any time (n = 178), Palliative (n = 27), Neoadjuvant (n = 3), Unknown (n = 3). g Patients who received any form of chemotherapy at any time (note that in the training set, chemotherapy was only given for palliation of symptoms). h Analysis compares those patients who received > 3 cycles of chemotherapy versus those who received less or no therapy. ' Analysis compares those patients who received radiotherapy at any time to all others. i Positive expression of S100A4 was defined as nuclear staining of any intensity in > 1 % of cells. k Positive expression of S100A2 was defined as cytoplasmic staining intensity 3+ in > 30% of cells.

Table 2: Multivariate analyses

Training Set Variable Hazard Ratio (95% Cl) P Value

A. Clinico-pathologic Only Tumor Location (Body/Tail) 3.00(1.52-5.92) 0.0015

(n = 75) Margin Involvement (Positive) 2.27(1.29-4.02) 0.0047

Vascular Invasion (Positive) 1.90 (1.09-3.32) 0.0237

B. Clinico-pathologic & Lymph Node Metastases (Positive) 2.20(1.16-4.17) 0.0154

S100A4(n = 58) S100A4 Expression (Positive) 3.39(1.64-6.99) 0.0010

Validation Set Variable Hazard Ratio {95% Cl) P Value

C. Clinico-pathologic Only Tumor Size (>20 mm) 1.79(1.28-2.50) 0.0007

(n = 294) Margin Involvement (Positive) 1.71 (1.30-2.25) 0.0001

Lymph Node Metastases (Positive) 1.57(1.18-2.08) 0.0018

Adjuvant Chemotherapy (> 3 cycles) 0.65 (0.45-0.93) 0.0181

D. Cinteo-pathologic & Tumor Size (> 20 mm) 1.74(1.22-2,47) 0.0020

S1D0A4(n = 294) Margin involvement (Positive) 1.79(1.34-2.39) <0.0001

Lymph Node Metastases (Positive) 1.42(105-1.92) 0.0224

Adjuvant Chemotherapy (> 3 cycles} 0,65(0.44-0.94) 0.0224

S100A4 Expression (Positive) 1.61(1.19-2.17) 0.0019

E. Clinico-pathologic, Tumor Size (>20 mm) 1.63(1.14-2.34) 0.0077

S100A4&S100A2 Margin Involvement (Positive) 1.81 (1.35-2.42) <0.0001

(n = 294) Lymph Node Metastases (Positive) 1.48(1.09-2.02) 0.0120

Adjuvant Chemotherapy (> 3 cycles) 0.67(046-0.99) 0.0441

S100A4 Expression (Positive) 1.49 (1.10-2.02) 0.0102

S100A2 Expression (Positive) 1.89 (1.24-2.90) 0.0033

Supplementary Table 1: Multivariate analyses for Training Cohort

Multivariate Analysis for Training Set

Variable Hazard Ratio (95% Cl) P Value

A. Clinico-pathologic Only Differentiation (Poor) 1.22(0.70-2.14) 0.4836

(n = 75) Tumor Location (Body/Tail) 2.89(1.45-5.78) 0.0026

Tumor Size (>20 mm) 1.26(0.68-2.34) 0.4577

Margin Involvement (Positive) 1.86 (0.96-3.61) 0.0657

Lymph Node Metastases (Positive) 1.17(0.61 -2.26) 0.6352

Perineural Invasion (Positive) 1.15(0.66-2.01) 0.6123

Vascular Invasion (Positive) 1.87(1.03-3.38) 0.0393

B. Glinico^athologic Only Differentiation (Poor) 1.25(0.72-2.17) 0.4242

Tumor Location (Body/Tail) 2.93(1.47-5.85) 0.0022

Tumor Size (> 20 mm) 1.26(0.68-2.33) 0.4686

Margin involvement (Positive) 1.98(1.07-3.66) 0.0287

Perineural Invasion (Positive) 118(0.68-2,04) 0.5669

Vascular Invasion (Positive) 1.93(1.08-3.45) 0.0275

C. Clinico-pathologic Only Differentiation (Poor) 1.25(0.72-2.16) 0.4330

Tumor Location (Body/Tail) 2.95(1.49-5.88) 0.0020

Tumor Size (>20 mm) 1.26(0.68-2.34) 0.4582

Margin Involvement (Positive) 2.02(1.10-3.73) 0.0236

Vascular Invasion (Positive) 2.01(1.14-3.55) 0.0154

D. Ctinico-pathologic Only Differentiation (Poor) 1.25(0.72-2.17) 0.4312

Tumor Location (Body/Tail) 3.07(1.55-6.06) 0.0013

Margin involvement (Positive) 2.08(1.13-3.83) 0.0193

Vascular Invasion (Positive) 197(1.12-147) 0.O164

E. Clinico-pathologic Only Tumor Location (Body/Tail) 3.00(152-5.92) 0.0015

(Final Model) Margin Involvement (Positive) 2.27(1.29-4.02) 0.0047

Vascular Invasion (Positive) 190(109-3.32) 0.0237

F. Clintco-pathoiogic& Differentiation (Poor) 131(0.66-2.60) 0.4455

S100A4(n = 5S) Tumor Location (Body/Tail) 136(0.56-3,30) 0.4975

TumorSize(>20mm) 106(0.45-2.45) 0.8992

Margin involvement (Positive) 154(0.72-3.27) 0.2638

Lymph Node Metastases (Positive) 168(0.81-3.46) 0.1623

Perineural invasion (Positive) 138(0.68-2.76) 0,3700

Vascular Invasion (Positive) 136(0.60-3.10) 0.4571

S100A4 Expression (Positive) 2.82(1.04-7.63) 0.0416

G. Clinico-pathologic & Differentiation (Poor) 132(0.69-2.55) 0.4009

S100A4 Tumor Location (Body/Tail) 136(0.56-3.30) 0.5021

Margin Involvement (Positive) 1.54 (0.73 - 3.27) 0.2614

Lymph Node Metastases (Positive) 166(0.81-3.39) 0.1628

Perineural Invasion (Positive) 139(0.72-2.71) 0.3273

Vascular Invasion (Positive) 134(0.61-2.95) 0.4598

S100A4 Expression (Positive) 2.88(1.13-7.35) 0.0266

H. Ctinico-pathologic& Differentiation (Poor) 129(0.67-2.46) 0.4436

S100A4 Margin invoivement (Positive) 153(0.72-3.23) 0.2672 Lymph Node Metastases {Positive} 169(0,83-3.42) 0,1464 Perineural invasion (Positive) 1.46(0.76-2.81) 0.2610 Vascular Invasion (Positive) 1.21(0.58-2.50) 0.6108 S100A4 Expression (Positive) 3.24(1.34-7.81) 0.0092

I. Clinico-pathologic & Differentiation (Poor) 1.30(0.68-2.49) 0.4271 S100A4 Margin Involvement (Positive) 1.50(0.71 -3.15) 0.2833 Lymph Node Metastases (Positive) 1.68(0.83-3.40) 0.1507 Perineural Invasion (Positive) 1.56(0.86-2.85) 0.1460 S100A4 Expression (Positive) 3.58(1.63-7.87) 0.0092

J. Cfinico-pathoiogic & Margin involvement (Positive) 1.64(0.82-3.31) 0.1645 S100A4 Lymph Node Metastases (Positive) 1.77(0.89-3,52) 0.1037 Perineural invasion (Positive) 1.54(0.84-2.79) 0.1603 S10GA4 Expression (Positive) 3.39(1.56-7.35) 0.0020

K. Clinico-pathologic & Lymph Node Metastases (Positive) 2.14(1.14-4.00) 0.0176 S100A4 Perineural Invasion (Positive) 1.50(0.82-2.72) 0.1849 S100A4 Expression (Positive) 3.86(1.81-8.20) 0.0004

L. Clinico-pathologic & Lymph Node Metastases (Positive) 2.20(1.16-4,17) 0.0154 S100A4 (Final Mode!) S1Q0A4 Expression (Positive) 3,39(1.64-6.99) 0,0010

Supplementary Table 2: Multivariate Analyses for Validation Cohort

Multivariate Analysis for Validation Set

Variable Hazard Ratio (95% Cl) P Value

A. Clinico-pathologic Only Tumor Location (Body/Tail) 1.38(0.98-1.93) 0.0673

(n = 294) Tumor Size (>20 mm) 1.67 (1.19-2.34) 0.0029

Margin Involvement (Positive) 1.69(1.29-2.23) 0.0002

Lymph Node Metastases (Positive) 1.53 (1.15-2.03) 0.0037

Perineural Invasion (Positive) 1.32(0.97-1.82) 0.0816

Vascular Invasion (Positive) 1.23 (0.94-1.62) 0.1288

Adjuvant Chemotherapy (> 3 cycles) 0.63(0.44-0.90) 0.0124

B. Clinico-pathologic Only Tumor Location (Body/Tail) 1.36(0.97-1.92) 0.0758

Tumor Size (>20mm) 169(1.20-2.36) 0.OQ24

Margin involvement (Positive) 1.66(1.26-2.19) 0.0003

Lymph Node Metastases (Positive) 1.56(1.18-2.07) 0.0021

Perineural invasion (Positive) 1.35(0.99-1.86) 0.0605

Adjuvant Chemotherapy (> 3 cycles) 0.62(0.43-0,89) Q.0102

C. Clinico-pathologic Only Tumor Size (>20 mm) 1.73(1.23-2.42) 0.0014

Margin Involvement (Positive) 1.67 (1.27-2.19) 0.0003

Lymph Node Metastases (Positive) 1.56(1.18-2.07) 0.0021

Perineural Invasion (Positive) 1.36 (1.00 - 1.87) 0.0527

Adjuvant Chemotherapy (> 3 cycles) 0.64(0.45-0.92) 0.0153

C. Ctinico-pathologic Oniy Tumor Size (> 20 mm) 1.79(1.28-2.50) 0.0007

(Final Model) Margin involvement (Positive) 171(1.30-2.25) 0.OQO1

Lymph Node Metastases (Positive) 1.57(1.18-2.08) 0.0018

Adjuvant Chemotherapy (≥ 3 cycles) 0.65(0.45-0.93) 0.0181

D. Clinico-pathologic & Tumor Location (Body/Tail) 1.36(0.93-1.97) 0.1115

S100A4(n = 294) Tumor Size (>20 mm) 1.66(1.17-2.36) 0.0049

Margin Involvement (Positive) 1.74(1.30-2.32) 0.0002

Lymph Node Metastases (Positive) 1.43(1.06-1.94) 0.0200

Perineural Invasion (Positive) 1.24 (0.88- 1.74) 0.2131

Vascular Invasion (Positive) 1.10 (0.82- 1.46) 0.5314

Adjuvant Chemotherapy (> 3 cycles) 0.62(0.43-0.91) 0.0143

S100A4 Expression (Positive) 1.51 (1.11 -2.05) 0.0085

E. Clinico-pathologic & Tumor Locaiioπ (Body/Tail) 1.35(0.93-1.96) 0.1177

S100A4 Tumor Size (>20mm) 167(1.17-2.38) 0.0046

Margin involvement (Positive) 1.73(1.29-2,31) Q.O0O2

Lymph Node Metastases (Positive) 1.44(1.06-1.94) 0.0191

Perineural invasion (Positive) 125(0.89-1.75) 0.1918

Adjuvant Chemotherapy (> 3 cycles) 0.62(0.42-0.91) 0.0136

S1Q0A4 Expression (Positive) 153(113-2.08) 0.OQ58

F. Clinico-pathologic & Tumor Location (Body/Tail) 138(0.95-2.00) 0.0927

S100A4 Tumor Size (>20 mm) 1.72 (1.21 - 2.44) 0.0025

Margin Involvement (Positive) 176(132-2.36) 0.0001

Lymph Node Metastases (Positive) 145(107-195) 0.0172

Adjuvant Chemotherapy (> 3 cycles) 0.62(0.43-0.91) 0.0142

S100A4 Expression (Positive) 1.59 (1.18-2.15) 0.0024 G. Ciinico-pathoiogie & Tumor Size (> 20 mm) 1.74(1.22-2.47) 0.0020

S100A4 (Final Mode!) Margin involvement (Positive) 1.79(1.34-2.39) <0.0001

Lymph Node Metastases (Positive) 1.42(1.05-1.92) 0.0224

Adjuvant Chemotherapy (> 3 cycles) 0.65(0.44-0,94) 0.0224

S100A4 Expression (Positive) 1.61(1.19-2.17) 0.0019

H. Clinico-pathologic, Tumor Location (Body/Tail) 1.28(0.87-1.88) 0.2018

S100A4&S100A2 Tumor Size (>20 mm) 1.57 (1.09-2.25) 0.0148

(n = 294) Margin Involvement (Positive) 1.75(1.30-2.35) 0.0002

Lymph Node Metastases (Positive) 1.48 (1.09-2.01) 0.0126

Perineural Invasion (Positive) 1.24 (0.87-1.75) 0.2280

Vascular Invasion (Positive) 1.09(0.81-1.46) 0.5657

Adjuvant Chemotherapy (> 3 cycles) 0.65(0.44-0.96) 0.0310

S100A4 Expression (Positive) 1.42(1.04-1.93) 0.0293

S100A2 Expression (Positive) 1.80 (1.17-2.76) 0.0074 i, Ginico-pathoiogic Tumor Location (Body/Tail) 1.28(0.87-1.87) 0.2086

S100A4&S1QQA2 Tumor Size (> 20 mm) 1.57(1.10-2.26) 0.0140

Margin involvement (Positive) 174(1.30-2.34) 0.OQO2

Lymph Node Metastases (Positive) 1.48(1.09-2.01) 0.0126

Perineural invasion (Positive) 1.25(0.88-1.77) 0.2045

Adjuvant Chemotherapy (≥ 3 cycles) 0.65(0.44-0.96) 0.0302

S10OA4 Expression (Positive) 1.44(1.06-196) Q.O2O7

S100A2 Expression (Positive) 1.81(1.18-2.77) 0.0069

J. Clinico-pathologic, Tumor Size (>20 mm) 1.58(1.10-2.27) 0.0127

S100A4&S100A2 Margin Involvement (Positive) 1.76(1.31-2.37) 0.0002

Lymph Node Metastases (Positive) 1.46 (1.08- 1.99) 0.0148

Perineural Invasion (Positive) 1.27 (0.90 - 1.79) 0.1743

Adjuvant Chemotherapy (> 3 cycles) 0.67(0.46-0.99) 0.0425

S100A4 Expression (Positive) 1.44(1.06-1.96) 0.0196

S100A2 Expression (Positive) 1.86(1.21 -2.84) 0.0044

K. Clinico-pathologic, Tumor Size (> 20 mm) 1.63(1.14-2.34) 0.0077

S1QOA4&S100A2 Margin involvement (Positive) 181(1.35-2.42) <0.0001

(Finaf Mode!} Lymph Node Metastases (Positive) 1.48(1.09-2,02) 0.0120

Adjuvant Chemotherapy [≥ 3 cycles) 0.67(0.46-0.99) 0.0441

S100A4 Expression (Positive) 149(1.10-2.02) 0.0102

S100A2 Expression (Positive) 1.89(1.24-2.90) 0.0033

Table 3: Cohort Chamcteristics of Patients with Colorectal Cancer

cT1/2VsT3/4 'NOVsNΪ/2 5 ^eGrade l/ll Vs Grade III f Cytoplasmic + nuclear H-score > 200 β Peto-Peto-Wilcoxon

Table 4. Cohort Characteristics of Patients with Breast Cancer

Characteristics

Median Length of Follow-Up (months) 68 (range 0-159) Median Age (years) 54 (range 24-87) Tumor Size (mm)

0-10 38/228(17%) 11 -20 96/228 (42%) 21 -50 85/228 (37%) >50 9/228 I Tumor Grade

38/228(17%) 89/228(39%) 100/228 (44%)

Lymph Node Metastasis

0 128/228(56%)

1-3 68/228 (30%)

4-10 22/228 (10%)

>10 10/228 (4%)

Esfrogen Receptor Positive 156/227 (69%) Progesterone Receptor Positive 133/228(58%) HER-2 Positive (FISH) 38/218(17%) Total Recurrences 54/228 (24%) Breast Cancer Deaths 36/228(16%) Five-year Recurrence-Free Survival 79% Five-year Breast Cancer-Specific Survival 85% Notchi Positive 88/228 (39%)

FISH, fluorescence in situ hybridization.

Example 2: Expression of S100A4 Predicts Survival after Pancreatectomy

Positive expression of S100A4 co-segregated with poor disease-specific survival after pancreatectomy in both the training and validation sets (Figure 1). In the training set, 34 out of 58 patients (59%) with analyzable tissue had positive expression of S100A4 and had a significantly poor outcome (median survival 21.9 Vs 9.5 months; P = 0.0001). S100A4 expression was also an independent prognostic variable on multivariate analysis (HR = 3.39, 95% CI = 1.64 - 6.99; P = 0.0010). These findings were recapitulated in the validation set with 163 out of 260 patients (63%) with analyzable tissue demonstrating positive expression of S100A4 which was again associated with poor outcome (median survival 29.7 Vs 15.7 months; P = 0.0003). Positive S100A4 expression was also an independent prognostic variable on multivariate analysis (HR = 1.61, 95% CI = 1.19 - 2.17; P = 0.0019).

Example 3: Expression of S100A4 Predicts Response to Adjuvant Chemotherapy A subgroup analysis was performed to investigate the effect of S100A4 nuclear expression on chemotherapy response using the validation cohort (patients in the training cohort were chemo-naϊve). The patients were stratified according to S100A4 expression. The group of patients with nuclear expression of S100A4 did not benefit from adjuvant chemotherapy (median survival 19.4 Vs 13.5 months; P = 0.2094) (Figure 2). The group of patients from which samples showing no staining were derived showed significant survival benefits in response to adjuvant chemotherapy (median survival 36.5 Vs 18.4 months; P = 0.0488) (Figure 2).

Example 4: Expression of S100A4 and S100A2 Stratifies Risk Groups after Pancreatectomy

S100A2 calcium-binding protein was previously investigated and reported as an independent prognostic factor after pancreatectomy for pancreatic cancer. To investigate the combining effects of S100A4 and S100A2 in predicting prognosis after pancreatectomy, the patients in the validation cohorts were grouped according to S100A4 expression and stratified using S100A2 expression. It was found that there were three distinct prognostic groups after pancreatectomy according to S100A4 and S100A2 expressions. Patients with S100A4 negative/S100A2 negative expression had the best prognosis, followed by patients with S100A4 positive/S100A2 negative expression. The median survivals were 29.8 Vs 17.1 Vs 11.9 months (P < 0.0001). This same effect can be observed when the training and validation cohorts were combined together for the analyses. Example 5: Expression of S100A4 Predicts Response to Adjuvant Chemotherapy in Colorectal Cancer

The present inventors assessed the relationship of aberrant S100A4 calcium- binding protein (metastatin) expression, with disease specific survival and adjuvant chemotherapy response in a cohort of 382 patients who underwent operative resection for colorectal cancer.

Tumors with positive or high S100A4 expression were defined by reference to a combined (nuclear and cytoplasmic) H-score of > 200 (H-score = intensity x %, intensity = 0 to 3 and % = 0 to 100). Positive or high S100A4 (nuclear and/or cytoplasmic) expression was a poor prognostic factor in the colorectal cancer (5-yr survival 82.7% Vs 69.6%, P = 0.0021), the colon cancer cohort (5-yr survival 85.9% Vs 68.4%, P = 0.0339) and rectal cancer cohort (5-year survival 78.8% Vs 70.2%, P = 0.0387) cohort.

Measurement of S100A4 expression in resected colorectal cancer has the potential to better target individuals who will benefit from adjuvant chemotherapy.

Improved selection for adjuvant chemotherapy in colorectal cancer will improve overall outcomes through treating those that are most likely to benefit, and directing others to clinical trials of novel therapeutic strategies.

Example 6: S100A4 Expression Predicts Response to Adjuvant Endocrine Therapy after Curative Resection for Breast Cancer

The present inventors assessed the relationship of aberrant S100A4 calcium- binding protein (metastatin) expression, with disease specific survival and adjuvant endocrine therapy response in a cohort of 292 patients who underwent curative resections for breast cancer.

Tumors with positive or high S100A4 expression were defined by reference to stromal intensity of 3 and low or negative expression was defined as stromal intensity of 0 to 2 (scoring was performed with stromal intensity of 0 to 3).

Aberrant S100A4 expression was prognostic of a beneficial response to adjuvant endocrine therapy in the breast cancer cohort (Figure 5). Patients with no or low S100A4 expression had a survival benefit after adjuvant endocrine therapy (5-year survival 90.1% Vs 81.2%, P = 0.0809, borderline significant). Patients with positive or high S100A4 expression did not benefit from adjuvant endocrine therapy (5-year survival 74.0% Vs 71.7%, P = 0.8459). Example 7: S100A4 Expression Predicts Response to Adjuvant Chemotherapy after Surgery for Ovarian Cancer

The present inventors assessed the relationship of aberrant S100A4 calcium- binding protein (metastatin) expression, with disease specific survival and adjuvant chemotherapy response in a cohort of 201 patients who underwent operative resection for ovarian cancer (serous cancer Grade 1 to 3).

Tumors with positive or high S100A4 expression were defined as stain intensity of 3 and tumors with low or no S100A4 expression were defined as stain intensity of 0 to 2 (the tissue was scored as intensity of 1 to 3 and percentage of cells stained). Aberrant S100A4 expression was prognostic of a beneficial response response to adjuvant chemotherapy in the ovarian cancer cohort (Figure 6). Patients with positive or high S100A4 expression had significant survival benefit after adjuvant chemotherapy (median survival of 88.7 Vs 35.5 months P = 0.0036).

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Claims

1. A method for predicting a response to a treatment regime, for determining an optimal treatment regime, or for predicting the likelihood or duration of survival of a subject suffering from or suspected of suffering from cancer, the method comprising: performing an assay to detect expression and/or activity of a S100A4 gene product in a biological sample of the subject, using the amount of aberrant levels of S100A4 as a basis for predicting the subject's response to the treatment regime, for determining the optimal treatment regime, or for predicting the likelihood or duration of survival of the patient.

2. The method of claim 1, wherein a subject having expression and/or activity of the S100A4 gene product above a predetermined level is unlikely to have a beneficial response to the treatment regime, or wherein a subject having expression and/or activity of the S100A4 gene product at or below the predetermined level is likely to have a beneficial response to the treatment regime.

3. The method of claim 2, wherein the cancer is pancreatic cancer, colorectal cancer, or breast cancer.

4. The method of claim 1, wherein a subject having expression and/or activity of the S100A4 gene product at or above a predetermined level is likely to have a beneficial response to the treatment regime, or wherein a subject having expression and/or activity of the S100A4 gene product below the predetermined level is unlikely to have a beneficial response to the treatment regime.

5. The method of claim 4, wherein the cancer is ovarian cancer.

6. The method of any one of claims 1 to 5, wherein the S100A4 gene product is mRNA.

7. The method of claim 6, wherein the mRNA has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

8. The method of any one of claims 1 to 5, wherein the S100A4 gene product is a S 100A4 protein.

9. The method of claim 8, wherein the S100A4 protein comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 3.

10. The method of any one of claims 2, 3, or 6 to 9, wherein the expression and/or activity of the S100A4 gene product is elevated relative to a reference sample from a normal or responsive subject.

11. The method of claim 10, said method comprising:

(i) determining the level of expression and/or activity of the S100A4 gene product in a biological sample from the subject; and

(ii) comparing the level of expression and/or activity of the S100A4 gene product at (i) to the level of expression and/or activity of the S 100A4 gene product in a reference sample from a normal or responsive subject, wherein an elevated level of expression and/or activity of the S100A4 gene product in the biological sample relative to the reference sample indicates that the subject is unlikely to have a beneficial response to the treatment regime.

12. The method of any one of claims 4 to 9, wherein the expression and/or activity of the S100A4 gene product is reduced relative to a reference sample from a normal or responsive subject.

13. The method of claim 12, said method comprising:

(i) determining the level of expression and/or activity of the S100A4 gene product in a biological sample from the subject; and (ii) comparing the level of expression and/or activity of the S100A4 gene product at (i) to the level of expression and/or activity of the S100A4 gene product in a reference sample from a normal or responsive subject, wherein a reduced level of expression and/or activity of the S100A4 gene product in the biological sample relative to the reference sample indicates that the subject is unlikely to have a beneficial response to the treatment regime.

14. A method of any one of claims 2, 3, or 6 to 11, further comprising performing an assay to detect expression and/or activity of a S100A2 gene product in a biological sample of the subject, the method comprising: performing an assay to detect expression and/or activity of a S100A2 gene product in a biological sample of the subject, using the amount of aberrant levels S100A4 and S100A2 as a basis for predicting the subject's response to a treatment regime, for determining the optimal treatment regime, or for predicting the likelihood or duration of survival of the patient.

5 15. The method of claim 14, wherein a subject having expression and/or activity of the S100A2 and S100A4 gene products above predetermined levels is unlikely to have a beneficial response to the treatment regime, or wherein a subject having expression and/or activity of the S100A2 and S100A4 gene products at or below predetermined levels is likely to have a beneficial response to the treatment regime. 10

16. The method of any one of claims 1 to 15, wherein the treatment regime comprises surgery and/or administering a therapeutic agent.

17. The method of claim 16, wherein the surgery comprises a pancreatectomy, 15 partial colectomy, breastectomy, or and oophorectomy.

18. The method of claim 16, wherein the therapeutic agent comprises a nucleoside analog.

20 19. The method of claim 18, wherein the nucleoside analog is a pyrimidine analog.

20. The method of claim 19, wherein the pyrimidine analog is a 2', T- difluoronucleoside.

25 21. The method of claim 20, wherein the 2', 2'-difluoronucleoside is gemcitabine or a derivative thereof.

22. The method of claim 16, wherein the therapeutic agent is an LHRH analogue, an aromatase inhibitor, tamoxifen, or fulvestrant.

30

23. The method of claim 16, wherein the therapeutic agent is oxaliplatin, paclitaxel, docetaxel, or irinotecan.

24. A method of treating cancer in a subject, the method comprising: performing the method according to any one of claims 1 to 23 to determine whether or not the subject is likely to have a beneficial response to a treatment regime; and treating the subject according to the treatment regime if they are likely to have a beneficial response to the treatment regime.

25. Use of a therapeutic agent to treat a subject who has been previously determined to be likely to have a beneficial response to treatment with the therapeutic agent by virtue of having aberrant levels of expression and/or activity of a S100A4 gene product in a biological sample therefrom.