WO2010127399A1 - Profils d'expression génétique et leurs utilisations - Google Patents

Profils d'expression génétique et leurs utilisations Download PDF

Info

Publication number
WO2010127399A1
WO2010127399A1 PCT/AU2010/000524 AU2010000524W WO2010127399A1 WO 2010127399 A1 WO2010127399 A1 WO 2010127399A1 AU 2010000524 W AU2010000524 W AU 2010000524W WO 2010127399 A1 WO2010127399 A1 WO 2010127399A1
Authority
WO
WIPO (PCT)
Prior art keywords
ilmn
genes
kit
luminal
cyp24a1
Prior art date
Application number
PCT/AU2010/000524
Other languages
English (en)
Inventor
Geoffrey Lindeman
Gordon Smyth
Jane Visvader
Di Wu
Original Assignee
Walter And Eliza Hall Institute Of Medical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2009901989A external-priority patent/AU2009901989A0/en
Application filed by Walter And Eliza Hall Institute Of Medical Research filed Critical Walter And Eliza Hall Institute Of Medical Research
Publication of WO2010127399A1 publication Critical patent/WO2010127399A1/fr

Links

Classifications

    • 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
    • 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
    • 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
    • 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
    • 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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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

Definitions

  • the specification describes cell-specific molecular markers, targets and methods useful for the stratification and treatment of cancers including breast cancers.
  • Gene and protein profiling technologies now use sophisticated algorithms to allow simultaneous characterization of multiple genes or proteins in an individual. This information, when linked to accurate prognostic algorithms promises to enhance our ability to optimise treatment options. Furthermore, information concerning changes in the expression of single or multiple genes over time within a single cell type or between cell types, when correctly interpreted, promises to provide new targets for therapeutic intervention and new diagnostic options. However, the problem still exists of selecting relevant targets and accurate markers.
  • One field where this problem is particularly acute is the field of cancer where transformed cells of uncertain origin exist along side normal and pre-neoplastic cells of various lineages.
  • Luminal epithelial cells form the> milk producing secretory cells of the lobules which are surrounded by an outer layer of contractile basal (myoepithelial) cells.
  • the art recognises several different cell types within the breast including luminal and basal (myoepithelial) progenitors, bipotent progenitors, stem cells and stromal cells. The different cells may be categorised by a range of morphological, genetic or proteinaceous markers.
  • genes and proteins associated with breast cancer have been identified, reliable prognostic tools and therapeutic treatments are highly sought after.
  • genes associated with breast, ovarian, prostate, pancreatic, colon and various other forms of cancer are the BRCAl and BRC A2 genes.
  • certain mutations in BRCAl or BRCA2 are linked to a substantial proportion of familial forms of breast cancer, which account for approximately 5-10% of breast cancers.
  • Reduced expression of BRCAl and BRCA2 genes are also found in sporadic (non-familial) forms of cancers.
  • breast tumor samples may be tested for oestrogen receptor expression and, if positive, subjects may be treated with oestrogen antagonists.
  • breast tumor samples may be tested for human epidermal growth factor receptor 2 (ErbB2) expression and positive subjects may be treated with ErbB2 antagonists.
  • ErbB2 human epidermal growth factor receptor 2
  • Breast cancer appears to be essentially a group of diseases that display widely different clinical course and response to treatment.
  • Various different and fairly imprecise breast cancer categories are described according to mainly morphologic, genetic (including surface marker, gene or protein profile), immunophenotypic and clinical features.
  • the relationship between normal and pre-neoplastic breast cells and these tumor subtypes is poorly understood and this confounds the process of identifying markers and targets and determining effective intervention strategies.
  • the difficulty of accurately subtyping breast cancer leads to inaccurate prognosis and much uncertainty for medical practitioners and their patients.
  • Gene expression profiling information derived using DNA microarrays to analyse expression from multiple thousands of genes has led to the subdivision of breast cancer into at least five different subtypes based upon patterns of expression segregating into different clusters (See, for example, Sorlie et al, Proc. Nat. Acad. Sci, USA 98 (19): 10869-10874, 2001; Neilsen et al, Clinical Cancer Research 70:5367-5374, 2004; Herschkowitz et al, Genome Biol. 8 R76, 2007, Hu et al, BMC Genomics 7: 96, 2006).
  • One subtype is the most aggressive (carries the poorest prognosis) "basal-like" tumor type thought to arise from basal epithelial cells and to • have a gene expression profile similar to normal breast basal (myoepithelial or mammary stem cell (MaSC)) cells.
  • the breast tumor subtypes, "Luminal A”, “Luminal B”, “Her2+/ER-”, “basal-like” and “normal breast like” subtypes are recognised by Hu et al. ⁇ supra).
  • Herschkowitz et al, 2007 ⁇ supra further describe a "claudin-low” subgroup that is oestrogen receptor negative, progesterone receptor negative and human epidermal growth factor (HER2/neu) overexpression negative (referred to as “triple negative") and expresses Claudin 3 and E-cadherin.
  • the "basal-like" breast cancer subtype has also been characterised as “triple negative”.
  • "basal-like" breast tumor cells are typically epidermal growth factor receptor (EGFR/HER1) positive or cytokeratin 5/6 positive and may I express p63.
  • Basal-like breast cancers typically exhibit poor prognostic features such as a high rate of mitosis, lymphovascular invasion, absence of steroid hormone receptor (estrogen and progesterone receptor) expression, loco-regional lymph node metastasis. These cancers are estimated to account for 15 to 20 percent of breast cancers. Subjects with mutations in BRCAl have a greater risk for developing breast cancer, and the tumors that arise in BRCAl mutation carriers are commonly "basal-like".
  • BRCAl Whilst multiple functions for BRCAl have been described, such as in the DNA damage response, X-chromosome inactivation, transcriptional and cell cycle control, the precise mechanisms by which BRCAl mutations lead to tumorigenesis remain to be elucidated. Within the mammary gland, targeted disruption of BRCAl leads to developmental defects, and in mammary epithelial cells, BRCAl has been implicated in the control of differentiation in vitro.
  • the present invention relates to the provision of stratifying and therapeutic protocols for cancer and further provides methods of testing for BRCAl function or early diagnosis of cancer risk in a sample from an individual.
  • the subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary.
  • the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
  • a cell includes a single cell, as well as two or more cells; reference to “an LPT gene or polypeptide” includes one gene or polypeptide, as well as two or more genes or polypeptides; and so forth.
  • the present invention is predicated upon the discovery by the inventors that gene expression in human and mammalian mammary luminal progenitor epithelial cells is very similar to that of the so-called "basal-like" subtype of breast cancer. Additionally, the characteristic luminal progenitor gene expression profile or signature is also apparent in BRCA 1 +/" breast tissue, e.g. breast tissue harbouring a heterozygous mutation in the BRCAl gene.
  • the heterozygous mutation may be one or more point mutations, a deletion, insertion or the like, as known by the person skilled in the art.
  • the observation of a luminal progenitor epithelial cell signature, and not the observation of a basal epithelial cell signature in tumor cells or in a breast tissue sample was found to be indicative, at least, of cellular BRCAl deficiency function and further indicates that the subject has an increased risk of breast cancer exhibiting characteristics of basal-like breast cancers including, without limitation, rapid or invasive growth or metastasis.
  • the present invention provides a method of stratifying cancer in a subject, said method comprising: (i) determining gene-set expression in a biological sample from a subject wherein the gene set is preselected from genes which are: (a) selectively or differentially expressed in CD49f f EpCAM + luminal epithelial progenitor cells compared to basal epithelial (MaSC enriched) and mature luminal epithelial cells; or (b) selectively or differentially expressed in CD49f h 'EpCAM ' basal epithelial progenitor cells compared to luminal progenitor and mature luminal epithelial cells; or (c) selectively or differentially expressed in CD49f EpCAM + mature luminal epithelial cells compared to basal epithelial and luminal progenitor epithelial cells; or (d) selectively or differentially expressed in CD49fEpCAM " stromal fibroblasts cells compared to basal epithelial and luminal epithelial cells
  • the method further comprises (ii) determining a measure of selective or differential gene expression of the gene set relative to controls; and (iii) wherein the measure indicates whether or not the individual has an increased risk of cancer associated with a particular cancer subtype.
  • the progenitor cell population is Lineage-negative (Lin-).
  • cancer subtypes include “claudin-low”, “normal”, “basal (MaSC-enriched)", “HER-2”, “Luminal A” and “Luminal B”.
  • the method comprising: determining expression of a set of genes in a biological sample from an individual wherein the gene set is preselected to comprise genes which are selectively or differentially expressed in CD49f + EpCAM + epithelial luminal progenitor cells compared to their expression by control cells such as at least basal epithelial cells or mature luminal epithelial cells.
  • a measure of selective or differential gene expression is determined which indicates whether or not an individual has an increased risk of "basal-like" breast cancer (perhaps to be renamed as a "Luminal Progenitor" type or profile) or a BRCAl -deficiency associated form of cancer.
  • LPT luminal progenitor target
  • BMS genes genes that are selectively or differentially expressed in basal or mature luminal or stromal mammary cell populations, as disclosed herein, are referred to collectively as BMS genes, gene sets or nucleic acid molecule or polynucleotide, RNA etc as the context demonstrates.
  • Their proteinaceous expression products are referred to as BMS polypeptides or prbteinaceous expression products.
  • BMS proteinaceous expression products
  • each BMS molecule is used herein to characterise (the BMS is either upregulated or downregulated relative to controls) only one cell type, i.e. basal, luminal or stromal.
  • the cancer is breast or ovarian cancer or a cancer associated with BRCAl deficiency. In a particular embodiment, the cancer is breast cancer.
  • determining expression or “gene expression” is meant broadly determining production by transcription of an RNA product or production by translation of a proteinaceous product of the gene. The phases encompass determination of the presence or absence of expression as well as increased or diminished expression relative to a standard or other control.
  • differential expression refers to production of the product at a higher or lower level in one sample compared to the other.
  • Selective expression refers to production of the product in one cell type or sample and not the other.
  • the gene-set expression is expression of RNA.
  • the gene-set expression is expression of protein.
  • the sample is a cellular sample, such as a mammary or ovarian sample.
  • the sample comprises a preparation of Lin " cells. Accordingly, in some embodiments, the method comprises preparing Lin " cells for determining gene-set expression.
  • the Lin- subset includes cells selected by removing cells expressing CD45, CD31 and CD235a.
  • the sample comprises or is known to comprise a pathological BRCAl gene mutation.
  • the method comprises determining gene set expression in Lin " CD49f f EpCAM + luminal epithelial progenitor cells isolated from the sample.
  • the gene set comprises or consists essentially of at least two or more LPT genes selected from the group consisting of the genes set out in Table 10 or 11. In some embodiments the set comprises between 2 to 10, or 8 to 20 or 15 to 30 genes.
  • the gene set comprises or consists of two or more genes selected from the first or last 50 genes set out in Table 12.
  • the gene set comprises or consists of two or more genes selected from the first and last 50 genes set out in Table 12.
  • “Two or more genes selected from the first and/or last 50 genes” includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 genes from the 50 most upregulated genes or from the bottom 50 most downregulated genes as set out in Table 12. Although more than 50 upregulated or downregulated genes are described in the specification for each cell type, this number encompasses the most selectively expressed genes and use of more than this number of genes is unlikely to provide much more information. Use of more than 50 genes is in no way excluded. In particular, Figure 4 represents a barcode plot using approximately 150 genes upregulated and 150 downregulated genes.
  • gene sets capable of distinguishing between or categorising basal epithelial cells, luminal progenitor epithelial cells and mature luminal epithelial cells have been defined using freshly isolated cell populations.
  • a gene set definitive of CD49f + EpCAM + luminal epithelial progenitor cells when compared to basal and mature luminal epithelial cells has been defined. Expression of this gene set was determined in six recognised subtypes of breast cancer, "basal-like”, “claudin-low”, HER2+/ER-”, “luminal A”, “luminal B”, and "normal breast-like” (See, for example, Herschkowitz et al, 2007 (supra)).
  • the profile of gene expression within this .predetermined subset of LPT genes was more similar to "basal-like" cancers than the other cancer subtypes. Specifically, upregulated luminal progenitor signature genes were more highly expressed in basal-like tumors than in other tumor subtypes. Similarly, down regulated luminal progenitor signature genes were found to be least expressed in the basal tumors compared to other tumor types. Furthermore, the luminal progenitor signature was most selectively expressed (either up-regulated or down regulated) in BRCAl +7" tissue.
  • the data were represented in a barcode plot (see Figures 4 and 8) which demonstrate the ability of the luminal progenitor signature genes (LPT gene) to distinguish between cancer types including between BRCAl and patients and non-BRCAl patients or to distinguish between basal-like breast cancer and other cancer types.
  • LPT gene luminal progenitor signature genes
  • the gene set is a set of at least two upregulated LPT genes comprising KIT and CYP24A1, a set of at least three genes comprising KIT, CYP24A1 and HSDl IBl, a set of at least four genes comprising KIT, CYP24A1, HSDl IBl and GSTAl, a set of at least five genes comprising KIT, CYP24A1, HSDI lBl, GSTAl and PIK3R1, a set of at least six genes comprising KIT, CYP24A1, HSDI lBl, GSTAl, PIK3R1 and LPL, a set of at least seven genes comprising KIT, CYP24A1, HSDI lBl, GSTAl, PIK3R1, LPL and UBE2C, a set of at least eight genes comprising KIT, CYP24A1, HSDI lBl, GSTAl, PIK3R1, LPL,.
  • the gene set is a set of at least two LPT genes comprising CYP24A1 and HSDI lBl, a set of at least three genes comprising CYP24A1, HSDI lBl and GSTAl, a set of at least four genes comprising CYP24A1, HSDl IBl, GSTAl and PIK3R1, a set of at least five genes comprising CYP24A1, HSDI lBl, GSTAl, PIK3R1 and LPL, a set of at least six genes comprising CYP24A1, HSDl IBl, GSTAl, PIK3R1, LPL and UBE2C, a set of at least seven genes comprising CYP24A1, HSDl IBl, GSTAl, PIK3R1, LPL, UBE2C and MATK, a set of at least eight genes comprising CYP24A1, HSDI lBl, GSTAl, PIK3R1, LPL, U
  • the gene set comprises c-kit.
  • Downregulated genes where employed may be selected from those set out in Table 12.
  • the downregulated genes are selected from two or more genes selected from the bottom 50 genes set out in Table 12.
  • LPT genes are identified and further gene sets defined. As the gene sets are tested on more samples, a further selection of informative gene sets will be possible.
  • the method comprises detection of RNA expression products using complementary oligonucleotide probes.
  • the method comprises hybridisation to oligonucleotide probes to detect RNA products from a predetermined gene set.
  • the gene set comprises one of the herein-described gene sets.
  • the gene set comprises between 5 and 50 or between 10 and 30 LPT genes, or between 20 and 30 LPT genes, or between 25 and 35 LPT genes.
  • the level of expression of each LPT gene in the gene set is used to give a measure of gene expression of the sample for comparison with reference measures.
  • the measure is a profile or signature of gene expression that may be represented numerically or graphically to facilitate interpretation of the sample data.
  • the methods comprise determining the expression level of one or more markers of luminal epithelial progenitor cells including, CD49f, EpCAM and cytokeratin 5/6.
  • the subject methods comprise determining the expression level of one or more markers of an active progesterone pathway, such as the presence of progesterone receptors on the surface of luminal progenitor cells or the presence of amphiregulin in the biological sample.
  • signature profiles were also determined for primary basal (MaSC-enriched), mature luminal, and stromal cell populations from mammary tissue.
  • the profile of gene expression for the signature genes of the basal epithelial cells most closely resembled the "claudin low" and "normal-like" tumor types.
  • the present invention provides a method of diagnosis, prognosis or treatment of "claudin low" or "normal-like” breast cancer, said method comprising: determining expression of a set of genes in a biological sample from a individual wherein the gene set is selected from genes which are selectively or differentially expressed in basal epithelial cells compared to at least epithelial luminal progenitor cells or mature luminal epithelial cells.
  • the gene set that is selectively or differentially expressed in basal epithelial cells comprises at least two or more genes set out in the first and/or last 50 genes set out in Table 13.
  • the profile of gene expression for the signature genes of the mature luminal epithelial cells most closely resembled the "luminal A” and "luminal B” tumor types.
  • the mature luminal epithelial cell population is expanded in BRCA2 patients and the mature luminal gene set profile or signature is useful in determining or defining the BRCA2 status of an individual.
  • the BRCA2 phenotype is an expanded mature luminal progenitor population, and this appears to reflect an expansion of a more differentiated cell than the luminal progenitor set.
  • the present invention provides a method of diagnosis, prognosis or treatment of "luminal A”, “luminal B”, or BRCA2-associated breast cancer, said method comprising: determining expression of a set of genes in a biological sample from a individual wherein the gene set is selected from genes which are selectively or differentially expressed in mature luminal epithelial cells compared to their expression by at least epithelial luminal progenitor cells or basal epithelial cells.
  • the gene set that is selectively or differentially expressed in mature luminal epithelial cells comprises at least two or more genes set out in the first and/or last 50 genes set out in Table 14.
  • the present invention provides a method of diagnosis, prognosis or treatment of fibroblast-associated cancer, said method comprising: determining expression of a set of genes in a biological sample from a individual wherein the gene set is selected from genes which are selectively or differentially expressed in stromal cells compared . to their expression by at least epithelial luminal progenitor cells or basal epithelial cells.
  • the gene set that is selectively or differentially expressed in stromal cells comprises at least two or more genes set out in the first and/or last 50 genes set out in Table 15.
  • the stromal cell gene set is probed in order to determine the presence of cancer associated fibroblasts in a clinical sample.
  • the steps of the method are repeated after treatment in order to reassess the prognosis, diagnosis or treatment. In some embodiments, the steps of the method are repeated at a later stage of disease progression.
  • a profile of expression of the predetermined gene set is represented as an overall signature expression score. In other embodiments, the profile of expression of the gene set is represented as a barcode or other graphical representation.
  • the sample is a biopsy such as a fine needle aspirate or core biopsy.
  • the sample may be a blood or other biological sample.
  • RNA, polypeptide or proteinaceous product are thus targets for therapeutic intervention and in this context are described herein as a "luminal progenitor target” or an "LPT” gene or expression product (RNA, polypeptide or proteinaceous product).
  • LPT genes or their expression products are also two or more genes selected from the first and/or last 50 genes listed in Table 12.
  • the method further comprises matching the sample profile or measure of selective or differential expression of the gene set with a treatment program comprising one or more agents that target and modulate the activity of one or more genes in the gene set or one or more LPT genes to modulate the level or activity of an RNA or proteinaceous expression product of one or more genes in the gene set or one or more LPT genes.
  • Matching may conveniently be achieved using a computer program product comprising code to process the data obtained from conduct of the method as input data and a computer readable medium that stores the code.
  • the steps (i) to (iii) of the method are repeated after treatment.
  • selective targeting of luminal progenitor cells and/or one or more genes within the gene set may eliminate these cells or modulate the expression profile of these cells leading to a reduced risk of cancer development, metastases or poor prognosis.
  • the present invention provides a method for the treatment or prevention of "basal-like" breast cancer or a BRCAl -deficiency associated cancer, said method comprising administering one or more agents that modulate the activity of one or more LPT genes set out in Table 10 or 11 or that modulate the activity of an RNA or proteinaceous expression product of one or more LPT genes set out in Table 10 or 11.
  • an LPT gene or LPT expression product is also selected from the first or last 50 genes listed in Table 12.
  • an LPT gene is selected whose proteinaceous expression product comprises a cell surface portion that may be bound by protein binding agents such as an antibody or a molecule comprising an antigen binding fragment thereof.
  • a plasma membrane protein from Table 10 is selected such as a plasma membrane protein selected from the group comprising PTCHDl, GPRI lO, PROMl, CXCR4 and DNER.
  • the plasma membrane protein is selected from the group comprising GAPRP, PIGR, SLC34A2, VNN3, ADORAl, MARCO, VNNl and CLDN8.
  • a suitable agent is an antagonist of c-kit, polymeric immunoglobulin receptor (PIGR) or vitamin D 24-hydroxylase (CYP24A1).
  • agents modulate the activity of one or more of c-Kit, PIGR and CYP 24Al in genetic or proteinaceous form.
  • the agent is a small molecule, an antibody, a nucleic acid or a protein or peptide, such as a stapled peptide.
  • the antagonist is an antibody or comprises an antigen-binding moiety.
  • the cancer is basal-like cancer. In some embodiments, the cancer is BRACl +7" associated.
  • the present invention provides a method for the treatment or prevention of luminal (luminal A or luminal B) or BRCA2-deficiency associated cancer, said method comprising administering one or more agents that modulate the activity of one or more genes set out in Table 14 or that modulate the activity of an RNA or proteinaceous expression product of one or more genes set out in Table 14.
  • the present invention provides a method for the treatment or prevention of "claudin low" or "normal-like” breast cancer, said method comprising administering one or more agents that modulate the activity of one or more genes set out in Table 13 or that modulate the activity of an RNA or proteinaceous expression product of one or more genes set out in Table 13.
  • the present invention provides a method for the treatment or prevention of f ⁇ broblast-associated cancer, said method comprising administering one or more agents that modulate the activity of one or more genes set out in Table 15 or that modulate the activity of an RNA or proteinaceous expression product of one or more genes set out in Table 15.
  • the therapeutic target is selected from the 1 to 3, 2 to 5, 4 to 30 or 10 to 50 gene(s) comprising groups of most upregulated or most downregulated genes set out in Table 13.
  • the present invention provides a diagnostic or prognostic probe set comprising two or more polynucleotides (oligonucleotides) each capable of selectively hybridising to mRNA of one of two or more of the genes listed in Table 10 or 11.
  • the present invention provides a diagnostic or prognostic probe set comprising two or more polynucleotides (oligonucleotides) each capable of selectively hybridising to mRNA of one of two or more of the genes selected from the first and/or last 50 genes listed in Table 12.
  • the present invention provides a diagnostic or prognostic probe set comprising two or more polynucleotides (oligonucleotides) each capable of selectively hybridising to mRNA of one of two or more of the genes selected from the first and/or last 50 genes listed in Table 13.
  • the present invention provides a diagnostic or prognostic probe set comprising two or more polynucleotides (oligonucleotides) each capable of selectively hybridising to mRNA of one of two or more of the genes selected from the first and/or last 50 genes listed in Table 14.
  • the present invention provides a diagnostic or prognostic probe set comprising two or more polynucleotides (oligonucleotides) each capable of selectively hybridising to mRNA of one of two or more of the genes selected from the first and/or last 50 genes listed in Table 15.
  • the probe set comprises two or more polynucleotides each capable of hybridising to mRNA of one of two or more of the genes selected from the first and/or last 50 genes selected from the group consisting of c-Kit, PIGR and CYP24A1 (as referenced in Table 10).
  • kits comprising probe sets complementary to an LPT gene set RNA product as described herein, or antibodies directed to LPT gene set polypeptides.
  • kits comprising probe sets complementary to a mammary basal epithelial cell signature gene set RNA product as described herein, or antibodies directed to mammary basal epithelial cell signature gene set polypeptides.
  • kits comprising probe sets complementary to a mature mammary luminal cell signature gene set RNA product as described herein, or antibodies directed to mature mammary luminal cell signature gene set polypeptides.
  • kits comprising probe sets complementary to a mammary stromal cell signature gene set RNA product as described herein, or antibodies directed to mammary stromal cell signature gene set polypeptides.
  • the diagnostic probe set comprises two or more antibodies or antigen binding molecules each capable of binding to one of two or more proteinaceous expression products of the genes listed in Table 10 or 11. Antigen binding may be used to quantify target protein expression on the surface of cells in the sample.
  • genes are selected from genes encoding plasma membrane proteins of Table 10, such as but not limited to PTCHDl, GPRI lO, PROMl, CXCR4 and DNER.
  • the plasma membrane protein is one or more selected from the group comprising GAPRP, PIGR, SLC34A2, VNN3, ADORAl, MARCO, VNNl and CLDN8.
  • the diagnostic probe set comprises two or more antibodies or antigen binding molecules each capable of binding to one of two or more proteinaceous expression products of the genes selected from the first and/or last 50 genes listed in Table 13.
  • the diagnostic probe set comprises two or more antibodies or antigen binding molecules each capable of binding to one of two or more proteinaceous expression products of the genes selected from the first and/or last 50 genes listed in Table 14.
  • the diagnostic probe set comprises two or more antibodies or antigen binding molecules each capable of binding to one of two or more proteinaceous expression products of the genes selected from the first and/or last 50 genes listed in Table 15.
  • the diagnostic probe set is for use in the diagnosis, prognosis and treatment of aggressive ("basal-like") or BRCAl +7" associated cancer.
  • the LPT gene set comprises at least c-Kit, PIGR and CYP24A1.
  • all or part of the gene set or probes therefore are immobilised on a solid surface, such as without limitation a bead, particle, chip, well, strip etc.
  • the LPT gene set comprises at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 36 of the genes listed in Table 10.
  • each of the listed genes having an average fold change of more than 10 is included in the set.
  • each of the listed genes having an average fold change of more than a fold change of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, or 20 is included in the set.
  • pre-neoplastic BRCA l +/ ⁇ subjects exhibit a primary defect in their luminal progenitor population which results in increased colony forming ability in the presence of B27 growth factor medium containing EGF, insulin and hydrocortisone in a 3D "Matrigel" assay.
  • pre-neoplastic BRCAl +/ ⁇ subjects exhibit factor-independent growth of luminal epithelial progenitor cells that is not affected by progesterone receptor antagonists.
  • normal luminal progenitor cells exhibited colony forming ability when contacted with progesterone or B27 growth factor medium containing EGF, insulin and hydrocortisone. That is, normal luminal epithelial progenitor cell colony formation and growth is factor dependent.
  • the luminal progenitor gene expression profile was able to distinguish between prophylactic BRCA1 +/" breast tissue and normal subject tissue.
  • the BRCA 1 +/' breast tissue also displayed factor independent growth of luminal progenitor cells.
  • the present invention provides methods for detecting proliferative changes associated with neoplastic transformation in BRCAl -deficient tissue. In some embodiments, these changes are detected at an early stage allowing more informed decisions regarding treatment options, or the identification of a subject harbouring a BRCAl or similar mutation. In other embodiments these changes detect an early stage change in breast tissue for a subject potentially at increased risk for the development of breast cancer, in particular "basal-like" breast cancer.
  • the present specification provides a method of diagnosis, prognosing or treating breast cancer, said method comprising determining the activity such as the proliferative capacity of luminal progenitor cells in a subject.
  • factor independent growth of luminal progenitor cells in vitro is determined in a biological sample from a human subject.
  • the method comprises determining the colony forming ability of luminal progenitor epithelial cells.
  • any conventional method for assessing cellular proliferation of the luminal progenitor cells may be employed. Cells may be conveniently selected for testing by preparing CD49f + EpCAM + epithelial cells. Alternatively, cells types may be determined after colony forming ability has been determined.
  • cells may be maintained in the presence of a progesterone antagonist.
  • the invention further contemplates an isolated population of human luminal progenitor cells for use in the diagnosis of breast cancer.
  • the cells are isolated using cell surface markers for at least CD49f and EpCAM.
  • the invention provides a method of diagnosis, prognosis or treatment of basal-like breast cancer and/or a BRCAl -deficiency associated form of cancer, said method comprising (i) determining expression of a set of genes in a biological sample from an individual wherein the gene set is preselected to comprise genes which are selectively or differentially expressed in CD49f f EpCAM + epithelial luminal progenitor cells compared to their expression by control cells such as at least basal epithelial cells or mature luminal epithelial cells.
  • the gene set comprises at least two or more LPT genes selected from the group consisting of the genes set out in Table 10 or 11. In other embodiments, the LPT gene set comprises two or more genes selected from the genes set out in Table 16 and/or 17.
  • the LPT gene set comprises 8 to 20 genes selected from the genes set out in Table 16 and/or 17.
  • the LPT gene set comprises KIT, CYP24A1 and ELF5. In other embodiments, the gene set comprises KIT and CYP24A1. In other embodiments, the " LPT gene set comprises CYP24A1 and ELF5. In other embodiments, the LPT gene set comprises KIT or CYP24A1 or ELF5. In a preferred embodiment, the LPT gene set comprises KIT.
  • the invention provides a method for the treatment or prevention of "basal-like" breast cancer and/or a BRCAl -deficiency associated cancer in a subject, said method comprising administering to the subject one or more agents that down modulate the activity of KIT polypeptide.
  • agents that down modulate or inhibit the activity of KIT polypeptide will predominantly down modulate the proliferation of luminal progenitor cells present in normal, pre-neoplastic and cancerous tissue.
  • treatment may be commenced prophylactically and selectively inhibit proliferation in target luminal progenitor cells thereby reducing the risk of cancer development or reducing cancer cell proliferation in selected subject.
  • the invention provides a method for the treatment or prevention of "basal-like" breast cancer and/or a BRCAl -deficiency associated cancer in a subject, said method comprising administering to the subject one or more agents that down modulate the activity of KIT selectively or differentially expressed on luminal progenitor cells.
  • the present methods further comprise, either before, during or after therapeutic or prophylactic administration, a method comprising (i) determining expression of a set of genes in a biological sample from an individual wherein the gene set is preselected to comprise genes which are selectively or differentially expressed in CD49f + EpCAM + epithelial luminal progenitor cells compared to their expression by control cells such as at least basal epithelial cells or mature luminal epithelial cells.
  • the pre-selected gene set preferably comprises KIT.
  • Targeted therapy to CD49f f EpCAM + expressing luminal progenitor cells is particularly provided.
  • the present invention provides a diagnostic or prognostic probe set or kit comprising same comprising two or more polynucleotides each capable of selectively hybridising to mRNA of one of the genes in the sets of genes defined hereinbefore.
  • the probe set comprising one or two or more polynucleotides each capable of hybridising to mRNA of KIT gene.
  • the diagnostic probe set or kit is for use or when used in the diagnosis, prognosis and treatment of aggressive ("basal-like") and/or BRCAl deficiency associated cancer.
  • the present invention provides a method of reducing the proliferative activity of normal luminal progenitor cells in human mammary epithelium comprising administering an effective amount of a KIT inhibitor to a subject for a time and under conditions sufficient to reduce normal (non-cancerous) luminal progenitor cell proliferation.
  • the present invention provides a method of reducing the proliferative activity of pre-neoplastic or neoplastic luminal progenitor cells in human mammary epithelium comprising administering an effective amount of a KIT inhibitor to a subject for a time and under conditions sufficient to reduce proliferation.
  • the pre-neoplastic tissue may in some embodiments further comprise a BRCAl deficiency. However, as described herein, in some embodiments, the pre-neoplastic tissue does not comprise a BRCAl or BRC A2 deficiency.
  • agent/s are delivered to CD49f f EpCAM + expressing luminal progenitor cells.
  • Figure IA to D are representations of results showing that CD49f and EpCAM define distinct subpopulations in the human mammary epithelium.
  • A Top. Expression analysis of lineage markers CD45 (hematopoietic cells), CD31 (endothelial cells) and CD235a (erythrocyte precursors) in human mammary tissue was carried out on live, single-cell gated populations. Bottom. These combined markers defined the Lineage-negative (Lin " ) population.
  • B Expression of CD49f and EpCAM in the Lin " population of a 27 year-old woman.
  • CD49f h 'EpCAM " subpopulation described here is similar to the CD49f + EpCAM neg low subset recently described by Eirew et al, Nat Med 14: 1384-1389, 2008.
  • C Expression of CD 133 and CD24 in the four Lin " populations defined by CD49f and EpCAM, depicted as FACS dot plots.
  • D Immunohistochemical analysis of cells isolated from normal reduction mammoplasties.
  • Outgrowths derived from transplantation of CD49f hi EpCAM " cells top: 25,000 cells from a 53 year-old woman; bottom: 45,000 cells from a 35 year-old woman). Bars: 250 ⁇ m.
  • B Outgrowths were sectioned and stained with antibodies against K8/18, vimentin and p63. Anti-K8/18 and -vimentin specifically recognize human antigens. The top image is a composite of two contiguous fields. Bars: 75 ⁇ m (K8/18), 60 ⁇ m (p63, Vim).
  • C Differential in vitro growth characteristics of specific epithelial subsets. Cells (1000) from each of the populations defined in Figure IB were cultured in Matrigel for 14 days.
  • Figure 3 A to G are representations of results showing that BRCA l +/ ⁇ luminal progenitor cells demonstrate factor-independent growth in vitro.
  • A Representative FACS dot plots showing the expression of CD49f and EpCAM from age-matched normal and BRCA I + ' ' breast tissues. Identical number of events/cells are displayed in each plot.
  • B Bar chart depicting the relative proportion of epithelial cell subpopulations (CD49f hi EpCAM " , CD49f " ⁇ EpCAM + or CD49f " EpCAM + ) in normal (white bars) and BRCA l +/ ⁇ (black bars) epithelia.
  • Figure 4A to C are graphical representations showing comparison of gene expression profiles of normal human mammary epithelial subsets with the major subtypes of breast cancer and with preneoplastic tissue from BRCA l +/ ⁇ patients.
  • MaSC represents the MaSC-enriched cell population.
  • B Boxplots of signature expression scores by BRCAl mutation status for each epithelial subset. The luminal progenitor signature is highest in breast tissue from BRCAl mutation carriers.
  • Figure 5A and B are photographic representations of results showing that orthotopic xenotransplantation of Lin " cells gives rise to outgrowths when transplanted into de- epithelialized mammary fat pads of NOD-SCID-IL2R ⁇ "/” mice.
  • A H&E section of an outgrowth, eight weeks following transplantation of 25,000 CD45 " CD235a " CD3r (Lin " ) cells derived from a reduction mammoplasty from a 41 year-old woman, admixed with 500,000 hTERT-immortalized breast stromal fibroblasts. The image represents a composite of two contiguous fields. Bar: 100 ⁇ m.
  • FIG. 6 is a photographic representation of results showing that CD49f hl EpCAM " cells have limited self-renewing activity.
  • a single cell suspension was prepared from mammary fat pads eight weeks following primary transplantation of CD49f hl EpCAM " cells. Each was secondarily transplanted into multiple cleared fat pads. Only occasional secondary mammary epithelial structures were observed (normal tissue, 1/6 and 2/6 fat pads; BRCA l +/ ⁇ tissue, 2/4 fat pads).
  • the human secondary outgrowth depicted is from a BRCA 1 +/ - carrier. Sections were stained with antibodies against K8/18, vimentin, p63 and K14. Staining for K8/18 and vimentin (both human-specific) proved donor origin. Bars: 50 ⁇ m.
  • Figure 7 A and B are representations of results showing that murine luminal progenitor cells from Brcal -deficient mammary glands exhibit B27 factor-independence.
  • Sorted luminal progenitor cells (CD29 lo CD24 + CD61 + ) from control and MMTV-cre-Brcal f/f mammary glands were • embedded in Matrigel and cultured for 14 days in media with or without B27 supplement. The images correspond to a 3 mm diameter Matrigel plug.
  • Figure 8 provides barcode plots demonstrating the ability of luminal progenitor signature genes to distinguish basal-like, 'normal breast-like', claudin-low, ERBB2, luminal A and luminal B subtypes of breast cancer, with corresponding one-sided mean-rank gene set test P- values. Red bars designate upregulated signature genes while blue bars designate downregulated signature genes.
  • Figure 9A to C are photographic representations of results showing that increased expression of c-KIT in CD49f l ⁇ pCAM + luminal progenitor cells and BRCA 1 -associated tumors.
  • B Heterogeneous c-KIT staining of luminal cells in a normal duct and Terminal Ductal Lobular Unit (TDLU).
  • C c-KIT immunostaining of BRCA 1 -associated and nonBRCAl/2 breast tumors.
  • Figure 10 is a graphical representation of data showing the results of quantitative RT-PCR analysis of specific genes that define human and mouse mammary epithelial subsets. Histograms depicting the relative fold difference in RNA expression between specific mammary epithelial cell subsets relative to other subsets in mouse and human mammary tissue. Expression analysis was relative to 18S rRNA. Examples of genes primarily expressed in the (a) MaSC-enriched subset, (b) Luminal progenitor subset, including c-Kit, and (c) Mature luminal subset. At least three independent samples from either mouse or human mammary cell populations were evaluated for each gene. Data represent mean ⁇ s.e.m.
  • FIG. 11 is a graphical representation of data showing c-KIT is expressed in Luminal Progenitor cells in human breast tissue.
  • A Dot plot depicting EpCAM and CD49f expression in Lin ⁇ cells from human breast tissue. The four subsets characterized by these markers are shown. Luminal progenitor cells are Lin ⁇ CD49f + EpCAM + .
  • B Histograms depicting c-KIT expression for each subpopulation (black line), compared to control (red).
  • Figure 12 is a graphical representation of data showing c-Kit marks the luminal progenitor cell. Histograms showing the MaSC-enriched (left) and Luminal subpopulations (right) in mouse mammary tissue. The expression of c-kit and CD61 are shown for each subset. The CD61 -positive cells in the luminal population (which is associated with progenitor activity) are also c-kit-positive (right panel). In contrast to human breast tissue, c-kit is expressed in a subset of the MaSC-enriched population, as defined by CD29 and CD24.
  • Figure 13 is a graphical representation of data showing masitinib (mas) inhibits colony formation by human breast luminal progenitor cells.
  • A MaSC-enriched (Lin " CD49 hl EpCAM + ) and Luminal Progenitor (Lin " CD49 hl EpCAM + ) cells were plated in Matrigel and colony formation determined after 11 days.
  • B Representative ' images from Experiment #2.
  • Figure 14 is a photographic representation of data showing inhibition of KIT reduces colony formation by MMTV-neu mammary tumor cells.
  • A Tumor cells were plated in Matrigel and treated with the c-KIT inhibitors Masitinib (mas), the pyridone compound 25 (ckit25) or ACK2 monoclonal antibody. Tumor colonies were scored after 9 days.
  • B Representative images from Experiment #3.
  • Figure 15 is a graphical representation of data showing MMTV-neu tumors express the luminal progenitor markers CD61 and c-Kit.
  • A A representative dot plot of a MMTV-neu tumor expressing both CD24 and CD61
  • B A histogram of an MMTV-neu tumor revealing c-kit expression (black), detected with ACK2 monoclonal antibody compared to control staining (red).
  • Figure 16 is a graphical and photographic representation of data showing tumor cells from the 878T xenograft express KIT.
  • A Flow cytometric analysis of 878T tumors. The majority of the tumor population appears to be EpCAM + CD49f +/1 °.
  • B KIT expression in EpCAM + CD49f f/1 ° tumor cells (black) compared to control staining, revealing high c-KIT expression levels.
  • C Immunohistochemical staining of 838T cells for KIT expression (DAKO).
  • Figure 17 is a graphical representation of data showing the KIT inhibitor masitinib results in improved tumor response and survival when combined with docetaxel therapy.
  • Table 1 provides a description of the SEQ ID NOs provided herein.
  • Table 2 provides an amino acid sub-classification.
  • Table 3 provides exemplary amino acid substitutions.
  • Table 4 provides a list of non-natural amino acids contemplated in the present invention.
  • Table 5 provides an immunohistochemical analysis of Lin " populations defined by CD49f and
  • EpCAM expression The mean percentage . of cells staining positively for the relevant antibodies, with the standard error of the mean ( ⁇ s.e.m.), are shown. A minimum of three experiments from independent normal breast reduction mammoplasty samples was used for each marker. For HER2/ErbB2 staining, breast tumors exhibiting HER2 amplification served as positive controls.
  • Table 6 provides limiting dilution analysis of Lin " subpopulations.
  • Normal human mammary cells sorted from the Lin " gate ( Figure IA) were injected at the indicated (f ) number (based on machine counts) into cleared mammary fat pads (MFPs), together with 500,000 hTERT fibroblasts, as described in the Methods. MFPs were analysed and the mammary repopulating frequency was calculated by limiting dilution analysis as described in Supplementary
  • Table 7 provides the classification of pathogenic BRCAl mutations in prophylactic mastectomy samples.
  • Table 8 provides the number of samples used for each breast tumor subtype.
  • Table 9 provides the number of probes and unique genes identified in each subpopulation signature set.
  • Table 10 provides examples of upregulated genes in the luminal progenitor gene signature from select ontology groups.
  • Table 11 provides preferred examples in order of preference of upregulated genes in the luminal progenitor gene signature from select ontology groups.
  • Table 12 provides the gene signatures for LPT subsets.
  • Table 13 provides the gene signatures for MaSC-enrich subsets.
  • Table 14 provides preferred the gene signatures for Mature Luminal subsets.
  • Table 15 provides the gene signatures for Stromal subsets.
  • Table 16 provides conserved genes upregulated in luminal progenitor cells. The conserved genes between mouse and human were selected by using the nested F multiple testing adjustments with FDR ⁇ 0.1 and at least 1.5 fold change. Mouse signature genes for a subset were first selected, then multiple testing adjustments were performed for the human data of these subsets of the ortholog genes. The mouse signature genes that were also significantly differentially expressed in human were defined as the conserved genes. The conserved genes represent those consistently up or down in one subpopulation across the two species.
  • Table 17 provides conserved genes downregulated in luminal progenitor cells. The conserved genes between mouse and human were selected by using the nested F multiple testing adjustments with FDR ⁇ 0.1 and at least 1.5 fold change.
  • Mouse signature genes for a subset were first selected, then multiple testing adjustments were performed for the human data of these subsets of the ortholog genes.
  • the mouse signature genes that were also significantly differentially expressed in human were defined as the conserved genes.
  • the conserved genes represent those consistently up or down in one subpopulation across the two species.
  • antibody encompasses a whole antibody, or a fragment thereof, for example a F(ab')2, Fab, Fab', Fv, VH or VK fragment, a single-chain antibody, a multimeric monospecific antibody or fragment thereof, or a bi- or multispecif ⁇ c antibody or fragment thereof.
  • An antibody as used herein may be a polyclonal or a monoclonal antibody.
  • the antibody may belong to any immunoglobulin class, and may be for example an IgG, for example IgGl, IgG2, IgG3, IgG4, IgE, IgM or IgA antibody.
  • the antibody may be of animal, for example mammalian origin, and may be for example a murine, rat or human antibody.
  • the antibody may be a chimeric antibody.
  • the term chimeric antibody is used herein to mean any antibody containing portions derived from different animal species. Particular non-limiting examples include those antibodies having a variable region derived from a murine or other antibody constant region, and those antibodies in which one or more CDR sequences and optionally one or more variable region framework amino acids are derived from a murine or other antibody and the remaining portions of the variable and the constant regions are derived from a human immunoglobulin.
  • subject refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the medical protocol of the present invention.
  • a subject regardless of whether a human or non-human animal or embryo may be referred to as an individual, subject, animal, patient, host or recipient.
  • the term "gene” as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions.
  • the gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the DNA sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • match and mismatch refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • hydrogen bonding which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • nucleobases nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances as known to those of skill in the art.
  • Nucleic acid sequence identity can be determined in the following manner.
  • the subject nucleic acid sequence is used to search a nucleic acid sequence database, such as the GenBank database (accessible at web site http://www.ncbi.nln.nih.gov/blast/), using the program BLASTM version 2.1 (based on Altschul et al, Nucleic Acids Research 25:3389- 3402 (1997)).
  • the program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity. The default parameters of BLASTM are used.
  • Amino acid sequence identity can be determined in the following manner.
  • the subject polypeptide sequence is used to search a polypeptide sequence database, such as the GenBank database (accessible at web site http://www.ncbi.nln.nih.gov/blast/), using the BLASTP program.
  • GenBank database accessible at web site http://www.ncbi.nln.nih.gov/blast/
  • BLASTP program is used in the ungapped mode.
  • Default filtering is used to remove sequence homologies due to regions of low complexity.
  • the default parameters of BLASTP are utilized. Filtering for sequences of low complexity may use the SEG program.
  • hybridize under stringent conditions refers to the ability of a nucleic acid molecule to hybridize to a target nucleic acid molecule (such as a target nucleic acid molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • target nucleic acid molecule such as a target nucleic acid molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot
  • typical stringent hybridization conditions are no more than 25 0 C to 30 0 C (for example, 1O 0 C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al.
  • exemplary stringent hybridization conditions are 5°C to 10 0 C below Tm.
  • oligonucleotide refers to a nucleic acid molecule of up to 100 bases.
  • complement when used in connection with a nucleic acid molecule refers to the complementary nucleic acid sequence as determined by Watson-Crick base pairing.
  • complement of the nucleic acid sequence 5'CCATG3' is 5'CATGG3'.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an "isolated polynucleotide”, as used herein, refers to a polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an "isolated peptide” or an “isolated polypeptide” and the like, as used herein refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.
  • an isolated composition, complex, polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means.
  • modulation or modulator in relation to a particular target is meant generally directly or indirectly up-regulating or down-regulating the level, effects or activity of the target.
  • sample means any biological fluid, cell, tissue, organ or portion thereof, that includes, or potentially includes, an LPT or BMS nucleic acid molecule or polypeptide.
  • the term includes samples present in an individual as well as samples obtained or derived from an individual.
  • a sample can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture.
  • a sample can be a subcellular fraction or extract, or a purified or crude nucleic acid or polypeptide preparation.
  • the present invention is not limited to any particular method for measuring gene expression and the addressee may measure the level or activity of the gene or part of the gene or its expression products such as mRNA or protein. Considerations such as the sample type, availability and amount will also influence selection of a particular method. For example, if only a small amount of cellular material is available, then methods which measure the amount of RNA by, for example, PCR amplification, can be an appropriate choice for determining gene set expression. Alternatively, an Enzyme Linked Immunoabsorbent Assay (ELISA), which measures the amount of polypeptide can be an appropriate choice for determining the expression level of one or two or more gene set polypeptides.
  • ELISA Enzyme Linked Immunoabsorbent Assay
  • Gene expression may be determined using hybridisation or sequencing based methods known in the art.
  • convenient methods include, without limitation, Northern blotting and in situ hybridisation dot-blots or other membrane-based technologies, RNAse protection assays, realtime PCR methods, reverse transcriptase PCR methods, e.g.
  • TaqMan RT-PCR differential display methods
  • MassARRAY-based gene expression profiling (Sequenom)
  • Bead arrays for detection of gene expression such as Luminex multicolour coded microspheres, Illumina bead array systems
  • microarrays such as Affymetrix GenChip or Incyte microarray technology
  • hicoverage expression profiling HiCEP
  • IHC immunohistochemistry
  • methods using mass spectrometry such as MALDI-TOF analysis
  • SAGE serial analysis of gene expression techniques
  • MPSS massively parallel signature sequencing
  • PCR or RT-PCR can be used with isolated RNA or crude cell lysate preparations. As described previously, PCR is useful when there is little starting material.
  • a further description of traditional PCR methods can be found in, for example, Dieffenbach, C. W., and Dveksler, G. S., PCR Primer: A Laboratory Manual, Cold Spring Harbor Press, Plainsview, N.Y. (1995).
  • RNA from a sample is contacted with a probe under conditions which allow annealing (hybridisation) of the probe to RNA.
  • annealing hybridisation
  • the sample is optionally washed and the signal is measured and compared with a suitable control or standard value.
  • the magnitude of the hybridization signal may be directly proportional to the expression levels of the LPT or BMS gene for which the probe was specific.
  • a suitable control for comparison can be, for example, the expression level of the gene set in a sample obtained from a normal individual or normal sample.
  • control sample for comparison can be measured simultaneously with one or more test samples or, alternatively, expression levels can be established for a particular type of sample and standardized to internal or external parameters such as polypeptide or polynucleotide content, cell number, cell type, or mass of sample. Such standardized control samples can then be directly compared with results obtained from the test sample. An increase (such as, by way of non-limiting example, an increase of two-fold or more) of expression levels of an LPT or BMS gene set indicates increased risk of cancer in the tested individual.
  • proteomics may be used to measure the level or activity of proteinaceous products of one or more of the genes identified herein.
  • activity is meant the characteristic activity of the proteinaceous product, such as its activity as an enzyme, i.e., kinase activity, or activity as a transcription factor, plasma membrane protein i.e., receptor or binding target for a ligand or antibody etc.
  • the sample is assessed by one or two dimensional electrophoresis to detect expression products of one of the gene sets disclosed herein.
  • Efficient computer assisted methods are available in the art to characterise proteinaceous molecules separated electrophoretically.
  • proteins may be stained with a fluorescent dye and imaged with a fluorescent scanner.
  • the sample may be assessed in an immunoassay.
  • the methods comprise contacting a cell or tissue sample, or lysate thereof, or fractionated sample thereof, from a subject with a binding agent and determining the amount of selective binding of the agent.
  • the fractionated sample can be, for example, a cell lysate or lipid membranes and the binding agent can be an antibody or a ligand or substrate or an analog depending upon which LPT/LPTs or BMS is to be assayed.
  • affinity binding assay All modes of affinity binding assay are applicable for use in determining the amount of a polypeptide in a sample. Suitable methods are rapid, efficient and sensitive. Affinity binding methods are convenient and can be adjusted to be performed in a variety of clinical settings and under conditions to suit a variety of particular needs. Affinity binding assays which are known and can be used in the methods of the invention include both soluble and solid phase formats. In one embodiment, a soluble phase affinity binding assay is immunoprecipitation using antibodies which are selective for one or more of PTCHDl, GPRl 10, PROMl, CXCR4 and DNER. Solid phase affinity binding assays are convenient as they are amenable to high throughput screening and automation.
  • solid phase affinity binding assays include immunoaff ⁇ nity binding assays such as an ELISA and radioimmune assay (RIA), immunochromatographic devices, dip sticks etc.
  • affinity binding assays are generally formatted for use with an antibody that is selective for the analyte or ligand of interest, essentially any binding agent can be substituted.
  • Suitable binding agents include, for example, steroids, steroid derivatives, macromolecules such as polypeptides, peptides, nucleic acids, aptamers, lipids and sugars as well as small molecule compounds. Methods are known in the art for identifying such molecules which bind selectively to a particular analyte or ligand and include, for example, screens of combinatorial libraries.
  • affinity binding formats are similarly known which can be used in the diagnostic methods of the invention.
  • particular embodiments of such affinity binding assays will be described further in reference to immunoaffinity binding assays.
  • Affinity binding methods are described in common laboratory manuals such as Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1999).
  • the diagnostic formats employing affinity binding can be used in conjunction with a variety of detection labels and systems conventional in the art to detect or quantify amounts of an LPT or BMS polypeptide in the analyzed sample.
  • Detection systems include the detection of bound polypeptide of the invention by both direct and indirect means.
  • Direct detection methods include labeling of an antibody or binding agent that binds specifically to a polypeptide of the invention.
  • Indirect detection systems include, for example, the use of labeled secondary antibodies and binding agents.
  • Secondary antibodies, labels and detection systems are well known in the art and can be obtained commercially or by conventional techniques.
  • Suitable detectable labels include, for example, enzymes, radioisotopes, fluorochromes as well as chemi- and bioluminescent compounds.
  • Specific examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, urease and luciferase.
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable by measuring absorbance at 450 run.
  • TMB chromogenic substrate tetramethylbenzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable by measuring absorbance at 405 ran.
  • a beta-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG), which yields a soluble product detectable by measuring absorbance at 410 nm, or a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, Mo.).
  • Luciferin is the substrate compound for luciferase which emits light following ATP-dependent oxidation. Fluorochrome detection labels are rendered detectable through the emission of light of ultraviolet or visible wavelength after excitation by light or another energy source.
  • DAPI fluorescein, Hoechst 33258, R-phycocyanin, B- phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine are specific examples of fluorochrome detection labels that can be utilized in the affinity binding formats of the invention.
  • Particularly useful fluorochromes include fluorescein and rhodamine.
  • Chemiluminescent as well as bioluminescent detection labels are convenient for sensitive, non-radioactive detection of the inventive polynucleotides and polypeptides and can be obtained commercially. Radioisotopes can alternatively be used as detectable labels for use in the binding assays of the invention.
  • Iodine- 125 is a specific example of a radioisotope useful for a detectable label.
  • Signals from detectable labels can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a fluorometer to detect fluorescence in the presence of light of a certain wavelength; or a radiation counter to detect radiation, such as a gamma counter for detection of iodine- 125.
  • a quantitative analysis of the amount of bound agent can be made using a spectrophotometrically.
  • the detection marker comprises a visually detectable reporter molecule and a positive result may be essentially immediately observed in the test and/or control portions of an immunochromatographic device.
  • the detection marker may be detected using further detection protocols and devices such as will be well known to those of ordinary skill in the art.
  • colloidal gold may be used or another colloidal metal or metal oxide particles or colloidal non-metal particles or dyes or coloured latex are conveniently used.
  • LPT or BMS gene set protein product may be evaluated in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays.
  • Rapid point of care diagnostics using, for example, immunochromatographic protocols together with colloidal material, etc., are known in the art.
  • 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 LPT or BMS polypeptide is determined by observation of a signal produced by the detectable marker (reporter molecule). The results may be qualitative or quantitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of LPT or BMS polypeptide. 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 an LPT or BMS is either covalently or passively bound to a solid or semi-solid support.
  • the support is typically glass or a polymer, the most commonly used polymers being nitrocellulose, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene or mixture or derivatives of these.
  • the solid supports may be in the form of tubes, beads, discs or 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 to the solid surface which is then 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 or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37°C including 25°C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and incubated with a second antibody specific for a portion of the antigen.
  • the second antibody is linked to a detectable marker which is used to indicate the binding of the second antibody to the antigen.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a detectable marker. Depending on the amount of target and the strength of the signal from the detectable marker, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • the method is a liquid phase method.
  • a liquid phase immunoassay see, for example, United States Patent No. 6,632,603 the sample is contacted with an agent capable of binding LPT or BMS polypeptide and a detector agent comprising a visually detectable agent such as colloidal gold or silver labelled.
  • the test sample is applied by flowing onto a defined zone of an insoluble porous support film having a pore size impassable to a complex formed between the LPT or BMS, if present, with the binding substance and the detector substance, but passable to the binding substance and detector substance while remaining uncomplexed in the absence of the desired LPT or BMS. If the LPT or BMS is present in the test specimen, the detector substance binds with the LPT or BMS and the binding substance to form a visually inspectable complex on the surface of the porous support film. After application of the test sample to the porous support, the surface of the porous support is visually inspected for colour to determine the presence and quantity or the absence of the LPT or BMS being assayed.
  • a liposome immunomigration, liquid-phase competition strip immunoassay is, for example, described in Glorio-Paulet et al, J Agric Food Chem 48 (5): 1678-1682, 2000.
  • antibodies according to the invention may be prepared by conventional immunization and recombinant DNA techniques.
  • polyclonal antibodies may be obtained from the sera of animals immunised with a LPT or BMS protein or fragment thereof.
  • Any suitable host for example BALB/c mice where it is desired to obtain a mouse polyclonal antibody, may be injected with the immunogen, the serum collected and the antibody recovered therefrom.
  • Monoclonal antibodies may be obtained from hybridomas derived form the spleen cells of an animal immunised as just discussed and fused to an appropriate "immortal" B-tumour cell.
  • the antibody may be recovered from either the serum or the hybridoma by making use of standard purification and or concentration techniques, for example by chromatography, using for example Protein A or by other affinity chromatography employing a protein of the invention or fragment thereof.
  • a cell line for example a hybridoma, expressing a suitable antibody
  • chimeric antibodies may be obtained by preparing one or more replicable expression vectors containing at least the DNA sequence encoding the variable domain of the antibody heavy or light chain and optionally other DNA sequences encoding remaining portions of the heavy and/or light chains as desired, and transforming an appropriate cell line, e.g., a non-producing myeloma cell line, such as a mouse NSO line, in which production of the antibody will occur.
  • an appropriate cell line e.g., a non-producing myeloma cell line, such as a mouse NSO line
  • Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous processes such as the production of synthetic antibodies, the screening of recombinant immunoglobulin libraries for specific-binding molecules or the in vitro stimulation of lymphocyte populations.
  • the present invention provides a method of identifying targets for the treatment of breast cancer. Specifically by comparing the profile of gene expression between normal and abnormal luminal progenitor cells, pathways that have been switched on or are driving tumor formation may be identified using the methods described herein.
  • agents that modulate the activity of the target can be identified.
  • LPT or BMS gene can be screened for mutations therein that affect gene expression.
  • Antibodies are particularly useful agents where the target has a binding site that is accessible from outside the cell. In this way, agents need not cross or fully traverse the cell membrane.
  • Antibodies specific to an LPT or BMS polypeptide can be used, for example, directly as an antagonist.
  • the antibodies can be generated using methods that are well known in the art and include, for example, polyclonal, monoclonal, chimeric * humanized single chain, Fab fragments, and fragments produced by a Fab expression library.
  • the antibodies of the present invention are CDR-grafted antibodies.
  • CDR-grafted antibody refers to an antibody molecule wherein the heavy and/or light chain contains one of more CDRs from a donor antibody (e.g., a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g., human antibody). Construction of CDR-grafted antibodies is fully described in European Patent Application EP- A-0239400, which publication is incorporated herein by reference. Some criteria for selecting which framework residues need to be altered are described in International Patent Application WO 90/07861, incorporated herein by reference.
  • LPT or BMS modulators include small chemical molecules which can penetrate a cell membrane or via an ion channel or other pore and an antigen binding agent which has the capacity for intracellular transmission such as cartilage fish-derived antibodies (e.g. shark antibodies; see for example, Liu et ah, BMC Biotechnol. 7: 78, 2007).
  • cartilage fish-derived antibodies e.g. shark antibodies; see for example, Liu et ah, BMC Biotechnol. 7: 78, 2007.
  • An antigen binding agent, or functionally active fragment thereof, which has the capacity for intracellular transmission also includes antibodies such as camelids and llama antibodies, scFv antibodies, intrabodies or nanobodies, e.g. scFv intrabodies and VHH intrabodies.
  • antigen binding agents can be made as described by Harmsen & De Haard in Appl. Microbiol. Biotechnol. Nov; 77(1): 13-22, 2007; Tibary et al, Soc. Reprod. Fertil. Suppl. 64: 297-313, 2007; Muyldermans, J. Biotechnol. 74: 277-302, 2001; and references cited therein.
  • scFv intrabodies which are able to interfere with a protein- protein interaction are used in the methods of the invention; see for example, Visintin et al, J. Biotechnol, /35:1-15, 2008 and Visintin et al, J. Immunol. Methods, 290(1-2): 135-53, 2008 for methods for their production.
  • modulatory agents may comprise a cell-penetrating peptide sequence or nuclear-localizing peptide sequence such as those disclosed in Constantini et al., Cancer Biotherm. Radiopharm. • 25(1): 3-24, 2008. Also useful for in vivo delivery are Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al., Biochem J. 390(pt2): 407-418, 2005 and Meyer- Losic et al., J Med Chem. 49(23): 6908-6916, 2006.
  • the invention provides the therapeutic use of fusion proteins of the agents (or functionally active fragments thereof), for example but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to a cell- penetrating peptide or nuclear-localizing peptide sequence.
  • a covalent bond e.g. a peptide bond
  • Natural products, combinatorial synthetic organic or inorganic compounds, peptide/polypeptide/protein, nucleic acid molecules and libraries or phage or other display technology comprising these are all available to screen or test for suitable agents. Natural products include those from coral, soil, plant, or the ocean or Antarctic environments. Libraries of small organic molecules can be generated and screened using high-throughput technologies known to those of skill in this art. See for example United States Patent No. 5,763,623 and United States Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesized having different substitutions of a common or subset of parent structures.
  • Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function, for example, varying in chain length, ring size or number or substitutions.
  • Virtual libraries are also contemplated and these may be constructed and compounds tested in silico (see for example, US Publication No. 20060040322) or by in vitro or in vivo assays known in the art. Libraries of small molecules suitable for testing are already available in the art (see for example, Amezcua et al., Structure (London) 10: 1349-1361, 2002).
  • Yeast SPLINT antibody libraries are available for testing for intrabodies which are able to disrupt protein-protein interactions (see Visintin et al., (supra)).
  • agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvol ⁇ tion; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997; United States Patent No. 5,738,996; and United States Patent No. 5,807,683).
  • Libraries of compounds may be presented, for example, in solution (e.g.
  • Nucleic acids including DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), double-stranded RNAs (dsRNA), short hairpin RNAs (shRNAs), piwi-interacting RNAs (PiRNA), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs), small nuclear (SnRNAs) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes are conveniently employed. Methods of producing chimeric constructs capable of producing dsRNA in eukaryotic cells are described in the art.
  • RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, Clin. Chem., 45(9): 1628-1650, 1999; Morris et al, Proc. Natl. Acad. Sci., USA, 95(6): 2902-2907, 1998).
  • Aptamers are nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing. Aptamers are described, for example, in United States Patent Nos. 5,475,096; 5,270,163; 5,589,332; 5,589,332; and 5,741,679.
  • agents that down modulate the formation, expression or activity of an LPT or BMS may be derived from the LPT or BMS polypeptide or their encoding sequences or are variants or analogs thereof.
  • agents may be hydrocarbon-stapled peptides or minature proteins which are alpha-helical and cell- penetrating, and are able to disrupt protein-protein interactions (see for example, Wilder et ai, ChemMedChem. 2(8): 1149-1151, 2007; & for a review, Henchey et al, Curr. Opin. Chem. Sept 12, 2008).
  • the agents are derived from nucleic acid molecules such as the nucleotide sequences of an LPT or BMS gene or corrected version thereof or variants thereof.
  • Variants include nucleic acid molecules sufficiently similar to naturally occurring forms of these molecules or their complementary forms over all or part thereof such that selective hybridisation may be achieved under conditions of medium or high stringency, or which have about 60% to 90% or 90 to 98% sequence identity to the nucleotide sequences defining a naturally occurring LPT or BMS sequences over a comparison window comprising at least about 15 nucleotides.
  • the hybridisation region is about 12 to about 18 nucleobases or greater in length.
  • the percent identity between a particular nucleotide sequence and the reference sequence is at least about 80%, or 85%, or more preferably about 90% similar or greater, such as about 95%, 96%, 97%, 98%, 99% or greater. Percent identities between 80% and 100% are encompassed.
  • the length of the nucleotide sequence is dependent upon its proposed function. For example, short interfering RNAs are generally about 20 to 24 nucleotides in length, whereas molecules designed to provide dominant negative functions may require full length or substantially full length molecules.
  • the term "homolog” or “homologs” refers broadly to functionally and structurally related molecules including those from other species. Homologs and orthologs are examples of variants.
  • the present invention contemplates the use of full length LPT or BMS polypeptides or biologically active portions or stapled peptides of one or more of these molecules as antagonists.
  • Biologically active portions or stapled peptides comprise one or more binding domains.
  • a biologically active portion or stapled peptide of a full length polypeptide can be a polypeptide which is, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350, 400, 450, 500, 550, 600 to about 640 or about 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400 to about 3000 or more amino acid residues in length.
  • Variant polypeptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess at least one biological activity of the native protein.
  • Antagonist variants are selected on the basis that they inhibit or antagonise the biological activity of the LPT or BMS. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native LPT or BMS polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by contemporary sequence alignment programs using default parameters.
  • a biologically active variant of an LPT or BMS polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • LPT or BMS polypeptide/peptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of an LPT or BMS polypeptides can be prepared by introducing mutations in the encoding DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985), Kunkel et al, (Methods in Enzymol., 154: 367-382, 1987), United States Patent No. 4,873,192, Watson et al.
  • Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify useful polypeptide variants (Arkin et al., Proc. Natl. Acad. Sci. USA, 89: 7811-7815, 1992; Delgrave et al, Protein Engineering, 6: 327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable. .
  • Variant LPT or BMS polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to reference amino acid sequences.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic.
  • amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 5.
  • Conservative amino acid substitution also includes groupings based on side chains. Whether an amino acid change results in a functional LPT polypeptide can readily be determined by assaying its activity. Activities that can readily be assessed are known to those of skill and include assays to determine binding or dimerization or oligomerization detected by, for example, nuclear magnetic resonance spectroscopy (NMR) where heteronuclear single quantum coherence (HSQC) spectra are observed, Biacore, kinetic, affinity and pull-down analyses. Conservative substitutions are shown in Table 6 below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
  • analogs of antagonists of LPT or BMS polypeptides have enhanced stability and activity or reduced unfavourable pharmacological properties. They may also be designed in order to have an enhanced ability to cross biological membranes or to interact with only specific substrates. Thus, analogs may retain some functional attributes of the parent molecule but may posses a modified specificity or be able to perform new functions useful in the present context i.e., for administration to a subject.
  • Analogs of peptide or polypeptide agents 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 molecule or their analogs.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid contemplated herein is shown in Table 4.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids and the introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids.
  • c-kit alone is not considered a target encompassed by the present invention as it pertains to modulation of neoplastic luminal progenitor cell levels, although a c-kit antagonist may be used in combination with another LPT antagonist or in conjunction with other therapies.
  • the antibodies and other agents that are effective to down modulate, antagonise or inhibit the activity of an LPT are used in the treatment, including therapy or prophylactic treatment, of cancer.
  • agents will typically be administered in the form of a pharmaceutical composition.
  • the present invention provides a use of an LPT modulator that binds to and modulates the activity of an LPT polypeptide or binds to and modulates the activity of an agent from which an LPT polypeptide is producible or a LPT analog or mimetic in the manufacture of a medicament for the treatment of cancer such as breast cancer, ovarian cancer or a BRCAl -deficiency associated cancer in a subject.
  • the BRCAl -associated cancers include cancers of the reproductive tissue such as fallopian tube carcinoma and primary peritoneal cancer.
  • An increase risk of other cancers such as melanoma, pancreatic cancer and stomach cancer are all associated with BRCA- deficiencies.
  • the present invention provides a use of an LPT antagonist that binds to and down modulates the activity of an LPT polypeptide or binds to and down modulates the activity of an agent from which an LPT polypeptide is producible or a LPT analog or mimetic in the manufacture of a medicament for the treatment of cancer such as breast cancer, ovarian cancer or other BRCAl -deficiency associated cancer in a subject.
  • the agent is an antibody or comprises antigen binding fragment thereof.
  • the medicament is suitable for local or systemic administration by any route, such as without limitation by patch, cellular transfer, implant, orally, intravenously, intravesicaly, intracerebrally, intradermally, intramuscularly, intraperitoneally, intrathecally, subcutaneously, sublingually, rectally, vaginally, intraocularly, nasally, respiratorialy, nasopharyngeal, subcutaneously, cutaneously, topically and transdermally.
  • any route such as without limitation by patch, cellular transfer, implant, orally, intravenously, intravesicaly, intracerebrally, intradermally, intramuscularly, intraperitoneally, intrathecally, subcutaneously, sublingually, rectally, vaginally, intraocularly, nasally, respiratorialy, nasopharyngeal, subcutaneously, cutaneously, topically and transdermally.
  • compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract. See for example, International Patent Publication No. WO 96/11698.
  • the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the actual amount of active agent administered and the rate and time-course of administration will depend on the nature and severity of the burn injury. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes into account the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences ⁇ supra).
  • the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7 ng, 0.8 ng. 0.9 ng, or 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg. 0.9 mg to about 1 to 10 mg or from 5 to 50 mg of LPT modulator or agent may be administered per kilogram of body weight per day or per week.
  • Therapeutic antibodies are typically administered at a dosage of about 1 to 10 mg/kg however dosages above or below this amount are contemplated. 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 agents may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules).
  • the agent or composition comprising the agent 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, sulfate, 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.
  • an effective amount in the context of treating cancer is meant the administration of that amount of active to a subject, either in a single dose or as part of a series or slow release system, that is effective for treatment.
  • the effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual tcf be treated, the formulation of the composition, 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.
  • C-KIT emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA 1 -associated pre-neoplastic tissue and tumors.
  • the data represented here implicate an aberrant luminal progenitor cell as the target cell of transformation in BRCA 1 -associated basal tumors and provide a more targeted approach to diagnosis and treatment.
  • MMTV-Cre (Wagner et al, Nucleic Acids Res 25: 4323-4330, 1997) and Brcal f/f mice (Xu et al, Nat Genet 22: 37-43, 1999) were obtained from the NCI, Frederick. Fl and F2 matings and genotyping was performed as described (Xu et al, 1999 (supra)).
  • nonBRCAl/2 carriers were individuals with a strong family history of breast cancer (kConFab Category 1, where no mutation in BRCAl or BRC A2 has been identified in the family by high sensitivity testing of an individual affected by breast or ovarian cancer. Normal breast samples refer to reduction mammoplasty specimens, where family history is generally not known.
  • Samples from human donors were minced and digested with 75 U/ml collagenase (Sigma) and 25 U/ml hyaluronidase (Sigma) and 100 U/ml DNase (Worthington Biochemical) in DME-HAM supplemented with 5% FCS, 5 ⁇ g/ml insulin, 2 mM glutamine, 10 ng/ml epidermal growth factor and 500 ng/ml hydrocortisone for 5 to 8 h at 37°C.
  • the resulting organoid suspension was sequentially digested with 0.25% trypsin/1 mM EGTA (1 min, 37°C) and 5mg/ml dispase (Roche Diagnostics; 1 min, 37 0 C) with intervening wash steps in PBS containing 2% FCS.
  • a single cell suspension was obtained by filtration through a 40 ⁇ m cell strainer (BD-Falcon) and, where required, red blood cells were removed by lysis.
  • Mouse mammary cell suspensions were prepared as described (Shackleton et al, Nature 439: 84-88, 2006).
  • Sorted cells were injected into cleared inguinal mammary fat pads of 3 - 4 week old NOD-SCID-IL2R ⁇ "A female mice that had been cleared of endogeneous epithelium as described (De Ome et al, Cancer Res 19: 515-520, 1959) with an adaptation of the' 'humanization' method (Kuperwasser et al, Proc Natl Acad Sci USA 101: 4966-4971, 2004).
  • transplantation buffer 50% FCS, 0.04% trypan blue in PBS
  • hTERT-immortalised human mammary stromal fibroblasts Typically, 500,000 fibroblasts comprising a 50:50 mix of unirradiated: irradiated cells (0.3 Gy) were coinjected.
  • Estrogen pellets 0.7 mg
  • Estrogen pellets prepared by mixing estrogen powder (Sigma) with a silicone elastomer (Nusil Silicone Technology) (Laidlaw et al, Endocrinology 136: 164-171, 1995), were implanted subcutaneously at the time of surgery.
  • Recipient mammary fat pads were removed for evaluation 8 - 10 weeks post transplantation.
  • Wholemounts were analysed for outgrowths under a dissecting microscope: structures were excised and processed for haematoxylin and eosin or immunohistochemical staining to evaluate morphology and demonstrate human origin, respectively, as described below.
  • individual transplanted glands from virgin mice were digested with 300 U/ml collagenase and 100 U/ml hyaluroriidase for 45 min. The cell suspension was passed through a 40 ⁇ m strainer and washed with PBS containing 2% FCS.
  • the resulting pellet was resuspended in 60 - 80 ⁇ l PBS containing 50% FCS, 0.04% trypan blue and then injected in a 10 ⁇ l volume into de-epithelialised mammary fat pads.
  • a 0.7 mg estrogen pellet was implanted subcutaneously at the time of surgery.
  • Wholemount analyses were performed 5 - 8 weeks post-transplantation.
  • antibodies for flow cytometry were obtained from BD- Pharmingen.
  • Antibodies against human antigens were: biotin-conjugated anti-CD24 (Stem Cell Technologies), PE-conjugated anti-CD31, PE-conjugated anti-CD45, PE-Cy5- conjugated anti-CD49f, APC-conjugated anti-CD 133 (Myltenyi), PE-conjugated anti- CD235a, FITC-conjugated anti-EpCAM (Stem Cell Technologies) and APC-Cy7- conjugated streptavidin.
  • Antibodies against mouse antigens were: PE-conjugated anti- CD24, FITC-conjugated anti-CD29 (WEHI Monoclonal Antibody Facility), biotin- conjugated anti-CD31, -CD45 and -TER-119, and APC-conjugated anti-CD61.
  • Sorted cells were spun at 700 rpm for 3 minutes onto glass slides using a Cytospin4 Centrifuge (Shandon, Thermo). Cells were then fixed in 4% PFA, 10% NBF or acetone and an antigen retrieval step was carried out in citrate buffer pH 6.0 using a DAKO pressure cooker (125°C, 30 sec). After cooling, the cells were blocked with 10% normal goat serum for 1 hour at room temperature, followed by incubation with the primary antibody for 1 hour at room temperature or overnight at 4°C.
  • Biotinylated secondary antibodies (anti -mouse and anti-rabbit) were incubated for 30 minutes at room temperature, followed by Vectastain RTU ABC reagent (Vector) for 30 minutes at room temperature, then developed with DAB (DAKO) before counterstaining with haematoxylin. Immunohistochemical staining of these sections was carried out as described below.
  • antibodies for immunohistochemical staining were obtained from Novocastra.
  • Antibodies against human antigens were: anti- ALDHl (BD- Pharmingen), anti-c-erbB-2 (DAKO), anti-cytokeratin 5/6 (DAKO), anti-keratin 44, anti-keratin 8/18, anti-keratin 19 (Abeam), anti-EGFR, anti-ER ⁇ , anti-Muc-1 (Stem Cell Technologies), anti-nestin (Abeam), anti-p63 (DAKO), anti-c-KIT (DAKO), anti- progesterone receptor A and anti-vimentin.
  • the media was supplemented, as indicated, with either B27 (Gibco), pituitary extract (Gibco), mifepristone (RU486, Sigma) or prolactin (a gift from A Parlow, National Hormone and Pituitary Program, National Institute of Diabetes, Digestive and Kidney Diseases). Gels were photographed, harvested, then fixed in 4% paraformaldehyde prior to processing for immunostaining.
  • Quantitative RT-PCR was performed using a Rotorgene RG-6000 (Corbett Research, Sydney, Australia) and SensiMix (dT) DNA Kit (Quantace, London, UK), under the following conditions: 10 min at 95°C, followed by 35 cycles consisting of: 15 sec at 95°C, 20 sec at 62°C and 20 sec at 72°C. A melt curve was generated at the end of each run to ensure product quality. From the amplification plot of each sample, a threshold cycle value C 1 was calculated from the exponential phase and a standard curve for each gene was plotted (C t versus log cDNA concentration). Gene expression was determined using the Rotor-Gene software (version 1.7).
  • Primers used for q-RT-PCR are as follows (F, forward; R, reverse): BRCAl F: 5'-GAAGAAACCACCAAGGTCCA-S' (SEQ ID NO: 1) and BRCAl R: 5'-GTTGATCTGTGGGCATGTTG-S' (SEQ ID NO: 2).
  • probe cocktail cRNA @ 0.05 ⁇ g/ ⁇ l
  • GEX-HYB Hybridization Buffer GEX-HYB Hybridization Buffer
  • EpCAM EpCAM + and CD49f 1 ⁇ pCAM + subsets expressed luminal lineage markers including K8/18, Kl 9 and Mucl ( Figure ID, Table 5).
  • the highest proportion of ER- and PgR-expressing cells was observed in the CD49f " EpCAM + subpopulation, indicating that it was enriched for mature luminal cells.
  • K5/6 which is considered to be a basal marker, was expressed in both the basal and luminal subpopulations.
  • the CD49f " EpCAM " cell population comprised stromal fibroblasts that expressed the highest levels of ALDHl, in contrast to the epithelial populations ( Figure • ID, Table 5).
  • CFC colony forming cell
  • Analysis of BRCAl mRNA expression amongst the different subpopulations by quantitative RT- PCR revealed substantially higher levels in the two luminal subsets than the basal stem/progenitor-enriched population, consistent J with a role for BRCAl in luminal progenitor cells (Figure 3E).
  • B27-independent progenitor growth was also observed in 1 out of 6 prophylactic mastectomy samples from BRCA2 mutation carriers (data not shown), and in 3 out of 19 reduction mammoplasties, where minimal family history was available (Figure 3D).
  • the assay may therefore also represent a 'read-out' for perturbed luminal progenitor cells in preneoplastic tissue other than that from BRCA 1 +/" women.
  • microarray profiling was used to derive gene expression signatures representative of the MaSC-enriched (basal), luminal progenitor and mature luminal epithelial populations using freshly sorted cells (>90% purity) from normal breast tissue. Genes characteristic of each population were found by subtraction of genes common to the other epithelial subsets, including both upregulated and downregulated genes (Tables 8, 9). These signature gene sets were then used to interrogate the expression profiles of the six distinct molecular subtypes of human breast cancer described thus far (Herschkowitz et al, Genome Biol 8: R76, 2007).
  • the MaSC-enriched signature genes were generally more concordant with claudin-low and 'normal-like' than the basal subtype.
  • the luminal progenitor cell signature was next compared with the expression profiles of pathologically normal preneoplastic tissue from BRCAl mutation carriers and 'nonBRCAl/2' individuals (refer Methods), as well as normal breast tissue.
  • the luminal progenitor expression signature score was highest in prophylactic BRCA L +/ ⁇ tissue compared to normal breast tissue or nonBRCAl/2 mutation carriers ( Figure 4B).
  • Gene set enrichment tests confirmed that the luminal progenitor signature genes were highly ranked in differential expression comparisons between the BRCA l +/ ⁇ tissues and the other two groups ( Figure 4C).
  • ERa was expressed by a substantial fraction of human luminal progenitor cells. ERa may therefore directly mediate the partial efficacy provided by prophylactic oophorectomy in the prevention of basal breast tumors in BRCAl mutation carriers (Kauff et al, J Clin Oncol 26: 1331-1337, 2008; Kauff et al, N Engl J Med 346: 1609-1615, 2002; Rebbeck et al, N Engl J Med 346: 1616-1622, 2002), compatible with reports suggesting that tamoxifen chemoprophylaxis may be protective (Narod, Oncogene 25: 5832-5836, 2006).
  • Luminal progenitor cells represent a likely cancer-initiating population in BRCA l +/ ⁇ patients and there appear to be fewer functional MaSCs in mouse mammary glands deficient in Brcal (data not shown), suggesting that the luminal progenitor cell is the primary target for transformation in BRCAl*' ' preneoplastic tissue.
  • the luminal progenitor gene signature contains a number of highly expressed genes within 'targetable' functional groups including c-KIT tyrosine kinase and CYP 24 Al, encoding a vitamin D3 metabolising enzyme (Table 10).
  • molecules such as c-KIT provide novel targets for the elimination or modulation of luminal progenitor cells that are the harbinger of BRCAl -associated and other basal-like breast tumors.
  • Microarray profiling was undertaken for four cell subpopulations (identified as MaSC-enriched, Lum Prog, Mat Lum and Stroma) from three patients.
  • Data analysis used the lumi and limma packages of the Bioconductor open-source software project (http://www.bioconductor.org).
  • Raw intensities were normexp background corrected with offset 16 (Ritchie et al, Bioinformatics 23: 2700-2707, 2007), quantile normalized (Bolstad et al, Bioinformatics 19: 185-193, 2003) then Iog 2 -transformed. Probes were filtered if not detected in any sample (detection p- value 0.01).
  • Microarray profiles were available for breast tissue from 15 patients (6 with BRCAl mutations, 5. normal controls, and 4 patients with no BRCAl/2 mutation but with a family history of breast cancer). Normalization and probe filtering was as for the cell population arrays. Sample quality was assessed in three ways using the expression profile data. First, samples were checked for keratin gene expression levels as a marker for epithelium tissue. One BRCAl sample was removed because most keratin genes lacked detectable expression (BeadStudio detection p- value 0.01). Second, the whole-genome distribution of fluorescent intensities was examined. Another BRCAl sample was removed because it showed a very low range of intensities suggesting poor RNA quality.
  • the cell subpopulation microarray data was used to identify a set of signature probes whose expression, or lack of expression, characterizes each of the three cell subpopulations (MaSc-enriched, luminal progenitor and mature luminal).
  • signature probes were defined as those which were significantly differentially expressed in the same direction versus both of the other two cell subpopulations (Table S5).
  • Each signature gene was then associated with an average log- fold change x g as a measure of its discriminatory strength, defined as the average Iog 2 -fold change for that probe versus the other two cell populations.
  • an expression signature score was computed to measure concordance of that sample with each cell subpopulation. Higher scores indicate that the expression signature of the cell subpopulation is found in the breast sample. Expression signature scores are defined as weighted averages,
  • x g is the average log-fold-change for that gene from the cell population data
  • y g is Iog 2 -expression for the same gene in the breast tissue sample.
  • Mean-rank gene set enrichment tests (Michaud (supra) were used to assess the rankings of the signature probes in the various differential expression analyses described above, i.e., between the tumor subtypes for the cancer samples and between the BRCAl mutation and normal groups for the breast tissue.
  • One-sided p-values were evaluated (by Wilcoxon's method) for the mean-rank of each up-regulated or down-regulated signature set under random permutation of probes.
  • Repopulating frequencies were calculated using the limdil webtool.
  • KIT is a luminal progenitor marker
  • KIT human
  • c-Kit mouse
  • KIT is a definitive luminal progenitor cell marker in mammary tissue, raising the possibility that targeting KIT could represent a method to target luminal progenitor cells, which represent a key target population of basal-like tumor development (for both BRCAl -associated and sporadic forms of the disease).
  • KIT inhibitors A number of KIT inhibitors have recently been developed. The majority of these, including Imatinib (Gleevec) have pleiotropic activity as they also inhibit other tyrosine kinase receptors such as c-abl and PDGFR. In addition, they appear to selectively inhibit activated forms of the KIT receptor, with high levels required to inhibit wild-type KIT. Several inhibitors have been developed that appear to effectively target wild-type KIT in human cell-based assays.
  • mice These include masitinib (developed by AB Sciences) (Mitry et al, Cancer Chemother Pharmacol 2010; Humbert et al, PLoS One 5: e9430, 2010; Dubreuil et al, PLoS One 4: e7258, 2009; Hahn et al, J Vet Intern Med 22: 1301-9, 2008) and the Aryl Aminoquinazoline Pyridone compound 25 (developed by Amgen) (Hu et al, J Med Chem 51: 3065-8, 2008).
  • the monoclonal antibody ACK2 appears to selectively inhibit wild-type KIT (Nishikawa et al, EMBOJ lO: 2111-8, 1991).
  • KIT contributes to the proliferative activity of normal luminal progenitor cells in human mammary epithelium.
  • mice A number of human primary breast tumor xenografts in NOD-SCID-IL2R ⁇ nu " mice have been derived that faithfully recapitulate the primary tumor phenotype on serial passage in mice for at least 3 passages.
  • a number of xenografts have been derived from women harbouring basal-like breast tumors as determined by their 'triple-negative' status for estrogen receptor, progesterone receptor and HER2 expression, and expression of EGFR and cytokeratin 5/6.
  • 838T was selected for further study as it was shown to express KIT both immunohistochemistry and flow cytometry (Figure 16).
  • the 838T xenograft model was also selected as optimised dosage schedules for docetaxel, a cytotoxic commonly used to treat breast cancer, in NOD-SCID-IL2R ⁇ nu " mice were available.
  • mice were transplanted with 1 - 3 x 10 5 838T cells (a cell suspension, passage 2) into the right mammary fat pads (cleared of endogenous epithelium) of female NOD-SCID-IL2R ⁇ nu mice. Mice were monitored for tumor development. Once tumors were established (as determined by a tumor volume of -100 - 150 mm 3 ), mice were treated with either vehicle, masitinib (30 mg/kg i.p. daily), docetaxel (5 mg/kg i.p. on day 1), or a combination of masitinib and docetaxel (30 mg/kg i.p. daily and 5 mg/kg i.p. on day 1, respectively).
  • masitinib (30 mg/kg i.p. daily)
  • docetaxel 5 mg/kg i.p. on day 1
  • a combination of masitinib and docetaxel (30 mg/kg i.p. daily and 5 mg/kg i.p. on day 1,
  • masitinib a tyrosine kinase inhibitor that targets c-KIT (both wild-type and constitutively active c-KIT mutants) as well as PDGFR and Lyn and to a lesser extent FGFR3 and the FAK pathway, can potentiate the cytotoxicity of docetaxel in KIT-positive tumors.
  • targeting KIT is an effective treatment or as an adjunct to chemotherapy in the treatment of breast cancer:
  • it is proposed to modulate normal or pre-neoplastic luminal progenitor cell activity, for example, as a prevention strategy against breast cancer development, particularly in high risk women such as BRCAl mutation carriers.
  • Non-conventional amino acid Code " Non-conventional amino acid Code ⁇ -aminobutyric acid Abu L-N-methylalanine Nmala ⁇ -amino- ⁇ -methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- .
  • Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
  • Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid .
  • Brackets refer to the number of different genes represented by the probes.
  • Average fold-change represents the average of the fold-change in expression between the luminal progenitor subset and the other two epithelial subsets (MaSC-enriched and mature luminal).
  • the complete datasets for each population are available via GEO.
  • Table 11 Examples of upregulated genes in the luminal progenitor gene signature from select ontology groups.
  • Average fold-change represents the average of the fold-change in expression between the luminal progenitor subset and the other two epithelial subsets (MaSC-enriched and mature luminal).
  • the complete datasets for each population are available via GEO.
  • NCBI RefSeq accession numbers in the Table are from RefSeq Release 29 (released 4 May 2008).
  • ILMN 2260392 NM 020445.4 7 ACTR3B 1.77 .
  • ILMN 2368530 NM 001012633.1 16 IL32 1.40

Abstract

Cette invention concerne une méthode de stratification des patients atteints d'un cancer consistant à déterminer l'expression d'un groupe de gènes dans un échantillon prélevé chez un sujet. Le groupe est présélectionné parmi des gènes qui sont : (a) exprimés sélectivement ou différentiellement dans des cellules progénitrices épithéliales luminales CD49f+EpCAM+ par rapport à des cellules épithéliales luminales matures ou épithéliales basales (enrichies en MaSC) ; et/ou (b) exprimés sélectivement ou différentiellement dans des cellules progénitrices épithéliales basales CD49fhiEpCAM- par rapport à des cellules épithéliales luminales matures et progénitrices luminales ; et/ou (c) exprimés sélectivement ou différentiellement dans des cellules épithéliales luminales matures CD49f-EpCAM+ par rapport à des cellules épithéliales progénitrices luminales et épithéliales basales ; et/ou (d) exprimés sélectivement ou différentiellement dans des cellules de fibroblastes du stroma CD49f-EpCAM- par rapport à des cellules épithéliales luminales et épithéliales basales. L'invention concerne également une méthode de diagnostic, de pronostic et de traitement du cancer du sein de phénotype basal et/ou une forme de cancer en rapport avec des anomalies du gène BRCA1, ladite méthode consistant à (i) déterminer l'expression d'un groupe de gènes dans un échantillon où le groupe de gènes est choisi de manière à englober les gènes qui sont exprimés sélectivement ou différentiellement dans des cellules progénitrices luminales épithéliales CD49f+EpCAM+ par rapport à leur expression par des cellules témoins, par exemple des cellules épithéliales basales ou des cellules épithéliales luminales matures. Ces groupes de gènes sont indiqués dans les tableaux 10 à 17. Les gènes préférés sont ceux dotés de l'activité c-Kit. Les méthodes de traitement ou de prévention consistent à administrer des agents modulant l'activité des produits d'expression des groupes de gènes, par exemple le polypeptide c-Kit. L'invention concerne également des sondes et des kits de diagnostic. Des méthodes de réduction de la prolifération des cellules progénitrices luminales néoplasiques ou prénéoplasiques ou normales dans l'épithélium chez l'homme sont décrites et consistent à administrer un agent qui régule à la baisse l'activité c-Kit.
PCT/AU2010/000524 2009-05-06 2010-05-06 Profils d'expression génétique et leurs utilisations WO2010127399A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2009901989 2009-05-06
AU2009901989A AU2009901989A0 (en) 2009-05-06 Gene expression profiles and uses therefor

Publications (1)

Publication Number Publication Date
WO2010127399A1 true WO2010127399A1 (fr) 2010-11-11

Family

ID=43049846

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2010/000524 WO2010127399A1 (fr) 2009-05-06 2010-05-06 Profils d'expression génétique et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2010127399A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013002490A1 (fr) * 2011-06-30 2013-01-03 충남대학교 산학협력단 Biomarqueur pour le diagnostic du cancer du sein
US20140141996A1 (en) * 2011-06-22 2014-05-22 Oncocyte Corporation Methods and Compositions for the Treatment and Diagnosis of Cancer
WO2014081313A1 (fr) * 2012-11-20 2014-05-30 University Of Tromsoe Profil d'expression génique dans des diagnostics
JP2014532428A (ja) * 2011-11-08 2014-12-08 ジェノミック ヘルス， インコーポレイテッド 乳癌の予後を予測する方法
US20140377779A1 (en) * 2013-06-25 2014-12-25 Samsung Electronics Co., Ltd. Composition for diagnosing breast cancer including material specifically binding to polymeric immunoglobulin receptor protein or fragment thereof, and method of diagnosing breast cancer by using the composition
EP2780472A4 (fr) * 2011-11-15 2015-10-28 Oncocyte Corp Procédés et compositions pour le traitement et le diagnostic du cancer de la vessie
EP3006560A1 (fr) * 2014-10-09 2016-04-13 European Molecular Biology Laboratory Miwi2 comme marqueur pour des cellules souches spermatogoniales et procédé de diagnostic de cellules souches spermatogoniales basé sur ce marqueur
US9562906B2 (en) 2010-06-10 2017-02-07 National University Of Singapore Methods for detection of gastric cancer
US10174289B2 (en) 2014-05-28 2019-01-08 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer
CN111175484A (zh) * 2018-09-26 2020-05-19 欧蒙医学实验诊断股份公司 神经自身免疫性疾病的诊断
US10781425B2 (en) 2010-05-06 2020-09-22 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
WO2020205627A1 (fr) * 2019-03-29 2020-10-08 Rain Therapeutics Inc. Utilisation de biomarqueur dans la thérapie du cancer
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
CN114127314A (zh) * 2019-07-19 2022-03-01 公立大学法人福岛县立医科大学 用于对乳腺癌的亚型(Subtype)进行鉴别或者分类的鉴别标志物遗传基因组、方法和套件
WO2022216846A1 (fr) * 2021-04-06 2022-10-13 Berg Llc Marqueurs protéiques pour le cancer du sein du type positif aux récepteurs des oestrogènes (er) et du type négatif aux récepteurs des oestrogènes (er)
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
RU2795720C1 (ru) * 2023-02-07 2023-05-11 Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") Способ прогнозирования риска развития люминального подтипа рака молочной железы
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same
CN117607443A (zh) * 2024-01-23 2024-02-27 杭州华得森生物技术有限公司 用于诊断乳腺癌的生物标志物组合

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GE, X. ET AL.: "Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues.", GENOMICS, vol. 86, no. 2, August 2005 (2005-08-01), pages 127 - 141 *
HERSCHKOWITZ, J. ET AL.: "Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors.", GENOME, BIOL., vol. 8, no. 5, May 2007 (2007-05-01), pages R76.1 - R76.17 *
LIM, E. ET AL.: "Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers.", NAT. MED., vol. 15, no. 8, August 2009 (2009-08-01), pages 907 - 913 *
PARK, J.-S. ET AL.: "Gene expression analysis in SV40-immortalized human breast luminal epithelial cells with stem cell characteristics using a cDNA microarray.", INT. J. ONCOL., vol. 24, no. 6, June 2004 (2004-06-01), pages 1545 - 1558 *
SORLIE, T. ET AL.: "Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.", PNAS., vol. 98, no. 19, September 2001 (2001-09-01), pages 10869 - 10874 *
SORLIE, T. ET AL.: "Repeated observation of breast tumor subtypes in independent gene expression data sets.", PNAS., vol. 100, no. 14, July 2003 (2003-07-01), pages 8418 - 8423 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10781425B2 (en) 2010-05-06 2020-09-22 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
US9562906B2 (en) 2010-06-10 2017-02-07 National University Of Singapore Methods for detection of gastric cancer
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer
US20140141996A1 (en) * 2011-06-22 2014-05-22 Oncocyte Corporation Methods and Compositions for the Treatment and Diagnosis of Cancer
WO2013002490A1 (fr) * 2011-06-30 2013-01-03 충남대학교 산학협력단 Biomarqueur pour le diagnostic du cancer du sein
JP2014532428A (ja) * 2011-11-08 2014-12-08 ジェノミック ヘルス， インコーポレイテッド 乳癌の予後を予測する方法
EP2780472A4 (fr) * 2011-11-15 2015-10-28 Oncocyte Corp Procédés et compositions pour le traitement et le diagnostic du cancer de la vessie
AU2012340393B2 (en) * 2011-11-15 2018-06-14 Oncocyte Corporation Methods and compositions for the treatment and diagnosis of bladder cancer
WO2014081313A1 (fr) * 2012-11-20 2014-05-30 University Of Tromsoe Profil d'expression génique dans des diagnostics
US9213029B2 (en) * 2013-06-25 2015-12-15 Samsung Electronics Co., Ltd. Method for diagnosing breast cancer by detection of polymeric immunoglobulin receptor in vesicles isolated from patients
US20140377779A1 (en) * 2013-06-25 2014-12-25 Samsung Electronics Co., Ltd. Composition for diagnosing breast cancer including material specifically binding to polymeric immunoglobulin receptor protein or fragment thereof, and method of diagnosing breast cancer by using the composition
US10174289B2 (en) 2014-05-28 2019-01-08 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11053477B2 (en) 2014-05-28 2021-07-06 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
EP3006560A1 (fr) * 2014-10-09 2016-04-13 European Molecular Biology Laboratory Miwi2 comme marqueur pour des cellules souches spermatogoniales et procédé de diagnostic de cellules souches spermatogoniales basé sur ce marqueur
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same
CN111175484A (zh) * 2018-09-26 2020-05-19 欧蒙医学实验诊断股份公司 神经自身免疫性疾病的诊断
CN111175484B (zh) * 2018-09-26 2024-04-16 欧蒙医学实验诊断股份公司 神经自身免疫性疾病的诊断
WO2020205627A1 (fr) * 2019-03-29 2020-10-08 Rain Therapeutics Inc. Utilisation de biomarqueur dans la thérapie du cancer
CN114127314A (zh) * 2019-07-19 2022-03-01 公立大学法人福岛县立医科大学 用于对乳腺癌的亚型(Subtype)进行鉴别或者分类的鉴别标志物遗传基因组、方法和套件
EP4001431A4 (fr) * 2019-07-19 2023-09-27 Public University Corporation Fukushima Medical University Ensemble de gènes marqueurs de distinction, procédé et kit permettant chacun de distinguer ou de classer un sous-type de cancer du sein
WO2022216846A1 (fr) * 2021-04-06 2022-10-13 Berg Llc Marqueurs protéiques pour le cancer du sein du type positif aux récepteurs des oestrogènes (er) et du type négatif aux récepteurs des oestrogènes (er)
RU2795720C1 (ru) * 2023-02-07 2023-05-11 Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") Способ прогнозирования риска развития люминального подтипа рака молочной железы
CN117607443A (zh) * 2024-01-23 2024-02-27 杭州华得森生物技术有限公司 用于诊断乳腺癌的生物标志物组合
CN117607443B (zh) * 2024-01-23 2024-04-16 杭州华得森生物技术有限公司 用于诊断乳腺癌的生物标志物组合

Similar Documents

Publication Publication Date Title
WO2010127399A1 (fr) Profils d'expression génétique et leurs utilisations
US10744116B2 (en) Detection and treatment of anti-PD-1 therapy resistant metastatic melanomas
Kohashi et al. Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor
Bianchini et al. Comparative study of gene expression by cDNA microarray in human colorectal cancer tissues and normal mucosa
US8211634B2 (en) Compositions, kits, and methods for identification, assessment, prevention, and therapy of cancer
US8168602B2 (en) Compositions, kits and methods for identification, assessment, prevention and therapy of cancer
Colombo et al. Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma
Laurent et al. Patient-derived xenografts recapitulate molecular features of human uveal melanomas
Dong et al. LncRNA COL1A1-014 is involved in the progression of gastric cancer via regulating CXCL12-CXCR4 axis
Pan et al. Role of L2DTL, cell Cycle-Regulated nuclear and centrosome protein, in aggressive HepatocellularCarcinoma
CA2568413A1 (fr) Compositions, kits et procedes pour identifier, evaluer, prevenir et traiter un cancer
US20190367964A1 (en) Dissociation of human tumor to single cell suspension followed by biological analysis
Mosbeh et al. Nuclear BAP1 loss is common in intrahepatic cholangiocarcinoma and a subtype of hepatocellular carcinoma but rare in pancreatic ductal adenocarcinoma
US20100298158A1 (en) Compositions, Kits, and Methods for Identification, Assessment, Prevention, and Therapy of Cancer
Chawla et al. An intestinal stem cell niche in Apc mutated neoplasia targetable by CtBP inhibition
CN111454948A (zh) LncRNA EPB41L4A-AS1作为诊断和治疗急性髓系白血病标志物的应用
KR20110015013A (ko) 결장직장암의 평가 방법 및 여기에 사용하기 위한 조성물
Fei et al. CircKIF5B promotes hepatocellular carcinoma progression by regulating the miR-192 Family/XIAP axis
EP2557159B1 (fr) Méthode de pronostic d'adénocarcinome pulmonaire, kit de détection d'adénocarcinome pulmonaire et composition pharmaceutique pour le traitement d'un adénocarcinome pulmonaire
Faumont et al. C-rel is the pivotal NF-κB subunit in germinal center diffuse Large b-cell lymphoma: a LYSA study
Dong et al. The role and gene expression profile of SOCS3 in colorectal carcinoma
CN117286251A (zh) 生物标志物nedd4在小细胞肺癌中的应用
EP2419532A1 (fr) Procédés et outils pour prédire l'efficacité d'anthracyclines dans un cancer
WO2010119133A1 (fr) Procédés et outils pour prédire l'efficacité d'anthracyclines dans un cancer
Takahashi et al. Expression of adhesion molecules and epithelial-mesenchymal transition factors in medullary carcinoma of the colorectum

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10771901

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10771901

Country of ref document: EP

Kind code of ref document: A1