WO2012030234A1

WO2012030234A1 - Methods and compositions for treating cancer

Info

Publication number: WO2012030234A1
Application number: PCT/NZ2011/000177
Authority: WO
Inventors: Swee Thong Tan; Darren John Day; Tinte Itinteang
Original assignee: Swee Thong Tan; Darren John Day; Tinte Itinteang
Priority date: 2010-09-01
Filing date: 2011-09-01
Publication date: 2012-03-08

Abstract

This invention relates to the treatment of cancer, particularly to methods of treating head and neck cancer. The invention provides for the use of modulators of the renin-angiotensin system, such as β-blockers, including Propranolol, for diagnosing and treating cancers, including head and neck cancers.

Description

METHODS AND COMPOSITIONS FOR TREATING CANCER

FIELD OF THE INVENTION

The invention relates to the treatment of cancer, particularly to methods of treating head and neck cancer. The invention provides for the use of modulators of the renin-angiotensin pathway, such as one or more β-blockers, including Propranolol, for diagnosing and treating cancers, including head and neck cancers.

BACKGROUND OF THE INVENTION

Cancer remains a prevalent cause of death in society. There is a continuing need for new approaches to improving the treatment of cancer, including the development of new therapeutics as well as new approaches to restore or enhance the anti-cancer activity of existing agents, chemotherapeutics and other cancer therapeutics. It is an object of the present invention to go some way to achieving the above improved anti-cancer treatments, or to at least provide the public with useful choice.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of inhibiting growth or proliferation of a neoplastic population of cells, the method comprising contacting the neoplastic population of cells with a therapeutically effective amount of a β-blocker.

In one embodiment the neoplastic population of cells is in a subject.

Accordingly the invention provides a method of inhibiting growth or proliferation of a neoplastic population of cells in a subject, the method comprising administering a therapeutically effective amount of a β-blocker to the subject in need thereof.

In another aspect the present invention provides a method of inhibiting tumour formation, inhibiting tumour growth or inhibiting tumour metastasis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a β-blocker to the subject.

In one embodiment the tumour is a head and neck cancer.

In another aspect the present invention provides a method of inhibiting differentiation of a protomesodermal neoplastic cell capable of differentiating into a nonvascular cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a β-blocker to the subject.

In another aspect the present invention provides a method of inhibiting differentiation of a totipotent stem cell capable of differentiating into a nonvascular cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a β-blocker to the subject. In one embodiment the neoplastic cells express one or more of the following cell markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.

In one embodiment the neoplastic cells express two or more of the following cell markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.

In one embodiment the neoplastic cells express three or more of the following cell markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD 133, ACE, angiotensin II receptor and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.

In one embodiment the neoplastic cells express one or more of the following cell markers: haemoglobin ζ chain, Oct4, SSEA4 or angiotensin II receptor.

In one embodiment the neoplastic population of cells is present in a solid tumour.

In one embodiment the neoplastic population of cells is a squamous cell carcinoma, melanoma, glioblastoma or sarcoma.

In one embodiment the β-blocker is a non-selective β-blocker.

In one embodiment the β-blocker is selected from the group comprising Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Propranalol, Sotalol, or Timolol.

In one embodiment the β-blocker is propranolol.

In one embodiment the β-blocker is formulated for oral, parenteral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal or topical administration.

In one embodiment the above methods of the invention further comprise separate, simultaneous or sequential administration of at least one additional cancer therapy.

In one embodiment the cancer therapy is an anti-tumour agent or an anti-tumour therapy.

In one embodiment the anti-tumour therapy is selected from therapies such as, but not limited to, surgery, chemotherapies, radiation therapies, hormonal therapies, biological

therapies/immunotherapies, anti-angiogenic therapies, cytotoxic therapies, vaccines, nucleic acid- based vaccines (eg nucleic acids expressing a cancer antigen such as DNA vaccines including pl85 vaccines), viral-based therapies (eg adeno-associated virus, lentivirus), gene therapies, small molecule inhibitor therapies, nucleotide-based therapies (eg RNAi, antisense, ribozymes etc), antibody-based therapies, oxygen and ozone treatments, embolization, and/ or chemoembolization therapies. In one embodiment the anti-tumour agent is a chemotherapeutic agent or an anti- tumourigenic agent.

In one embodiment the anti-tumour agent comprises chemotherapeutic agents,

immunotherapeutic agents, anticachectic agents, antimucositic agents, hematopoietic agents. In one embodiment the β-blocker is formulated for separate, simultaneous or sequential administration with an anti-tumour agent.

In one embodiment the anti-tumour agent is a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is selected from tubulin disruptors, DNA intercalators, and mixtures thereof. In one embodiment the chemotherapeutic agent is paclitaxel, doxorubicin, epirubicin, fluorouracil, cyclophosphamide or methotrexate.

In one embodiment the anti-tumour agent is an immunotherapeutic agent. In one embodiment the immunotherapeutic agent is an expression plasmid encoding the T cell co- stimulator B7-1, a T cell co-stimulator, or a functionally related molecule, for example a soluble B7- Ig chimera. In one embodiment the anti-tumour agent comprises immune cell therapy.

In one embodiment the β-blocker and the anti- tumour agent are administered orally or parentally. In one embodiment the administration is by intravenous injection or intratumoural injection.

In one embodiment the above methods of the invention further comprise separate, simultaneous or sequential administration of at least one additional modulator of the reriin- angiotensin pathway.

In one embodiment the at least one additional modulator is one or more of β-blockers, one or more ACE-inhibitors, or one or more angiotensin receptor blockers.

In one embodiment the β-blocker is propranolol.

In one embodiment, the ACE inhibitor is selected from the group comprising Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril, or

Trandolapril.

In one embodiment, the angiotensin-receptor blocker is selected from the group comprising

Valsartan, Telmisartan, Losartan, Irbesartan, Candesartan or Olmesartan.

In one embodiment the β-blocker and the at least one additional modulator are administered orally or parentally. In one embodiment the administration is by intravenous injection or intratumoural injection. In one embodiment the β-blocker, optionally with at least one additional anti-tumour agent or optionally with at least one additional modulator, or optionally with a combination of both, is administered daily for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks.

In one embodiment the β-blocker, or optionally with at least one additional anti-tumour agent or optionally with at least one additional modulator, or optionally with a combination of both, is administered for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or at least about 1, 2, 3, 4, 5 , 6, 7 or 8 weeks or at least about 1, 2, 3, 4, 5 or 6 months.

In one embodiment the β-blocker, optionally with at least one additional anti-tumour agent or optionally with at least one additional modulator, or optionally with a combination of both, is administered at least once daily (or continuously over a day) orally or by parenteral drip, or a combination of both administration routes.

Another aspect of the invention relates to a method of inhibiting growth or proliferation of a neoplastic population of cells in a subject, the method comprising:

a) identifying in a sample from the subject a population of cells wherein one or more of the cells is one or more of the following:

i) positive for the presence of one or more of the following markers: brachyury,

haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE or angiotensin II receptor; ii) optionally positive for the presence of one or more of the following markers:,

VEGFR-2, CD31, Tal- , SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA- 2;

iii) negative for the presence of CD45; or

iv) any combination of any two or more of i) to iii);

b) administering to the subject a modulator of the renin-angiotensin pathway.

In one embodiment the method is a method of treating or preventing cancer in the subject, including but not limited to head and neck cancer.

Another aspect of the invention relates to a method of diagnosing cancer in a subject, the method comprising:

VEGFR-2, CD31, Tal-1 , SMA, Glut-1 , aldehyde dehydrogenase, vimentin or GATA-

2; .

iii) negative for the presence of CD45; or iv) any combination of two or more of i) to iii);

b) diagnosing cancer on the basis of the identification.

In one embodiment the cancer is head and neck cancer.

Another aspect of the invention relates to a method of inhibiting growth or proliferation of a neoplastic population of cells in a subject, the method comprising administering a therapeutically effective amount of a β-blocker to the subject in need thereof, wherein the subject is a subject for whom the presence or absence in a sample from the subject of a population of cells having one or more of the cell markers selected from the group comprising: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor, and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA- 2., has been determined.

Another aspect of the invention relates to a method of identifying a population of cells likely to be responsive to treatment with a modulator of the renin-angiotensin pathway, the method comprising determining the presence or absence on one or more of the cells of one or more cell markers selected from the following group: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor, and optionally one or more of the following cell markers: VEGFR-2, CD3 , Tal-1 , SMA, Glut-1 , aldehyde dehydrogenase, vimentin or GATA-2.; and assessing the likely responsiveness to treatment on the basis of the determination.

In one embodiment the population of cells is present in a sample taken from a subject.

Accordingly, the invention provides a method of identifying a subject likely to be responsive to or suitable for treatment with a modulator of the renin-angiotensin pathway, the method comprising determining the presence or absence on one or more of the cells of one or more cell markers selected from the following group: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor, and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.; and determining the likely responsiveness to or suitability for treatment on the basis of the identification.

In one embodiment the identification, selection or determination is by contacting the sample or the cells with a labelled detection agent. In one example, the labelled detection agent is an antibody, including a labelled antibody, or a conjugated antibody.

In one embodiment, the identification is by FACS, or by in situ hybridisation.

In one embodiment the subject has been diagnosed with head and neck cancer.

In one embodiment the subject is predisposed to head and neck cancer. In one embodiment the renin-angiotensin modulator is selected from a β-blocker, angiotensin- converting enzyme (ACE) inhibitor, or angiotensin-receptor blocker.

In one embodiment the the β-blocker is selected from the group comprising Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Propranalol, Sotalol, or Timolol.

In one embodiment, the β-blocker is Propranolol.

In one embodiment, the ACE inhibitor is selected from the group comprising Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, amipril, or

Trandolapril.

Valsartan, Telmisartan, Losartan, Irbesartan, Candesartan or Olmesartan.

The use of a β-blocker in the preparation of a medicament for use in any of the methods described above, such as for inhibiting growth or proliferation of a neoplastic population of cells in a subject, is also contemplated.

A β-blocker for use in any of the methods or treatments described above is also contemplated.

For example, a β-blocker for inhibiting growth or proliferation of a neoplastic population of cells in a subject in need thereof is specifically contemplated.

Additionally, a composition comprising a β-blocker and one or more anti-tumour agents or anti-tumour therapies for use in any of the methods or treatments described above is also contemplated. For example, a composition comprising a β-blocker and one or more anti-tumour agents or anti-tumour therapies for inhibiting growth or proliferation of a neoplastic population of cells in a subject in need thereof is also contemplated.

Additionally, a composition comprising a β-blocker and one or more additional modulators of the renin-angiotensin pathway for use in any of the methods or treatments described above is also contemplated. For example, a composition comprising a β-blocker and one or more β-blockers, one or more ACE-inhibitors, or one or more angiotensin-receptor blcokers for inhibiting growth or proliferation of a neoplastic population of cells in a subject in need thereof is also contemplated.

The following embodiments may relate to any of the above aspects.

In various embodiments the cancer or the head or neck cancer is malignant.

In various embodiments the cancer or the head or neck cancer is a solid tumour or a lymphoma.

In various embodiments the solid tumour is a squamous cell carcinoma, melanoma, glioblastoma or sarcoma.

In various embodiments the lymphoma includes, but is not limited to, Hodgkin's disease or Non-Hodgkin's disease. In various embodiments the head or neck cancer is of the oral cavity, nasopharynx, oropharynx, hypopharynx, larynx or trachea.

In various embodiments the oral cavity head or neck cancer is present in the lip, tongue, buccal region, gingivae, the soft palate or hard palate.

In various embodiments the cancer or the head or neck cancer is an adenocarcinoma, adenoid cystic carcinoma or mucoepidermoid carcinoma.

In various embodiments the subject is suffering from or is susceptible to cancer o head and neck cancer; has undergone therapy, but is in relapse or is susceptible to relapse; has a tumour or a head and neck tumour refractory to therapy with a chemotherapeutic or anti-tumourigenic agent; or has previously undergone surgery, unsuccessful surgery or unsuccessful therapy with a

chemotherapeutic or anti-tumourigenic agent.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a representative histological section under analysis with Chalkley graticle as described herein in Example 2.

Figure 2 is a graph depicting Chalkley counts for the non-RASM group compared to counts in the RASM group, as described herein in Example 2.

Figure 3 shows a comparison of the rates of squamous cell carcinoma recurrence in the non- RASM group compared to the RASM group (Overall), together with recurrence rates in early stage cases (stages I and II, Early) and in late stage cases (stages III and IV j

Late), as described herein in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification and isolation of a population of neoplastic cells from a mammalian subject with head and neck cancer, and inhibition of proliferation of the population of neoplastic cells.

1. Definitions

The term "comprising" as used in this specification means "consisting at least in part of. When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

An "effective amount" is the amount required to confer therapeutic effect. The

interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, et al. (1966). Body surface area can be approximately determined from height and weight of the subject. See, e.g., Scientific Tables, Geigy

Pharmaceuticals, Ardley, New York, 1970, 537. Effective doses also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, and the like.

The term "inhibiting tumour formation" is intended to mean that tumours do not form, or that tumours form but do not establish or grow, or that tumours form but remain small, benign and do not become cancerous or metastasize, or that tumours grow more slowly. Tumour formation may be monitored through CT scans and tumor markers where available.

The term "inhibiting tumour growth" is intended to mean that tumours do not form in a subject treated according to the invention, or that one or more tumours that may be present in a subject treated according to the invention do not grow in size or become cancerous or metastasize, or that one or more tumours present in a subject treated according to the invention reduce in size (preferably by at least about 20, 30, 40, 50, 60, 70, 80, 90 or 100% by volume) or that one or more tumours present in a subject treated according to the invention are eradicated. Tumour size may be monitored through CT scans and tumor markers where available.

The term "oral administration" includes oral, buccal, enteral and intra-gastric administration.

The term "parenteral administration" includes but is not limited to topical (including administration to any dermal, epidermal or mucosal surface), subcutaneous, intravenous, intraperitoneal, intramuscular and intratumoural (including any direct administration to a tumour) administration.

The term "protomesodermal neoplastic cell" as used herein means a cell capable of differentiating into a cell selected from one or more of the following cell types: melanoma cells, squamous cell carcinoma cells and the like. In one embodiment the protomesodermal neoplastic cell is capable of differentiating into cell of the mesenchymal or neuronal cell lineage. In one embodiment the protomesodermal stem cells of the present invention are pluripotent. In one embodiment the protomesodermal stem cells of the present invention are multipotent. In one embodiment the protomesodermal stem cells of the present invention are totipotent. The term "subject" is intended to refer to an animal, preferably a mammal, more preferably a mammalian companion animal or human. Preferred companion animals include cats, dogs and horses.

The term "treat" and its derivatives should be interpreted in their broadest possible context. The term should not be taken to imply that a subject is treated until total recovery. Accordingly, "treat" broadly includes maintaining a subject's disease progression or symptoms at a substantially static level, increasing a subject's rate of recovery, amelioration and/or prevention of the onset of the symptoms or severity of a particular condition, or extending a patient's quality of life. The term "treat" also broadly includes the maintenance of good health for sensitive individuals and building stamina for disease prevention.

2. Representative diseases amenable to treatment

It is contemplated that the methods of the invention can be used in the treatment of a range of cellular proliferative and/ or differentiative disorders, including head and neck cancers, as described in more detail below.

Examples of cellular proliferative and/ or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or haematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumour can arise from a multitude of primary tumour types, including but not limited to those of head and neck, prostate, colon, lung, breast and liver origin.

As used herein, the term "cancer" (also used interchangeably with the terms,

"hyperproliferative" and "neoplastic") refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterised by rapidly proliferating cell growth. Cancerous disease states may be categorised as pathologic, i.e., characterising or constituting a disease state, e.g., malignant tumour growth, or may be categorised as non-pathologic, i.e., a deviation from normal but not associated with a disease state, e.g., cell proliferation associated with wound repair. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/ or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the oesophagus. The term "carcinoma" is art recognised and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas,

gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumour cells form recognisable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumours of mesenchymal derivation.

2.1 Head and neck cancers

Head and neck cancers include a group of biologically similar cancers that originate from the upper aerodigestive tract. They affect the lip, oral cavity, nasal cavity, paranasal sinuses, pharynx and larynx.

Many head and neck cancers are classified as squamous cell carcinomas that originate from the mucosal linings of the upper aerodigestive tract. Adenocarcinomas, originating from the columnar epithelium can also develop. Less common head and neck cancers include adenoid cystic carcinomas and mucoepidermoid carcinomas.

Causes of head and neck cancers include alcohol and tobacco use, diet (over consumption of processed meats and red meat), human papillomarvirus and Epstein-Barr virus, gastroesophageal reflux disease and ethnicity.

Current treatment and management strategies for head and neck cancers include surgical resection of the tumour and radiation therapy. The existence of extensive primary tumours or regional metastases often require combined pre- or post-operative radiation therapy and in some cases concomitant chemotherapy treatment.

3. Tumourigenesis

Tumourigenesis is the process of tumour development, involving a multistep process characterised by numerous defects in the homeostatic mechanisms that control cell growth, proliferation and differentiation. It is thought that mutations to a number of cellular control mechanisms drive a population of normal cells to become a cancer. These include self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis (Spencer et al, 2006). The genetic instability observed in those cells that transform into neoplastic cells is thought to facilitate in the acquisition of other mutations, thus leading to cancer development.

3.1 Neoplastic cells and neoplasia

As used herein, a "neoplastic population of cells" as used herein can be any population of cells that has or is capable of differentiating into a cancer. A population of neoplastic cells are thought to be descendents from a single progenitor cell and this clonality is often considered to be necessary for identification of neoplastic cell populations.

Neoplasia can be defined as an abnormal proliferation of cells, resulting in the formation of a neoplasm.

4. Methods for isolating and identifying populations of neoplastic cells

The invention provides methods of providing a population of neoplastic cells from a mammalian subject with head and neck cancer. In some embodiments, the population of neoplastic cells is capable of expressing brachyury the expression of which may be used in the methods of the invention so as to provide this population of cells.

In various embodiments, the population of stem cells is isolated from a head and neck tissue sample. In other embodiments, the population of stem cells is isolated from a blood sample from a mammalian subject with head and neck cancer.

In one embodiment, a population of cells consisting of brachyury-expressing cells is provided. In one embodiment, each cell in the population of cells is capable of expressing brachyury. In one embodiment, essentially all cells in the population express brachyury.

In one embodiment, a population of cells consisting of ACE-expressing cells is provided. In one embodiment, each cell in the population of cells is capable of expressing ACE. In one embodiment, essentially all cells in the population express ACE.

In various embodiments, the presence of a cell marker is determined by determining the presence, absence, activity or amount of one or more gene products of the gene encoding the cell marker.

In various embodiments, the gene product is a cell marker polypeptide. In one example, the cell marker polypeptide expressed by a neoplastic cell of the invention is bound to a detection agent to facilitate the isolation, identification, or selection process.

For example, cells can be assayed in vitro or in situ with a binding partner, antibody, or nucleic acid that binds brachyury, including a labelled binding partner, antibody or nucleic acid. In embodiments where a neoplastic cell is attached to a solid support, assays may employ other types of signal molecules, where unbound signal molecule can be separated from signal molecule bound to the cell. For example, a signal molecule may be labelled with a radioactive isotope (e.g., ¹²³I, ^{1 1}1, ³'S, 32P, C or ³H), a light scattering label, an enzymatic or protein label (e.g., GFP or peroxidase), or a chromogenic label or dye (e.g., Texas Red). In addition, FACS or other cell sorting mechanisms well known in the art may be used to isolate cells.

In other embodiments, the gene product is a cell marker polynucleotide. In one example, the cell marker polynucleotide expressed by a neoplastic cell of the invention is bound to a detection agent to facilitate the isolation, identification, or selection process. Expression of such a

polynucleotide, including for example messenger RNA or cDNA derived therefrom, and the presence, absence, or amount of such a polynucleotide, can be used as an indicator of the presence of the cell marker polypeptide.

Exemplary methods for measuring the expression of a cell marker polynucleotide of the invention are well known and include but are not limited to Northern analysis, RT-PCR and dot- blot analysis (Sambrook et l, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). One embodiment of the invention thus includes a step in which ascertaining whether a sequence of the polynucleotide encoding a cell marker of the invention is present, and this may include amplifying the polynucleotide in the presence of primers based on a nucleotide sequence encoding a cell marker as described herein.

In one embodiment determination of the presence of the cell marker includes ascertaining whether a sequence of the mRNA encoding a protein having biological activity of the cell marker is present, and may include determining the amount of mRNA. The absence of the mRNA encoding such a protein, or a decrease in the amount of such mRNA indicates the absence of the cell marker. The reverse association holds true.

Again, if an amplification method such as PCR is used in ascertaining whether the mRNA encoding a cell marker such as brachyury is present, the method includes amplifying the mRNA, for example in the presence of a pair of primers complementary to a nucleotide sequence encoding the cell marker polypeptide or other gene product, or in the presence of a pair of primers

complementary to a nucleotide sequence encoding a variant of the cell marker. It will be appreciated that in embodiments of the invention reliant on assessing the amount of cell marker mRNA present in a sample, quantitative amplification methods well known in the art may be employed, for example quantitative RT-PCR, microarray analysis, real-time PCR, and other methods described herein. - Primers suitable for use in the present invention, such as those used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Likewise, a probe of the present invention, is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which^" selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection in the presence of nucleic acid molecules having differing sequences. The target nucleic acid sequence can in whole or in part be identified in a sequence database such as Genbank via reference to the cell maker, and used in the design of the probes or primers for use in the present invention. Other methods for determining whether a particular nucleotide sequence is present in a sample may include the step of restriction enzyme digestion of a nucleotide sample. Separation and visualisation of the digested restriction fragments by methods well known in the art may form a diagnostic test for the presence of a particular nucleotide sequence. The nucleotide sequence digested may be a PCR product amplified as described above.

Still other methods for determining whether a particular nucleotide sequence is present in a sample include a step of hybridisation of a probe to a sample nucleotide sequence. Thus, methods for detecting the presence of cell marker-specific nucleotides may comprise the additional steps of hybridisation of a probe derived from the nucleotide sequence encoding the cell marker.

Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the cell marker gene sequence, including the nucleotide sequence of any mRNA gene product or cDNA derivative therefrom, to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection of the presence or absence of the cell marker specific nucleotides and thus the presence of the cell markers described herein.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30°C (for example, 10°C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., 1989; Ausubel et al., 1987). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+).

With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10°C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) °C.

Such probes and primers may be hybridised with genomic DNA, mRNA, or cDNA produced form mRNA, derived from a sample under test. For expressed cell markers, those skilled in the art will recognise that samples comprising mRNA or cDNA are preferred.

Probes, and in certain embodiments primers, suitable for use in the present invention may additionally comprise a detectable label or other indicia of the presence of the pf be when bound to sample nucleotide sequence. Methods for labelling probes such as radiolabelling are well known in the art (see for example, Sambrook et al., 1989).

Other nucleic acid detection strategies amenable to use in the present invention include the Ligase Chain Reaction (LCR); various isothermal equivalents of PCR including the strand displacement amplification (SDA) method as described in JP-B 7-114718 and the various modified SDA methods as described in U.S. Pat. No. 5,824,517, and PCT International patent application publications WO 99/09211, WO 95/25180 and WO 99/49081; the LAMP method described in PCT International patent application publication WO 00/28082; the "Rolling Circle Amplification" (RCA) reaction scheme; the isothermal amplification method reliant on nicking endonucleases described in U.S. Pat. No.s 7,112,423 and 6,884,586 and PCT International patent application PCT/US02/22657 published as WO03/008622 and the related "exponential amplification reaction" (EXPAR) method; the Helicase Dependent Amplification (HDA) method such as the IsoAmp II Universal HDA kits available from New England Biolabs; the self-sustained sequence replication (3SR) method and the nucleic acid sequence based amplification (NASBA) method as described in Japanese Patent No. 2650159, and the transcription-mediated amplification (TMA) method; and various detection strategies dependent on Fluorescent Resonance Energy Transfer (FRET), including the "molecular beacons" approaches and the widespread TaqMan real-time PCR system.

Those skilled in the art will recognise that the presence of any of the cell markers, described herein, including brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD 133, ACE, angiotensin II receptor, angiotensinogen, VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, Sox O, vimentin or GATA-2., may be determined by any of these methods. Thus, in various embodiments, the presence of brachyury may be determined using methods reliant on the presence, absence, activity or amount of one or more brachyury gene products.

Other cell markers or phenotypic characteristics, including for example morphological markers, can be used to isolate or characterise a population of neoplastic cells, in accordance with the description and examples provided herein.

The cells may be isolated using excised tissue and separation methods, such as those described herein, but as will be appreciated by those skilled in the art other methods of isolation are also contemplated. For instance, cells may be harvested from a subject in situ.

When used for diagnosing a head or neck cancer, the cells may alternatively be isolated using a blood sample obtained from a mammalian subject with head and neck cancer, including processed blood samples including cell concentrates such as those obtained during the preparation of plasma.

4.1 Cell markers

It will be appreciated that cells and cell populations may be identified and isolated via the presence (including the expression) or absence of one or more markers. As used herein the phrase "positive for the presence of [a cell marker]" includes cells that are at the time of the assessment capable of expressing the recited cell marker and those that do express the recited cell marker or a gene product (including mRNA or other RNA or one or more proteins) encoded by the gene coding for the recited cell marker. Similarly, the phrase "negative for the presence of [a cell marker]" includes cells that are at the time of the assessment not capable of expressing the recited cell marker and those that do not express the recited cell marker or a gene product (including mRNA or other RNA or one or more proteins) encoded by the gene coding for the recited cell marker. For the sake of clarity, the term "[cell marker]" as used above includes one of the cell markers specifically identified herein.

4.1.1 Haemoglobin ζ chain

Haemaglobin ζ is an embryonic haemoglobin, produced in the yolk sac of an early embryo, and is thus reported to be associated with early embryonic tissue.

Agents for detecting the presence of haemoglobin ζ chain are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Santa Cruz Biotechnology Ltd, USA.

4.1.2 Brachyury

Brachyury is a transcription factor involved in the formation of mesoderm. It is reportedly used as a cell marker of human embryonic stem cells that have differentiated into mesoderm.

Brachyury is expressed during the gastrula and neurula stages in involuting mesoderm and in the notochord, and has been associated with cells of the primitive streak.

Agents for detecting the presence of brachyury are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.3 Oct4

Oct4 is a transcription factor of the POU family and is reported to be associated with undifferentiated embryonic stem cells.

Agents for detecting the presence of Oct4 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.4 SSEA4

Stage-specific embryonic antigen 4 is a glycolipid carbohydrate epitope that is reported to be expressed on the surface of human teratocarcinoma stem cells, human embryonic germ cells and human embryonic stem cells. Agents for detecting the presence of Oct4 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.5 CD34

CD34 is a cell surface glycoprotein and is reported to be expressed in early hematopoietic and vascular-associated tissue. CD34+ cells include hematopoietic progenitor stem cells, mesenchymal stem cells, endothelial progenitor cells, vascular endothelial cells, mast cells and dendritic cells.

Agents for detecting the presence of CD34 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.6 CD133

CD133 (also known as Prominin 1) act as a hematopoietic stem cell marker, and is reported to be expressed in hematopoietic stem cells, endothelial progenitor cells, glioblastomas, and neuronal and glial stem cells.

Agents for detecting the presence of CD133 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.7 ACE

Angiotensin converting enzyme (also known as CD143) is reported as a marker for endothelial cells and haemangioblasts differentiating from ES cells.

Agents for detecting the presence of ACE are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Santa Cruz Biotechnology Ltd, USA.

4.1.8 Angiotensin II receptor Angiotensin II receptors are a class of G-protein coupled receptors and are reported to be expressed in a number of tissues, including the kidney and heart.

Agents for detecting the presence of angiotensin II receptor are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is ProSci Incorporated, USA.

4.1.9 VEGFR-1 and VEGFR-2

Vascular endothelial growth factor receptors are signalling proteins reported to be involved in vasculogenesis and angiogenesis. VEGF expression is typically restricted to vascular endothelial cells.

Agents for detecting the presence of VEGFR-1 or VEGFR-1 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.10 CD31

CD31 (also known as platelet endothelial cell adhesion molecule) serves as an endothelial cell marker. Expression of CD31 has been reported in a number of vascular tumours, including epithelioid hemangioendothelioma.

Agents for detecting the presence of CD31 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.11 CD45

CD45 (also known as the common leukocyte antigen) is a protein tyrosine phosphatase reported to be expressed in cells of the hematopoietic lineage, including hematopoietic stem cells. It is not expressed in platelets or erythrocytes.

Agents for detecting the presence of CD45 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.12 GATA-2

GATA binding protein 2 is a transcription factor, reportedly involved in the regulation of gene expression in hematopoietic stem cells. It is associated with hematopoietic progenitor cells, including early erythroid cells, myeloid cells (e.g., mast cells) and megakaryocytes. It is also expressed in non-haematopoietic ES cells.

Agents for detecting the presence of GATA-2 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.113 Tal-1

T-cell acute leukemia 1 (also known as stem cell leukemia) is an important regulator of the haematopoietic lineage, and is reportedly expressed in haematopoietic stem cells, myeloid, erythroid and the megakaryocyte lineages of both the adult and embryonic brain.

Agents for detecting the presence of Tal-1 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.14 Smooth muscle actin

Smooth muscle actin (also known as alpha-actin 2 or cell growth-inhibiting gene 46) is expressed in vascular smooth muscle, and is a marker of differentiation of smooth muscle cells and pericytes.

Agents for detecting the presence of smooth muscle actin are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.15 Glut-1 Glucose transporter 1 (also known as SLC2A1) is widely expressed in fetal tissues, however in adult tissue its expression is greatest in erythrocytes and endothelial cells of barrier tissues, such as the blood-brain barrier. Recently, expression of Glut-I has been reported in perineural cells.

Agents for detecting the presence of Glut-1 are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA.

4.1.16 Aldehyde dehydrogenase

Aldehyde dehydrogenase (ALDH) has been reported to be expressed in embryonal tissue, including stem and progenitor cells isolated from bone marrow, brain, breast and other tissue. Aldehyde dehydrogenase expression has also reportedly been associated with stem cells present in certain cancers.

Agents for detecting the presence of aldehyde dehydrogenase are well known in the art. Suitable agents include those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Abeam, USA. Enzymatic assays may also be used to detect the activity of ALDH and are well known in the art. See for example Sondergaard CS, et al., "Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction." / Trans! Med. 2010 Mar 9;8:24. Examples of enzymatic assays include fluorimetric assays such as that available from is GenScript, USA.

4.1.17 Vimentin

Vimentin (gi/ 4507895) is a homodimeric intracellular phosphoprotein with a reported molecular mass of 54 kDa, whose phosphorylation is reportedly enhanced during cell division.

While some have suggested that the expression of vimentin is characteristic of cells of mesenchymal origin, others suggest vimentin synthesis is characteristic of all proliferating cells in vitro regardless of their embryonal origin, and that expression is downregulated upon differentiation. Vimentin gene expression is reportedly upregulated in some metastatic tumour cells, and it has thus been proposed as a marker for oncogenic progression and used in the histological classification of human tumours.

Agents for detecting the presence of vimentin are well known in the art, including for example those described in US Patent No. 7,670,604, incorporated by reference herein in its entirety. Suitable agents include, those adapted for use in cell identification, (whether in situ or otherwise), such as the primary antibodies described herein in the examples for use in conjunction with labelled secondary antibodies in manual or automated cell identification or selection techniques. One commercial source for such antibodies is Epitomics, Inc., USA.

5. Modulators of the Renin-Angiotensin pathway

5.1 The renin-angiotensin pathway

The renin-angiotensin pathway, also known as the renin-angiotensin-aldosterone pathway, is involved in the regulation of blood pressure and fluid balance in the body. A drop in blood pressure of loss of blood stimulates the juxtaglomerular apparatus in the kidney to release renin. This enzyme acts by cleaving angiotensinogen (an inactive peptide released from the liver), thus converting it to angiotensin I. Angiotensin I is converted to angiotensin II by Angiotensin-converting enzyme (ACE). Angiotensin II acts on numerous effects within the body. These include constriction of glomerular arterioles , reabsorption of Na⁺ and Cl excretion of K⁺, retention of H₂0, release of aldosterone from the adrenal gland, release of anti-diuretic hormone (ADH) from the pituitary gland and sympathetic nervous system activation.

5.2 β-blockers

Beta blockers are commonly known to be effective in the treatment and management of cardiovascular conditions, such as cardiac arrhythmias, myocardial infarction, angina and hypertension, β-blockers act as beta adrenergic receptor antagonists, blocking the action of endogenous catecholamines, in particular epinephrine and norepinephrine. Three different beta adrenergic receptors exist (β,, β₂ and β₃), to which various β-blockers act. β-blockers can be classified as β,-selective,

β,-selective or non-selective.

Example of various β-blockers include Alprenblol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Propranolol, Sotalol, Timolol (nonselective); Acebutolol, Atenolol, Betaxolol, Bisoprolol, Celiprolol, Esmolol, Metoprolol and Nebivolol (β,-selective);

Butaxamine and ICI-188,551 ₂-selective); and SR 59230A ^-selective).

5.3 Angiotensin-converting enzyme (ACE) inhibitors

ACE inhibitors, like β-blockers, are known to be effective in the treatment of hypertension and congestive heart failure. Other uses of ACE inhibitors include renal disease and systemic sclerosis. ACE inhibitors act on the renin-angiotensin-aldosterone system, by blocking the conversion of angiotensin I to angiotensin II.

ACE inhibitors are classified into three groups based on their molecular structure— sulfhydryl- containing, dicarboxylate-containing and phosphonate-containing.

Examples of ACE inhibitors include Captopril and Zofenopril (sulfhydryl-containing);

Enalapril, Ramipril, Quinapril, Perindopril, Lisinopril and Benazepril (dicarboxylate-containing); and W

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Fosinopril (phosphonate-containing). Naturally occurring ACE inhibiting agents include casokinin, lactokinins and lactotripep tides.

5.4 Angiotensin receptor blockers (ARBs)

Angiotensin receptor blockers, also known as angiotensin II receptor antagonists , AT receptor antagonists or sartans, are commonly known to be effective in the treatment of hypertension, diabetic nephropathy (kidney damage as a result of diabetes) and congestive heart failure. Angiotensin receptor blockers modulate the renin-angiotensin-aldosterone system by blocking the activation of angiotensin II receptors. To ensure efficacy, ARBs must exhibit a combination of three pharmocodynamic and pharmacokinetic parameters, including pressor inhibition, AT, affinity and biological half life.

Examples of ARBs include Valsartan, Telmisartan, Losartan, Irbesartan, Olmesartan, Candesartan and Eprosartan.

6. Methods of treating, preventing or diagnosing head and neck cancers

The invention contemplates using a modulator of the renin-angiotensin pathway, including β- blockers, ACE inhibitors and Angiotensin receptor blockers, in the treatment of head and neck cancers. Specifically, the invention contemplates using modulators of the renin-angiotensin pathway for inhibiting proliferation of a neoplastic population of cells in a subject suffering from or susceptible to developing head and neck cancer.

The invention also contemplates using the neoplastic cells in vitro to determine the effects of various modulators of the renin-angiotensin pathway that may be suitable for use in the diagnosis of head and neck cancers.

Compositions useful according to the invention may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected with regard to the intended route of administration and standard pharmaceutical practice. For example, a composition useful according to the invention can be administered orally as a powder, liquid, tablet or capsule, or parenterally as a liquid. Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, and may be adapted for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release.

Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. Active ingredients can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tabletting agent. Pharmaceutical compositions can also be administered via the parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipients. Cyclodextrins, or other solubilising agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.

The efficacy of a composition useful according to the invention can be evaluated both in vitro and in vivo. See, e.g., the examples below. Briefly, the composition can be tested in vitro or in vivo for efficacy. For in vivo studies, the composition can be fed to or injected into an animal (e.g., a mouse) and its effects assessed. Based on the results, an appropriate dosage range and

administration route can be determined.

The compositions useful herein may be used alone or in combination with one or more other therapeutic agents, as described herein.

When used in combination with another therapeutic agent, the administration of a composition useful herein and the other therapeutic agent may be simultaneous or sequential.

Simultaneous administration includes the administration of a single dosage form that comprises all components or the administration of separate dosage forms at substantially the same time.

Sequential administration includes administration according to different schedules, preferably so that there is an overlap in the periods during which the composition useful herein and other therapeutic agent are provided.

Suitable agents with which the compositions useful herein can be . co-administered are described herein.

As will be appreciated, the dose of the composition administered, the period of

administration, and the general administration regime may differ between subjects depending on such variables as the severity of symptoms of a subject, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/ or general health of a subject. It should be appreciated that administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate.

6.1 In vivo treatment or prevention of cancers

The invention provides a method of inhibiting tumour formation, inhibiting tumour growth or inhibiting tumour metastasis in a subject, the method comprising administering an effective amount of β-blocker to the subject.

The present invention also relates to methods of inhibiting growth or proliferation of a neoplastic population of cells in a subject, the method comprising administering an effective amount of β-blocker to the subject. The present invention further relates to a method of inhibiting growth or proliferation of a neoplastic population of cells, the method comprising contacting the neoplastic population of cells with an effective amount of β-blocker.

Accordingly, the present invention has utility in preventing cancer, particularly head and neck cancer, particularly in preventing relapse (tumour growth) after surgery such as often results from growth and proliferation of secondary tumours, or preventing tumour spread after diagnosis.

In one embodiment the β-blocker is a non-selective β-blocker.

In one embodiment the β-blocker is Propranolol.

In one embodiment the β-blocker is formulated for oral, parenteral, sublingual, intranasal, . intraocular, transdermal, mucosal or topical administration.

In various embodiments the neoplastic population of cells is present in a subject suffering from a head and neck cancer. In various embodiments the subject is suffering from or is susceptible to head and neck cancer; has undergone therapy, but is in relapse or is susceptible to relapse; has a head and neck tumour refractory to therapy with a chemotherapeutic or anti-tumourigenic agent; or has previously undergone surgery, unsuccessful surgery or unsuccessful therapy with a

chemotherapeutic or anti-tumourigenic agent.

Where appropriate, these methods may further comprise separate, simultaneous or sequential administration of at least one additional anti- tumour agent or anti-tumour therapy as described herein.

In one embodiment the β-blocker, optionally with at least one additional anti-tumour agent or anti-tumour therapy are formulated for co-administration with the at least one anti-tumour agent or anti-tumour therapy described herein.

In one embodiment the β-blocker, optionally with at least one additional anti-tumour agent or anti-tumour therapy are formulated for sequential administration with the at least one anti-tumour agent or anti-tumour therapy described herein.

In one embodiment β-blocker is included as or is delivered as an adjuvant for the anti-tumour agent or anti-tumour therapy in that the β-blocker enhances or potentiates the effects of the anti- tumour agent or anti-tumour therapy. At least one additional therapeutic agent may be delivered separately.

When used in combination with another therapeutic agent, the administration of a composition useful herein and the other therapeutic agent may be simultaneous or sequential. Simultaneous administration includes the administration of a single dosage form that comprises all components or the administration of separate dosage forms at substantially the same time.

Suitable agents with which the compositions of the invention can be co-administered include chemotherapeutic agents, immunotherapeutic agents, anticachectic agents, antimucositic agents, hematopoietic agents, and other suitable agents known in the art. Such agents are preferably administered parenterally, preferably by intravenous, subcutaneous, intramuscular, intraperitoneal, intramedullar, epidural, intradermal, transdermal (topical), transmucosal, intra-articular, and intrapleural, as well as oral, inhalation, vaginal and rectal administration.

Additionally, it is contemplated that a composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a subject in particular instances. For example, therapeutic agents that target the same or different facets of the disease process may be used.

Suitable agents with which the compositions useful herein can be co-administered include alpha v beta 3 integrin receptor antagonists, antiestrogens or SERMs (Selective Estrogen Receptor Modulators) (including but not limited to tamoxifen, raloxifene, lasofoxifene, toremifene, azorxifene, clomiphene, droloxifene, idoxifene, levormeloxifene, zuclomiphene, enclomiphene, nafoxidene, and salts thereof), antiresorptive agents, bisphosphonates (including but not limited to alendronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, olpadronate, pamidronate, piridronate, risedronate, tiludronate, zoledronate, and pharmaceutically acceptable salts thereof), calcium receptor antagonists, calcium supplements, cathepsin K inhibitors, Dual Action Bond Agents (DAB As) (including but not limited to strontium ranelate), estrogen and estrogen derivatives (including but not limited to 17 beta-estradiol, estrone, conjugated estrogen, equine estrogen, and 17 beta-ethynyl estradiol), flavonoids, folic acid, osteoanabolic agents, osteoprotegerin, progestin and progestin derivatives (including but not limited to norethindrone and medroxyprogesterone acetate), vacuolar ATPase inhibitors, antagonists of VEGF, thiazolidinediones, calcitonin, protein kinase inhibitors, parathyroid hormone (PTH), PTH analogs, recombinant parathyroid hormone, growth hormone secretagogues, growth hormone releasing hormone, insulin- like growth factor, bone morphogenetic protein (BMP), inhibitors of BMP antagonism,

prostaglandin derivatives, fibroblast growth factors, vitamin B6, vitamin D, vitamin D derivatives (including but not limited to 1,25-dihydroxycholecalciferol), vitamin K, vitamin K derivatives, soy isoflavones, calcium salts, fluoride salts, statins (including but not limited to lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, and pitavastatin), and combinations thereof, and other suitable agents known in the art. In one embodiment the anti-tumour therapy is selected from therapies such as, but not limited to, surgery, chemotherapies, radiation therapies, hormonal therapies, biological

therapies/immunotherapies, anti-angiogenic therapies, cytotoxic therapies, vaccines, nucleic acid- based vaccines (eg nucleic acids expressing a cancer antigen such as DNA vaccines including pi 85 vaccines), viral-based therapies (eg adeno-associated virus, lentivirus), gene therapies, small molecule inhibitor therapies, nucleotide-based therapies (eg RNAi, antisense, ribozymes etc), antibody-based therapies, oxygen and ozone treatments, embolization, and/ or chemoembolization therapies.

In one embodiment the anti-tumour agent is a chemotherapeutic agent or an anti- tumourigenic agent.

Examples of chemotherapeutic agents include, but are not limited to, cisplatinum, carboplatinum, taxanes, 5-fluorouracil, methotrexate, and ifosphamide.

In one embodiment the anti-tumour agent is an immunotherapeutic agent. Preferably the immunotherapeutic agent is an expression plasmid encoding the T cell co-stimulator B7-1, a T cell co-stimulator, or a functionally related molecule, for example a B7-Ig chimera.

In one embodiment the anti-tumour agent or therapy comprises dendritic cell therapy.

In one embodiment the radiation therapy includes external beam radiation therapy (including gamma-ray and x-ray therapy) and internal radiation therapy using radioisotopes. Radioisotopes may also be used as anti-tumour agents according to the invention.

In one embodiment the β-blocker and the at least one anti-tumour agent are administered orally or parenterally although the preferred route depends on the anti-tumour agent selected. In one embodiment the at least one anti-tumour agent is administered by intravenous or intratumoural injection.

In one embodiment the β-blocker, optionally with at least one additional anti-tumour agent or optionally with at least one additional modulator, or optionally with a combination of both, is administered daily for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks.

For example, the β-blocker, optionally with at least one additional anti-tumour agent, is administered at least once daily including continuously over a day orally, by parenteral drip or by a combination of administrative routes.

6.2 In vitro screening The methods of the invention provide neoplastic cells suitable for use in drug discovery programs, including for example screening methods to identify agents suitable for use in the treatment or prevention of head and neck cancers. In vitro screening assays, with reliable positive and negative controls, are useful as a first step in the development of drugs to treat or prevent head and neck cancers, as well as in the derivation of further information on the disease process.

Accordingly, in yet a further aspect the present invention provides a method of treating or preventing head and neck cancer in a subject, the method comprising:

identifying in a sample from a subject a population of cells wherein the cells is one or more of the following:

i) positive for the presence of brachyury;

ii) positive for the presence of one or more of the following markers:, VEGFR-2, CD31, CD34, CD133, GATA-2, ACE, Tal-1, SMA, Glut-1, aldehyde dehydrogenase;

iii) negative for the presence of CD45; or

iv) any combination of two or more of i) to iii);

administering to the subject a modulator of the renin-angiotensin pathway.

Exemplary characteristics of the cells may include the ability of the cells to undergo a morphological or anatomical change, such as for example, tumourigenesis, angiogenesis or vasculogeneis, the ability of the cells to respond to a stimulus, including response to the presence of a molecule such as a signalling molecule, the ability of the cells to secrete a factor, or the

upregulation or downregulation of a gene or gene product, such as an increase or decrease in the amount or activity of an enzyme, receptor, or other cell marker.

In one embodiment the screening is for toxicity of the renin-angiotensin modulator. In various embodiments the screening is for teratogenicity or tetragenicity, oncogenicity or

tumourogenicity, or angiogenicity.

In one exemplary embodiment, the characteristic is tumourigenesis

In another exemplary embodiment, the characteristic is abnormal angiogenesis, including for example abnormal vasculogenesis, including for example tumour vasculogenesis.

In another aspect, the present invention provides a method of diagnosing head and neck cancer in a subject, the method comprising:

identifying in a sample from the subject a population of cells wherein the cells are one or more of the following:

i) positive for the presence of brachyury;

diagnosing head and neck cancer on the basis of the identification.

In one embodiment the renin-angiotensin modulator is a β-blocker, ACE inhibitor or an Angiotensin receptor blocker.

Examples of β-blockers include Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol,

Nadolol, Penbutolol, Pindolol, Propranalol, Sotalol, or Timolol.

In one embodiment the β-blocker is Propranolol.

Examples of ACE inhibitors include Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril, or Trandolapril.

Examples of angiotensin receptor blockers include Valsartan, Telmisartan, Losartan,

Irbesartan, Candesartan or Olmesartan.

In one representative example, the tissue is vascular tissue and the development is of blood vessels. EXAMPLES

EXAMPLE 1 - Characterisation of a population of primitive haematopoietic cells in head and neck squamous cell carcinoma and sarcomas such as osteosarcoma.

This example describes the characterisation of primitive cells in head and neck tumours using an array of cell markers.

Materials and Methods

1. Tissue preparation for Immunohistochemistry

Biopsies from head and neck mucosal and cutaneous squamous cell carcinoma and osteosarcoma weres obtained from 16 patients with squamous cell carcinoma or osteosarcoma.

The biopsies underwent tissue processing and paraffin embedding and slides were mounted with 4 μπι paraffin embedded sections.

Rehydration and antigen retrieval was performed on the paraffin embedded tissue samples.

2. Cell staining

Primary antibodies as described in Table 1 were used for immunohistochemical labelling in accordance with the manufacturer's recommendations.

Cells were initially labelled with CD34 antibody. CD34+ cells were then subsequentiy labelled using Brachyury, CD133, Tal-1, ACE and AT2R antibodies.

Table 1. Antibodies for immunohistochemical labelling

Antibody Description Dilution

CD34 Hematopoietic Progenitor Cell Antigen 1:50 - 1:1000 Transcription factor and marker of mesodermal

Brachyury 1:50 - 1:1000

stem cells

Glycoprotein expressed in neural and

CD133 1:50 - 1 :1000

embryonic stem cells

Tal-1 T-cell Acute Lymphocytic Leukaemia Protein 1 1:50 - 1:1000

ACE Angiotensin Converting Enzyme 1:50 - 1:1000

AT2R Angiotensin II Receptor 1:50 - 1:1000

Results

Strong immunoreactivity for CD34 was observed in the endothelium of the head and neck squamous cell carcinomas and osteosarcomas.

All sections showed immunoreactivity for each of the cell markers tested.

Discussion

The results of this example indicate that the endothelial cells present in head and neck squamous cell carcinomas and osteosarcomas express primitive haematopoietic associated proteins. These results further suggest the tumours exhibit an embryonic-like process of tumourigenesis.

EXAMPLE 2

Introduction

This example investigates the relationship between renin-angiotensin system modulators (RASM) and the extent of vasculogenesis in patients treated with oral cavity squamous cell carcinoma (SCC).

Methods

Patients with oral cavity SCC were identified from the applicant's clinical head and neck database recording clinical cases from 1990 to 2010 (n=78). Those patients who had been receiving RASM were identified (n=30), and tumour recurrence was documented for all patients.

Immunohistochemistry for CD34+ was performed on sections of SCC as described above in Example 1. A Chalkley count, an established measure of tumour vasculogenesis, was performed in which the Chalkley count of 3 high CD34+ regions was done and the average number of CD34+ cells was taken.

Results

As can be seen in Table 2 below, the average Chalkley count for SCC patients exposed to RASM was substantially lower than that observed in patients who had not been receiving RASM treatment. Also, recurrence rate was lower among those patients receiving RASM treatment Table 2. Chalkley analysis of CD34+ cells

Conclusions

This data shows that patients with oral cavity SCC with higher recurrence rates had higher Chalkley counts. Notably, oral cavity SCC in patients treated with RASM had a lower Chalkley counts, and these patients evidenced lower recurrence, compared to patients who were not receiving RASM treatment.

The applicants believe, without wishing to be bound by any theory, that RASM confers a low recurrence rate of oral cavity SCC, possibly by inhibiting tumour vasculogenesis.

EXAMPLE 3

Introduction

This example investigates the effect of renin-angiotensin system modulators (RASM) on the cancer recurrence rate in patients with intra-oral squamous Cell carcinoma (SCC).

Methods

Patients with oral cavity SCC were identified from the applicant's clinical head and neck database recording clinical cases from 1990 to 2010. Those patients who had been receiving RASM were identified, and tumour recurrence was documented for all patients. This information was supplemented by medical notes and GP records. All primary oral cavity SCC patients, whether treated by surgery and/ or RT were included. Patients with recurrent or new cancer, or those undergoing palliative treatment, were excluded.

Results

208 eligible patients were identified, and data was collected on 84% (174/208). 37% (70/191) of these patients were undergoing RASM treatment. As can be seen in Table 3 below, the recurrence rate in patients undergoing RASM treatment was 14.3% (10/70), substantially lower than the rate of recurrence in patients who were not undergoing RASM treatment, 24.8% (30/ 121). Table 3. Rates of recurrence in SCC patients

Recurrence rates were also analysed depending on the stage of SCC:

Early stages (I & II) Late stages (III & TV)

RASM: 16.1% (5/31) RASM: 21.9% (7/32)

No RASM: 22.9% (11/48) No RASM: 38.5% (25/65)

Relative Risk = 0.7 Relative Risk = 0.57

P = 0.46 P = 0.103

Conclusions

This data shows that oral cavity SCC in patients treated with RASM evidenced lower recurrence, compared to patients who were not receiving RASM treatment.

The applicants believe, without wishing to be bound by any theory, that RASM confers a low recurrence rate of oral cavity SCC, and that this effect may be more significant for late stage disease. INDUSTRIAL APPLICABILITY

The present invention provides methods to diagnose and treat cancer, particularly methods of diagnosing and treating head and neck cancer, as well as methods to identify individuals who would benefit from particular treatment strategies. The invention is accordingly expected to provide the public with useful medical and social benefits.

Claims

1. A method of inhibiting growth or proliferation of a neoplastic population of cells, the

method comprising contacting the neoplastic population of cells with a therapeutically effective amount of a β-blocker.

2. A method of claim 1 wherein the neoplastic population of cells is in a subject.

3. A method of inhibiting growth or proliferation of a neoplastic population of cells in a

subject, the method comprising administering a therapeutically effective amount of a β- blocker to the subject in need thereof.

4. A method of inhibiting tumour formation, inhibiting tumour growth or inhibiting tumour metastasis in a subject in need thereof, the method comprising administering a

therapeutically effective amount of a β-blocker to the subject.

5. A method of claim 4 wherein the tumour is a head and neck cancer.

6. A method of inhibiting differentiation of a protomesodermal neoplastic cell capable of differentiating into a nonvascular cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a β-blocker to the subject.

7. A method of inhibiting differentiation of a totipotent stem cell capable of differentiating into a nonvascular cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a β-blocker to the subject.

8. A method of any one of claims 1 to 7 wherein the neoplastic cells express one or more of the following cell markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.

9. A method of any one of claims 1 to 7 wherein the neoplastic cells express two or more of the following cell markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.

10. A method of any one of claims 1 to 7 wherein the neoplastic cells express three or more of the following cell markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.

11. A method of any one of claims 1 to 10 wherein the neoplastic cells express one or more of the following cell markers: haemoglobin ζ chain, Oct4, SSEA4 or angiotensin II receptor.

12. A method of any one of claims 1 to 11 wherein the neoplastic population of cells is present in a solid tumour.

13. A method of any one of claims 1 to 12 wherein the neoplastic population of cells is a squamous cell carcinoma, melanoma, glioblastoma or sarcoma.

14. A method of any one of claims 1 to 13 wherein the β-blocker is a non-selective β-blocker.

15. In one embodiment the β-blocker is selected from the group comprising Alprenolol,

Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Propranalol, Sotalol, or Timolol.

16. A method of claim 15 wherein the β-blocker is propranolol.

17. A method of any one of claims 1 to 16 wherein the β-blocker is formulated for oral,

parenteral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal or topical administration.

18. A method of any one of claims 1 to 17 further comprising separate, simultaneous or

sequential administration of at least one additional cancer therapy.

19. A method of claim 8 wherein the cancer therapy is an anti-tumour agent or an anti-tumour therapy.

20. A method of claim 19 wherein the anti-tumour therapy is selected from therapies such as, but not limited to, surgery, chemotherapies, radiation therapies, hormonal therapies, biological therapies/immunotherapies, anti-angiogenic therapies, cytotoxic therapies, vaccines, nucleic acid-based vaccines (eg nucleic acids expressing a cancer antigen such as DNA vaccines including pi 85 vaccines), viral-based therapies (eg adeno-associated virus, lentivirus), gene therapies, small molecule inhibitor therapies, nucleotide-based therapies (eg RNAi, antisense, ribozymes etc), antibody-based therapies, oxygen and ozone treatments, embolization, and/ or chemoembolization therapies.

21. A method of claim 1 wherein the anti-tumour agent is a chemo therapeutic agent or an anti- tumourigenic agent.

22. A method of any one of claims 1 to 21 further comprising separate, simultaneous or

sequential administration of at least one additional modulator of the renin-angiotensin pathway.

23. A method of claim 22 wherein the at least one additional modulator is one or more of β- blockers, one or more ACE-inhibitors, or one or more angiotensin receptor blockers.

24. A method of claim 23 wherein the β-blocker is selected from the group comprising

Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Propranalol, Sotalol, or Timolol.

25. A method of claim 24 wherein the β-blocker is propranolol.

26. A method of inhibiting growth or proliferation of a neoplastic population of cells in a

subject, the method comprising: a) identifying in a sample from the subject a population of cells wherein one or more of the cells is one or more of the following:

i) positive for the presence of one or more of the following markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD 133, ACE or angiotensin II receptor;

ii) optionally positive for the presence of one or more of the following markers:,

VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA- 2;

iii) negative for the presence of CD45; or

iv) any combination of any two or more of i) to iii);

b) administering to the subject, a modulator of the renin-angiotensin pathway.

27. A method of claim 26 wherein the method is a method of treating or preventing cancer in the subject, including but not limited to head and neck cancer.

28. ' A method of diagnosing cancer in a subject, the method comprising:

i) positive for the presence of one or more of the following markers: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD 33, ACE or angiotensin II receptor;

VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA- 2;

iii) negative for the presence of CD45; or

iv) any combination of two or more of i) to iii);

b) diagnosing cancer on the basis of the identification.

29. A method of claim 28 wherein the cancer is head and neck cancer.

30. A method of inhibiting growth or proliferation of a neoplastic population of cells in a subject, the method comprising administering a therapeutically effective amount of a β- blocker to the subject in need thereof, wherein the subject is a subject for whom the presence or absence in a sample from the subject of a population of cells having one or more of the cell markers selected from the group comprising: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor, and optionally one or more of the following cell markers: VEGFR-2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2., has been determined.

A method of claim 30 wherein the method is a method of treating or preventing cancer in the subject, including but not limited to head and neck cancer.

A method of identifying a population of cells likely to be responsive to treatment with a modulator of the renin-angiotensin pathway, the method comprising determining the presence or absence on one or more of the cells of one or more cell markers selected from the following group: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor, and optionally one or more of the following cell markers: VEGFR- 2, CD31, Tal-l, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.; and assessing the likely responsiveness to treatment on the basis of the determination.

A method of claim 32 wherein the population of cells is present in a sample taken from a subject.

A method of identifying a subject likely to be responsive to or suitable for treatment with a modulator of the renin-angiotensin pathway, the method comprising determining the presence or absence on one or more of the cells of one or more cell markers selected from the following group: brachyury, haemoglobin ζ chain, Oct4, SSEA4, CD34, CD133, ACE, angiotensin II receptor, and optionally one or more of the following cell markers: VEGFR- 2, CD31, Tal-1, SMA, Glut-1, aldehyde dehydrogenase, vimentin or GATA-2.; and determining the likely responsiveness to or suitability for treatment on the basis of the identification.

A method of claim 34 wherein the identification, selection or determination is by contacting the sample or the cells with a labelled detection agent.

A method of claim 34 or 35 wherein the subject has been diagnosed with head and neck cancer or is predisposed to head and neck cancer.

A method of any one of claims 26 to 36 wherein the renin-angiotensin modulator is selected from a β-blocker, angiotensin-converting enzyme (ACE) inhibitor, or angiotensin-receptor blocker.

A method of claim 37 wherein the β-blocker is selected from the group comprising Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Propranalol, Sotalol, or Timolol.

A method of claim 38 wherein the β-blocker is Propranolol.

A method of claim 37 wherein the ACE inhibitor is selected from the group comprising Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril, or Trandolapril.

A method of claim 37 wherein the angiotensin-receptor blocker is selected from the gtoup comprising Valsartan, Telmisartan, Losartan, Irbesartan^ Candesartan or Olmesartan.

42. A method of any one of claims 1 to 41 wherein the cancer or the head or neck cancer is malignant.

43. A method of any one of claims 1 to 41 wherein the cancer or the head or neck cancer is a solid tumour or a lymphoma.

44. A method of an one of claims 1 to 41 wherein the solid tumour is a squamous cell carcinoma, melanoma, glioblastoma or sarcoma.