WO2011077165A1

WO2011077165A1 - Prognostic marker

Info

Publication number: WO2011077165A1
Application number: PCT/GB2010/052205
Authority: WO
Inventors: Julie Gormley; Shauna Hegarty; Shane Olwill
Original assignee: Fusion Antibodies Limited
Priority date: 2009-12-23
Filing date: 2010-12-23
Publication date: 2011-06-30
Also published as: GB0922553D0

Abstract

The invention provides a method to predict prognostic outcome in a cancer patient, the method comprising determining expression of Cathepsin S in sample of cells from a tumour from said patient to determine an expression value, comparing the expression value with one or more reference expression values; and correlating the determined expression value in said sample of cells with a predicted prognostic outcome. In a patient who is not subject to chemotherapy treatment, a higher determined level of expression of Cathepsin S is indicative of poorer prognostic outcome (i.e. decreased survival rate) than a lower determined level of expression of Cathepsin S. In contrast, in a patient who is subject to chemotherapy treatment, a higher determined level of expression of Cathepsin S is indicative of an improved prognostic outcome for said patient.

Description

Prognostic marker Field of the Invention

This application relates to methods for prediction of therapeutic outcome in the treatment of cancer, for example colorectal cancer. Background to the Invention

Colorectal cancer is one of the principal causes of cancer deaths in the Western world. The long-term survival of colorectal cancer patients depends on a number of factors, including the cancer stage and the development of metastatic disease. In some cases, neoadjuvant and adjuvant chemotherapeutic and radiotherapy treatments are employed to prevent and treat metastatic disease, either alone or in conjunction with surgery.

Fluoropyrimidines such as 5-FU are used in the treatment of many cancers, including gastrointestinal, breast and head and neck cancers. 5- FU is converted intracellular^ to fluorodeoxyuridine monophosphate FdUMP, which, together with 5,10-methylene tetrahydrofolate (CH₂THF) forms a stable ternary complex with thymidylate synthase (TS), resulting in enzyme inhibition. TS catalyses the reductive methylation of deoxyuridine monophosphate (dUMP) by CH₂THF to produce deoxythymidine monophosphate (dTMP) and dihydrofolate (Longley et al Nat Rev Cancer, 3:330-338, 2003). As this reaction provides the sole de novo intracellular source of dTMP, which is essential for DNA replication and repair, TS inhibition results in DNA damage. Non-TS-directed mechanisms of cytotoxicity have also been described for 5-FU, such as misincorporation of fluoronucleotides into DNA and RNA (Longley et al Nat Rev Cancer, 3:330-338, 2003).

Despite recent improvements in chemotherapy and the development of more effective combination treatments, the response to chemotherapy remains comparatively low. In view of the potentially deleterious side- effects of these treatments, as well as their cost, there is a need for more effective prediction of responsiveness to such chemotherapy and to be able to predict which particular chemotherapy will be most effective in a particular patient.

Cathepsin S (Cat S) is a member of the papain superfamily of lysosomal cysteine proteases. As described in the present inventors' applications, PCT/GB2007/001312 and PCT/GB2007/050634, Cathepsin S inhibitors, such as antibodies, may be used in the treatment of cancer and other angiogenesis related conditions. Summary of the Invention

The present inventors have investigated the expression of Cathepsin S in colorectal cancer tumours and have demonstrated that Cathepsin S expression varies dependent on the grade of tumour. Moreover, the inventors have demonstrated correlations between survival and Cathepsin S expression in both cancer patients treated by surgery alone and cancer patients treated with surgery and adjuvant chemotherapy. Of particular surprise, was the demonstration that the correlation between survival and Cathepsin S expression differs between such chemotherapy treated and chemotherapy untreated populations of colorectal cancer patients. Accordingly, in a first aspect of the invention, there is provided a method to predict prognostic outcome in a cancer patient, said method comprising

(a) providing a sample of cells from a tumour from said patient,

(b) determining expression of Cathepsin S in said sample of cells to determine an expression value,

c) comparing the expression value with one or more reference expression values; and

correlating the determined expression value in said sample of cells with a predicted prognostic outcome.

The inventors have identified a correlation between expression of

Cathepsin S in tumour cells and survival prognosis in patients treated with surgery alone (in the absence of chemotherapy), such that colorectal cancer patients whose tumour samples comprise relatively high levels of Cathepsin S compared to an average for all tumour patients were found to have poorer survival rates, whereas patients whose tumour samples comprise low levels of Cathepsin S were found to have increased survival rates. Accordingly, in one embodiment of the first aspect of the invention, in which the patient is a patient who is not subject to chemotherapy treatment, a higher determined level of expression of Cathepsin S is indicative of poorer prognostic outcome (i.e. decreased survival rate) than a lower determined level of expression of Cathepsin S.

The inventors further investigated the relationship between Cathepsin S expression level and responsiveness to treatment with adjuvant chemotherapy. In contrast to the situation where no chemotherapy was administered, where patients were treated with chemotherapy, the reverse relationship between Cathepsin S expression in tumour cells and survival outcome was observed. As described in the Examples, the inventors have demonstrated that, in subpopulations of patients subject to chemotherapy, the relationship between Cathepsin S expression and predicted survival is such that a higher determined level of expression of Cathepsin S is indicative of improved survival outcome compared to patients also subject to chemotherapy but in the tumours of whom expression of Cathepsin S is lower.

Accordingly in one embodiment of the first aspect of the invention, in which the patient is a patient who is subject to chemotherapy treatment, a higher determined level of expression (or expression value) of Cathepsin S in said tumour relative to a lower determined level of expression (or expression value) in said tumour is indicative of an improved prognostic outcome for said patient.

The invention further provides as a second aspect a method to predict prognostic outcome in a cancer patient subject to chemotherapy, said method comprising:

(a) providing a sample of cells from a tumour from said patient,

(b) determining expression of Cathepsin S in said sample of cells to determine an expression value, and

correlating the determined expression value in said sample of cells with a predicted prognostic outcome with chemotherapy treatment.

In the context of the present inventions cancer patient "subject to chemotherapy" should be understood to refer to a cancer patient who is currently receiving chemotherapy treatment or a cancer patient who will undergo chemotherapy treatment. In one embodiment, where the patient is not currently receiving chemotherapy treatment but is to undergo chemotherapy treatment, said treatment may be initiated within 1 year, for example within 9 months, such as within 6 months, for example within 3 months, such as within 1 month from the provision of the sample employed in the method.

In a particular embodiment of this aspect of the invention, a higher determined level of expression (or expression value) of Cathepsin S in said tumour relative to a lower determined level of expression (or expression value) in said tumour is indicative of positive response to said chemotherapy and an improved prognostic outcome for said patient.

The invention may therefore be used in discriminating between

subpopulations of cancer patients who will benefit or not benefit from treatment with chemotherapy.

Accordingly, in a third aspect of the invention, there is provided a method for predicting the response of a patient to chemotherapy, the method comprising

(a) providing a sample of cells from a tumour from said patient,

(c) correlating the expression value of Cathepsin S in said sample of cells with likely responsiveness of the patient to chemotherapy.

In one embodiment, the patient is a patient who has undergone or will undergo surgical treatment for said tumour.

The inventors have found that there is a correlation between high expression of Cathepsin S and a positive response to treatment with chemotherapy. Accordingly, in one embodiment of the third aspect of the invention, relative to a lower determined expression value or level of Cathepsin S in said tumour, a higher determined expression value or level in said tumour is predictive of improved clinical outcome in the presence of chemotherapy treatment compared to in the absence of chemotherapy treatment.

Accordingly, where it is determined that the tumour from a cancer patient has high expression of Cathepsin S, treatment with chemotherapy should be considered. In contrast, in our patient in which cells of the tumour tissue displays low expression of Cathepsin S, chemotherapy may not be beneficial. Indeed, the use of chemotherapy in such patients may be contraindicated, for example to avoid unnecessary side-effects. The decision as to whether or not to employ chemotherapy in such patients may take account of a number of factors other than the

expression level of Cathepsin S, for example clinical symptoms, existing or previous therapeutic regimens, pathological data and the stage of cancer in the patient at the time of testing.

In one embodiment, the sample of cells from a tumour is a sample of tumour cells. In an alternative embodiment, said sample may comprise, in addition to tumour cells, tumour-associated cells. The cells for which Cathepsin S expression is determined may be tumour cells and/or tumour- associated cells. In a particular embodiment of the methods of the invention, the cells for which Cathepsin S expression is determined are tumour cells.

The finding that, in patients with high expression of Cathepsin S in tumours, clinical outcome is much more favourable in the presence of adjunct chemotherapy enables the early intervention with such adjunct chemotherapy at a stage of disease in which such treatment may not normally be considered as required. Accordingly, the invention may find particular use in determining whether or not to employ chemotherapeutic agent(s) at early stages of disease, for example in stage I or stage II cancer.

Accordingly, in one embodiment of any of the invention, the cancer is a stage I or stage II cancer.

In one embodiment of the invention, the cancer is colorectal cancer.

In the methods of the invention, expression may be measured by any suitable means. For example, expression levels can be measured using a molecular biological-based assay method, for example, northern blot methods, Southern blot methods, western blot methods, transcription mediated amplification etc or an immunological assay method such as ELISA, dot blot methods, microArray techniques, immunohistochemistry techniques, or radioimmunoassay techniques.

The reference expression value against which the determined expression is compared may be an average expression value for a matched healthy tissue or indeed an average expression value for Cathepsin S in a population of cells from a tumour of the same type as the tumour cell being investigated.

In one embodiment, the expression value comprises a score assigned to the degree of expression of Cathepsin S in the sample of cells from the tumour with the reference values comprising a discreet number of score values, from which prognostic outcome has been determined for a population of samples.

A fourth aspect of the invention provides a method of treatment of cancer in a patient in need thereof, wherein said cancer is characterised by high expression of Cathepsin S, said method comprising administration of a chemotherapeutic agent to said patient.

In a fifth aspect of the invention, there is provided the use of a

chemotherapeutic agent in the preparation of a medicament for the treatment of cancer in a patient in which said cancer is characterised as expressing a high level of Cathepsin S relative to a predetermined value or level. A sixth aspect of invention provides a chemotherapeutic agent for use in the treatment of cancer in a patient whose cancer is a cancer expressing a high level of Cathepsin S relative to a predetermined value or level.

In any of the fourth to sixth aspects of the invention, the cancer may be a stage I or stage II cancer.

The second or third aspects of the invention may be used to predict outcome of or suitability for treatment with any chemotherapeutic agent. Accordingly, the chemotherapeutic agents for use in any aspect of the present invention may be any chemotherapeutic agent, for example a thymidylate synthase inhibitor, a topoisomerase inhibitor, a platinum cytotoxic drug, or a folate inhibitor. In a particular embodiment of the invention, the chemotherapeutic agent is a thymidylate synthase inhibitor, for example a fluoropyrimidine inhibitor such as 5-FU. The chemotherapeutic agents for use in any aspect of the invention may be given as a first line treatment, for example after biopsy. However, in a particular embodiment, the chemotherapeutic treatment is given as adjuvant chemotherapeutic treatment post surgery.

In one embodiment of the invention, the cancer is colorectal cancer.

Furthermore, the discovery by the present inventors that, in a large proportion of cases, there is a correlation between tumour grade and expression of Cathepsin S enables the use of measurement of cathepsin- S expression as at least one criteria in the classification of the grade of particular tumour. Accordingly, in a seventh aspect the invention, there is provided a method of determining the grade of a tumour, the method comprising providing a sample of cells of said tumour, determining the expression of Cathepsin S in said sample, comparing the expression with one or more control samples, wherein a low expression of Cathepsin S is indicative of a poorly differentiated tumour and high expression of Cathepsin S is indicative of a highly differentiated tumour.

In any of the fourth, fifth, sixth or seventh aspects of the invention, the predetermined value or level may be the value or level of Cathepsin S expression in non-cancerous tissue of the same type as the cancer. For example, where the cancer is colorectal cancer, the Cathepsin S level in the cancer may be compared to the Cathepsin level in non-cancerous colorectal tissue. In one embodiment of the invention, a high level of Cathepsin S relative to a non-cancerous tissue is a level in which the detected Cathepsin S expression is more than 1 .5 times or greater, for example greater than 2 times, such as greater than 3 times, for example greater than 10 times that in a non-cancerous tissue.

In one embodiment, a low level of Cathepsin S relative to a non-cancerous tissue is a level in which the detected Cathepsin S expression is less than 1 .5 times, for example less than 1 .25 times that in a non-cancerous tissue.

The control samples which may be used may be from high, moderately or poorly differentiated tumours, or indeed may be from normal tissue from the same patient. The invention may be used to monitor disease progression, for example using biopsy samples at different times. In such embodiments, instead of comparing the expression of Cathepsin S against a control sample from e.g. a different tissue source known not to have enhanced Cathepsin S expression, the expression of the Cathepsin S may be compared against a biological sample obtained from the same tissue at an earlier time point, for example from days, weeks or months earlier.

Preferred and alternative features of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.

Detailed Description

The present invention is based on the demonstration that the degree of expression of Cathepsin S in tumour cells is indicative of prognostic outcome in a patient, the correlation being dependent on whether or not the patient receives chemotherapy, and, moreover, that the degree of expression of Cathepsin S may be indicative of responsiveness of the tumour to a particular chemotherapeutic agent. Accordingly, Cathepsin S may be used as a biomarker for predicted clinical outcome for a patient.

Thus, by determining expression of Cathepsin S in cells of said tumour to determine an expression value, and comparing the expression value with one or more reference expression values; the prognostic outcome for a particular patient may be assessed.

The cells for which Cathepsin S expression may be determined in the methods of the invention may be tumour cells and/or tumour-associated cells. In a particular embodiment of the methods of the invention, the cells for which Cathepsin S expression is determined are tumour cells.

In the context of the present invention a tumour cell is any neoplastic cell, Tumour-associated cells are any cell found in the immediate environment of tumour cells, including tumour-associated stromal cells, tumour associated inflammatory cells, for example tumour-associated

macrophages, and tumour associated endothelial cells.

Cathepsin S expression may be measured using any means known in the art and may, for example, be based on the measurement of Cathepsin S polypeptides or nucleic acid encoding Cathepsin S. Thus, detection of Cathepsin S may involve the determination of mRNA, genomic DNA, or cDNA. mRNA may be detected by, for example, northern blot analysis, nucleotide array detection, PCR methods etc. Genomic DNA may be measured using, for example, Southern hybridisations. In an alternative embodiment, detection of Cathepsin S polypeptides may be carried out. In such methods, any known means of protein detection may be used to detect the entire or partial amino acid sequence of any of Cathepsin S proteins or polypeptides. Such means of protein detection may include, for example, Western blot analysis, protein array detection methods, ELISA based methods, immunoassays, such as

immunoprecipitation and/or immunofluorescence etc.

In one embodiment of the invention, antibodies, or antibody fragments, may be used to detect the expression of Cathepsin S. In such

embodiments, the antibodies or proteins may be immobilised on a solid support. Such supports are well-known in the art and include glass, polypropylene, polystyrene, dextrans etc. Although, in one embodiment of the invention, the expression of Cathepsin S is determined in vitro, in vivo methods may also be used. For example, Cathepsin S polypeptide expression may be determined using a labelled antibody directed against Cathepsin S, which is administered to the subject being investigated. The antibody may be labelled with, for example, a radioactive marker, the presence or location of which in a patient can then be detected using conventional imaging techniques.

Having measured the expression level of Cathepsin S in tumour cells of a patient, either in vitro or in vivo, the prognostic outcome for that patient may be predicted by comparing the determined expression level of

Cathepsin S with one or more reference expression values for Cathepsin S. In one embodiment of the invention, the reference expression value(s) against which the determined expression level of Cathepsin S is compared is/are control expression levels determined from "normal" non-cancerous tissue. Such control expression levels may be determined from "normal" tissue from the same patient, or indeed from an average determined from "normal" non-cancerous tissue from a population of subjects.

In an alternative embodiment, the control expression levels may be determined from cells of tumour tissue, preferably of the same cancer type, from a population of subjects. In such an embodiment, one or more average values or expression levels may be determined and used in the assessment of expression in a particular patient. In comparing the determined expression level of Cathepsin S from a particular patient with a reference expression value, the expression level being compared may be absolute expression levels, or alternatively, normalised expression levels. Such normalised expression levels may be obtained by determining the expression level of Cathepsin S with the expression of a constitutively expressed gene such as the actin gene.

In another embodiment, the control expression levels are determined from analysis of a population of subjects with the expression of Cathepsin S determined for tumour cells from each of the population in subjects and the population divided into a number of subsets dependent on the relative expression of Cathepsin S in the tumour from each subject. In such a way, a score may be applied to each range of expression. For example, a scoring system may be arranged in which 0 correlates to no expression, 1 correlates to low expression, 2 correlates to moderate expression, and 3 correlates to high expression. Thus on determining the expression of Cathepsin S from a particular sample, a score may be applied to the expression and this score used as the expression value to be compared with the reference expression value(s) in order to predict prognostic outcome. The range of cathepsin S expression levels considered to be low, moderate, high etc may depend on a number of factors including the type of cancer being considered, the patient population, the method of assessment etc. In one embodiment, a high level of Cathepsin S relative to a non-cancerous tissue is a level in which the detected Cathepsin S expression is more than 1 .5 times or greater, for example greater than 2 times, such as greater than 3 times, for example greater than 10 times that in a non-cancerous tissue. In one embodiment, a low level of Cathepsin S relative to a non-cancerous tissue is a level in which the detected

Cathepsin S expression is is less than 1 .5 times, for example less than 1 .25 times that in a non-cancerous tissue.

In one embodiment, a scoring scheme may be employed in which no detected expression of Cathepsin S is considered to be 0% expression and maximal expression of Cathepsin S in a population of samples is considered to be 100% expression. Each sample being assessed can then be assigned a % expression value relative to the maximal value. A scoring scheme may be employed dividing the % values into a number of discrete categories, such as no expression, low expression, moderate expression and high expression. For example, in one embodiment, a sample displaying more than 0% but <25% of the maximal Cathepsin S

expression may be considered as displaying low expression, a sample displaying expression in the range 25%- 75% of the maximal expression may be considered as displaying moderate expression and a sample displaying expression of greater than 75% of the maximal expression may be considered as displaying high expression. As noted above, depending on the type of cancer, patient population etc, the ranges considered low, moderate, high etc may differ. For example in another embodiment, a scoring scheme may be such that low expression in a sample is

considered a detected Cathepsin S expression which is <30% such as <20%,for example <15%, such as <10% relative to the maximal value and high expression in a sample is considered a detected Cathepsin S expression which is >60%,for example >70%, such as >80%, such as >90% expression relative to the maximal value in a population.

In one embodiment, the invention may be used to monitor disease progression, for example using biopsy samples at different times. In such embodiments, instead of comparing the expression of Cathepsin S against a reference expression value for Cathepsin S from e.g. a different tissue source known not have enhanced Cathepsin S expression, or indeed from a population of other subjects, the expression of Cathepsin S may be compared against a biological sample obtained from the same tissue at an earlier time point, for example from days, weeks or months earlier. Any suitable biological sample may be used in the methods of the invention; the nature of the tumour may determine the nature of the sample which is to be used in the methods of the invention. The sample may be, for example, a sample from a tumour tissue biopsy, bone marrow biopsy or circulating cells in e.g. blood. Other sources of biological sample may include plasma, serum, cerebrospinal fluid, urine, interstitial fluid, ascites fluid, sputum etc. Alternatively, e.g. where for example, the tumour is a gastrointestinal tumour, tumour cells may be isolated from faeces samples. Solid tumour samples may be collected, for example, in complete tissue culture medium with antibiotics. Cells may be manually teased from the tumour specimen or, where necessary, are enzymatically disaggregated by incubation with collagenase/DNAse and suspended in appropriate media containing, for example, human or animal sera. The biological sample may be obtained from biopsy or resected tissue, for example, by excision aspiration or punctuation, or any other suitable surgical method. In other embodiments, biopsy samples may be isolated and frozen or fixed in fixatives such as formalin. The samples may then be tested for expression levels. In an alternative embodiment, the biological samples may be obtained by "smear", obtained by any suitable means.

Antibody molecules

In a particular embodiment of the invention, antibodies may be used to determine the level of expression of Cathepsin S in a tumour sample. Any antibody molecule with specificity for Cathepsin S may be used in such an embodiment.

Antibody molecules include but are not limited to polyclonal, monoclonal, monospecific, polyspecific antibodies and fragments thereof and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. Intact (whole) antibodies comprise an immunoglobulin molecule consisting of heavy chains and light chains, each of which carries a variable region designated VH and VL, respectively. The variable region consists of three complementarity determining regions (CDRs, also known as hypervariable regions) and four framework regions (FR) or scaffolds. The CDR forms a complementary steric structure with the antigen molecule and determines the specificity of the antibody.

Fragments of antibodies may retain the binding ability of the intact antibody and may be used in place of the intact antibody. Accordingly, for the purposes of the present invention, unless the context demands otherwise, the term "antibodies" should be understood to encompass antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, dAb, and Fv fragments, scFvs, bispecific scFvs, diabodies, linear antibodies (see US patent 5, 641 , 870, Example 2 ; Zapata et al., Protein Eng 8 (10) : 1057-1062 [1995]) ; single-chain antibody molecules ; and multispecific antibodies formed from antibody fragments.

The Fab fragment consists of an entire L chain (VL and CL), together with VH and CH1 . Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. The F(ab') 2 fragment comprises two disulfide linked Fab fragments. Fd fragments consist of the VH and CH1 domains. Fv fragments consist of the VL and VH domains of a single antibody. Single-chain Fv fragments are antibody fragments that comprise the VH and VL domains connected by a linker which enables the scFv to form an antigen binding site (see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Diabodies are small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a multivalent fragment, i.e. a fragment having two antigen-binding sites (see, for example, EP 404 097 ; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90 : 6444-6448 (1993)). Further encompassed by fragments are individual CDRs.

In one embodiment of the present invention, the methods employ a

Cathepsin S antibody molecule, such as the antibody 1 E1 1 as described herein and in the applicant's WO2006/109045. The inventors have identified the amino acid sequences of the VH and VL regions of the intact antibody 1 E1 1 . The antibody V_H domain of this antibody comprises the amino acid sequence Seq ID No: 1 and /or the antibody V_L domain comprises the amino acid sequence Seq ID No: 3: Seq ID No: 1 :

VQLQESGGVLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWV AYITTGGVNTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA RHSYFDYWGQGTTVTVSS Seq ID No: 2:

DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQS

PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTH

VPPTFGSGTKLEIKR In another embodiment antibodies which may be used in the invention include antibodies or fragments thereof which bind to the binding region on Cathepsin S which has the amino acid sequence shown as Sequence ID No: 1 : ELPYGREDVLKEAVANKGPVSVGVDARHP (Sequence ID No: 3).

Details of such antibodies, for example the 1 E4 antibody, are provided in WO2008/044076. Antibodies for use in the invention herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U. S. Patent No. 4, 816, 567 ; and Morrison et ai, Proc. Natl. Acad. Sci. USA, 81 : 6851 -6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen- binding sequences derived from a non-human primate(e. g. Old World Monkey, Ape etc), and human constant region sequences.

Antibody molecules for use in the present invention may be produced in any suitable way, either naturally or synthetically. Such methods may include, for example, traditional hybridoma techniques (Kohler and Milstein

(1975) Nature, 256 :495-499), recombinant DNA techniques (see e.g. U.

S. Patent No. 4,816, 567), or phage display techniques using antibody libraries (see e.g. Clackson et ai (1991 ) Nature, 352: 624-628 and Marks et al. (1992) Bio/ Technology, 10: 779-783). Other antibody production techniques are described in Using Antibodies: A Laboratory Manual, eds.

Harlow et ai., Cold Spring Harbor Laboratory, 1999.

Chemotherapeutic Agents

As described above, in some embodiments of the invention, the

determined expression of Cathepsin S may be used to determine a treatment regimen including or excluding one or more chemotherapeutic agents.

The methods of the invention may be used to determine the suitability of use of any chemotherapeutic agent. For example such chemotherapeutic agents may include thymidylate synthase inhibitors, antimetabolites, nucleoside analogs, platinum cytotoxic agents or topoisomerase inhibitors. Examples of thymidylate synthase inhibitors which may be used in the invention include 5-FU, MTA and TDX. In one embodiment, the

thymidylate synthase inhibitor is 5-FU. An example of an antimetabolite which may be used is tomudex (TDX). Examples of platinum cytotoxic agents which may be used include cisplatin and oxaliplatin. In one embodiment of the invention, the chemotherapeutic agent is cisplatin. Examples of nucleoside analogs which may be used include but are not limited to gemcitabine and cytarabine.

In a particular embodiment of the invention, the chemotherapeutic agent is a topoisomerase inhibitor. In a particular embodiment, the topoisomerase inhibitor is a topoisomerase I inhibitor, for example a camptothecin. A suitable topoisomerase I inhibitor, which may be used in the present invention is irinotecan (CPT-1 1 ) or its active metabolite SN-38. CPT-1 1 specifically acts in the S phase of the cell cycle by stabilizing a reversible covalent reaction intermediate, referred to as a cleavage or cleavage complex and may also induces G2-M cell cycle arrest.

Chemotherapeutic agents which may be used in the present invention in addition or instead of the specific agents recited above, may include alkylating agents; alkyl sulfonates; aziridines; ethylenimines;

methylamelamines; nitrogen mustards; nitrosureas; anti-metabolites; folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti- adrenals; folic acid replenishers; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;

defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; ionidamine; mitoguazone; mitoxantrone.

In a particular embodiment of the invention, the chemotherapeutic agent is a fluoropyrimidine, e.g. 5-FU, or a metabolite thereof. Treatment may involve a single chemotherapeutic agent or a combination. In one embodiment of the invention, a combination of 5-FU and folinic acid (FA) is used. In another embodiment, the chemotherapeutic agents used may be a combination of a thymidylate synthase inhibitor, e.g. 5-FU, and a platinum cytotoxic agent, e.g. oxaliplatin.

Where reference is made to specific chemotherapeutic agents, it should be understood that analogues including biologically active derivatives and substantial equivalents thereof, which retain the antitumour activity of the specific agents, may be used. For example, a derivative of 5-FU, which may be used in the invention is the oral 5-FU analogue capecitibine (Xeloda ®).

Treatment

Treatment" includes any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.

"Treatment of cancer" includes treatment of conditions caused by cancerous growth and/or vascularisation and includes the treatment of neoplastic growths or tumours. Examples of cancers/tumours which the invention may find use include, for instance, sarcomas, including osteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , cervical and ovarian carcinoma, non-small cell lung cancer, hepatocellular carcinoma, lymphomas, including Hodgkin and non- Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, and leukemias, including acute lymphoblastic leukaemia and acute myeloblasts leukaemia, astrocytomas, gliomas and retinoblastomas.

In a particular embodiment of the invention, the cancer is colorectal cancer.

The invention may be particularly useful in the assessment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery.

Pharmaceutical Compositions

Pharmaceutical compositions for use in accordance with the present invention may comprise, in addition to active ingredients such as chemotherapeutic agents, a pharmaceutically acceptable excipient, a carrier, buffer stabiliser or other materials well known to those skilled in the art (see, for example, (Remington: the Science and Practice of Pharmacy, 21 ^st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.). Such materials may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such aspolyvinylpyrrolidone ; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine ; carbohydrates; chelating agents; tonicifiers; and surfactants.

The pharmaceutical compositions may also contain one or more further active compounds selected as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect the activity of the antibody molecule, nucleic acid or composition of the invention. The active ingredients (e.g. chemotherapeutic agents) may be

administered via microspheres, microcapsules liposomes, other microparticulate delivery systems. For example, active ingredients may be entrapped within microcapsules which may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. For further details, see Remington: the Science and Practice of Pharmacy, 21 ^st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.

Sustained-release preparations may be used for delivery of active agents. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the active agent, which matrices are in the form of shaped articles, e. g. films, suppositories or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl- methacrylate), or poly (vinylalcohol)), polylactides (U. S. Pat. No. 3, 773,

919), copolymers of L-glutamic acid and ethyl-Lglutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D- (-)-3-hydroxybutyric acid. Dose

The chemotherapeutic agents for use in the invention, as appropriate, are suitably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual dosage regimen will depend on a number of factors including the determined expression level of Cathepsin S, the condition being treated, its severity, the patient being treated, the agents being used, and will be at the discretion of the physician. The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration. The invention will now be described further in the following non-limiting examples. Reference is made to the accompanying figures in which:

Figure 1 shows examples of sections scored according to the scoring system for Cathepsin S in colorectal tissue microarrays (TMAs) with percentage of cases that are scored at each different level in brackets for NI240 cohort (n=212);

Figure 2 shows representative images of Cathepsin S expression patterns from normal colonic mucosa, colorectal cancer and lymph node metastatic tissue samples;

Figure 3 shows Kaplan-Meier survival analysis according to Cathepsin S expression level in (i) an untreated population and (ii) adjuvant- chemotherapy-treated population; the number of patients (n) in each group at the time of randomisation is shown; log-rank P values for comparison between low and high antigen expression levels are shown;

Figure 4 shows Kaplan-Meier analysis of survival according to adjuvant chemotherapy status in patients with (i) low Cathepsin S expression, (ii) moderate Cathepsin S expression and (iii) high Cathepsin S expression; the number of patients (n) in each group at the time of randomisation is shown; log rank P values for comparison between treated and untreated populations are shown; Figure 5 shows a Risk Plot to illustrate the relative risk of different interactions between antigen expression level and adjuvant chemotherapy treatment status at different stages of disease; and

Figure 6. shows Kaplan-Meier analysis of 8-year RFS and 8-year OS in the NI240 cohort according to (A) Cats expression level in the untreated group of patients; (B) treatment status in patients with low Cats

expression and; (C) treatment status in patients with high Cats

expression. Log rank P values are shown. Abbreviations: RFS,

recurrence-free survival; OS, overall survival; Cats, Cathepsin S; n, sample size; events, incidents of disease recurrence or death due to CRC.

Methods

Patient Samples

Three cohorts of patient samples were analysed for expression of

Cathepsin S. The NI240 cohort consisted of 232 cases of CRC and matched normal tissue (4 replicate cores per case). These samples were collected from Belfast City Hospital from 1994 until 1997. As described in the Examples, one sample which was identified as a Stage IV cancer was included in the analysis presented as Examples 1 -4 but was subsequently excluded from the analysis presented in Examples 5-8. The HB cohort consisted of 70 cases of Stage 3 or 4 CRC (12 replicates per case) and matched lymph nodes (4 replicates per case). These samples were collected at Beaumont Hospital, Dublin. The CO6161 TMA, obtained from US Biomax, 1 100 Taft St, Rockville, MD20850, USA, consisted of 296 cases of CRC (colorectal cancer), 1 1 cases of adjacent normal tissue and 17 cases of normal colonic mucosa (2 replicate cores per case) and was used to assess prevalence and associations with disease stage and grade. All samples were taken under the appropriate local ethical guidance with full consent from all patients. Due to local and institutional ethical constraints, different levels of information were available for each cohort. Among the clinicopathological data provided were clinical (TNM) stage, grade, extent of lymph node involvement, lymphvascular invasion (LVI) status, tumour location, age and gender of patients, recurrence, overall and disease-free survival.

Clinical Trial Structure and Treatment information

The NI240 cohort was designed as a randomised controlled phase III study to compare 16 weeks of De Gramont schedule FU/FA (5- fluorouracil/ folinic acid) adjuvant therapy to observation alone following potentially curative surgery. Two hundred and fifty four patients with stages II and III CRC were recruited in 1994 - 1997 from hospitals throughout Northern Ireland. Tissue was obtained from the initial resection specimen. There was full approval from the local research ethics committee and all involved hospitals, and all patients gave consent for the use of their specimens in research, according to the Declaration of

Helsinki. In arm 1 , protocol defined follow-up alone occurred. In arm 2, 8 cycles of intravenous FA 200 mg/nn² as a 2-hour infusion followed by bolus FU 400 mg/nn² and 22-hour infusion FU 400 mg/im² for 2 consecutive days every 2 weeks was used. Rectal cancer patients received post-operative adjuvant radiotherapy as clinically indicated. Patient age, sex, tumor stage and site were well balanced between arms. Median follow-up was 6.8 years. Of the 254 patients enrolled in study, only 212 were included in final IHC analysis; 42 cases could not be scored due to lack of availability of tissue or insufficient tissue. The median follow-up period for outcome was 6.08 years (Examples 1 -4) or 6.8 years (Examples 5-8).

Immunohistochemistry

All tissue microarrays were stained at the same time under identical conditions. Deparaffinization, antigen retrieval and immunohistochemistry (IHC) were performed on paraffin-embedded 4 μιτι tissue microarray sections and on whole sections using an automated IHC platform (Bond Max™, Leica Microsystems, Newcastle, U.K.). A polymer-based detection system (Refine cat#DS9800) was used with 3',3-Diaminobenzidine (DAB) as the chromogen resulting in a brown end colour. Anti-Cathepsin S monoclonal antibody (1 E1 1 ) was diluted 1/1000 in antibody diluent (cat#AR9352). On-board antigen retrieval was carried in ER 1 solution (cat#AR9961 ) for 10 minutes. Following detection of the antibody-antigen complex, sections were counterstained with haematoxylin. The positive controls included a sample of colonic adenocarcinoma and inflamed tonsil and the negative control was performed on all cases by excluding the primary antibody. All procedures were covered under ethical approval by NREC (08/NIR03/122).

Scoring

All cases were independently scored by two pathologists, who were blinded to clinical data. Tumour and normal colonic mucosa samples were scored as 0, 1 +, 2+ or 3+ for intensity of staining. In order to increase reliability and repeatability, this scoring regime was agreed by both investigators, based on observation of the staining, prior to independent scoring. Repeatability was assessed by the analysis of multiple cores from each specimen, which were randomly positioned throughout the series of arrays. In any cases of discordance (4%), cores were reviewed until a consensus was reached and scoring was further spot-checked by a third investigator Polarisation to the apical or basal membrane was noted.

Modal scores were determined for replicates of each case. For statistical analysis, scores were reclassified as low (0 and 1 +), moderate (2+) and high (3+) expression. In cases were biphasic or multiphasic distribution of staining occurred within the tumours or normal colonic mucosa samples, the intensity that covered the higher extent of the core was chosen.

Criteria were set to determine if sufficient tissue was available for reliable determination of Cats score and cases were excluded if insufficient tissue was available. All TMAs were stained at the same time under identical conditions using a previously established protocol with appropriate positive and negative controls, ensuring consistency in staining intensity across all cohorts.

Figure 1 illustrates examples of a section assigned to each score.

Statistics

Ordinal regression was used to evaluate the significance of difference in Cathepsin S expression level in matched tumour vs normal and metastatic vs primary tumour tissue. Ordinal regression was also used to test for association between Cathepsin S expression level and grade and stage of disease. Association between Cathepsin S expression and lymphvascular invasion status or location of tumour was analysed using the Pearson Chi- squared test. The primary clinical outcome variable for the NI240 cohort was overall survival with an event classified as death due to disease. As only one case of Stage 4 disease was found in this cohort; this case was grouped with Stage 3 cases for the purposes of statistics in Examples 1 -4, but was excluded from the analysis presented in Examples 5-8 In survival analyses, the primary clinical outcome variables were recurrence-free survival (RFS), defined as the time from randomisation to radiologically or histologically proven recurrence of CRC; and overall survival (OS), defined as the time from randomisation to CRC-related death. The follow-up time was censored at 100 months. As the aim of this retrospective study was to specifically investigate any potential links between Cats expression and cancer progression to death, non-CRC related death resulted in censoring of data at that time point. Survival times according to different variables were compared by the Kaplan-Meier method and univariate log rank test. Multivariate analysis was performed using Cox Regression modelling. Univariate and multivariate hazard ratios (HR) were calculated using Cox Proportional Hazards modelling. Cathepsin S expression level was treated as a continuous linear variable in the multivariate analysis. All reported P values were two-sided and P values of less that 0.05 were considered to be statistically significant. Statistics were performed using SPSS 17.0.

Results

Example 1 Aberrant expression of Cathepsin S in colorectal carcinoma and metastases compared to normal colorectal mucosa. (Prevalence of Cathepsin S in CRC)

Representative images of Cathepsin S expression patterns from normal colonic mucosa, colorectal cancer and lymph node metastatic tissue samples are shown in Figure 1 . Cathepsin S was found to be expressed in >95% CRC tumour samples (n=561 over 3 cohorts of patients). Low expression was found in 38% cases ('low expression' population was grouped with 'no expression' population), moderate expression was found in 43% cases and high expression was found in 19% cases. A low to negligible level of finely granular expression was displayed in normal colonic mucosa. A more coarsely granular expression was observed in tumour samples. Cathepsin S is highly expressed in subpopulations of stromal cells, predominantly tumour-associated macrophages, serving as an internal positive control for staining in all samples. The NI240 cohort contained matched normal colon for 176 samples of CRC. Ordinal regression analysis revealed a significant increase in Cathepsin S staining intensity in tumour compared to matched normal tissue (Table 1 ; p=0.000). Ordinal regression analysis revealed a significantly lower Cathepsin S expression intensity in lymph node metastatic tissue compared to matched primary tumour tissue (Table 1 ; p=0.03).

Table 1 Ordinal regression analysis to compare Cats expression levels 96% cases stain positive.

Example 2 Association of Cats expression with clinicopatholoqical characteristics

The inventors investigated the potential association of Cathepsin S expression intensity with well-known clinicopathological features. A significant association of decreasing Cathepsin S expression intensity with increasing grade of tumour was discovered (Table 2; p=0.000) Similar results were found (p=0.005) when the analysis was repeated; In some cases, loss of Cathepsin S expression with transition from moderately differentiated to poorly differentiated tumour was clearly visible. There was no significant association of Cathepsin S expression intensity with stage of disease, tumour location or lymphvascular invasion (LVI).

Table 2. Ordinal regression analysis to compare cathepsin S (CatS) expression levels:

Example 3 Association between CatS expression and survival (for overall survival where death is due to disease)

(Figure 3. - Kaplan-Meier survival curve and univariate log rank test for significance/ Table 3. Multivariate analysis by Cox regression. Figure 5. Risk Plot with increasing stage of disease)

The Belfast City (NI240) cohort had survival data with a median follow up time of 6.08 years. By the end of follow-up period 43% of patients had died, 34% directly due to disease. When all patients (212) in the NI240 cohort were considered, univariate analysis revealed no significant association between Cathepsin S expression and overall survival. However, in a multivariate model adjusted for the stage of disease, location of tumour and LVI status, which are heavily related to outcome, increasing Cathepsin S intensity level was significantly associated with reduced survival (hazard ratio for death 1 .779 (95 percent confidence interval, 1 .140-2.775); p=0.01 1 ).

Taking into consideration that selection for treatment in this trial was totally random, it is possible to analyse the untreated (i.e. surgery only) and treated (i.e. surgery and chemotherapy) populations independently. When the untreated population alone was interrogated, a trend of increasing Cathepsin S expression correlating to reduced survival was observed (Figure 3). This trend was not observed in the treated population, however.

Table 3. Multivariate analysis for all patients adjusted for Stage. Grade LVI status and tumour location. Hazard ratios are calculated by Cox

Regression modelling. (NB. One case of stage 4 grouped with stage 3 and 2 cases of synchronous location have been removed)

Example 4 Association between Cats Expression and benefit of adiuvant chemotherapy.

(Figure 4. Kaplan-Meier survival curve and univariate log rank test for significance/ Table 3. Multivariate analysis by Cox regression). as the effect of treatment or Cathepsin S expression level was shown not to differ according to the stage of disease, patients from all stages were pooled for the following analyses. In a pooled analysis for all patients, no significant benefit from adjuvant chemotherapy was observed. Upon stratification by stage of disease (or even when each stage was analysed independently) there remained no significant benefit of treatment with adjuvant chemotherapy. A significant interaction was observed, however, between Cathepsin S expression level and the benefit of treatment. By univariate analysis, for those patients with a high expression level of Cathepsin S, treatment with adjuvant chemotherapy had a significant benefit for survival (p=0.013). For those patients with a moderate

Cathepsin S expression level, there was no significant benefit of adjuvant chemotherapy and for those with a low Cathepsin S expression level, there is a trend of reduced benefit after treatment with chemotherapy. In a multivariate model, adjusted for stage of disease, the interaction between increasing Cathepsin S expression and treatment with chemotherapy was shown to have a significant effect on survival (hazard ratio for death 0.389, 95 percent confidence interval 0.198-0.763, p=0.006). In effect, interaction between these two variables, was shown to reverse the trend seen when analysing the effect of Cathepsin S expression alone. The relative risk upon interaction between these two variables with increasing stage of disease is illustrated in Figure 5.

The trend remained the same with stratification for stage of disease. When each stage subgroup was interrogated independently, the trend remained for stages 2 and 3 but was only significant for stage 3 (p= 0.262 and p=0.01 1 respectively).

The samples were further analysed after exclusion of a single sample from the NI240 cohort., which had been confirmed as being from a patient with Stage IV disease. The results are presented in Examples 5- 8. For these analyses, the follow-up time was extended from that of the analyses summarised in Examples 1 -4. Example 5 Prevalence of Cats in Colorectal Carcinoma and Metastatic Tissue (2)

After exclusion of the Stage 4 sample from the NI240 cohort, Cats expression in tumour tissue was again assessed across cohorts (n being 560). Low expression was found in 31 % of cases, moderate expression in 52% and high expression in 17% of cases. As in Example 1 , low to negligible level of finely granular cytoplasmic staining was displayed in normal colonic mucosa, whereas an intense, coarsely granular

cytoplasmic staining pattern was observed in tumour samples and matched metastatic tissue. Epithelial cells in 60% of the tumours contained Cats diffusely expressed throughout the cytoplasm but in the remainder of tumours, the expression was alternatively polarised to either the apical or basal pole of the epithelium. A decrease in Cats staining with transition from moderately to poorly differentiated tumour was observed Subpopulations of stromal cells, possibly tumour-associated macrophages, stained positive for Cats.

Cats expression in matched pairs of tumour and adjacent normal tissue could be compared for 175 out of 21 1 cases in the Nl CRC cohort, with remaining cases excluded due to insufficient tissue. A 1 .3-fold increase in Cats expression was found in tumour compared to normal tissue (P < 0.001 ). Matched lymph node metastatic tissue was available for 67 samples of CRC in the Beaumont Hospital cohort. Cats was found to be expressed in >95% cases of metastatic tumour tissue found in lymph nodes, with a significantly higher (1 .2-fold) expression in the primary tumour tissue compared to involved nodal tissue (P = 0.03). Example 6 Association of Cats Expression with Clinicopatholoqical Traits Table 4 summarises the clinicopathological information analysed with Table 5 summarising the association of cathepsin S expression with disease stage and tumour grade in all cohorts. Twenty-one cases of unknown grade were removed from the grade analysis. Association of Cats with tumour site and LVI status was assessed in the Beaumont Hospital and Nl Cancer Centre cohorts only. Three cases of synchronous location were removed in the tumour site analysis and 55 cases of unknown LVI status were removed from the LVI analysis. (Calculated P values from " rdinal regression and†Pearson chi-squared tests are shown;†Proximal (caecum, ascending colon, hepatic flexure, transverse colon); Distal (descending colon, sigmoid colon), Rectal (rectosigmoid colon, rectum); abbreviations: Cats, Cathepsin S; n, number; LVI, lymphovascular invasion). The results were almost identical to those described in Example 2, with a significant association of increasing Cats expression with decreasing tumour grade being noted (P = 0.005; Table 5) with no correlation between Cats expression and other pathological features being found.

Table 4. Clinicopathological information for Nl CRC adjuvant chemotherapy trial, Beaumont Hospital and US Biomax cohorts

Nl CRC

adjuvant US Beaumont chemotherapy Biomax n (%) = Hospital trial C06161

Patients 560 70 21 1 279

Matched normal samples 175 0 175 0

Matched lymph node 0 0 samples 67 67

65 (35-80) 57 (26-

Median (range) age (yrs) - 86)

Gender -Male 353 (63) 50 126 177

-Female 207 (37) 20 85 102

- Proximal 1 14 (41 ) 33 81

*Tumour - Distal 91 (32) 21 70

Site - Rectum 73 (26) 16 57

- Synchronous 3 (1 ) - 3

-1 22 (7) 0 0 22

Stage -II 318 (53) 0 136 182

(TNM) -III 206 (37) 64 75 67

-IV 14 (3) 6 - 8

-1 46 (8) 0 19 27

-II 403 (72) 60 160 183

Grade

-III 90 (16) 10 24 56

-Unknown 21 (4) - 8 13

-Y 79 (28) 34 45

LVI

-N 127 (45) 16 1 1 1 -

Status

-Unknown 75 (27) 20 55

NOTE: Information was not available where indicated (-).

*Proximal (caecum, ascending colon, hepatic flexure, transverse colon), Distal (descending colon, sigmoid colon), Rectal (rectosigmoid colon, rectum)

Abbreviations: LVI, lymphovascular invasion. Table 5. Association of Cats expression with clinicopathological features

Antigen Expression Level

Moderate

n (%) = Low (%) (%) High (%)

Grade: 539

Well differentiated (1) 46 (8) 16 (35) 17 (37) 13 (28)

0.005*

Moderately differentiated(ll) 403 (75) 142 (35) 180 (45) 81 (20)

Poorly differentiated (III) 90 (17) 46 (51 ) 32 (36) 12 (13)

Stage (TNM) 558

- 1 22 (4) 13 (59) 7 (32) 2 (9)

- II 318 (57) 120 (38) 141 (44) 57 (18)

0.15*

- Ill 206 (37) 72 (35) 87 (42) 47 (23)

- IV 14 (3) 7 (50) 5 (36) 2 (14)

Tumour Site† 278

Proximal 1 14 (41 ) 34 (30) 59 (52) 21 (18)

- Distal 91 (33) 26 (29) 47 (51 ) 18 (20) 0.98i

Rectum 73 (26) 23 (32) 35 (48) 15 (20)

LVI Status 206

- Yes 78 (38) 23 (30) 37 (47) 18 (23) 0.53i

- No 128 (62) 37 (29) 69 (54) 22 (17)

NOTE: Association of CatS expression with disease stage and tumour grade was assessed in all cohorts. Twenty cases of unknown grade were removed from the grade analysis. Association of CatS with tumour site and LVI status was assessed in the Beaumont Hospital and Nl Cancer Centre cohorts only. Three cases of synchronous location were removed in the tumour site analysis and 55 cases of unknown LVI status were removed from the LVI analysis. Calculated P values from Ordinal regression and ^Pearson chi- squared tests are shown.

†Proximal (caecum, ascending colon, hepatic flexure, transverse colon), Distal (descending colon, sigmoid colon), Rectal (rectosigmoid colon, rectum)

Abbreviations: CatS, Cathepsin S; n, number; LVI, lymphovascular invasion. Example 7 Association of Cats Expression with Survival (2)

Of the 254 patients enrolled in the Nl CRC trial, 21 1 were included in the survival analyses, 106 in the surgery alone ("untreated") group and 105 in the adjuvant FU/FA-treated ("treated") group. RFS and OS were monitored with a median follow-up time of 6.8 years. Forty three of the 57 patients (75%) with rectal cancer received adjuvant post-operative radiation therapy as per clinical guidelines extant at the time of the trial. Seven cases (16%) of rectal cancer recurred primarily locally, which is

comparable to standard post-surgical incidence rates for this time period (30). Since the relative levels of Cats (from low to moderate to high) were found to be similarly distributed among rectal patients compared to the entire patient cohort, rectal cases were included in the analysis.

As with the analysis presented for the cohort described in Example 3, in a pooled analysis for all patients, by the end of the follow-up period 43% of patients had died, 34% from CRC, and disease had recurred in 37% of cases. There was no evidence of an association between Cats expression and RFS or OS in this pooled group. Among untreated patients Cats expression was associated with poor 8- year RFS (P = 0.03; Fig 6A), with an estimated HR of 1 .72 (95% CI, 1 .13 to 2.66; P = 0.01 , Table 6). This trend, although apparent, was not found to be significant for 8-year OS (P = 0.08; Fig 6A), with an estimated HR of 1 .62 (95% CI, 1 .05 to 2.51 ; P = 0.03; Table 6). Among treated patients, there was no association between Cats and RFS or OS. Table 6. Univariate analysis for 8-year RFS and OS in (a) all patients according to Cats or treatment status; (b) treatment subgroups according to CatS; and (c) Cats stratified subgroups according to treatment

8-year RFS 8-year OS

% HR 95% CI P = % HR 95%CI P =

(a ) CatS (n=21 1 )

Low 63 63

Moderate 63 1.22 0.88 to 1.69 0.23 62 1.1 1 0.79 to 1.55 0.54

High 54 62

Treatment (n=21 1 )

Untreated 56 0.75 0.48 to 1.17 0.20 55 0.71 0.45 to 1.13 0.15

Treated 64 68

(b ) Untreated (n=106)

Low 67 65

Moderate 57 1.72 1.13 to 2.66 0.01 53 1.62 1.05 to 2.51 0.03

High 32 38

Treated (n=105)

Low 60 61

Moderate 68 0.84 0.51 to 1.39 0.50 70 0.74 0.44 to 1 .26 0.27

High 64 73

(c ) Low (n=66)

Untreated 67 65

1.34 0.60 to 3.19 0.45 1.33 0.56 to 3.15 0.52 Treated 60 61

Moderate (n=109)

Untreated 57 53

0.69 0.37 to 1.30 0.25 0.70 0.37 to 1.34 0.28 Treated 68 70

High (n=36)

Untreated 32 38

0.33 0.12 to 0.89 0.03 0.25 0.08 to 0.81 0.02 Treated 64 73

Abbreviations: RFS, recurrence-free survival; OS, overall survival; CatS, Cathepsin S; HR, hazard ratio; n, total number; CI, confidence interval; untreated, patients receiving no adjuvant chemotherapy; treated, patients treated with adjuvant FU/FA. Example 8 Association of Cats Expression with Benefit of Adjuvant chemotherapy

There was a non-significant trend towards improved RFS and OS in the 105 treated patients compared to the 106 untreated patients. A significant interaction was found between Cats expression and the RFS benefit from adjuvant FU/FA (P = 0.03) and this trend remained upon stratification for disease stage (P = 0.01 ). Similarly, there was a trend for OS (P = 0.02), which remained when stratified by stage (P = 0.01 ).

Among the 36 patients (17%) with high Cats expression, there was a significant benefit from treatment (P = 0.02 for RFS and P = 0.01 for OS; Fig 2C). The 8-year RFS HR was 0.33 (95% CI, 0.12 to 0.89; P = 0.03) and the 8-year OS HR was 0.25 (95% CI, 0.08 to 0.81 ; P = 0.02; Table 6). There was no evidence of treatment benefit in the 52% of patients with moderate Cats expression; the 8-year RFS HR was 0.69 (95% CI, 0.37 to 1 .30; P = 0.25) and the 8-year OS HR was 0.70 (95% CI, 0.37 to 1 .34; P = 0.28; Table 6). In the 31 % of patients with low Cats expression, again there was no evidence of treatment benefit. The 8-year RFS HR was 1 .34 (95% CI, 0.60 to 3.19; P = 0.45) and the 8-year OS HR was 1 .33 (95% CI, 0.56 to 3.15; P = 0.52; Table 6).

In a multivariate model, adjusted for disease stage, LVI status and tumour site, the interaction between Cats expression and treatment status was significant for both RFS (HR 0.46; 95% CI, 0.24 to 0.90; P = 0.02) and OS (HR 0.49; 95% CI, 0.50 to 0.96; P = 0.04; Table 7). The trend held when stratified for stage; RFS HR was 0.45 (95% CI, 0.23 to 0.88; P = 0.02) and OS HR was 0.46 (95% CI, 0.23 to 0.91 ; P = 0.03). When dichotomised stage subgroups were interrogated independently, the trend only reached significance in the stage III subgroup. The low patient numbers in stage subgroups limited the power of these analyses. The estimated HR for the interactive term was 0.57 (95% CI, 0.20 to 1 .61 ; P = 0.29) for RFS and 0.45 (95% CI, 0.15 to 1 .36; P = 0.16) for OS for stage II and 0.38 (95% CI, 0.15 to 0.93; P = 0.03) for RFS and 0.45 (95% CI, 0.18 to 1 .12; P = 0.09).

Table 7. Multivariate Cox Regression analysis of 8-year RFS and OS

8-year RFS 8-year OS

HR 95% CI P HR 95% CI P

CatS expression 1.81 1.17 to 2.82 0.08 1.47 0.96 to 2.26 0.08

Treatment status 1.25 0.59 to 2.66 0.56 1.22 0.57 to 2.61 0.60

†Treatment status oc

CatS expression 0.46 0.24 to 0.90 0.02 0.49 0.50 to 0.96 0.04

<0.0 <0.0

Stage 3.12 1.96 to 4.95 01 2.79 1.72 to 4.53 01

LVI- (Yes versus No/ 0.00 0.00

NOS) 1.90 1.19 to 3.03 7 2.04 1.24 to 3.35 5

*Tumour site (Proximal

versus Distal) 2.00 1.1 1 to 3.61 0.02 2.00 1.08 to 3.71 0.03

*Tumour site (Rectal 0.00

versus Distal) 2.36 1.28 to 4.36 6 2.10 1.1 1 to 3.96 0.03

NOTE: Tumour site was considered as a categorical variable comparing Proximal and Rectal locations to Distal. Three cases of synchronous location were excluded from the model.

†, interactive term for CatS expression and treatment status.

*Proximal (caecum, ascending colon, hepatic flexure, transverse colon), Distal (descending colon, sigmoid colon), Rectal (rectosigmoid, rectum).

Abbreviations: RFS, recurrence-free survival; OS, overall survival; CatS, Cathepsin S; HR, hazard ratio; CI, confidence interval; LVI, lymphovascular invasion; NOS, not otherwise specified. Table 8. Multivariate Cox Regression analysis of 8-year RFS and OS after Stratification for Disease Stage

8-year RFS 8-year OS

HR 95% CI P HR 95% CI P

CatS expression 1.83 1.18 to 2.85 0.008 1.52 0.98 to 2.34 0.06

Treatment status 1.29 0.61 to 2.76 0.51 1.29 0.60 to 2.77 0.51

†Treatment status oc

0.45 0.23 to 0.88 0.02 0.46 0.23 to 0.91 0.03 CatS expression

LVI- (Yes vs No/ NOS) 1.88 1.17 to 3.00 0.009 2.06 1.25 to 3.39 0.04

*Tumour site

1.99 1.10 to 3.59 0.02 2.05 1.10 to 3.82 0.02 (Proximal vs Distal)

*Tumour site (Rectal

2.38 1.29 to 4.42 0.006 2.16 1.14 to 4.09 0.02 vs Distal)

NOTE: Tumour site was considered as a categorical variable comparing Proximal and

Rectal locations to Distal. Three cases of synchronous location were excluded from the model.

†, interactive term for CatS expression and treatment status.

*Proximal (caecum, ascending colon, hepatic flexure, transverse colon), Distal

(descending colon, sigmoid colon), Rectal (rectosigmoid, rectum).

Abbreviations: RFS, recurrence-free survival; OS, overall survival; CatS, Cathepsin S;

HR, hazard ratio; CI, confidence interval; LVI, lymphovascular invasion; NOS, not otherwise specified.

Table 9. Multivariate Cox Regression analysis of 8-year RFS and OS for stage subgroups

8-year RFS 8-year OS

Stage II HR 95% CI P HR 95% CI P

CatS expression 2.18 1.07 to 4.47 0.03 2.25 1.09 to 4.68 0.03

Treatment status 1.47 0.40 to 5.38 0.56 1.43 0.38 to 5.41 0.60

†Treatment status oc

0.57 0.20 to 1.61 0.29 0.45 0.15 to 1.36 0.16 CatS expression

LVI- (Yes versus No/

1.57 0.78 to 3.15 0.20 2.1 1 1.01 to 4.38 0.05 NOS)

*Tumour site (Proximal

1.20 0.51 to 2.81 0.67 1.18 0.48 to 2.87 0.72 versus Distal)

*Tumour site (Rectal

1.95 0.82 to 4.63 0.13 1.86 0.75 to 4.63 0.18 versus Distal)

Stage III HR 95% CI P HR 95% CI P

CatS expression 1.59 0.90 to 2.83 0.1 1 1.16 0.66 to 2.02 0.61

Treatment status 1.25 0.49 to 3.20 0.65 1.28 0.50 to 3.30 0.61

† Treatment status oc

0.38 0.15 to 0.93 0.03 0.45 0.18 to 1.12 0.09 CatS expression

LVI- (Yes versus No/

2.20 1.12 to 4.32 0.02 2.29 1.12 to 4.68 0.02 NOS)

*Tumour site (Proximal

3.30 1.37 to 7.95 0.008 3.70 1.49 to 9.19 0.005 versus Distal)

*Tumour site (Rectal

3.29 1.31 to 8.23 0.01 2.93 1.15 to 7.43 0.02 versus Distal)

†, interactive term for CatS expression and treatment status.

*Proximal (ascending colon, caecum, transverse colon), Distal (descending colon, sigmoid colon), Rectal (rectosigmoid, rectum).

HR, hazard ratio; CI, confidence interval; LVI, lymphovascular invasion; NOS, not otherwise specified. All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1 . A method to predict prognostic outcome in a cancer patient, said method comprising (a) providing a sample of cells from a tumour from said patient,

correlating the determined expression value in said sample of cells with a predicted prognostic outcome;

wherein, where the patient is a patient who is subject to

chemotherapy treatment, a higher determined expression value of Cathepsin S in said tumour cells relative to a lower determined expression value is indicative of an improved prognostic outcome for said patient, wherein, where the patient is a patient who is not subject to chemotherapy treatment, a higher determined expression value of

Cathepsin S in said tumour cells relative to a lower determined expression value is indicative of poorer prognostic outcome for said patient.

2. The method according to claim 1 , wherein the patient is a patient who is presently undergoing chemotherapy treatment or is to begin chemotherapy treatment within 3 months.

3. A method for predicting the response of a cancer patient to chemotherapy, the method comprising

(a) providing a sample of cells from a tumour from said patient,

(b) determining expression of Cathepsin S in said sample of cells to determine an expression value, and (c) correlating the expression value of Cathepsin S in said sample of cells with likely responsiveness of the patient to chemotherapy.

4. The method according to claim 3, wherein, relative to a lower determined expression value in said tumour, a higher determined expression value in said tumour is predictive of improved clinical outcome in the presence of chemotherapy treatment compared to in the absence of chemotherapy treatment.

5. The method according to claim 3, wherein, relative to a higher determined expression value in said tumour, a lower determined expression value of cathepsin S in said tumour is predictive of poorer clinical outcome in the presence of chemotherapy treatment compared to in the absence of chemotherapy treatment.

6. The method according to any one of claims 3 to 5, wherein the method further comprises determining a therapeutic regimen for said patient based on the correlation of expression with likely responsiveness.

7. The method according to any one of claims 2 to 6, wherein the chemotherapy treatment comprises a thymidylate synthase inhibitor.

8. The method according to claim 7 wherein the chemotherapy treatment is 5-FU.

9. The method according to any one of claims 2 to 8, wherein said chemotherapy treatment is adjuvant chemotherapy treatment.

10. Use of a chemotherapeutic agent in the preparation of a

medicament for the treatment of cancer in a patient in which said cancer is characterised as expressing a high level of cathepsin S relative to a predetermined value.

1 1 . The use according to claim 10, wherein said patient is a patient identified as being suitable for such treatment using the method of any one of claims 3 to 6.

12. A chemotherapeutic agent for use in the treatment of cancer in a patient whose cancer is a cancer expressing a high level of cathepsin S relative to a reference value.

13. The chemotherapeutic agent according to claim 12, wherein said patient is a patient identified as being suitable for such treatment using the method of any one of claims 3 to 6.

14. A method for evaluating in vitro the grade of tumour cells from a subject with cancer, which method comprises:

(a) providing an in vitro sample of cells from a tumour from said subject;

(b) determining expression of Cathepsin S in said cells, wherein enhanced expression in the sample of cells compared to a predetermined value is indicative that said tumour is highly differentiated and a low expression in the sample of cells compared to a predetermined value is indicative that said tumour is poorly differentiated.

15. The method according to any one of claims 1 to 9 or 14, the use according to claim 10 or claim 1 1 or the chemotherapeutic agent according to claim 12 or claim 13, wherein the cancer is colorectal cancer.

16. The method according to any one of claims 1 to 9, claim 14, or claim 15, the use according to claim 10, claim 1 1 , claim 14, or claim 15, or the chemotherapeutic agent according to claim 12, claim 13, claim 14, or claim 15,wherein the cancer is Stage I or Stage II cancer.