Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/112—Disease subtyping, staging or classification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4703—Regulators; Modulating activity
  • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/52—Assays involving cytokines
  • G01N2333/555—Interferons [IFN]
  • G01N2333/56—IFN-alpha
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
  • G01N2333/7156—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]

Definitions

• the present invention relates generally to a method of diagnosing, prognosing or monitoring the development or progress of metastatic cancer, more particularly bone metastatic cancer.
• the method of the present invention more particularly provides a method for detecting metastatic cancer, or a predisposition thereto, by screening for the differential expression of a panel of genes which comprise an IRF7 binding site.
• the present invention provides a method of therapeutically or prophyiacticaliy treating metastatic cancer, in particular bone metastatic cancer. More particularly, the present invention provides a means of therapeutically or prophyiacticaliy treating metastatic cancer by upregulating type I IFN levels.
• a neoplasm is an abnormal mass or colony of cells produced by a relatively autonomous new growth of tissue. Most neoplasms arise from the clonal expansion of a single cell that has undergone neoplastic transformation. The transformation of a normal cell to a neoplastic cell can be caused by a chemical, physical, or biological agent (or event) that alters the cell genome.
• Neoplastic cells are characterized by the loss of some specialized functions and the acquisition of new biological properties, foremost, the property of relatively autonomous growth. They pass on their heritable biological characteristics to progeny cells. Neoplasms may originate in almost any tissue containing cells capable of mitotic division.
• a malignant neoplasm manifests a greater degree of autonomy, is capable of invasion and metastatic spread, may be resistant to treatment, and may cause death.
• a benign neoplasm exhibits a lesser degree of autonomy, is usually not invasive and does not metastasize. Cancer is second only to heart disease as the most common cause of death in western countries. The estimated incidence of cancer in the US, for example, is about 1 x 10 6 new cases annually. Nearly 80% of all malignant neoplasms arise in 10 anatomical sites, namely lung, breast, colon and rectum, prostate, lymph nodes, uterus, bladder, pancreas, blood and stomach.
• Metastatic tumours are very common in the late stages of cancer.
• the spread of metastases may occur via the blood, the lymphatics or through both routes, with the most common places for metastases to arise being the lymph nodes, lungs, liver, brain and the bones.
• tumours there is a propensity for certain tumours to seed in particular organs. This was first discussed as the "seed and soil" theory by Stephen Paget over a century ago in 1889.
• prostate cancer usually metastasises to the bones.
• colon cancer has a tendency to metastasise to the liver while in women, stomach cancer often metastasises to the ovary.
• the "seed and soil” theory it is difficult for cancer cells to survive outside their region of origin, so in order to metastasise they must find a location with similar characteristics.
• breast tumour cells which gather calcium ions from breast milk, metastasise to bone tissue, where they can gather calcium ions from bone.
• Malignant melanoma spreads to the brain, presumably because neural tissue and melanocytes arise from the same cell line in the embryo.
• Metastasis involves a complex series of steps in which cancer cells leave the original tumour site and migrate to other parts of the body via the bloodstream or the lymphatic system. To do so, malignant cells break away from the primary tumour and attach to and degrade proteins that make up the surrounding extracellular matrix, which separates the tumour from adjoining tissue. By degrading these proteins, cancer cells are able to breach the extracellular matrix and escape.
• metastasis suppressors The body resists metastasis by a variety of mechanisms through the actions of a class of proteins known as metastasis suppressors, of which about a dozen are known. It has also been determined that one of the critical events required is the growth of a new network of blood vessels, that is tumour angiogenesis. Significant research has therefore focussed on angiogenesis inhibitors as a means to prevent the growth of metastases. Despite these findings, however, the effective treatment of metastatic cancer has been elusive and largely still relies on the application of very non-specific and highly toxic chemotherapy based methods. Equally elusive has been the development of a method for the early diagnosis of metastatic cancer.
• metastatic cancer is found at the same time as the primary tumour, often in the context of surgery, the prognosis for the patient is usually very poor due to the advanced stage of disease.
• confirmation of metastatic cancer can be virtually impossible to make due to the limitations of current diagnostic techniques.
• the primary tumour will be removed and the patient may nevertheless be subjected to a full course of chemotherapy in the hope that this will be effective to kill any metastatic tumours which may be present.
• tumours will necessarily metastasise and it is virtually impossible, based just on histological analysis, to predict which tumours will metastasise and which will not.
• a molecular signature has been identified which, if present in a primary tumour, is characteristic of the propensity of that tumour to metastasise.
• downregulation in a tumour of the level of expression of genes which comprise an IRF7 binding site, relative to the levels present in a non-metastatic tumour is indicative of the presence of a metastatic phenotype.
• the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
• One aspect of the present invention provides a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of one or more genes by said tumour, which genes comprise an IRF7 binding site in their promoter, wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding . non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the expression of one or more genes, of Table 1, by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the functional level of IRF7,
• IRF9 or STATl in said tumour wherein a decrease in the functional level of said IRF7, IRF9 or STATl relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the expression of one or more genes of Table 1 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour and wherein the metastases are bone metastases.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the functional level of IRF7, IRF9 or STATl in said tumour wherein a decrease in the functional level of said IRF7, IRF9 or STATl is indicative of the metastatic phenotype of said tumour and wherein the metastases are bone metastases.
• said tumour is a tumour of the breast, colon, kidney, lungs, skin, ovary, pancreas, prostate, rectum, stomach, thyroid or uterus.
• said screening is directed to at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes, at least 60 genes, at least 70 genes, at least 80 genes, at least 90 genes, at least 100 genes, at least 1 10 genes, at least 120 genes, at least 130 genes, at least 140 genes, at least 150 genes, at least 160 genes, at least 170 genes, at least 180 genes, at least 190 genes, at least 200 genes or at least 208 genes.
• said screening is directed to one or more genes selected from Table 2.
• a further aspect of the present invention is directed to a method of treating metastatic cancer in an individual, which cancer is characterised by aberrant IRF7 functionality, said method comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of Type I IFN in said individual.
• the present invention is more particularly directed to a method of treating a breast, kidney or prostate metastatic cancer in an individual, which cancer is characterised by aberrant IRP7 functionality, said method comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of Type I IFN in said individual.
• a method of treating metastatic cancer, which metastases are present in the bone and which cancer is characterised by aberrant IRF7 functionality, in an individual comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of Type I IFN in said individual.
• a method of treating metastatic cancer in a patient which cancer is characterised by aberrant IRF7 functionality comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of IFN-a in said patient.
• a method of treating metastatic cancer in an individual which cancer is characterised by aberrant IRF7 functionality comprising administering an effective amount of a composition wherein said composition comprises an agent which upregulates the level of IFN- ⁇ in said individual.
• Type I IFN in the manufacture of a medicament for the treatment of a metastatic cancer in an individual, which cancer is characterised by aberrant IRF7 functionality.
• Figure 1 Bone metastases of breast carcinoma origin downregulate interferon pathways and other immune genes. Heat maps generated using hierarchical clustering applying Euclidean distance and centroid linkage rule. The colored scale bar represents fold change Log 2 . The grey bar represents the CLOVER enrichment score as described in Methods. IRF7 was identified as a significant regulatory factor in the intersection of the suppressed genes and the INTERFEROME genes.
• FIG. 2 Expression of the transcription factor IRF7 is suppressed in bone metastases,
• (a) Real-time RT-PCR analysis for IRF7 expression in mRNA isolated from 4T1.2 primary tumours and spine metastases (error bars represent SEM, n 4). IRF7 transcript abundance is relative to GAPDH.
• CFSE labelled naive Balb/c splenocytes were activated with anti-CD3/CD28 antibodies and co-cultured with or without CDl lb + Ly6G + MDSCs (ratio 1 :1) derived from the 4T1.2-BV tumour-bearing mice.
• Total splenocytes, CD4 + and CD8 + T cell proliferation at 72 h was measured by flow cytometry.
• a two-tailed student t-test was performed and the mean fluorescence intensity (MFI) for CFSE was compared between the splenocytes ⁇ MDSC of 7 replicate samples.
• MFI mean fluorescence intensity
• Y axis on Kaplan-Meier curve represents "Bone Relapse-free survival"
• X axis is time in months.
• Purple, black and blue represents highest, intermediate and lowest tertile of expression of the IRF7 gene signature respectively.
• Log-rank p value is calculated pooled across the three strata, (d) The IRF7 gene signature is not significantly associated with non- bony relapses (brain, liver, lung).
• Figure 6 The effect of type 1 1FN treatment on metastasis, (a) Real-time RT-PCR analysis of 4T1.2 cells treated with lOOOIU/mL recombinant IFN-ci
• . (n 3, * p ⁇ 0.05, error bars are S.E.M.). (b) Proliferation of 4T1.2 cells treated with lOOOIU/mL of recombinant IFN scenery
• Figure 7 Visualization of the TRANSFAC Match predicted IRF IRF7.JSRE and IRF2 sites in the putative promoter regions (- 1 ,500bp-200bp) of a subset of interferon regulated metastases suppressed genes.
• FIG. 8 Promoter analysis framework applied in the identification and visualization of Interferon signature enrichment and IRF promoter binding sites from the suppressed gene list.
• Type I IFN signature was identified using the Interferons database.
• Promoter analysis was performed on sequence extracted from Ensembl v59 and analysed using the CLOVER algorithm and the TRANSFAC database. Visualization was achieved using the Interferome interface.
• FIG. 9 Tm plots (negative first derivative of the high resolution melting curves) obtained from MS-HRM experiments for the DNA methylation analysis of IRF7 and STAT1.
• the Tm curves were partly smoothened by applying a light digital filter available in the analysis software.
• the curves for the unmethylated and fully methylated control DNAs are shown in green and red respectively.
• the DNA methylation standards 50% (turquoise dotted curves) and 10% (grey dotted curves) are shown.
• the black curves correspond to the normal tissue sample 841/07 and the blue curves correspond to the spine metastasis 2,375.07.
• the normal tissue and all tumour samples were unmethylated for all regions analysed.
• FIG. 10 Enforced expression of IRF7 restores expression of Type I IFN pathway, a. IRF7 expression in parental cells (4T1.2) or cells expressing a base vector construct (B V) or an IRF7 expression construct (IRF7). 5 individual clones were pooled in each group, b.
• Figure 11 Enforced IRF7 expression in 4T1.2 cells restores the IRF7-signature.
• Heat map representation of expression values for differentially expressed genes between 4T1.2BV and 4T1.2-IRF7 cells (fold change > 2, P ⁇ 0.05) hierarchically clustered on gene expression value. Red coloration represents induction while blue indicates reduction of expression relative to base vector control.
• Figure 14 Measurement of CD1 lb + Ly6G + cells in tumour bearing mice.
• Figure 17 Quantitation of lung metastases in mice bearing 4T1.2 tumours. Q-PCR detection of metastatic tumour burden in spines of 4T1.2-BV and 4T1.2-IRF7 tumour burdened mice (endpoint, day 32), or 4T1.2-BV mice treated with saline or IFNal (endpoint, day 33).
• Figure 18 Measurement of MDSC and G-CSF in blood of 4T1.2 tumour bearing mice treated with IFNal . Mice were treated with IFN-al or saline control one day after 4T1.2 tumour cell inoculation into the mammary gland. Five days after tumour inoculation, MDSC percentage (a) and absolute numbers (b) were measured, along with G-CSF levels by ELISA (c).
• Figure 19 Changes in immune cells in the mammary gland, lung and bone
• a) Mice were treated with IFN-al or saline control on day 1 , 3 and 5 after 4T1.2 tumour cell inoculation into the mammary gland. Ten days after tumour inoculation MDSC, NK, CD4 + and CD8 + T cell percentages were measured in fatpad/primary tumour, lung and bone marrow. N 5. Difference between treatment groups was assessed using an unpaired Ttest and significance is indicated, b) Cells isolated from the mammary gland, lung and bone marrow of naive Balb/c mice were stained for DX5, TCR, CD1 lb and CD27. Shown are representative profiles for CD1 lb and CD27 expression on gated DX5 + TCR. Percentage of CD 1 1 b low hi6h and/or CD27 low hi6h are indicated.
• Figure 20 Treatment with the IFN agonist poly I:C suppresses metastasis. Mice bearing 4T1.2 cells were treated with 25ug poly I:C 3 x weekly by tail vein injection beginning at day 3.
• Figure 21 Combination of poly I:C and doxorubicin reduces metastasis in an early treatment setting.
• 4T1.2 cells were inoculated into the 4* mammary gland of Balb/c mice. Primary tumours were resected when they reached 0.2g and 2 days later treatment was started for a period of 2 weeks. Mice were treated with vehicle alone, 25 ug poly I:C every 2 days, 4mg/kg doxorubicin every 4 days, or the combination of poly I:C and doxorubicin. Metastatic burden was visualised in all mice at a set time point using bioluminescence imaging.
• FIG. 22 Blocking type I IFN signaling using mice deficient in Ifnarl enhances bone metastasis in weakly (4T1 ) and non-metastatic (66cl4) breast cancer models, (a) primary tumour growth of 4T1 and 66cl4 tumours, (b) Metastatic burden in mice bearing 4T1 tumours (day 32 without resection), (c) Metastatic burden in mice bearing 66cl4 tumors (day 21 after resection). RTB, relative tumour burden, (d) Representative H&E-stained sections of femurs from mice bearing 4T1 and 66cl4 tumors at endpoint. Scale bar, 50 ⁇ . T represents a tumour region.
• the present invention is predicated, in part, on the determination both that the loss of IRF7 gene functionality in a tumour induces a shift to a metastatic phenotype and, further, that this loss of functionality is routinely identifiable by virtue of the presence of a unique gene signature in the tumour, this signature being a downregulation in the level of expression of one or more genes comprising an IRF7 binding site in their promoter.
• This finding has facilitated not only the development of a method for screening a tumour to determine its propensity to metastasise, but has also led to the development of a means of therapeutically or
• prophylactically treating patients presenting with a primary tumour which exhibits a metastatic phenotype said method being based on upregulating levels of Type 1 IFN in the patient.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the expression of one or more genes by said tumour, which genes comprise an IRF7 binding site in their promoter, wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the expression of one or more genes, of Table 1 , by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the functional level of IRF7, IRF9 or STATl in said tumour wherein a decrease in the functional level of said IRF7, IRF9 or STATl relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• Tumour is used herein to describe an abnormal mass or growth of cells or tissue that is characterized by uncontrolled cell division.
• Tumours may be benign (not cancerous) or malignant (cancerous). Tumours may be identified, monitored or assessed through clinical screening or diagnostic procedures, including, but not limited to, palpation, biopsy, cell proliferation index, endoscopy, mammography, digital mammography, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), radiography, radionuclide evaluation, CT- or MRI-guided aspiration cytology, and imaging-guided needle biopsy, among others.
• CT computed tomography
• MRI magnetic resonance imaging
• PET positron emission tomography
• radiography radionuclide evaluation
• CT- or MRI-guided aspiration cytology CT-guided aspiration cytology
• imaging-guided needle biopsy among others.
• said tumour is a primary tumour.
• references to the "metastatic phenotype" of a tumour should be understood as a reference to the capacity of the cells of the subject tumour to spread from the organ or tissue of origin to another organ or tissue, typically via the lymphatics or the blood circulation. It should also be understood that the subject cells, although exhibiting a metastatic phenotype, may or may not have actually travelled to another organ in the patient in whom the tumour has developed.
• metastatic "status” should be understood to mean that the tumour is assessed to determine whether or not it exhibits a metastatic phenotype.
• the subject method is particularly effective in detecting the metastatic phenotype of tumours which exhibit a predisposition to metastasising to the bone. This is an extremely valuable finding since bone metastases are generally regarded as particularly aggressive and difficult to treat. Accordingly, developing a means to identify tumours exhibiting a metastatic phenotype provides a mechanism of potentially identifying these bone metastases at a much earlier stage than is currently possible. This may then significantly improve the prognostic outcome for the patient.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the expression of one or more genes of Table 1 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour and wherein the metastases are bone metastases.
• a method of assessing the metastatic status of a tumour from an individual comprising detecting the functional level of 1RF7, IRF9 or ST ATI in said tumour wherein a decrease in the functional level of said IRF7, IRF9 or STAT1 is indicative of the metastatic phenotype of said tumour and wherein the metastases are bone metastases.
• cancer metastases are the lungs, bone and liver. Although most cancers have the ability to spread to many different parts of the body, they will often spread to one site more often than others. Listed below are the three most common sites for metastases, excluding lymph nodes, for several types of cancer: Cancer Type Main sites of metastasis
• said tumour is a tumour of the breast, colon, kidney, lungs, skin, ovary, pancreas, prostate, rectum, stomach, thyroid or uterus.
• a method of assessing the metastatic status of a breast, kidney or prostate tumour from an individual comprising detecting the expression of one or more genes of Table 1 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• a method of assessing the metastatic status of a breast, kidney or prostate tumour from an individual comprising detecting the functional level of IRF7, IRF9 or STAT1 in said tumour wherein a decrease in the functional level of said IRF7, IRF9 or STAT1 relative to the level of expression in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said breast, kidney or prostate tumour.
• the method of the present invention does not conclusively identify the site or likely site of metastases.
• the positive diagnosis of a metastatic phenotype does enable one to then design an appropriate monitoring and treatment regime to deal with the patient's transition to metastatic cancer.
• interferon regulatory factors are a family of transcription factors with diverse functions which include host defense, cell cycle regulation, apoptosis, oncogenesis, and immune cell development and homeostasis.
• IRFs interferon regulatory factors
• the DNA binding domain is located at the amino termini of the IRFs and consists of a five-tryptophan repeat that binds to a specific GAAA genomic sequence that is similar to the IFN-stimulated response element (ISRE).
• the IRFs become activated via phosphorylation at their carboxyl termini, after which they translocate from the cytoplasm to the nucleus to effect transcription of ISRE-containing genes.
• the various IRFs differ in cellular localisation, structural properties, an activation-induced stimuli, thus conferring each IRF with unique functions.
• ENSG00000068366 ACSL4 Reference to each of the genes detailed above should be understood as a reference to all forms of these genes and variants thereof. As would be appreciated by the person of skill in the art, some genes are known to exhibit allelic variation between individuals or single nucleotide polymorphisms. SNPs encompass insertions and deletions of varying size and simple sequence repeats, such as dinucleotide and trinucleotide repeats. Variants include nucleic acid sequences from the same region sharing at least 90%, 95%, 98%, 99% sequence identity i.e. having one or more deletions, additions, substitutions, inverted sequences etc. relative to the genes described herein.
• the present invention should be understood to extend to such variants which, in terms of the present diagnostic applications, achieve the same outcome despite the fact that minor genetic variations between the actual nucleic acid sequences may exist between individuals.
• the present invention should therefore be understood to extend to all forms of DNA which arise from any other mutation, polymorphic or allelic variation.
• screening for the "level of expression” of these genes may be achieved in a variety of ways including screening for any of the forms of RNA transcribed from these genes or cDNA generated therefrom or the protein expression product.
• Reference to “screening for the level of RNA transcripts” should be understood as a reference to either screening the RNA directly or screening cDNA transcribed therefrom. Changes to the levels of any of these products is indicative of changes to the expression of the subject gene. As detailed hereinbefore, the loss of functional IRF7 in tumours which have undergone transition to a metastatic phenotype will lead to a reduction or loss in the expression of all or some of the genes of Table 1.
• nucleic acid molecule or protein which is identified and measured may be a whole molecule or a fragment thereof.
• nucleic acid molecule should be understood as a reference to both deoxyribonucleic acid molecules and ribonucleic acid molecules and fragments thereof.
• the present invention therefore extends to both directly screening for RNA levels in a sample or screening for the complementary cDNA which has been reverse-transcribed from an RNA population of interest. It is well within the skill of the person of skill in the art to design methodology directed to screening for DNA, RNA or protein.
• any of the genes listed in Table 1 above may be used in the method of the invention either alone or in combination with other genes in Table 1 or other diagnostic markers. Although information concerning the expression of as few as one gene is expected to provide useful information, confidence in the accuracy of the classification of a tumour as metastatic will increase when more markers are included. Tumours may be analysed with respect to the expression of groups of these genes, including from 1 to 10 of the of the genes listed in Table 1, in any combination. It is well within the ability of one of skill in the art to select groups of genes for analysis from among the genes listed in Table 1.
• said screening is directed to at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes, at least 60 genes, at least 70 genes, at least 80 genes, at least 90 genes, at least 100 genes, at least 1 10 genes, at least 120 genes, at least 130 genes, at least 140 genes, at least 150 genes, at least 160 genes, at least 170 genes, at least 180 genes, at least 190 genes, at least 200 genes or at least 208 genes.
• said screening is directed to one or more genes selected from Table 2.
• Still another embodiment is directed to a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of IFITM3, TLR3, IRF7, and IL13RA1 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• Yet another embodiment is directed to a method of assessing the metastatic status of the tumour from an individual, said method comprising detecting the expression of CALDI , RUNXl , YWHAZ and HBEGF by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• Still yet another embodiment is directed to a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of IL13RA1 , CSF2RB, STAT1, CD44, IRF7, IF144, TLR3 and IFITM3 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• said method comprises detecting the expression of L13RA1 , CD86, CSF2RB, STAT1, CD44, IRF7, IFI44, TLR3, IER3, IFITM3, RUNX3 and CTSS.
• Yet still another embodiment is directed to a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of IFI44, IRF7, CSF2RB, STAT1 , TLR3, IL13RA1 and IFITM3 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• said method comprises detecting the expression of IFI44, IRF7, CSF2RB, STAT1 , TLR3, IFI202B, IER3, RUNX3, CTSS, IL13RA1 , IFITM3 and CD86.
• a further embodiment is directed to a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of RUNX1 , SQLE, PDXK, YWHAZ, DDX3X, RBBP4, HBEGF, DAB2 and FAM120A by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• said method comprises detecting the expression of RU X1, SQLE, PDXK, HNMT, CALD1 , YWHAZ, DDX3X, RBBP4, THBS1 , HBEGF, DAB 2 and FAM120A. '
• Another further embodiment is directed to a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of PTPRK and SLC6A6 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• said method comprises detecting the expression of CSDE1, ATP6V1B2, PTPRK, LGMN, CXCL9, TGIF1 , NIPA2, SLC6A6, ARG1 , CFP, CTSH and ACSL4.
• Still another further embodiment is directed to a method of assessing the metastatic status of a tumour from an individual, said method comprising detecting the expression of DHX58, BST2, IFI44, IFIT3, IRF7, STAT1, DSP and USP18 by said tumour wherein a decreased level of expression of said genes relative to the level of expression of said genes in a corresponding non-metastatic tumour is indicative of the metastatic phenotype of said tumour.
• said screening method is directed to screening for mRNA or cDNA.
• said screening method is directed to screening for the encoded protein expression product.
• gene panels detailed hereinbefore may be screened for as isolated panels or they may form part of a larger panel. That is, one may elect to screen for a given panel together with one or more additional markers.
• the cellular defect which leads to metastatic transition is not necessarily a loss of IRF7, IRF9 or STAT1 expression per se.
• the defect may also be a loss of the functional form of these proteins. In this case, the protein may still be present, albeit not in a functional form.
• these proteins function by undergoing phosphorylation and thereafter translocation from the cytoplasm to the nucleus. Once in the nucleus, IRF7 and IRF9 bind to the gene promoter in order to induce transcription. Non-functional protein is therefore detectable either by screening for the localisation of the protein, with cytoplasmic localisation being indicative of non-functionality, or phosphorylation, wherein a lack of phosphorylation is indicative of non- functionality. This form of testing may be done together with testing for the absolute levels of protein or for the RNA transcripts of said proteins.
• the subject gene expression or functional protein levels are measured in the cells of the tumour. It would be appreciated by the person of skill in the art that the testing of a tumour to determine its metastatic status will often occur after the tumour has been surgically excised. However, to the extent that surgical excision may not be possible or desirable or to the extent that an immediate result is sought, a biopsy specimen can be harvested either at or immediately after initial diagnosis and the testing can be performed on this specimen.
• the results obtained from the tumour of the individual in issue are assessed relative to the level which is present in a corresponding non-metastatic tumour.
• This is the control level.
• corresponding is meant a tumour of the same tissue type as the tumour which is the subject of testing.
• the control level of gene or protein expression will be that of a non-metastatic breast tissue tumour.
• the control level may be a standard result which reflects individual or collective results obtained from individuals other than the mammal in issue.
• This form of analysis is in fact a preferred method of analysis since it enables the design of kits which require the collection and analysis of a single biological sample, being a test sample of interest.
• control level may be calculated by any suitable means which would be well known to the person of skill in the art. For example, a population of non-metastatic tumour tissues can be assessed in terms of the level of gene or protein expression thereby providing a standard value or range of values against which all future test samples are analysed. It should also be understood that this control level may be determined from the subjects of a specific cohort and for use with respect to test samples derived from that cohort. Accordingly, there may be determined a number of standard values or ranges which correspond to cohorts which differ in respect of characteristics such as age, gender, ethnicity or health status. It is to be expected that standards would be developed for each tissue type in which a tumour can arise. Said "control level" may be a discrete level or a range of levels.
• the biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing.
• a tissue sample may require homogenisation prior to testing or it may require sectioning for in situ testing of the intracellular localisation of proteins.
• a cell sample may require permeabilisation prior to testing.
• a reagent such as a buffer
• the biological sample may be directly tested or else all or some of the nucleic acid or protein material present in the biological sample may be isolated prior to testing. To this end, it would be appreciated that when screening for changes to the level of expression of the genes in Table 1, one may screen for the RNA transcripts themselves or cDNA which has been transcribed therefrom. In yet another example, the sample may be partially purified or otherwise enriched prior to analysis. It is within the scope of the present invention for the target cell population or molecules derived therefrom to be pretreated prior to testing, for example, inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).
• the term "individual" or "patient” as used herein includes humans, primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g. kangaroos, deer, foxes).
• livestock animals e.g. horses, cattle, sheep, pigs, donkeys
• laboratory test animals e.g. mice, rats, guinea pigs
• companion animals e.g. dogs, cats
• captive wild animals e.g. kangaroos, deer, foxes.
• the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human. It should be understood that the decrease in the level of expression of the subject molecules, in a tumour which has transitioned to a metastatic phenotype, may be either a partial reduction in the level of
• the degree of reduction in expression may vary between genes. Accordingly to the extent that six or more of the genes of Table 1 are tested, the degree to which expression levels are reduced as between the genes may differ. However, the critical issue is that the levels of each gene will be reduced relative to its corresponding control. It should also be expected that as between different patients with the same type of primary tumour being tested, there may be observed variation in the degree of reduction of expression of the genes which are tested. However, provided that the observed level is below the non-metastatic tumour control level, this is indicative of the shift to a metastatic phenotype.
• Means for testing for changes to the expression levels of one or more of the genes listed in Table 1 in a tumour sample can be achieved by any suitable method, which would be well known to the person of skill in the art, such as but not limited to assessment of expression profiles of RNA by array technologies (Alon et al, Proc. Natl. Acad. Sci. USA: 96, 6745-6750, June 1999).
• a "microarray” is a linear or multi-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support. The density of the discrete regions on a microarray is determined by the total numbers of target polynucleotides to be detected on the surface of a single solid phase support.
• a DNA microarray is an array of oligonucleotide probes placed onto a chip or other surfaces used to detect complementary oligonucleotides from a complex nucleic acid mixture. Since the position of each particular group of probes in the array is known, the identities of the target
• polynucleotides can be determined based on their binding to a particular position in the microarray.
• DNA microarray technology makes it possible to conduct a large scale assay of a plurality of target nucleic acid molecules on a single solid phase support.
• RNA from the sample of interest is subjected to reverse transcription to obtain labelled cDNA. See U.S. Pat. No. 6,410,229 (Lockhart et al.)
• the cDNA is then hybridized to oligonucleotides or cDNAs of known sequence arrayed on a chip or other surface in a known order.
• the RNA is isolated from a biological sample and hybridised to a chip on which are anchored cDNA probes. The location of the oligonucleotide to which the labelled cDNA hybridizes provides sequence information on the cDNA, while the amount of labelled hybridized RNA or cDNA provides an estimate of the relative
• RNA or cDNA of interest See Schena et al. Science 270:467-470 (1995).
• use of a cDNA microarray to analyze gene expression patterns in human cancer is described by DeRisi, et al. ⁇ Nature Genetics 14:457-460 (1996)).
• nucleic acid probes corresponding to the subject nucleic acids are made.
• the nucleic acid probes attached to the microarray are designed to be substantially complementary to the nucleic acids of the biological sample such that specific hybridization of the target sequence and the probes of the present invention occurs.
• This complementarity need not be perfect, in that there may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. It is expected that the overall homology of the genes at the nucleotide level probably will be about 40% or greater, probably about 60% or greater, and even more probably about 80% or greater; and in addition that there will be corresponding contiguous sequences of about 8-12 nucleotides or longer.
• the sequence is not a complementary target sequence.
• substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions.
• a nucleic acid probe is generally single stranded but can be partly single and partly double stranded.
• the strandedness of the probe is dictated by the structure, composition, and properties of the target sequence.
• the oligonucleotide probes range from about 6, 8, 10, 12, 15, 20, 30 to about 100 bases long, with from about 10 to about 80 , bases being preferred, and from about 15 to about 40 bases being particularly preferred. That is, generally entire genes are rarely used as probes. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
• the probes are sufficiently specific to hybridize to a complementary template sequence under conditions known by those of skill in the art.
• the number of mismatches between the probe's sequences and their complementary template (target) sequences to which they hybridize during hybridization generally do not exceed 15%, usually do not exceed 10% and preferably do not exceed 5%, as-determined by BLAST (default settings).
• Oligonucleotide probes can include the naturally-occurring heterocyclic bases normally found in nucleic acids (uracil, cytosine, thymine, adenine and guanine), as well as modified bases and base analogues. Any modified base or base analogue compatible with hybridization of the probe to a target sequence is useful in the practice of the invention.
• the sugar or glycoside portion of the probe can comprise deoxyribose, ribose, and/or modified forms of these sugars, such as, for example, 2'-0-alkyl ribose.
• the sugar moiety is 2'-deoxyribose; however, any sugar moiety that is compatible with the ability of the probe to hybridize to a target sequence can be used.
• nucleoside units of the probe are linked by a phosphodiester backbone, as is well known in the art.
• internucleotide linkages can include any linkage known to one of skill in the art that is compatible with specific
• probe including, but not limited to phosphorothioate, methylphosphonate, sulfamate (e.g., U.S. Pat. No. 5,470,967) and polyamide (i.e., peptide nucleic acids).
• Peptide nucleic acids are described in Nielsen et al. (1991) Science 254: 1497-1500, U.S. Pat. No. 5,714,331, and Nielsen (1999) Curr. Opin. Biotechnol. 10:71-75.
• the probe can be a chimeric molecule; i.e., can comprise more than one type of base or sugar subunit, and/or the linkages can be of more than one type within the same primer.
• the probe can comprise a moiety to facilitate hybridization to its target sequence, as are known in the art, for example, intercalators and/or minor groove binders. Variations of the bases, sugars, and intemucleoside backbone, as well as the presence of any pendant group on the probe, will be compatible with the ability of the probe to bind, in a sequence-specific fashion, with its target sequence. A large number of structural modifications, are possible within these bounds.
• the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. ⁇ Nucleic Acids Symp. Ser., 24: 197-200 (1991)) or in the European Patent No. EP- 0225,807.
• synthetic methods for preparing the various heterocyclic bases, sugars, nucleosides and nucleotides that form the probe, and preparation of oligonucleotides of specific predetermined sequence are well-developed and known in the art.
• a preferred method for oligonucleotide synthesis incorporates the teaching of U.S. Pat. No. 5,419,966.
• probes may be designed for a particular target nucleic acid to account for polymorphism and/or secondary structure in the target nucleic acid, redundancy of data and the like.
• more than one probe per sequence either overlapping probes or probes to different sections of a single target gene are used. That is, two, three, four or more probes, are used to build in a redundancy for a particular target.
• the probes can be overlapping (i.e. have some sequence in common), or are specific for distinct sequences of a gene.
• polynucleotide is situated in a discrete area of the microarray.
• Probes may be in solution, such as in wells or on the surface of a micro-array, or attached to a solid support.
• solid support materials that can be used include a plastic, a ceramic, a metal, a resin, a gel and a membrane.
• Useful types of solid supports include plates, beads, magnetic material, microbeads, hybridization chips, membranes, crystals, ceramics and self-assembling monolayers.
• One example comprises a two- dimensional or three-dimensional matrix, such as a gel or hybridization chip with multiple probe binding sites (Pevzner et a/., J. Biomol. Struc. & Dyn. 9:399-410, 1991 ; Maskos and Southern, Nuc. Acids Res.
• Hybridization chips can be used to construct very large probe arrays that are subsequently hybridized with a target nucleic acid. Analysis of the hybridization pattern of the chip can assist in the identification of the target nucleotide sequence. Patterns can be manually or computer analyzed, but it is clear that positional sequencing by hybridization lends itself to computer analysis and automation.
• one may use an Affymetrix chip on a solid phase structural support in combination with a fluorescent bead based approach.
• one may utilise a cDNA microarray.
• the oligonucleotides described by Lockkart et al. i.e. Affymetrix synthesis probes in situ on the solid phase
• nucleic acids can be attached or immobilized to a solid support in a wide variety of ways.
• immobilized herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal.
• the binding can be covalent or non-covalent.
• non-covalent binding and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non- covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
• covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
• Nucleic acid probes may be attached to the solid support by covalent binding such as by conjugation with a coupling agent or by covalent or non-covalent binding such as electrostatic interactions, hydrogen bonds or antibody-antigen coupling, or by combinations thereof.
• Typical coupling agents include biotin/avidin, biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F c fragment, and streptavidin/protein A chimeras (T. Sano and C. R. Cantor, Bio/Technology 9:1378-81 (1991)), or derivatives or combinations of these agents.
• Nucleic acids may be attached to the solid support by a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond or a combination of these sorts of bonds.
• the array may also be attached to the solid support by a selectively releasable bond such as 4,4'-dimethoxytrityl or its derivative.
• Derivatives which have been found to be useful include 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or 4 [bis-(4- methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)j- hydroxymethyl-benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl- benzoic acid, and salts of these acids.
• the probes are attached to the microarray in a wide variety of ways, as will be appreciated by those in the art.
• the nucleic acids can either be synthesized first, with subsequent attachment to the microarray, or can be directly synthesized oh the microarray.
• the microarray comprises a suitable solid substrate.
• substrate or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
• the solid phase support of the present invention can be of any solid materials and structures suitable for supporting nucleotide hybridization and synthesis.
• the solid phase support comprises at least one substantially rigid surface on which the primers can be immobilized and the reverse transcriptase reaction performed.
• the substrates with which the polynucleotide microarray elements are stably associated and may be fabricated from a variety of materials, including plastics, ceramics, metals, acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber,
• Substrates may be two-dimensional or three-dimensional in form, such as gels, membranes, thin films, glasses, plates, cylinders, beads, magnetic beads, optical fibers, woven fibers, etc.
• a preferred form of array is a three- dimensional array.
• a preferred three-dimensional array is a collection of tagged beads. Each tagged bead has different primers attached to it.
• Tags are detectable by signalling means such as color (Luminex, Illumina) and electromagnetic field (Pharmaseq) and signals on tagged beads can even be remotely detected (e.g., using optical fibers).
• the size of the solid support can be any of the standard microarray sizes, useful for DNA microarray technology, and the size may be tailored to fit the particular machine being used to conduct a reaction of the invention. In general, the substrates allow optical detection and do not appreciably fluoresce.
• the surface of the microarray and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
• the microarray is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred.
• the probes can be attached using functional groups on the probes.
• nucleic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known.
• additional linkers such as alkyl groups (including substituted and heteroalkyl groups) may be used.
• the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support.
• either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
• the immobilization to the solid support may be very strong, yet non-covalent.
• biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
• the arrays may be produced according to any convenient methodology, such as preforming the polynucleotide microarray elements and then stably associating them with the surface.
• the oligonucleotides may be synthesized on the surface, as is known in the art.
• a number of different array configurations and methods for their production are known to those of skill in the art and disclosed in WO 95/251 16 and WO 95/35505
• Covalent chemical attachment of DNA to the support can be accomplished by using standard coupling agents to link the 5'- phosphate on the DNA to coated microspheres through a phosphoamidate bond.
• Methods for immobilization of oligonucleotides to solid-state substrates are well established. See Pease et al., Proc. Natl. Acad. Sci. USA 91(1 1):5022-5026 (1994).
• a preferred method of attaching oligonucleotides to solid-state substrates is described by Guo et al., Nucleic Acids Res.
• Immobilization can be accomplished either by in situ DNA synthesis (Maskos and Southern, supra) or by covalent attachment of chemically synthesized oligonucleotides (Guo et al., supra) in combination with robotic arraying technologies.
• gene expression can also be quantified using liquid-phase assays.
• PCR kinetic polymerase chain reaction
• Kinetic PCR allows for the simultaneous amplification and quantification of specific nucleic acid sequences.
• the specificity is derived from synthetic oligonucleotide primers designed to preferentially adhere to single-stranded nucleic acid sequences bracketing the target site. This pair of oligonucleotide primers form specific, non- covalently bound complexes on each strand of the target sequence. These complexes facilitate in vitro transcription of double-stranded DNA in opposite orientations.
• the probe is designed to selectively bind the target DNA sequence between the two primers.
• the fluorochrome is cleaved from the probe by the exonuclease activity of the polymerase resulting in signal dequenching.
• the probe signalling method can be more specific than the
• each type of quantification method can be used in multi-well liquid phase arrays with each well representing primers and/or probes specific to nucleic acid sequences of interest.
• an array of probe/primer reactions can simultaneously quantify the expression of multiple gene products of interest. See Germer et ai, Genome Res. 10:258-266 (2000); Heid et ai, Genome Res. 6:986-994 (1996).
• Means for testing for changes to the functional levels of the IRF7, IRF9 and STAT1 proteins can also be achieved by any suitable method which would be well known to the person of skill in the art. For example, screening for the intracellular localisation of a protein can be achieved by simple immunohistochemistry.
• suitable methods include, but are not limited to, antibody screening of tissue sections or biopsy specimens. '
• the presence of the marker protein may be determined in a number of ways such as by Western blotting, ELISA or flow cytometry procedures. These, of course, include both single-site and two-site or
• “sandwich” assays of the non-competitive types as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
• Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
• any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
• the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample.
• Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody.
• a first antibody having specificity for the marker or antigenic parts thereof is either covalently or passively bound to a solid surface.
• the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
• the binding processes are well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
• an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25°C) to allow binding of any subunit present in the antibody.
• the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the antigen.
• the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antigen.
• reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
• the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
• an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
• glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
• Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
• the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
• the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
• Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
• fluorescent compounds such as fluorecein and rhodamine
• fluorecein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
• the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
• the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
• Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
• the present findings have enabled the development of means for treating metastatic cancer.
• the method of this aspect of the present invention is best used as an adjunct therapy to whatever treatment regime has been selected to target the primary tumour.
• the primary tumour is surgically removed and the subject treatment method is subsequently applied in order to therapeutically treat metastatic disease.
• the primary tumour may not be surgically removable and is being treated by radiation therapy while nevertheless simultaneously treating the patient for metastatic tumours using the present method.
• the application of the method of the present invention is generally going to be indicated after analysis of the primary tumour has revealed a shift to a metastatic phenotype.
• the diagnostic method herein disclosed identifies the shift to a metastatic phenotype, in some cases metastatic spread may already have commenced while in other clinical situations, although the shift to a metastatic phenotype may have occurred, the actual spread of primary tumour cells to other organs may not yet have occurred. It is to be expected that other than in the more advanced stages of metastatic cancer, where the metastatic tumours become more highly visible, one may not be able to confirm whether or not metastatic spread of the primary tumour has occurred.
• the method of the present invention may be functioning prophylactically, such as where a primary tumour exhibiting a metastatic phenotype cannot be surgically removed but has not yet actually spread, or it may be functioning therapeutically, such as where metastatic spread has commenced, even if these metastases are not yet detectable by conventional diagnostic techniques.
• the treatment method of this aspect of the present invention has utility in the treatment of either advanced stage'or early stage metastatic disease
• its application in preventing or treating early stage metastases is particularly significant since this provides a means of potentially preventing a patient from reaching the point of advanced disease, which can be both debilitating and lead to poorer prognostic outcomes due to the range of other clinical problems which are associated with advanced stage disease and which can render late stage treatments less effective.
• Chemotherapy is the primary treatment used to date, this being very nonspecific and of only moderate effectiveness. Clinicians have also been reluctant to subject patients to this treatment regime, due to its side effects, where there has been no clinical indication of metastatic disease.
• the present invention is predicated on the determination that the loss of functional IRF7 in a primary tumour induces a shift to a metastatic phenotype. Accordingly, in addition to the fact that a diagnostic method has been developed based on screening for the status of the IRF7 related gene signature in primary tumours, it has also been determined that restoration of the IRF7 pathway provides an effective treatment regime.
• this aspect of the present invention is directed to a method of treating metastatic cancer in an individual, which cancer is characterised by aberrant IRF7
• said method comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of Type I IFN in said individual.
• references to "aberrant IRF7 functionality" should be understood as a reference to a metastatic cancer which derives from a primary tumour of the type diagnosable by the method of the first aspect of the present invention. That is, briefly, IRF7 has become non-functional or less functional within the primary tumour and has therefore resulted in the shift of the tumour to a metastatic phenotype. This is diagnostically characterised by a loss of expression of 6 or more genes of Table 1 by the tumour cells or a reduction in the functional levels of IRF7, IRF9 and STAT1. Without limiting the present invention to any one theory or mode of action, patients exhibiting this defect in a primary tumour have been determined to respond favourably to Type I IFN therapy which acts to restore IRF7 pathway functionality, thereby effectively treating metastatic disease.
• said metastatic cancer is a cancer of the breast, colon, kidney, lungs, skin, ovary, pancreas, prostate, rectum, stomach, thyroid or uterus.
• the present invention is more particularly directed to a method of treating a breast, kidney or prostate metastatic cancer in an individual, which cancer is characterised by aberrant IRF7 functionality, said method comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of Type I IFN in said individual.
• metastatic cancer should be understood as a reference to a metastatic cancer which has arisen from a primary tumour present in one of these organs.
• the present invention is not limited to these organs, however, can extend to metastatic cancers arising from any organ, such as those mentioned hereinbefore, which are characterised by a loss of IRF7 functionality, this now being routinely diagnosable.
• a method of treating metastatic cancer, which metastases are present in the bone and which cancer is characterised by aberrant IRF7 functionality, in an individual comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level if Type I IFN in said individual.
• human Type I IFNs bind to a specific cell surface receptor complex known as the IFN-a receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.
• IFNAR IFN-a receptor
• the mammalian Type I IFNs are designated IFN-a (alpha), IFN- ⁇ (beta), IFN- ⁇ (kappa), IFN- ⁇ (delta), IFN- ⁇ (epsilon), IFN- ⁇ (tau), IFN- ⁇ (omega), and IFN- ⁇ (zeta, also known as limitin).
• Type I IFN should therefore be understood as a reference to any interferon type which falls within this class including all precursor, proprotein, or intermediate forms, it also includes reference to any isoforms which may arise from alternative splicing of Type I IFN mRNA or polymorphic forms of a Type I IFN.
• Reference to Type I IFN extends to any Type I IFN protein, whether existing as a dimer, multimer or fusion protein. In one embodiment, said Type I IFN is IFN-a or IFN- ⁇ .
• a method of treating metastatic cancer in a patient which cancer is characterised by aberrant IRF7 functionality comprising administering an effective amount of a composition, wherein said composition comprises an agent which upregulates the level of IFN-a in said patient.
• a method of treating metastatic cancer in an individual which cancer is characterised by aberrant IRF7 functionality comprising administering an effective amount of a composition wherein said composition comprises an agent which upregulates the level of IFN- ⁇ in said individual.
• an “effective amount” means an amount necessary at least partly to attain the desired immune response, or to prevent or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the capacity of the individual's immune system to stimulate a specific immune response, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
• an agent which upregulates the level of Type I IFN in the manufacture of a medicament for the treatment of a metastatic cancer in an individual, which cancer is characterised by aberrant IRF7 functionality.
• said metastatic cancer is a cancer of the breast, colon, kidney, lungs, skin, ovary, pancreas, prostate, rectum, stomach, thyroid or uterus.
• said cancer is characterised by the onset of bone metastases.
• said Type I IFN is IFN-a or IFN- ⁇ .
• this can be any suitable molecule including, but not limited to:
• a proteinaceous or non-proteinaceous molecule which interacts with a Pattern Recognition receptor such as the Toll-like receptor including, for example, the TLR7/8 agonist imiquimod.
• the proteinaceous molecules described above may be derived from any suitable source such as natural, recombinant or synthetic sources and includes fusion proteins or molecules which have been identified following, for example, natural product screening.
• the reference to non-proteinaceous molecules may be, for example, a reference to a nucleic acid molecule or it may be a molecule derived from natural sources, such as for example natural product screening, or may be a chemically synthesised molecule.
• the present invention contemplates analogues of Type I IFN expression product or small molecules capable of acting as agonists. Chemical agonists may not necessarily be derived from the Type I IFN expression product but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to meet certain physiochemical properties.
• Screening for the modulatory agents hereinbefore defined can be achieved by any one of several suitable methods including, but in no way limited to, contacting a cell comprising the Type I IFN gene or functional equivalent or derivative thereof with an agent and screening for the modulation of Type I IFN protein production or functional activity, modulation of the expression of a nucleic acid molecule encoding Type I IFN or modulation of the activity or expression of a downstream Type I IFN cellular target. Detecting such modulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of Type I IFN activity such as luciferases, CAT and the like.
• Type I IFN gene or functional equivalent or derivative thereof may be naturally occurring in the cell which is the subject of testing or it may have been transfected into a host cell for the purpose of testing. Further, to the extent that a Type I IFN nucleic acid molecule is transfected into a cell, that molecule may comprise the entire Type I IFN gene or it may merely comprise a portion of the gene such as the portion which regulates expression of the Type I IFN product. For example, the Type I IFN promoter region may be transfected into the cell which is the subject of testing. In this regard, where only the promoter is utilised, detecting modulation of the activity of the promoter can be achieved, for example, by ligating the promoter to a reporter gene.
• the promoter may be ligated to luciferase or a CAT reporter, the modulation of expression of which gene can be detected via modulation of fluorescence intensity or CAT reporter activity, respectively.
• a CAT reporter the modulation of expression of which gene can be detected via modulation of fluorescence intensity or CAT reporter activity, respectively.
• Type I IFN binding sites ligated to a minimal reporter.
• These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as the proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the Type I IFN nucleic acid molecule or expression product itself or which modulate the expression of an upstream molecule, which upstream molecule subsequently modulates Type I IFN expression or expression product activity.
• these methods provide a mechanism of detecting agents which either directly or indirectly modulate Type I IFN expression and/or activity.
• the agents which are utilised in accordance with the method of the present invention may take any suitable form.
• proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules used, linked, bound or otherwise associated with the proteins such as amino acids, lipid, carbohydrates or other peptides, polypeptides or proteins.
• the subject non-proteinaceous molecules may also take any suitable form.
• Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound otherwise associated with any other proteinaceous or non-proteinaceous molecules.
• said agent is associated with a molecule which permits its targeting to a localised region.
• the subject proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the expression of Type 1 1FN or the activity of the Type 1 JFN expression product.
• Said molecule acts directly if it associates with the Type I IFN nucleic acid molecule or expression product to modulate expression or activity, respectively.
• Said molecule acts indirectly if it associates with a molecule other than the Type I IFN nucleic acid molecule or expression product which other molecule either directly or indirectly modulates the expression or activity of the Type I IFN nucleic acid molecule or expression product, respectively.
• the method of the present invention encompasses the regulation of Type I IFN nucleic acid molecule expression or expression product activity via the induction of a cascade of regulatory steps.
• Derivatives of the molecules herein described include fragments, parts, portions or variants from either natural or non-natural sources.
• Non-natural sources include, for example, recombinant or synthetic sources.
• recombinant sources is meant that the cellular source from which the subject molecule is harvested has been genetically altered. This may occur, for example, in order to increase or otherwise enhance the rate and volume of production by that particular cellular source.
• Parts or fragments include, for example, active regions of the molecule.
• Derivatives may be derived from insertion, deletion or substitution of amino acids.
• Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a
• Deletional variants are characterised by the removal of one or more amino acids from the sequence.
• Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above.
• Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
• Analogues of the molecules contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.
• nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
• the derivatives of the nucleic acid molecules utilised in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in
• nucleic acid sequences also include degenerate variants.
• a “variant” or “mutant” of Type I IFN should be understood to mean molecules which exhibit at least some of the functional activity of the form of Type I IFN of which it is a variant or mutant.
• a variation or mutation may take any form and may be naturally or non-natural ly occurring.
• a “homologue” is meant that the molecule is derived from a species other than that which is being treated in accordance with the method of the present invention. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of Type I IFN, for example, which exhibits similar and suitable functional characteristics to that of the Type I IFN which is naturally produced by the subject undergoing treatment.
• Chemical and functional equivalents should be understood as molecules exhibiting any one or more of the functional activities of the subject molecule, which functional equivalents may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening.
• functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening.
• libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used.
• a general synthetic scheme may follow published methods (eg., Bunin BA, et al. (1994) Proc. Natl. Acad. Sci. USA, 91 :4708-4712; DeWitt SH, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an array, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library.
• One suitable permutation strategy is outlined in US. Patent No. 5,763,263.
• Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occurring ligands of a biological target. In the present context, for example, they may be used as a starting point for developing Type I IFN analogues which exhibit properties such as more potent
• Type I IFN or a functional part thereof may according to the present invention be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein).
• U.S. Patent No. 5,763,263 By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.
• oligomeric or small- molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above.
• a selected biological agent such as a biomolecule, a macromolecule complex, or cell
• each member of the library is screened for its ability to interact specifically with the selected agent.
• a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction.
• the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.
• Analogues of Type I IFN contemplated herein include, but are not limited to, modifications to side chains, incorporating unnatural amino acids and/or derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the analogues.
• the specific form which such modifications can take will depend on whether the subject molecule is proteinaceous or non- proteinaceous. The nature and/or suitability of a particular modification can be routinely determined by the person of skill in the art.
• Modulation of said Type I IFN functional levels may be achieved via the
• Type I IFN a nucleic acid molecule encoding Type I IFN or an agent which effects modulation of Type I IFN activity or Type I IFN gene expression (herein collectively referred to as “modulatory agents").
• composition of the present invention in the form of a
• composition may be performed by any convenient means.
• the components of the pharmaceutical composition are contemplated to exhibit therapeutic or prophylactic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal.
• a broad range of doses may be applicable. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
• the composition may be administered in a convenient manner such as by the oral, inhaled, intraperitoneal, subcutaneous, suppository routes or implanting (e.g. using slow release molecules). It may also be administered via non-mucosal routes, where appropriate, such as via intravenous or other such routes.
• the composition may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application).
• acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
• the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
• a binder such as tragacanth, corn starch or gelatin
• a disintegrating agent such as alginic acid
• a lubricant such as magnesium stearate.
• the modulatory agents of the invention can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition.
• Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the invention.
• Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers.
• Detergents can also used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers.
• Pharmaceutically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of
• physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
• Various preservatives are well known and include, e.g., phenol and ascorbic acid.
• a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the peptide or polypeptide of the invention and on its particular physio-chemical characteristics.
• Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules.
• conventional nontoxic solid carriers can be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
• a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed.
• a non-solid formulation can also be used for enteral administration.
• the carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like.
• Suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.
• the composition of the invention, when administered orally, can be protected from digestion.
• compositions for oral delivery of therapeutic agents liposomal delivery is discussed in further detail, infra).
• composition of the invention can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally.
• sustained delivery or sustained release mechanisms can deliver the formulation internally.
• biodegradable microspheres or capsules or other biodegradable polymer configurations capable of sustained delivery of a peptide can be included in the formulations of the invention (see, e.g., Putney (1998) Nat. Biotechnol. 16:153- 157).
• composition of the invention can be delivered using any system known in the art, including dry powder aerosols, liquid delivery systems, air jet nebulisers, propellant systems, and the like. See, e.g., Patton (1998) Biotechniques 16: 141 -143; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura
• the Type I IFN formulation can be administered in the form ofan aerosol or mist.
• the formulation can be supplied in finely divided form along with a surfactant and propellant.
• the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes.
• Other liquid delivery systems include, e.g., air jet nebulisers.
• Type I IFN will be formulated in pharmaceutically acceptable compositions suitable for pulmonary or respiratory delivery to a patient.
• Particular formulations include dry powders, liquid solutions or suspensions suitable for nebulisation, and propellant formulations suitable for use in metered dose inhalers (MDI's).
• MDI's metered dose inhalers
• Liquid formulations of Type I IFN for use in nebuliser systems can include
• components to enhance or maintain chemical stability including chelating agents, protease inhibitors, isotonic modifiers, inert gases, and the like.
• the Type I IFN of the present invention will be dissolved or suspended in a suitable aerosol propellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC).
• a suitable aerosol propellant such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC).
• CFC chlorofluorocarbon
• HFC hydrofluorocarbon
• Suitable CFC's include trichloromonofluoromethane (propellant 1 1), dichlorotetrafluoroethane (propellant 1 14), and dichlorodifluoromethane (propellant 12).
• Suitable HFC's include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).
• the Type I IFN of the present invention will be processed into respirable particles as described below for the dry powder formulations.
• the particles are then suspended in the propellant, typically being coated with a surfactant to enhance their dispersion.
• Suitable surfactants include oleic acid, sorbitan trioleate, and various long chain diglycerides and phospholipids.
• Such aerosol propellant formulations may further include a lower alcohol, such as ethanol (up to 30% by weight) and other additives to maintain or enhance chemical stability and physiological acceptability.
• a lower alcohol such as ethanol (up to 30% by weight) and other additives to maintain or enhance chemical stability and physiological acceptability.
• Dry powder formulations will typically comprise the Type I IFN in a dry, usually lyophilized, form with a particular size within a preferred range for deposition within the alveolar region of the lung.
• Respirable powders of Type I IFN within the preferred size range can be produced by a variety of conventional techniques, such as jet-milling, spray-drying, solvent precipitation, and the like. Dry powders can then be administered to the patient in conventional dry powder inhalers (DPI's) that use the inspiratory breath through the device to disperse the powder or in air-assisted devices that use an external power source to disperse the powder into an aerosol cloud.
• DPI's dry powder inhalers
• Dry powder devices typically require a powder mass in the range from about 1 mg to 10 mg to produce a single aerosolized dose ("puff). Since the required dose of Type I IFN may be lower than this amount, the Type I IFN may be combined with a pharmaceutically acceptable dry bulking powder.
• Preferred dry bulking powders include sucrose, lactose, trehalose, human serum albumin (HSA), and glycine.
• Other suitable dry bulking powders include cellobiose, dextrans, maltotriose, pectin, sodium citrate, sodium ascorbate, mannitol, and the like.
• suitable buffers and salts may be used to stabilize the Type I IFN in solution prior to particle formation.
• Suitable buffers include phosphate, citrate, acetate, and tris-HCl, typically at concentrations from about 5 mM to 50 mM.
• Suitable salts include sodium chloride, sodium carbonate, calcium chloride, and the like.
• Other additives, such as chelating agents, peptidase inhibitors, and the like, which would facilitate the biological activity of the Type I IFN once it is dissolved within the lung would be appropriate.
• EDTA ethylenediaminetetraacetie acid
• compositions of the invention in vesicles composed of substances such as proteins, lipids (for example, liposomes, see below), carbohydrates, or synthetic polymers (discussed above).
• lipids for example, liposomes, see below
• carbohydrates for example, liposomes, see below
• synthetic polymers discussed above.
• compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical modulatory pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, etc. The amount of modulatory agent adequate to accomplish this is defined as a "therapeutically effective dose.”
• the dosage schedule and amounts effective for this use, i.e., the "dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or cGndition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like.
• the mode of administration also is taken into consideration.
• the dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington's; Egleton (1997) "Bioavailability and transport of peptides and peptide drugs into the brain” Peptides 18: 1431-1439; Langer (1990) Science 249:1527-1533.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyoi (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
• the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
• dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
• Immunopurification was performed according to the protocol developed by Parker and colleagues (Parker et al. 2008), with the epithelial selection antibody replaced by a-mouse-Ep- CAM antibody (BD Pharmingen, clone G818). Purity of populations was assessed by RT- PCR analysis for CD31 , vWF and P1 H12 (endothelial populations), CD45 (haemopoietic populations) and Ep-CAM, CK18 and CK 19 (epithelial populations) and NeoR (tumour specific populations) (data not shown).
• Genespring GT 1.0 (Agilent Technologies, Santa Clara, CA, USA) with the sequence data sourced from the MouseGenome9999 sequence file (Agilent Technologies, Santa Clara, CA, USA).
• Expression data was filtered by p value ⁇ 0.05 and fold-change > 2 to identify genes strongly differentially expressed between primary tumour epithelium and the metastatic epithelium. Genes were mapped to Ensembl release 59 gene models and the resulting gene list was analysed for IFN signatures using INTERFEROME, the database of IFN regulated genes. Significance was determined by the application of a Pearson chi-squared test.
• Putative proximal promoter sequences 1500bp 5' upstream and 200bp downstream relative to the annotated transcription start site were extracted from Ensembl release 59 through the Ensembl Bioperl API (Culhane et al. 2010, supra).
• the CLOVER algorithm (Frith et al., Nucleic Acids Res 32: 1372-1381 (2004)) utilizing the Transfac Professional (ver.
• Promoter enrichment analysis using and binding site identification was performed on the 540 suppressed genes identified through INTERFEROME analysis.
• Transfac Pro 2010.3 matrices were applied in both the CLOVER and MATCH algorithms.
• 66cl4 and 4T1 cell lines were kindly provided by Dr. Fred Miller (Karmanos Cancer Institute, Detroit, MI). Derivation of the 4T1.2 line has previously been described (Eckhardt et al. 2005, supra; Lelekakis et al. 1999, supra).
• Recombinant Interferon-ai was used at a dose of 1000 IU/mL.
• Interferon-a receptor blocking antibody (MAR1-5A3) was used at a concentration of ⁇ g/mL (Leinco, St Louis, MO).
• G-CSF ELISA was performed according to manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Flow cytometry was performed using standard techniques.
• MDSCs were assessed using Ly6G (clone IA8) and CD1 l b (clone Ml/70) specific antibodies (BD Pharmingen, Franklin Lakes, NJ, USA) at a dilution of 1 :200.
• Flow Cytometry was performed using an LSR-II (BD Pharmingen, San Jose, CA).
• Real-time RT-PCR was performed using standard techniques.
• In vitro proliferation was assessed using a sulforhodamine B binding assay as previously described (Eckhardt et al. 2005, supra).
• Immunohistochemistry was performed using standard techniques, using a- mouse Irf7 (Zymed Invitrogen, Carlsbad, CA, USA) a-human Irf7 (BD Pharmingen, Franklin Lakes, NJ, USA) at a concentration of 5 ⁇ g/mL.
• the mixture of MDSC and CFSE labelled splenocytes were plated in triplicates in a 96-well round-bottom plate.
• the suppressor and responder cells were cultured together at ratios of 1 : 1 , 1 :2, 1 :4, 1 :8 and 1 :16 with the same number of responder cells in each well (100,000 cells/well).
• Cells were stimulated using anti-CD3 (clone 145-2cl 1 , BD Biosciences, San Jose, CA, USA) and anti-CD28 (clone 37.51 , BD Biosciences, San Jose, CA, USA) antibodies at 40ng/ml.
• the total volume of medium in each well was adjusted to 175 uj.
• CD4 + and CD8 + T-lymphocytes were assessed using specific antibodies (anti-mouse CD4-APC, clone GK1.5, eBioscience; anti-mouse CD8a PE-Cy7, clone 53-6.7, eBioscience)
• mice Female Balb/c mice (6-8 weeks old) were obtained from ARC (Perth, WA, Australia) or Walter and Eliza Hall Institute (Kew, Vic, Australia) breeding facilities.
• Female NSG mice were obtained from WEH1 breeding facility under license from The Jackson Laboratory and • Balb/c Ifnarl ⁇ _ (Hwang et al. 1995, supra) were bred in-house.
• Recombinant IFN-al was injected intraperitoneally 3 times weekly at a dose of 10 5 IU/dose. All other mouse assays were performed using standard techniques. Metastasis was assessed using a previously described assay (Eckhardt et al. 2005, supra). Mouse investigations were performed with the approval of the Peter MacCallum Animal Ethics Experimentation Committee.
• Metastasis data has been logio transformed for visualization. Unless otherwise stated, all statistics are Student's T-test performed on untransformed data. Dot plots and histograms are means and error bars are S.E.M.. All statistics were performed using the data analysis package within Microsoft Excel or the analysis tool within GraphPad Prism 5. Kaplan-Meier survival analysis was performed using GraphPad Prism 5 and the Gehan-Breslow-Wilcoxon test was used to determine significance.
• Four publicly available breast cancer microarray datasets with known first site of distant relapse were used in the survival analyses: GSE2034, GSE12276, GSE2603, NKI295 as described in Harrell et al. (2012, supra).
• the Irf7 gene set was treated as a continuous variable and is defined as the averaged gene expression of the subgroup of 208 genes within the 540 IRGs that contained predicted Irf7 putative binding sites
• Tissues were disaggregated in DMEM containing 3mg/mL Collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) and digested for 90 min at 37°C prior to filtration through a ⁇ and a 70 ⁇ nylon filter and red blood cell lysis in an alkaline buffer (150mM NH4CI, ImM KHCO3, O.lmM EDTA). B lymphocytes and T lymphocytes were removed by incubation with pan-B and pan-T DynaBeads (Invitrogen, Carlsbad, CA, USA) for 20 min at 4°C.
• DMEM containing 3mg/mL Collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) and digested for 90 min at 37°C prior to filtration through a ⁇ and a 70 ⁇ nylon filter and red blood cell lysis in an alkaline buffer (150mM NH4CI, ImM KHCO3, O.lmM EDTA).
• Epithelial cells were then selected by incubation with sheep anti-rat DynaBeads (Invitrogen, Carlsbad, CA, USA) pre-conjugated to a rat anti-mouse EP-CAM antibody (BD Pharmingen, Franklin Lakes, NJ, USA) for 30 min at 4°C.
• sheep anti-rat DynaBeads Invitrogen, Carlsbad, CA, USA
• a rat anti-mouse EP-CAM antibody BD Pharmingen, Franklin Lakes, NJ, USA
• GAPDH Fwd 5'-TCCCACTCTTCCACCTTCGA-3' (SEQ ID NO: l )
• Expression level of each gene was calculated based on the cycle threshold (CT), set within the linear range of DNA amplification.
• mice were inoculated with 10 5 cells into the 4 th mammary fatpad of female 6-8 week old Balb/c mice. Tumour volumes were monitored by caliper measurement 3 times weekly. All animals were sacrificed by isoflurane overdose when any animal showed signs of distress (usually ds 28 -35). Metastatic burden in secondary tissues was detected by a Q-PCR based assay using a reporter sequence inserted into the genome of the 4T1.2 cells. Survival assays were conducted in the same way except at day 22 the mice were anaesthetized using either a combination of ketamine hydrochloride (12 ⁇ %1% of mouse)/xylaxil
• mice hydrochloride of mouse
• aerosol isoflurane in 100% oxygen and the primary tumour was surgically removed and weighed.
• Mice were harvested individually, not as a group, when they showed signs of distress. At time of sacrifice, all mice had evidence of bone and lung metastasis unless otherwise stated.
• Anti-CD8 (clone 53-6.7, produced in house) and the control antibody rat IgG2a (clone 2A3, produced in house) were injected intraperitoneal ly at 50 ⁇ g per dose on days -1, 0, 1 , 4, 7 and subsequently every 7 d.
• Rabbit anti-asialo GM1 was purchased from Wako chemicals and injected intraperitoneally according to the manufacturer's instructions.
• Bones were decalcified in 20% EDTA for 14 d at room temperature.
• AH tissues were paraffin embedded and sectioned by the Peter MacCallum Cancer Centre histology facility or Peter MacCallum Cancer Centre Pathology Division. H&E staining was also performed by the same histology facility. The exception was a separate series of tissue microarrays constructed by P.A. at The Johns Hopkins Hospital from paired primary breast carcinomas and hematogenous metastases from the same patients, most of which were harvested at rapid autopsy as previously described (Wu et al. 2008; Cimino et al , Breast Cancer Res Treat 123:701 -708 (2010)). Standard immunohistochemistry techniques were used to prepare the sections for staining.
• Heat antigen retrieval was performed in citrate buffer (lOmM tri-sodium citrate, pH 6.0) at 125°C for 3 min under pressure, a-mouse Irf7 antibody was obtained from Invitrogen (clone zm7), a-human/mouse Irf9 antibody from abeam (cat# ab51634) and a-human Irf7 antibody from BD Pharmingen (cat#AHPl 179).
• IFN-a ELISA was performed using standard molecular biology techniques.
• Antibody clone RMMA-1 was used as the capture antibody (PBL Interferon Source, Piscataway, NJ, USA) and a rabbit polyclonal anti-mouse IFN-a was used as the detection antibody (PBL Interferon Source, Piscataway, NJ, USA).
• Cells were seeded at a density of 50,000/mL and allowed to adhere for 4 h before being treated with vehicle control or 10 ⁇ g/mL of poly(I:C) for 24 h.
• Splenocyte single cell suspension was prepared. Cells were resuspended in 20 mL PBS containing 2 mM EDTA and subsequently labelled with 1 ⁇ CFSE (Invitrogen, Carlsbad, CA, USA). After a seven-min incubation at room temperature, the tube was filled up with ct- MEM containing 5% FCS and spun at 800 g for five min. After being washed twice, the cells were then resuspended in a-MEM containing 5% FCS. The labelling of cells by CFSE was analyzed by flow cytometry.
• Sections were cut from eight blocks containing formalin-fixed paraffin-embedded (FFPE) normal mammary tissue, primary tumours and matched bone metastases. One 3 ⁇ section from each block was stained with H&E to identify the tumour enriched area. The content of tumour cells in all cases was, assessed by a pathologist. Tissue areas consisting of at least 80% tumour cell content were selected for needle macrodissection. Seven ⁇ tissue sections of each sample were stained with methyl green and subsequently subjected to needle macrodissection.
• FFPE formalin-fixed paraffin-embedded
• DNA was quantified and its purity assessed using the NanoDrop spectrophotometer 2000 (NanoDrop Technologies, Thermo Fisher Scientific, Wilmington, DE). The purity of all DNA samples extracted had an absorbance ratio of between 1.8 and 1.9.
• Fully methylated control DNA was prepared from 500 ng of extracted lung DNA of a normal BALB/c mouse by utilising the CpG Methyltransferase M.SssI (New England BioLabs, Ipswich, MA). M.SssI treatment was performed as described elsewhere (Mikeska et al, Methods Mol. Biol. 791 :33-53 (201 1)). The preparation was stored at -35°C.
• MS-HRM Methylation-sensitive high resolution melting
• a DNA methylation standard series was prepared by diluting fully methylated control DNA in unmethylated control DNA.
• the DNA methylation standards comprised 100% 50%, 25%, 10% and 0% of fully methylated control DNA.
• MS-HRM Wijdacz and Dobrovic, Nucleic Acids Res 35:e41 (2007) was performed on a Rotor-Gene 6000 (Corbett, Sydney, Australia). Each DNA methylation standard and each sample was run in duplicate, while the bisulfite untreated genomic DNA control and the no template control were performed only once.
• the primer sequences for the analysis of the CpG rich region spanning exon 1 of Irp were: 5'-GAGTGGTTTAAGAGTTTTATATATTTGGTAT-3' (SEQ ID NO: l 1) and 5'- ACCACACCCTACCTAAACTCTA-3' (SEQ ID NO: 12) (Region 1).
• the amplified region corresponds to GenBank accession number AC 163434, nucleotides 62597-62727.
• the primer sequences for the analysis of the CpG island associated with the boundary of intron 1/exon 2 of Irp were: 5'-AGATAGCGGGAAGTTAGTAGTTAT-3' (SEQ ID NO: 13) and 5'- CTAAATAAACTATCACAAA-CTAAACCCTA-3 ' (SEQ ID NO: 14) for region 2 and 5'- GGTTTAGTTTGTGATAGTTTATTTAGGT-3' (SEQ ID NO: 15) and 5 '- CTCAATATAAATTCCTCTACCAAAATAACTA-3' (SEQ ID NO: 16) for region 3.
• the amplified regions correspond to GenBank accession number AC 163434, nucleotides 62250- 62388 (Region 2) and nucleotides 62152-62275 (Region 3).
• PCR was performed in 0.1 mL tubes with a final reaction volume of 20 ⁇ _ containing 200 nmol L of each primer, 200 ⁇ /L of each dNTP, 5 ⁇ /L SYTO 9 (Invitrogen, Life Technologies, Carlsbad, CA), 2.5 mmol/L MgCl 2 (2.0 mmol/L for region 3), 0.5 U HotStarTaq DNA Polymerase in its supplied buffer (l x) (Qiagen) and 1 uL (1.5 ⁇ for region 3) of bisulfite modified DNA.
• PCR amplification conditions were: 1 cycle of 95°C for 15 min, 50 cycles of 95°C for 25 s (20 s for region 3), 60°C (57°C for region 2 and 57.5°C for region 3) for 20 s (15 s for region 3) and 72°C (70°C for region 3) for 25 s (20 s for region 3). This was immediately followed by a hold at 97°C for 1 min, 60°C for 1.5 min and a HRM step from 60 to 95°C rising at 0.2°C per second, and holding for 1 sec after each stepwise increment.
• the primer sequences for the analysis of the CpG island associated with the promoter region of Stat] were: 5 ' -GGTGNGTTGATGGAATAGT-3 ' (SEQ ID N0.17) and 5'- CNAAAATCTCC-AAAAAACTTTAACAA-3' (SEQ ID NO: 18) (N corresponds to a mixture of the bases A, C, G and T).
• the amplified region corresponds to GenBank accession number AC 123752, nucleotides 146569-146709.
• PCR was performed in 0.1 mL tubes with a final reaction volume of 20 ⁇ , containing 200 nmol/L of each primer, 200 ⁇ /L of each dNTP, 5 ⁇ /L SYTO 9 (Invitrogen, Life Technologies), 1.75 mmol/L MgCl 2 , 0.5 U HotStarTaq DNA Polymerase in its supplied buffer (l ) (Qiagen) and 1 uL of bisulfite modified DNA.
• PCR amplification conditions were: 1 cycle of 95°C for 15 min, 50 cycles of 95°C for 25 s, 56°C for 20 s and 72°C for 25 s. This was immediately followed by a hold at 97°C for 1 min, 60°C for 1.5 min and a HRM step from 60 to 95°C rising at 0.2°C per second, and holding for 1 sec after each stepwise increment.
• Tumour epithelial cells were isolated from 4T1.2 primary tumours growing in the mammary gland and matched spine metastases (Eckhardt et al. 2005), as described previously (Parker et al, J Pathol 214:337-346 (2008)). Differences in gene expression profiles of tumour cells isolated from four matched pairs of primary tumours and bone metastases (Fig. 1) were interrogated by gene ontology analysis and found to be highly enriched for immune and defense response ontologies (confidence level P ⁇ 0.003) (Table 3a).
• Irf7 is a major regulator of the type I IFN pathway (Honda et al, Nature 434:772-777
• Irf7 restores type I IFN signalling and suppresses metastasis
• tumour cell IFN signalling driven by Irf7 reduces metastasis to bone, and the host response through Ifnarl is critical for metastasis suppression and associated prolonged survival.
• Irf7 expression restores anti-metastatic immune responses
• mice bearing 4T1 .2-BV tumours have a marked elevation of peripheral white blood cells (WBCs) and splenomegaly compared to naive mice. This response was reduced in mice bearing 4T1.2-Irf7 tumours
• MDSC they were isolated from 4T1.2-bearing mice and demonstrated to inhibit proliferation of CD4 + or CD8 + T-lymphocytes (Fig. 4d). These data demonstrate that the MDSC population reduced in 4T1 2-Irf7 tumour-bearing mice is functionally capable of suppressing lymphocyte activity.
• CD8 + T cells and NK cells were depleted from 4T1.2-Irf7 tumour- and 4T1.2-BV tumour-bearing mice.
• primary tumour growth was unaltered by depletion of either or both cell types (Fig. 4f).
• IFN-ai treatment increased expression of Irf7, as well as Irf9 and STAT1 in 4T1.2 cells in vitro (Fig. 6a), consistent with the data demonstrating that Irf7 increased Irf9 and STAT1 expression via IFN.
• IFN In contrast, IFN treatment significantly reduced metastasis to the spine and femur to undetectable levels (P ⁇ 0.007) (Fig. 6d, e).
• NK cells from the mammary gland and lungs were predominantly CD 1 l b high /CD27 ,0W ( Figure 19b), indicative of terminally differentiated cells that have a higher threshold for stimulation (Hayakawa and Smyth, J Immunol 176: 1517- 1524 (2006)).
• the majority of NK cells from the bone marrow were CD 1 1 b' ow /CD27 hi6h ( Figure 10b), representing NK cells with greater effector function that are reportedly more sensitive to stimulation by cytokines such as IFN.
• poly I:C The dose of poly I:C was reduced by mimicking an early treatment setting and initiating treatment after primary tumour removal for only 2 weeks, before the detection of large metastases. Also evaluated was the value of combining poly I:C treatment with a commonly used chemotherapeutic, doxorubicin. Although the single agents did not significantly impact metastasis alone in this short-treatment setting, the combination was very effective, with an obvious reduction in metastasis at end point ( Figure 21). This work indicates that combining IFN agonists with chemotherapeutics has potent anti -metastatic effects, even at reduced doses.
• IRF7 expression in normal human breast, primary tumors and distant metastases Archived, paraffin embedded sections of normal breast, primary breast tumors and matched metastases (including 7 bone metastases) were evaluated for IRF7 expression using immunohistochemistry. There was a significant reduction in IRF7 expression in metastases compared to the primary tumor (Fisher's exact test, p ⁇ 0.04).

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