WO2012031320A1

WO2012031320A1 - Cancer diagnostic

Info

Publication number: WO2012031320A1
Application number: PCT/AU2011/001148
Authority: WO
Inventors: John Stagg; Mark John Smyth; Sherene Loi
Original assignee: Peter Maccallum Cancer Institute
Priority date: 2010-09-06
Filing date: 2011-09-06
Publication date: 2012-03-15

Abstract

CD73 may be used as a prognostic marker to detect the recurrence of triple negative breast cancer in patients who have undergone a cancer therapy, in particular a chemotherapy based treatment. There is provided a method for diagnosing or predicting the recurrence of triple negative breast cancer in a subject who has undergone a cancer therapy, the method the comprising (i) obtaining a triple negative breast cancer cell sample from the subject; and (ii) detecting CD73 activity or expression in a cell of the sample, wherein increased activity or expression of CD73 in the cell, when compared to a normal breast tissue cell, indicates that the subject has or is at risk of developing recurrent triple negative breast cancer. There is also provided a method for predicting the efficacy of a triple negative breast cancer therapy in a subject comprising: (i) administering a cancer therapy to the subject; (ii) obtaining a triple negative breast cancer cell sample from the subject; and (iii) detecting CD73 activity or expression in a cell of the sample, wherein decreased activity or expression of CD73 in the triple negative breast cancer cell, when compared to a triple negative breast cancer cell of the same type prior to therapy, indicates that the therapy is efficacious.

Description

CANCER DIAGNOSTIC

FIELD OF THE INVENTION The present invention relates generally to a diagnostic for cancer detection. In particular, the present invention provides a method for diagnosing or predicting recurrence of triple negative breast cancer in a subject who has previously undergone a cancer therapy.

BACKGROUND OF THE INVENTION

Breast cancer is a complex and intrinsically heterogeneous disease that has been typically defined and classified using numerous clinical and pathologic features to predict outcome and treatment response. These clinical features include age, tumour size, axillary node involvement, angio-lymphatic invasion, histologic grade, hormonal receptor status and HER-2/neu (also known as ERBB2) amplification.

Triple negative breast cancer (TNBC) is a particular type of breast cancer defined by absence of oestrogen receptor (ER) and progesterone receptor (PR) expression as well as absence of HER-2/neu. It is characterized by its biological aggressiveness, worse prognosis and lack of a therapeutic target in contrast with hormonal receptor positive and HER-2/neu+ breast cancers. Given these characteristics, triple-negative breast cancer is a challenge in today's clinical practice.

Triple negative breast cancer patients with the same clinical and pathologic features may exhibit highly variable responses to therapy and outcome, which generates new questions such as how to explain the differences between patients who relapse from those that do not or which are the molecular mechanisms underlying the chemo-sensitivity /resistance within this subgroup. Triple negative breast cancer constitutes 12-24% of all breast cancers, and provides a challenge for clinicians since they lack a specific treatment target that, when inhibited, ameliorates the prognosis of the patients. Furthermore, as well as lacking a target, triple negative tumours have a worse relapse-free and overall survival.

Breast cancer, including triple negative breast cancer, recurs at distant sites in a substantial number of women who receive adjuvant chemotherapy after surgical removal of the primary breast tumour. De novo resistance mechanisms within tumour cells before treatment are key factors leading to the failure of chemotherapeutic drugs to prevent metastatic recurrence. Accordingly, the discovery of genomic alterations and genes contributing to de novo chemoresistance to specific drugs is needed. Although a number of multiresistant genes have been discovered, their over-expression is often induced during drug treatment and expression of these genes in tumours before treatment is not generally useful for guidance of drug selection. Gene signatures generated from responses of tumour cell lines to drugs are reported to predict drug responses in patients, however, some researchers have found that cell-line derived signatures are not predictive of response in clinical cases. Repeatedly observed genomic gain or loss has helped to identify genomic regions that may harbour genes contributing to malignant behaviour and poor outcome. However, which genomic regions harbour genes that may contribute to de novo resistance to therapy is currently unknown.

Accordingly, there exists a need to develop reliable diagnostics which can be used to detect the recurrence of triple negative breast cancer in patients who have undergone cancer treatment.

In this respect, the identification of genes crucial for tumour response to specific chemotherapy drugs is a challenge, but is necessary to improve therapy outcomes. Recently, Li et al. identified a small number of over expressed and amplified genes from chromosome 8q22 that were associated with early disease recurrence in breast cancer sufferers despite anthracycline-based adjuvant chemotherapy. Over-expression of the antiapoptotic gene YWHAZ and the lysosomal gene LAPTM4B was associated with poor tumour response to anthracycline treatment in a neoadjuvant chemotherapy trial in women with primary breast cancer. As such, over expression of the YWHAZ and LAPTM4B genes may be used to predict anthracycline resistance and influence chemotherapy selection.

CD73 or ecto-5'nucleotidase (5'-NT) is ubquitously expressed protein in a number of tissues. This protein is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) linkage, has ecto-cnzyme activity and plays a role in signal transduction. The primary function of CD73 is its conversion of extracellular nucleotides (e.g., 5 -AMP), to which cells are generally impermeable, to their corresponding nucleosides (e.g., adenosine), which can readily enter most cells. Thus, ecto-5 '-nucleotidase catalyzes the dephosphorylation of purine and pyrimidine ribo- and deoxyribonulceoside monophosphates to the corresponding nucleoside. Although CD73 has broad substrate specificity, it prefers purine ribonucleoside. Specifically, CD73 has been reported to dephosphorylate nucleoside monophosphates (AMP) into adenosine in mammalian cells. Hydrolysis of AMP to adenosine is a major signaling pathway in cells. Rather little is known about the regulation of CD73. As an enzyme that produces nucleosides, particularly adenosine, in the extraxcellular space, CD73 is thought to modulate neuronal signaling, vascular perfusion, drug metabolism and immune response. It is also thought to have anti-inflammatory and immunosuppressive capabilities. However, like other GPI- anchored proteins, CD73 also has cellular functions that are independent of its enzymatic activity. For instance, CD73 has been reported to bind to the intracellular filament protein actin and the extracellular matrix proteins laminin and fibronectin, suggesting a possible role for 5 '-NT in cell adhesion.

CD73 expression is tissue and cell type specific, however, it is not clear how such specificity is regulated. Several factors have been reported to regulate the expression of CD73. Protein kinase C (PKC) has been shown to mediate adrenergic stimulation of CD73 activation in the heart, and adenosine release in rat cardiomyocyte (Kitakaze et al. (1995), Kitakaze et al. (1996)). Parathyroid hormone has also been shown to stimulate CD73 by a mechanism involving PKC (Siegfried et al. (1 95)). Some cytokines were also shown to regulate CD73 activity. In rat, IL-1P and TNF-a induces CD73 activity (Savic et al. (1990)), and in monocytes, interferon-γ and IL-4 reduced its activity (Armstrong et al. (1988)).

CD73 production of adenosine by the dephosphorylation of AMP, has been shown to regulate adenosine receptor engagement in many tissues (Resta et al. (1 98)). This further indicated that adenosine functions in cytoprotection, cell growth, angiogenesis and immunosuppression, and plays a role in tumorigenesis (Spychala (2000)). Recently, Stagg et al. (2010) targeted CD73 using a CD73-specific monoclonal antibody and showed that the growth of breast tumours in a mouse model was significantly inhibited.

SUMMARY OF THE INVENTION

The present inventors have determined that CD73 may be used as a prognostic marker to detect the recurrence of triple negative breast cancer in patients who have undergone a cancer therapy, in particular a chemotherapy based treatment.

Accordingly, in one aspect of the present invention there is provided a method for diagnosing or predicting the recurrence of triple negative breast cancer in a subject who has undergone a cancer therapy, the method the comprising

(i) obtaining a triple negative breast cancer cell sample from the subject; and

(ii) detecting CD73 activity or expression in a cell of the sample,

wherein increased activity or expression of CD73 in the cell, when compared to a normal breast tissue cell, indicates that the subject has or is at risk of developing recurrent triple negative breast cancer.

In another aspect of the present invention there is provided a method for predicting the efficacy of a triple negative breast cancer therapy in a subject comprising:

(i) administering a cancer therapy to the subject;

(ii) obtaining a triple negative breast cancer cell sample from the subject; and (iii) detecting CD73 activity or expression in a cell of the sample,

wherein decreased activity or expression of CD73 in the triple negative breast cancer cell, when compared to a triple negative breast cancer cell of the same type prior to therapy, indicates that the therapy is efficacious.

In yet another aspect of the present invention there is provided a method for diagnosing or predicting development of triple negative breast cancer in a subject comprising:

(i) obtaining a breast tissue cell sample from the subject; and

(ii) detecting CD73 activity or expression in a cell of the sample,

wherein increased activity or expression of CD73 in the cell, when compared to a normal breast tissue cell, indicates that the subject has or is at risk of developing triple negative breast cancer.

In yet another aspect of the present invention there is provided a kit comprising a CD73 protein or nucleic acid detecting agent, together with instructions for use. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the expression distribution in the various breast cancer subtypes.

These data show that CD73 expression is the highest in triple negative breast cancer subtype as compared to the other breast cancer subtypes.

Figure 2 shows that CD73 expression is strongly associated with doxorubicin resistance in ER7HER2^" breast cancer tissue. These data are based on tissue samples obtained from 120 patients treated with anthracycline only neoadjuvant chemotherapy. Figure 3 shows that CD73 expression is strongly associated with doxorubicin resistance in ER7HER2^" breast cancer tissue. These data are based on tissue samples obtained from 120 patients treated with anthracycline only neoadjuvant chemotherapy. DETAILED DESCRIPTION OF THE INVENTION

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

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

It is to be understood that unless otherwise indicated, the subject invention is not limited to specific manufacturing methods, formulation components, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent, as well as two or more agents; reference to "the composition" includes a single composition, as well as two or more compositions; and so forth. Gene expression studies have well established that breast cancer, in addition to being clinically heterogeneous, is also a molecular heterogeneous disease. Early gene expression studies classified breast cancer into at least three clinically relevant molecular subtypes: basal-like, HER2-enriched, and luminal tumors, with each subtype exhibiting different prognosis and response to therapies. Demonstration of the molecular heterogeneity within breast cancer has changed the way clinicians perceive the disease and clinical trials in breast cancer are now conducted in each individual subtype and not in the breast cancer population as a whole.

Anthracyclines are among the most widely used chemotherapeutic drags in breast cancer. However, their clinical use is associated with rare but severe toxicities, such as long-term hematological disorders and congestive heart failure. In addition, the efficacy of anthracyclines is restricted to a subset of the breast cancer patient population. Therefore, identifying molecular markers to predict the response of breast tumors to anthracycline- based chemotherapy remains a priority. Several studies have suggested that gene expression profiles might have the potential to refine or to identify prognostic and predictive markers of response/resistance to anti-cancer treatments. One of the main messages arising from these gene expression studies is that measuring gene expression versus immunohistochemistry may be more objective because assays are automated and quantitative, as opposed to the ones currently used in the clinic, and would appear to be more robust in predicting clinical outcome and relative response to chemotherapy.

The present inventors have determined that CD73 may be used as a prognostic marker to detect the recurrence of triple negative breast cancer in patients who have undergone a cancer therapy, and in particular chemotherapy based treatments. Often patients having triple negative breast cancer will develop resistance to certain chemotherapeutic agents, such as doxorubicin, causing relapse or recurrence of the disease.

(i) obtaining a triple negative breast cancer cell sample from the subject; and

(ii) detecting CD73 activity or expression in a cell of the sample,

wherein increased activity or expression of CD73 in the cell, when compared to a normal breast tissue cell, indicates that the subject has or is at risk of developing recurrent triple negative breast cancer. In this specification, the term "expression" or "gene expression" include transcription and/or translation of nucleic acid material. In this specification, the term "detecting" or "detected" includes any means of detecting, including direct or indirect detection of gene expression and changes therein.

In certain embodiments the present invention comprises detecting CD73 expression by measuring CD73 protein levels, or measuring CD73 transcript levels. In other embodiments, the present invention further comprises detecting CD73 activity. In yet a further embodiment, CD73 activity or expression can be detected at multiple time points.

Throughout this specification the term "protein" refers to a polymer of amino acid residues, and amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to both naturally and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. "Amino acid analogues" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is a carbon that is bound to a hydrogen, a carboxyl group, an amino group and an R group, e.g., homoserine, norleucine, methionine sulfoxide and methionine methyl sulphonate. Such analogues have modified R groups (e.g. norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but retain a function similar to that of a naturally occurring amino acid. Detecting CD73 expression or activity in a cell or sample, such as a tissue sample, may be achieved using techniques well known in the art which include, but are not limited to, northern blotting, quantative RT-PCR, western blotting or quantative immunohistochemistry.

In this specification, "recurrent" or "recurrence" means the reappearance of any sign or symptom of disease, and in particular, triple negative breast cancer, after a period of remission. The remission may be induced by chemotherapy such as doxyrubicin treatment(s).

In a further embodiment, the present invention provides a method of diagnosing or predicting recurrence of triple negative breast cancer in a subject comprising subjecting the subject to whole body scanning for CD73 activity or expression in a cell. A variety of methods known to those of ordinary skill in the art are available for detecting the activity or expression of a gene product in a cell, tissue sample or organism. The present invention embodies diagnostic methods and methods for detecting CD73 activity or expression comprising measuring CD73 protein or transcript levels. Methods of detecting for CD73 enzyme activity, or protein expression levels may also be employed. These methods are provided to identify subjects who may be at risk for redeveloping triple negative breast cancer. In addition, these same methods may be applied to detect the efficacy of a cancer therapy.

Assays to detecting the level of expression of a polypeptide are also well known to those of skill in the art. This can be accomplished also by assaying for CD73 mRNA levels, mRNA stability or turnover, as well as protein expression levels. It is further contemplated that any post-translational processing of CD73 may also be detected, as well as whether it is being localized or regulated properly. In some cases an antibody that specifically binds CD73 may be used. Assays for CD73 activity also may be used. 1. CD73 Expression

There are a number of techniques which can be used to detect CD73 expression from both a transcript and protein expression level.

Northern Blotting Techniques

The present invention employs northern blotting in assessing the expression of CD73 in a cancer or tumor cell. The techniques involved in northern blotting are commonly used in molecular biology and is well known to of one skilled in the art. These techniques can be found in many standard books on molecular protocols (e.g. Sambrook et al. (2001)). This technique allows for the detection of RNA i.e., hybridization with a labeled probe.

Briefly, RNA is separated bj gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with, (e.g) a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.

United States Patent 5,279,721, incorporated by reference herein, discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

Quantitative RT-PCR

The present invention also employs quantitative RT-PCR in assessing the expression or activity of CD73 in a cancer or tumor cell reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR) can be used to determine the relative concentrations of specific mRNA species, such as a CD73 transcript, isolated from a cell. By determining that the concentration of a specific mRNA species varies, it is shown that the gene encoding the specific mRNA species is differentially expressed. In PCR, the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting.

Thereafter, the rate of amplification becomes increasingly diminished until there is not an increase in the amplified target between cycles. If one plots a graph on which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, one observes that a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After some reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.

The concentration of the target DNA in the linear portion of the PCR is directly proportional to the starting concentration of the target before the PCR was begun. By determining the concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is only true in the linear range portion of the PCR reaction.

The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCR for a collection of RNA populations is that the concentrations of the amplified PCR products must be sampled when the PCR reactions are in the linear portion of their curves.

The second condition that must be met for an RT-PCR study to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard. The goal of an RT-PCR study is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample. In such studies, mRNAs for β-actin, asparagine synthetase and lipocortin II may be used as external and internal standards to which the relative abundance of other mRNAs are compared.

Most protocols for competitive PCR utilize internal PCR internal standards that are approximately as abundant as the target. These strategies are effective if the products of the PCR amplifications are sampled during their linear phases. If the products are sampled when the reactions are approaching the plateau phase, then the less abundant product becomes relatively over represented. Comparisons of relative abundances made for many different RNA samples, such as is the case when examining RNA samples for differential expression, become distorted in such a way as to make differences in relative abundances of RNAs appear less than they actually are. This is not a significant problem if the internal standard is much more abundant than the target. If the internal standard is more abundant than the target, then direct linear comparisons can be made between RNA samples.

The discussion above describes the theoretical considerations for an RT-PCR assay for clinically derived materials. The problems inherent in clinical samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target).

Both of the foregoing problems are overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target. This assay measures relative abundance, not absolute abundance of the respective mRNA species. Other studies are available that use a more conventional relative quantitative RT-PCR with an external standard protocol. These assays sample the PCR products in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling must be empirically determined for each target cDNA fragment. In addition, the reverse transcriptase products of each RNA population isolated from the various tissue samples must be carefully normalized for equal concentrations of amplifiable cDNAs. This is very important since this assay measures absolute mRNA abundance. Absolute mRNA abundance can be used as a measure of differential gene expression only in normalized samples. While empirical determination of the linear range of the amplification curve and normalization of cDNA preparations are tedious and time consuming processes, the resulting RT-PCR assays can be superior to those derived from the relative quantitative RT-PCR with an internal standard.

One reason for this is that without the internal standard/competitor, all of the reagents can be converted into a single PCR product in the linear range of the amplification curve, increasing the sensitivity of the assay. Another reason is that with only one PCR product, display of the product on an electrophoretic gel or some other display method becomes less complex, has less background and is easier to interpret.

Immunohistochemistr

The present invention also employs quantitative immunohistochemistry in assessing the expression of CD73 in a cancer or tumor cell.

Briefly, frozen-sections may be prepared by rehydrating frozen "pulverized" tumor at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections containing an average of about 500 remarkably intact tumor cells. Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 h fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.

Western Blotting

The present invention also employs the use of western blotting (immunoblotting) analysis to assess CD73 activity or expression in a cell such as a triple negative breast cancer cell. This technique is well known to those of skill in the art (e.g. refer to United States Patent No. 4,452,901 incorporated herein by reference and Sambrook et al. (2001)). In brief, this technique generally comprises separating proteins in a sample such as a cell or tissue sample by SDS-PAGE gel electrophoresis. In SDS-PAGE proteins are separated on the basis of molecular weight, then are transferring to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), followed by incubation of the proteins on the solid support with antibodies that specifically bind to the proteins. For example, in the present invention, anti-CD73 antibodies specifically bind to CD73 proteins on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g. labeled sheep, goat, or mouse antibodies) that specifically bind to the CD73.

ELISA

The present invention may also employ the use of immunoassays such as an enzyme linked immunosorbent assay (ELISA) in assessing the activity or expression of CD73 in a cancer or tumor cell. An ELISA generally involves the steps of coating, incubating and binding, washing to remove species that are non-specifically bound, and detecting the bound immune complexes. This technique is well known in the art (e.g. United States Patent No. 4,367,1 10 and Harlow and Lane (1988)).

In an ELISA assay, a CD73 protein sample may be immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder.

This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of the antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 2 to 4 or more hour to allow effective binding, at temperatures preferably on the order of 25°C to 37°C (or overnight at 4°C). Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting the sample to a second antibody having specificity for the first. To provide a detecting means, the second antibody preferably has an associated enzyme that generates a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS) and H₂0₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. The use of labels for immunoassays are described in United States Patent Nos. 5,310,687, 5,238,808 and 5,221 ,605.

Other immunodetection methods that may be contemplated in the present invention include radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay. These methods are well known to those of ordinary skill and have been described in Doolittle and Ben-Zeev (1999); Gulbis and Galand (1993); De Jager et al. (1993); and Nakamura et al (1987).

Tissue Microarray Immunohistochemistry

This is a recently developed technique that enables the simultaneous examination of multiple tissues sections concurrently as compared to the more conventional technique of one section at a time. This technique is used for high throughput molecular profiling of tumor specimen (Kononen et al. (1998)).

Determination of Circulating Cancer Cells

With the advent of enrichment techniques, detection of circulating cancer cells can be used for the early detection of cancer recurrence after treatment of a primary tumor, early diagnosis of metastasis, and use in selection and monitoring of treatment strategies for various tumors (Martin et al. (1998); Wang et al. (2000); Hu et al. (2003)). Anti-CD73 antibodies of the present invention may be used in conjunction with cancer cell enrichment techniques in the detection of circulating pancreatic cancer cells. One suitable cell enrichment methodology is the magnetic-activated cell separation system as distributed by Miltenyi Biotec Inc. (Auburn, CA). Alternatively, magnetically anti-CD73 antibodies may be used to enrich circulating pancreatic cancer cells.

An alternative enrichment technique is Circulating Cancer Cell Test (Cell Works Inc., Baltimore, MD; see Wang et al. (2000)). This procedure utilizes a double gradient sedimentation for the removal of most RBC and WBC as well as magnetic cell sorting for the additional removal of WBC before spreading the cancer cells onto a slide utilizing a cytospin apparatus.

The fixed cells on the slide are then stained with a suitably anti-CD73 antibody and positive cells are automatically scanned with an spectroscopic microscope system, first in low magnification, where the fluorescent digital image is captured at a resolution of 0.2 um using multiple excitation/emission wavelengths, then at higher resolution for further analysis. The system has automatic adjustment of exposure, focus and other parameters required for proper image acquisition and analysis to identify cancer cells and markers on the basis of intensity and blob analysis.

Whole Body Imaging

The present invention may further employ the use of whole body imaging techniques to identify subjects who have or may be at risk of developing cancer. Such diagnostic methods may employ positron emission tomography (PET) scanning, electron beam tomography (EBT) scanning, and MRI scanning. Essential to these methods is the use of labeled targeting agents, such as antibodies, that colocalize with ecto-5'-nucleotidase or CD73 in a quantitative fashion. 2. CD73 Activity 5 '-Nucleotidase Assays

Assays which measure CD73 activity, and in particular the inhibition of CD73 to dephosphorylate purine and pyrimidine ribo- and dexoyribonucleoside monophosphates, may also be used in the present invention. This procedure detects the amount of inorganic phosphate that is released from 5'-nucleotide, such as AMP, using a molybdate color reaction. CD73 activity may also be measured using the Berthelot indophenol reaction. In this procedure CD73 hydrolyzes 5 -AMP to produce adenosine, which is deaminated by adenosine deaminase (ADA) to yield inosine and ammonia. The ammonia that is produced is then measured colorimetrically by the Berthelot indophenol reaction. Such assays or systems are commercially available from suppliers such as Molecular Probes Inc., (Eugene, OR; EnzChek™ phosphate assay kit). Thin-layer chromatography using radio-labeled AMP, or HPLC using ethano-labeled AMP may also be used to measure CD73 activity. Furthermore, radio-labeled AMP could be theoretically injected to patients and PET-SCAN used to measure CD73 activity, based on the fact that adenosine uptake by tumors is CD73 -dependent (Cho et al. (2006)). In other embodiments of the present invention, the cancer therapy is chemotherapy, radiotherapy, immunotherapy, gene therapy, hormonal therapy or surgery.

Chemotherapeutic agents may include, for example cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, vincristine, vinblastine and methotrexate Temazolomide (an aqueous form of DTIC), or any analog or derivative thereof. One example of a suitable chemotherapeutic agent is doxorubicin. In yet a further embodiment of the present invention, the chemotherapy is anthracycline only neoadjuvant chemotherapy. In neoadjuvant chemotherapy (preoperative treatment) initial chemotherapy is designed to shrink the primary tumour, thereby rendering local therapy (surgery or radiotherapy) less destructive or more effective.

Doxorubicin is a commonly used ehemotherapeutic agent to treat patients having the various different breast cancer types. In the case of triple negative breast cancer, more often than not patients develop resistance to doxorubicin, causing relapse. The present invention therefore provides a reliable diagnostic marker which can be used to assess the efficacy of a chemotherapy treatment. Based on the CD73 marker assessment information obtained using the methods according to the present invention, clinicians can make more informed decisions regarding subsequent treatments.

Accordingly, in another aspect the present invention provides a method for predicting the efficacy of a triple negative breast cancer therapy in a subject comprising:

(i) administering a cancer therapy to the subject;

(ii) obtaining a triple negative breast cancer cell sample from the subject; and

(iii) detecting CD73 activity or expression in a cell of the sample,

wherein decreased activity or. expression of CD73 in the triple negative breast cancer cell, when compared to a triple negative breast cancer cell of the same type prior to therapy, indicates that the therapy is efficacious.

The present invention also contemplates initial diagnosis of triple negative breast cancer. Accordingly, in another aspect of the present invention there is provided a method for diagnosing or predicting development of triple negative breast cancer in a subject comprising:

(i) obtaining a breast tissue cell sample from the subject; and

(ii) detecting CD73 activity or expression in a cell of the sample,

wherein increased activity or expression of CD73 in the cell, when compared to a normal breast tissue cell, indicates that the subject has or is at risk of developing triple negative breast cancer. The present invention also contemplates a kit comprising a CD73 protein or nucleic acid detecting agent, together with instructions for use. Examples of suitable CD73 detecting agents include, but are not limited to, primers specific to amplify a nucleic acid encoding CD73, CD73 nucleic acid hybridization probe including chromophore-conjugated probes, antibodies that specifically bind CD73, and the like. The skilled person would be familiar with standard protein and nucleic acid detection agents as described in the art.

EXAMPLES

Methods & Materials Detecting CD73 activity

For thin layer chromatography (TLC), tumor cells are seeded overnight in a 24-well plate in complete medium. The next day, cells are replenished with fresh medium with 1 microCurie of tritiated AMP and incubated for 1 hour, before a 10 microlitre aliquot of substrate and products were separated on aluminum sheets (Silica gel 60 F254, Merck) using as solvent isobutanol:isoamylalcool:emoxyethanol:arnmonia: water (9:6:18:9:15), and autoradiography measured using a BETA-Imager 2000 counter (Biospace, Paris, France). CD73 activity was calculated using the formula: (cpm of adenosine in supernatant of treated cells/cpm of AMP in supernatant of treated cells/(cpm of adenosine in supernatant of untreated cells/cpm of AMP in supernatant of untreated cells).

Results

Triple negative breast cancer is a particular type of breast cancer defined by absence of oestrogen receptor (ER) and progesterone receptor (PR) expression as well as absence of HER-2/neu. The other breast cancer subtypes include ER7HER-2⁺, ER⁺/HER-2^" (high profile) and ER⁺/HER-2^" (low profile). Figure 1 shows the expression distribution of for each breast cancer subtype. These data show that CD73 expression is the highest in the triple negative breast cancer subtype as compared to the other breast cancer subtypes.

Figure 2 shows that CD73 expression is closely correlated with doxorubicin resistance in ER7Her-2^~ tissue (i.e. triple negative breast cancer subtype) following anthrocycline only neoadjuvant chemotherapy, and related statistical data provided in Table 1. These data show that the "Area Under the Curve" (AUC) can be used for prediction of pathological complete response (pCR) in ER^~HER2^~ breast cancer after four cycles of doxorubicin neoadjuvant therapy. The diagonal line indicates the performance of a random predictor

Substitute Sheet

(Rule 26) RO/AU and the upper line indicates the performance of the mean mRNA level of CD73 to predict pCR (AUC: 0.84 (0.7-0.98) p=0.004).

Table 1 - Area under curve (Fig. 2)

a Under the nonparametric assumption

b Null hypothesis: true area = 0.5

c HER2.bin = ERnegHER2neg

n=62

In contrast, Figure 3 shows that CD73 expression is not as closely correlated with doxorubicin resistance in a non-triple negative breast cancer subtype (i.e. ERHer^*). In this case the related statistical data is provided in Table 2. Collectively, these data demonstrate the specificity of CD73 expression in patients with recurring triple negative breast cancer post cancer treatment.

Table 2 - Area under curve (Fig. 3)

a Under the nonparametric assumption

b Null hypothesis: true area = 0.5

c HER2.bin = HER2pos

n=31

Substitute Sheet

(Rule 26) RO/AU REFERENCES

Armstrong et al. (1988) Ann. Neurol. 24:12-16.

Cho et al. (2006)) Nucl Med; 47:837-845

De Jager et al. ( 1993) Semin. Nucl. Med. 23(2): 165- 179.

Doolittle and Ben-Zeev (1999) etAo^ o/. Biol 109:215-237.

Gulbis and Galand (1993) Hum Pathol. 24(12):1271-85.

Harlow and Lane (1988) Antibodies: A Laboratory manual, Cold Spring Harbor

Laboratory.

Hu et al. (2003) Oncology 64:160-165.

Kitakaze et al. (1995) Circulation 91 :2226-2234.

Kitakaze et al. (1996) Circulation 93:781-791.

Kononen ttf a/. (1998) Nat. Med. 4(7):844-847.

Li et al. (2010) Nature Medicine 16(2):214-218.

Martin et al. ( 1998) Exp. Hematol. 26:252-264.

Nakamura et al. (1987) In: Handbook of Experimental Immunology (4^th Ed.), Weir et al,

(eds) 1 :27, Blackwell Scientific Publ, Oxford.

Resta et al.(l99%) Immunol. Rev. 161 :95-109.

Sambrook et al (2001) In: Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Savic et al. (1990) Immunology 70:321-326.

Siegfried et al. ( 1995) Endocrinology 136:1267- 1275.

Spychala (2000) Phamacol. Ther. 87: 161 - 173.

Stagg et al. (2010) Proc. Nat. Acad. Sci 107(4): 1547- 1552.

Wang et al. (2000) Cancer 88:2787-2795.

Claims

1. A method for diagnosing or predicting the recurrence of triple negative breast cancer in a subject who has undergone a cancer therapy, the method comprising

(i) obtaining a triple negative breast cancer cell sample from the subject; and

(ii) detecting CD73 activity or expression in a cell of the sample,

2. The method according to claim 1 wherein detecting comprises detecting CD73 expression.

3. The method according to claim 1 or claim 2 wherein detecting CD73 expression comprises measuring CD73 transcript levels.

4. The method according to claim 3 wherein detecting CD73 expression comprises northern blotting.

5. The method according to claim 3 wherein detecting CD73 expression comprises quantative RT-PCR.

6. The method according to claim 1 or claim 2 wherein detecting CD73 expression comprises measuring CD73 protein levels.

7. The method according to claim 6 wherein detecting CD73 expression comprises western blotting.

8. The method according to claim 6 wherein detecting CD73 expression comprises quantative immunohistochemistry.

9. The method according to claim 1 wherein detecting comprises assessing CD73 activity.

10. The method according to any one of claims 1 to 9 further comprising detecting CD73 expression or activity at multiple time points.

11. The method according to any one of claims 1 to 10 wherein the cancer therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, gene therapy, hormonal therapy and surgery.

12. The method according to claim 1 1 wherein the chemotherapy is anthracycline only neoadjuvant chemotherapy.

13. The method according to any one of claims 1 to 12 further comprising the step of administering a second cancer therapy to the subject.

14. The method according to claim 13 wherein the second cancer therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, gene therapy, hormonal therapy and surgery.

15. A method for predicting the efficacy of a triple negative breast cancer therapy in a subject comprising:

(i) obtaining a triple negative breast cancer cell sample from a subject who has undergone a cancer therapy; and

(ii) detecting CD73 activity or expression in a cell of the sample,

16. The method according to claim 15 wherein detecting CD73 expression comprises detecting CD73 protein levels.

17. The method according to claim 15 wherein detecting CD73 expression comprises detecting CD73 transcript levels.

18. The method according to any one of claims 15 to 17 further comprising detecting CD73 activity or expression at multiple time points.

19. A method for diagnosing or predicting development of triple negative breast cancer in a subject comprising:

(i) obtaining a breast tissue cell sample from the subject; and

(ii) detecting CD73 activity or expression in a cell of the sample,

20. A kit comprising a CD73 protein or nucleic acid detecting agent, together with instructions for use.

21. The kit according to claim 20 wherein the protein or nucleic acid detecting agent is selected from the group consisting of primers specific to a nucleic acid encoding CD73, a CD73 nucleic acid hybridization probe and antibodies that specifically bind CD73.