WO2005100593A1 - Procede destine a reguler la progression d'un cancer - Google Patents

Procede destine a reguler la progression d'un cancer Download PDF

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Publication number
WO2005100593A1
WO2005100593A1 PCT/AU2005/000542 AU2005000542W WO2005100593A1 WO 2005100593 A1 WO2005100593 A1 WO 2005100593A1 AU 2005000542 W AU2005000542 W AU 2005000542W WO 2005100593 A1 WO2005100593 A1 WO 2005100593A1
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Prior art keywords
activin
neoplasm
inhibin
follistatin
level
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PCT/AU2005/000542
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English (en)
Inventor
Gail Petuna Risbridger
Sally Louise Mellor
Emma Margaret Anne Ball
Hong Wang
John Stuart Pedersen
Catriona Mclean
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Monash University
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Priority to AU2005233212A priority Critical patent/AU2005233212A1/en
Priority to JP2007507627A priority patent/JP2007532889A/ja
Priority to US11/578,711 priority patent/US20080038729A1/en
Priority to EP05731618A priority patent/EP1756301A4/fr
Priority to CA002562835A priority patent/CA2562835A1/fr
Publication of WO2005100593A1 publication Critical patent/WO2005100593A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • 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 present invention relates generally to a method of diagnosing, predicting or monitoring the development or progress of a cancer and, in particular, to a method of diagnosing, predicting or monitoring the development or progress of prostate cancer in a mammal.
  • the present invention more specifically provides a method for delineating early stage and advanced stage cancers, or predispositions thereto, by screening for changes in the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin expression in a mammal.
  • the present invention further provides a method for diagnosing or monitoring conditions associated with or characterized by the onset of a cancer.
  • a neoplasm is an abnormal mass or colony of cells produced by a relatively autonomous new growth of tissue. Most neoplasms arise from the clonal expansion of a single cell that has undergone neoplastic transformation. The transformation of a normal cell to a neoplastic cell can be caused by a chemical, physical, or biological agent (or event) that alters the cell genome.
  • Neoplastic cells are characterized by the loss of some specialized functions and the acquisition of new biological properties, foremost, the property of relatively autonomous growth. They pass on their heritable biological characteristics to progeny cells. Neoplasms may originate in almost any tissue containing cells capable of mitotic division.
  • a malignant neoplasm manifests a greater degree of autonomy, is capable of invasion and metastatic spread, may be resistant to treatment, and may cause death.
  • a benign neoplasm exhibits a lesser deg-ree of autonomy, is usually not invasive and does not metastasize.
  • Cancer is a generic term which generally denotes malignant neoplasms. This is a disease which occurs worldwide and is second only to heart disease as the most common cause of death in western countries. The estimated incidence of cancer in the XJS, for example, is about 1 x 10 6 new cases annually. Nearly 80% of all malignant neoplasms arise in 10 anatomical sites, namely: lung, breast, colon and rectum, prostate, lymph nodes, uterus, bladder, pancreas, blood and stomach.
  • Prostate cancer is a disease that occurs in men mostly over the age o 50. It can occur in younger men but this is rare. Figures suggest that approximately one in four males above the age of 55 will suffer from a prostate disease of some form. The incidence in Australia ofprostatic cancer is high and similarly prevalent rates occur in most countries. Globally, prostate cancer now represents the third highest incidence of cancer a ter lung cancer (due largely to smoking) and stomach cancer. This represents a significant cost to health care systems and decreases the quality of life of men suffering from this disorder.
  • Ttiis may be partly due to a 'real' increased risk, but is certainly related to the increased likeli-hood of detection, via PSA tests, and the increased number of TU Ps operations.
  • a TURP is done when prostate tissue is removed to improve symptoms of a benign prostate condition. However, in doing so, subsequent pathology sometimes indicates the existence of cancer. Whether there is a real increase in risk or not, the numbers of cases of prostate cancer will rise due to the population at risk - older men - growing with the lengthening of life expectancy.
  • prostate cancer Although the causes of prostate cancer are not fully understood, men with a family history of prostate cancer in a first degree relative have two to three times the risk of developing the disease, indicating a role for genetic predisposition. However, the maj ority of prostate cancers are sporadic and unrelated to family history.
  • prostate cancer is usually a treatable disease. However, about half the men who are diagnosed with prostate cancer are unfortunately diagnosed at a late stage when the disease is less treatable. In this regard, early stage prostate cancer is generally localised to the prostate. Advanced prostate cancer, although having originated in the prostate, has generally spread beyond the prostate to other parts of the body signals a significantly less hopeful prognosis.
  • Prostate cancer pathologies are graded with a Gleason grading from 1 to 5 in order of increasing malignancy. Cribriform pathological patterns are sometimes observed at the stage of Gleason Grade 3 and 4. Cribriform pathologies are associated with prostate cancer progression and poor patient outcome.
  • Activins composed of two ⁇ -subunits, ⁇ A and/or ⁇ , and their antagonists the inhibins (combinations of an ⁇ - and either of the two ⁇ -subunits) are members of the transforming growth factor (TGF)- ⁇ superfamily [Vale et al. (1990) In Peptide growth factors and their receptors: Handbook of Experimental Physiology, Vol. 95 (Eds, Sporn, M. and Roberts, A.) Springer-Nerlag, Berlin, pp. 211-248].
  • TGF transforming growth factor
  • Activins regulate cell growth or differentiation by binding activin receptors and initiating a signaling cascade [Pangas et al. (2000), Trends Endocrinol Metab, 11 , 309-314] . Changes in expression of inhibin/activin subunits, activin receptors, or the activin-binding proteins follistatin (FS315 and FS288) have been shown to influence growth of a variety of types of cells. Tumor suppressor activity of inhibin- ⁇ in the gonads and adrenals has been recorded in transgenic mice bearing a targeted deletion of the inhibin- ⁇ subunit [Matzuk et al. (1992), Nature, 360, 313-9; Matzuk et al.
  • Inhibin- ⁇ and activin- ⁇ subunits are synthesized in the human prostate.
  • Activin ⁇ A- and ⁇ -subunits are also localized to the epithelium of benign tissues and poorly differentiated adenocarcinomas of the prostate [Thomas et al. (1997), J Clin Endocrinol Metab, 82, 3851-9; Thomas et al. (1998), Prostate, 34, 34-43]. Follistatin expression is noted in both benign epithelium and poorly differentiated cancer. Antibodies, raised to different isoforms, show distinct labelling patterns in malignant versus benign epithelia suggesting differential production by these prostatic compartments [Thomas 1997 TGFb, supra] .
  • One aspect of the present invention is directed to a method of detecting the onset of a neoplasm or a predisposition to developing a neoplasm in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage neoplasm or a predisposition thereto and an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an
  • Another aspect of the present invention provides a method of detecting the onset of a malignant neoplasm of the breast, ovary, thyroid, testis or adrenal gland or a predisposition to developing a malignant neoplasm of the breast, ovary, thyroid, testis or adrenal gland in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin, protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage malignant neoplasm or a predisposition thereto and an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin-
  • the present invention provides a method of detecting the onset of a malignant neoplasm of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or a predisposition to developing an advanced malignant neoplasm of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin-
  • the present invention provides a method of detecting the onset of a malignant neoplasm of the cervix, brain, skin, lymph node, lung, salivary gland, liver, gallbladder or pancreas or a predisposition to developing an advanced malignant neoplasm of the cervix, brain, skin, lymph node, lung, salivary gland, liver, gallbladder or pancreas in a mammal said method comprising screening for the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage malignant neoplasm
  • a further aspect of the present invention provides a method of detecting the onset of a prostate malignant neoplasm or a predisposition to developing a prostate malignant neoplasm in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , actrvin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage prostate malignant neoplasm or a predisposition thereto and an increase in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ s, activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or
  • Another further aspect of the present invention provides a method of detecting the onset of an early stage cancer of the breast, ovary, thyroid, testis or adrenal gland or a predisposition to developing an early stage cancer of the breast, ovary, thyroid, testis oorr adrenal gland in a mammal said method comprising screening for the level of two oorr more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin pprr ⁇ otein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said early stage cancer or a predisposition thereto.
  • the present invention provides a method of detecting the onset of an early stage cancer of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or a predisposition to developing an early stage cancer of oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/
  • the present invention provides a method of detecting the onset of an early stage cancer of the cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas or a predisposition to developing an early stage cancer of cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said early stage cancer or a predisposition.
  • the present invention provides a method of detecting the onset of an early stage prostate cancer or a predisposition to developing an early stage prostate cancer in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage prostate cancer or a predisposition thereto.
  • the present invention provides a method of detecting the onset of an advanced stage cancer of the breast, ovary, thyroid, testis or adrenal gland or a predisposition to developing an advanced stage cancer of the breast, ovary, thyroid, testis or adrenal gland in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ B , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said advanced stage cancer or a predisposition thereto.
  • the present invention provides a method of detecting the onset of an advanced stage cancer of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or a predisposition to developing an advanced stage cancer of oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and
  • the present invention provides a method of detecting the onset of an advanced stage cancer of the cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas or a predisposition to developing an advanced stage cancer of the cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said advanced stage cancer or a predisposition thereto.
  • the present invention provides a method of detecting the onset of an advanced stage prostate cancer or a predisposition to developing an advanced stage prostate cancer in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an advanced stage prostate cancer or a predisposition thereto.
  • the present invention provides a method of detecting the onset of metastatic prostate cancer or a predisposition to developing metastatic prostate cancer in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of metastatic prostate cancer or a predisposition to developing metastatic prostate cancer.
  • Another aspect of the present invention is directed to a method of monitoring for the onset or progression of a neoplasm in a mammal said method comprising screening for the modulation in the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin in said mammal wherein the level of said inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin relative to the normal level of inhibin is indicative of the onset or progression of said neoplasm.
  • Still another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein or encoding nucleic acid molecule and reagents useful for facilitating the detection by the agent in the first compartment. Further means may also be included, for example, to receive a biological sample.
  • Figure 1 is a graphical representation of inhibin- ⁇ , activin- ⁇ c, follistatin 315 and activin- ⁇ A expression in Gleason grade 4 or 5 prostate cancers.
  • Figure 2 shows the difference in inhibin-ot, activin- ⁇ c, follistatin 315 and activin- ⁇ A expression in cribriform prostate cancers compared to benign epithelium.
  • the horizontal bar represents the mean of each immunostaining score in either benign epithelium or cribriform cancer.
  • Figure 3 shows the effect on proportion of LNCaP cells in S-phase when activin- ⁇ c or inhibin- ⁇ is overexpressed.
  • FACS analysis was performed on LNCaP cells transiently transfected with activin- ⁇ c cDNA and LNCaP cells stably transfected with inhibin- ⁇ cDNA. In both cases, controls consisted of expression vector lacking the cDNA insert.
  • LTSTCaP cells overexpressing either activin- ⁇ c or inhib ⁇ n- ⁇ black bars
  • Figure 4 shows the effect on proportion of PC3 cells in S-phase when activin- ⁇ c or in-hibin- ⁇ is overexpressed. FACS analysis was preformed on PC3 cells transiently transfected with activin- ⁇ c cDNA and PC3 cells stably transfected with inhibin- ⁇ cDNA. In both cases, controls consisted of expression vector lacking the cDNA insert.
  • PC3 cells overexpressing either activin- ⁇ c or inhibin-oc demonstrated a significant increase in proportion of cells in S-phase compared to PC3 cells not expressing activin- ⁇ c or inhibin- ⁇ (p ⁇ 0.05; grey bars).
  • Figure 5 shows the detection of expression of activin- ⁇ c (and/or activin- ⁇ E ) [panel A], follistatin 315 [panel B] and activin- ⁇ A [panel C] in tissue from patients with prostate cancer metastases to the lymph node. Immunostaining for activin- ⁇ c, follistatin and activin- ⁇ A was localised to prostate cancer tumour cells with no staining observed in mouse IgG negative control [panel D].
  • Figure 6 is a graphical representation of activin- ⁇ c and follistatin 315 expression in metastatic prostate cancer compared to benign prostat ⁇ c secretory epithelium.
  • Figure 7 is a graphical representation of inhibin- ⁇ , activin- ⁇ c, follistatin 315 and activin- ⁇ .
  • Immunostaining for activin- ⁇ c, follistatin 315 and activin- ⁇ A significantly increased in Gleason grade 4 or 5 prostate cancer compared to Gleason grade 3 prostate cancer.
  • Figure 8 shows the difference in inhibin- ⁇ , activin- ⁇ c, follistatin 315 and activin- ⁇ A expression in cribriform prostate cancers compared to Gleason grade 3 prostate cancer.
  • Figure 9 is a graphical representation of the effect on monolayer wound healing when inhibin ⁇ is overexpressed in LNCaP cells.
  • Monolayers were wounded by scraping an approximately 1mm thick line along the middle of the wells. Measurements of the width of the wound were taken over time and expressed as a percentage of wound closure, where time 0 represents 100% wound.
  • the data from inhibin ⁇ transfected and empty vector transfected clones are pooled in the graph.
  • the symbols represent: closed grey, parental cell line; open grey, empty vector transfected clones, closed black, inhibin ⁇ transfected clones.
  • LNCaP cells overexpressing inhibin ⁇ black
  • Figure 10 is a graphical representation of the effect on subcutaneous tumor growth when inhibin ⁇ is overexpressed in LNCaP cells.
  • Male SCID mice were inoculated under the skin with parental LNCaP, three empty vector transfected clones and three inhibin ⁇ transfected clones.
  • Figure 11 is a graphical representation of the effect on intraprostatic tumor growth when inhibin ⁇ is overexpressed in LNCaP cells.
  • Male SCID mice prostates were inoculated with parental LNCaP, three empty vector transfected clones and three inhibin ⁇ transfected clones. The prostate tumors formed after orthotopical injections of all the cells.
  • the present invention is predicated, in part, on the surprising determination that analysis of the levels of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin provide a significantly more sensitive and accurate means of detecting and/or assessing a cancer and, even more particularly, determining whether the identified cancer corresponds to an early stage cancer or a late stage/advanced cancer, than any one of these markers alone.
  • one aspect of the present invention is directed to a method of detecting the onset of a neoplasm or a predisposition to developing a neoplasm in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage neoplasm or a predisposition thereto and an increase in the level of inhibin- ⁇ , activin- ⁇ A , actrvin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the
  • said method is directed to detecting two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ c or follistatin.
  • Reference to a "neoplasm” should be understood as a reference to an encapsulated or unencapsulated growth of neoplastic cells.
  • Reference to a “neoplastic cell” should be understood as a reference to a cell exhibiting abnormal growth.
  • the term “growth” should be understood in its broadest sense and includes reference to proliferation.
  • abnormal growth in this context is intended as a reference to cell growth which, relative to normal cell growth, exhibits one or more of an increase in the rate of cell division, an increase in the number of cell divisions, an increase in the length of the period of cell division, an increase in the frequency of periods of cell division or uncontrolled proliferation.
  • the common medical meaning of the term “neoplasia” refers to "new cell growth” that results as a loss of responsiveness to normal growth controls, eg. to neoplastic cell growth. Neoplasias include “tumours” which may be either benign, pre-malignant or malignant.
  • the term “neoplasm” should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.
  • neoplasm in the context of the present invention should be understood to include reference to all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of histopathologic type or state of invasiveness, where the tissue in issue expresses inhibin, activin subunit or follistatin either constitutively or subsequently to an appropriate stimulus.
  • the neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell.
  • the present invention is preferably directed to the diagnosis of malignant neoplasms, the diagnosis and/or monitoring of non-malignant neoplasms is not excluded.
  • the subject neoplasm is a neoplasm of the prostate, ovary, skin, breast, lymph node, lung, salivary gland, liver, gallbladder, pancreas, oesophagus, stomach, colon, rectum, kidney, bladder, endometrium, cervix, adrenal gland, thyroid, brain, small intestine, large intestine, larynx, nasal cavity, throat cancer, neural tumours or testis and even more preferably a malignant neoplasm.
  • the present invention provides a method of detecting the onset of a malignant neoplasm of the breast, ovary, thyroid, testis or adrenal gland or a predisposition to developing a malignant neoplasm of the breast, ovary, thyroid, testis or adrenal gland in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin, protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage malignant neoplasm or a predisposition thereto and an increase in the level of inhibin-c , activin- ⁇ A , activin- ⁇
  • said neoplasm is a neoplasm o-f the breast.
  • said neoplasm is a neoplasm of the ovary.
  • the present invention provides a met-hod of detecting the onset of a malignant neoplasm of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or a predisposition to developing an advanced malignant neoplasm of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal whierein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇
  • the present invention provides a method of detecting the onset of a malignant neoplasm of the cervix, brain, skin, lymph node, lung, salivary gland, liver, gallbladder or pancreas or a predisposition to developing an advanced malignant neoplasm of the cervix, brain, skin, lymph node, lung, salivary gland, liver, gallbladder or pancreas in a mammal said method comprising screening for the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage malignant neoplasm or
  • the present invention particularly provides a method of detecting the onset of a prostate malignant neoplasm or a predisposition to developing a prostate malignant neoplasm in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of ii hibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage malignant neoplasm or a predisposition thereto and an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , actrvin- ⁇ c, activin- ⁇ o, activin- ⁇ or follistatin protein and/or gene expression
  • the present invention is preferably directed to detecting two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ c or follistatin. More preferably, the present invention is directed to detecting any three of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ c or follistatin and still more preferably, all four of inhibin- ⁇ 3 activin- ⁇ A, activin- ⁇ c or follistatin.
  • inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c;, activin- ⁇ o, activin- ⁇ E and follistatin, and in particular inhibin- ⁇ , activin- ⁇ A , activin- ⁇ c and follistatin, are linked to the regulation of cellular proliferation, in the context of a switching mechanism.
  • cancers are generally multi-step processes involving an initial transition from non-malignant to malignant status and, following a shift to pre-malignant lesions and localized cancer, the shift to metastasis. In the non-malignant state tfcie activities of tumor suppressor molecules are thought to dominate.
  • neoplasm being an "advanced stage" neoplasm or cancer should be understood as a reference to a high grade cancer, which high grade cancer may or may not have metastasised or otherwise spread beyond the organ or tissue in which it has originated.
  • metastases can generally form via distribution of the neoplastic cells through the bloodstream or the lymphatic channels or across body cavities such as the pleural or peritoneal spaces, thus setting up secondary tumours at sites distant from the original tumours. Any individual primary tumour will exhibit its own pattern of local behaviour and metastases.
  • reference to an "advanced stage" neoplasm or cancer encompasses any level or degree of spreading of the neoplastic cells beyond the organ or tissue in which it originated, whether that be relatively localised spreading in the immediate vicinity of the organ or tissue in issue or the more significant spreading of the neoplastic cells to other regions of the body, which accords with the more commonly understood notion of "metastatic” cancer. It also encompasses the form of cancer which is characterised by the development of metastases subsequently to removal of the organ or tissue in which the cancer originated. Accordingly, for example, "advanced" prostate cancer may develop, or a predisposition to development may be found to exist, subsequently to removal of the prostate.
  • Reference to an "early stage” neoplasm or cancer is a reference to a low grade, non- metastatic neoplasm.
  • the method of the present invention is preferably directed to detecting and classifying early stage versus advanced stage prostate cancers.
  • a neoplasm "grade” (as it applies to the definition of "early stage” and “late stage") in the context of prostate cancer should be understood as a reference to the classification of the neoplasm in- accordance with the Gleason grading system.
  • the Gleason system is based on the architectural pattern of the glands of the prostate tumour. It evaluates how effectively the cells of any particular cancer are able to structure themselves into glands resembling those of the normal prostate.
  • the ability of a tumour to mimic normal gland architecture is termed its differentiation, and, in general, a tumour whose structure is nearly normal (well differentiated) generally exhibits biological behaviour closer to normal, that is, is not aggressively malignant.
  • the Gleason grading from very well differentiated (grade 1) to very poorly differentiated (grade 5) is usually assessed as follows:
  • Gleason grade 4 is characterized by a significant loss of architecture, specifically loss of the normal gland unit.
  • grade 4 is generally identified by loss of the ability to form individual, separate gland units, each with its separate lumen.
  • Gleason grade 5 usually predicts another significant step towards poor prognosis. This grade is also characterised by lack of evidence of any gland unit formation. Grade 5 is generally termed “undifferentiated”, due to its features not being significantly distinguishing from undifferentiated cancers which occur in other organs.
  • each patient is also given a Gleason score.
  • the Gleason score is based on the summation of the grades of the two most common architectural patterns in a tissue sample. This provides a slightly more refined means of classifying the neoplasm of a given patient. For example, the lowest possible Gleason score is 2 (1+1), where both the primary and secondary patterns exhibit a Gleason grade of 1. Very typical Gleason scores might be 5 (2+3), where the primary pattern has a Gleason grade of 2 and the secondary patterns has a grade of 3, or 6 (3+3), a pure pattern.
  • Another typical Gleason score might be 7 (4+3), where the primary pattern has a Gleason grade of 4 and the secondary pattern has a grade of 3. Finally, the highest possible Gleason score is 10 (5+5), when the primary and secondary patterns both have the most disordered Gleason grades of 5.
  • the method of the present invention provides a highly sensitive or accurate means of detecting the onset or predisposition to the onset of a neoplastic condition and, in particular, the delineation of early stage cancers and advanced stage cancers.
  • early stage prostate cancer should be understood to encompass a neoplasm comprising cells of Grade 1 or 2 or equivalent grade thereof, while advanced stage prostate cancer should be understood to encompass a neoplasm comprising cells of Gleason grade 4 or 5, or equivalent grade thereof.
  • any given prostate cancer may comprise cells of various grades
  • the present invention is directed to screening those cells which fall within the ambit of "early stage” or “advanced stage” cancer, irrespective of what the overall Gleason score may suggest about the classification of a prostate cancer.
  • a prostate cancer comprising Gleason grade 4 and 2 cells correlates to a Gleason score of 6, this being a score which is equated with a "moderate” grade cancer.
  • the present invention can be designed to analyse subgroups of cells if necessary, for example via analysis of tissue sections, for the purpose of the present invention it is not the overall grade of the neoplasm which is of relevance but the grade of the cells which are the subject of enquiry.
  • the present invention may be directed to analysing the grade 4 component of a neoplasm.
  • the advanced stage neoplasms as defined herein should be understood to correlate to neoplasms which are also routinely termed “advanced cancers", “aggressive cancers” and “metastatic cancers", although not all advanced cancers are necessarily metastatic cancers.
  • reference to an "advanced” cancer, in the context of prostate cancer encompasses any level or degree of spreading of the neoplastic cells beyond the prostate, whether that be relatively localised spreading in the immediate vicinity of the prostate or the more significant spreading of the neoplastic cells to other regions of the body, which accords with the more commonly understood notion of "metastatic” cancer.
  • it also encompasses the form of cancer which is characterised by the development of prostate derived metastases subsequently to removal of the prostate. Accordingly, "advanced" prostate cancer may develop subsequently to removal of the prostate.
  • the present invention provides a method of detecting the onset of an early stage cancer of the breast, ovary, thyroid, testis or adrenal gland or a predisposition to developing an early stage cancer of the breast, ovary, thyroid, testis or adrenal gland in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said early stage cancer or a predisposition thereto.
  • said neoplasm is a neoplasm of the breast.
  • said neoplasm is a neoplasm of the ovary.
  • the present invention provides a method of detecting the onset of an early stage cancer of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or a predisposition to developing an early stage cancer of oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and
  • the present invention provides a method of detecting the onset of an early stage cancer of the cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas or a predisposition to developing an early stage cancer of cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said early stage cancer or a predisposition.
  • the present invention provides a method of detecting the onset of an early stage prostate cancer or a predisposition to developing an early stage prostate cancer in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein a decrease in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an early stage prostate cancer or a predisposition thereto.
  • said markers are preferably inhibin- ⁇ , activin- ⁇ A , activin- ⁇ c or follistatin and said screening is directed to any 3 or all 4 of these markers.
  • the present invention provides a method of detecting the onset of an advanced stage cancer of the breast, ovary, thyroid, testis or adrenal gland or a predisposition to developing an advanced stage cancer of the breast, ovary, thyroid, testis or adrenal gland in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said advanced stage cancer or a predisposition thereto.
  • said neoplasm is a neoplasm of the breast.
  • said neoplasm is a neoplasm of the ovary.
  • said high grade cancer is metastatic cancer.
  • the present invention provides a method of detecting the onset of an advanced stage cancer of the oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or a predisposition to developing an advanced stage cancer of oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or
  • said high grade cancer is metastatic cancer.
  • the present invention provides a method of detecting the onset of an advanced stage cancer of the cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas or a predisposition to developing an advanced stage cancer of the cervix, brain, skin, lymph note, lung, salivary gland, liver, gallbladder or pancreas in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of said advanced stage cancer or a predisposition thereto.
  • said high grade cancer is metastatic cancer.
  • the present invention provides a method of detecting the onset of an advanced stage prostate cancer or a predisposition to developing an advanced stage prostate cancer in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of an advanced stage prostate cancer or a predisposition thereto.
  • said high grade prostate cancer is metastatic prostate cancer.
  • the present invention provides a method of detecting the onset of metastatic prostate cancer or a predisposition to developing metastatic prostate cancer in a mammal said method comprising screening for the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ B , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression in said mammal wherein an increase in the level of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ B , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin protein and/or gene expression is indicative of the onset of metastatic prostate cancer or a predisposition to developing metastatic prostate cancer.
  • said markers are preferably inhibin- ⁇ , activin- ⁇ A, activin- ⁇ c or follistatin and said screening is directed to any 3 or all 4 of these markers.
  • the present invention is predicated on the determination that changes in the level of expression of two or more of inhibin- ⁇ , activin- ⁇ A, actrvin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin, are indicative of the development of a cancer, in particular low grade or high grade prostate cancer.
  • activin ⁇ c should be understood as a reference to all forms of activin ⁇ c and to fragments, derivatives, mutants or variants thereof. "Activin ⁇ c” is also interchangeably referred to as “activin ⁇ c subunit”. It should also be understood to include reference to any isoforms which may arise from alternative splicing of activin ⁇ c mRNA or mutant or polymorphic forms of activin ⁇ c.
  • activin ⁇ c is not intended to be limiting and should be read as including reference to all forms of activin ⁇ c including any protein encoded by the activin ⁇ c subunit gene, any subunit polypeptide such as precursor forms which may be generated, and any activin ⁇ c protein, whether existing as a monomer, multimer or fusion protein.
  • Multimeric protein forms of activin ⁇ c include for example the homodimeric activin C ( ⁇ c- ⁇ c) or the heterodimeric activin AC ( ⁇ A- ⁇ c), activin BC ( ⁇ - ⁇ c), activin CD ( ⁇ c- ⁇ ) or activin CE ( ⁇ c- ⁇ ) proteins.
  • activin ⁇ c in its monomeric, homodimeric or heterodimeric form.
  • a corresponding definition applies with respect to "activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D , and activin- ⁇ E".
  • the structure of activins are similar to one another and other members of the TGF ⁇ superfamily and are based on the conservation of the number and spacing of the cysteines within each subunit and the disulphide linkages between the two subunits that form characteristic cysteine knots. Other similarities relate to dimer formation, the location of the bioactive peptide in the carboxy terminal region of the precursor activin subunit molecule and similar intracellular signalling mechanisms.
  • Human activin ⁇ c for example, in comparison with other TGF- ⁇ superfamily members, reveals a typical structure with 9 conserved cysteines and a large precursor molecule that contain a core of hydrophobic amino acids at the N terminus thought to be the secretion signal sequence (Hotten G et al, 1995, Biochem Biophys Res Commun, 206: 608-13).
  • the mouse activin ⁇ c also contains 9 conserved cysteines and N terminal hydrophobic amino acids that may serve as a signal peptide (Schmitt et al. 1996, Genomics, 32: 358-66).
  • inhibin ⁇ should be understood as a reference to all forms of inhibin ⁇ and to fragments, derivatives, mutants or variants thereof. Inhibin ⁇ is also interchangeably referred to as “inhibin ⁇ subunit”. It should also be understood to include reference to any isoforms which may arise from alternative splicing of inhibin ⁇ mRNA or mutant or polymorphic forms of inhibin ⁇ .
  • inhibin ⁇ is not intended to be limiting and should be read as including reference to all forms of inhibin ⁇ including any protein encoded by the inhibin ⁇ subunit gene, any subunit polypeptide, the precursor polypeptide forms pre, pro ⁇ N and ⁇ C, and any inhibin ⁇ protein, whether existing as a monomer, nrultimer or fusion protein.
  • Multimeric protein forms of inhibin ⁇ include for example the heterodimeric ⁇ polypeptide (for example ⁇ A, ⁇ , ⁇ c, ⁇ o, ct ⁇ and ⁇ ) and the dimeric precursor ⁇ C- ⁇ polypeptide.
  • the ⁇ N and/or ⁇ C regions of precursor ⁇ -subunit proteins are found to exist either as part of an existing precursor ⁇ -subunit protein or in isolation, for example, following cleavage of said region from a precursor ⁇ -subunit protein.
  • Precursor ⁇ -subunit proteins exist in many forms including, but not limited to, the forms pre- pro- ⁇ N - ⁇ C and pro- ⁇ C.
  • detection of ⁇ -inhibin proteins, including precursor ⁇ -subunit proteins includes the detection of the ⁇ N and/or ⁇ C regions both in isolation, and as part of one or more of the various forms of precursor ⁇ -subunit protein.
  • the inhibin- ⁇ proteins which are detectable in the tissues from patients, in particular the prostate from patients diagnosed with benign prostate hyperplasia or in the non-malignant regions of prostate may comprise for example, the ⁇ N and/or ⁇ C regions.
  • the present invention is exemplified, but not limited in any way, by reference to detection of inhibin- ⁇ levels via the detection of the ⁇ C regions of the inhibin- ⁇ protein.
  • Inhibin-cc proteins comprising ⁇ N and/or ⁇ C regions are also referred to as precursor ⁇ -subunit proteins.
  • the ⁇ N and/or ⁇ C regions of * precursor ⁇ -subunit proteins are found to exist either as part of an existing precursor ⁇ -sub ⁇ -mit protein or in isolation, for example, following cleavage of said region from a precursor ct-subunit protein.
  • Precursor ⁇ -subunit proteins exist in many forms including, but not limited to, the forms pre- pro- ⁇ N - ⁇ C and pro- ⁇ C.
  • detection of inhibin- ⁇ proteins, including precursor ⁇ -subunit proteins includes the detection of the ⁇ N and/or ⁇ C regions both in isolation, and as part of one or more of the various forms of precursor ⁇ -subunit protein.
  • inhibin- ⁇ may undergo different forms of processing and/or cleavage at the ⁇ -C region of the inhibin- ⁇ subunit. This has been evidenced by the fact that the commonly used diagnostic antibody Groome RI [Robertson et al, 2001, Mol Cell Endo.
  • the form of inhibin- ⁇ which is detected is the form of inhibin- ⁇ which comprises amino acids 73-96 of the ⁇ -C region.
  • said inhibin- ⁇ protein is detected utilising the PO#12 monoclonal antibody and said advanced cancer is metastatic cancer.
  • follistatin 1 should be read as including reference to all forms of follistatin and to fragments, derivatives, mutants or variants thereof including, by way of example, the three protein cores and six molecular weight forms which have been identified as arising from the alternatively spliced mRNAs FS315 and FS288. Accordingly, it should also be understood to include reference to any isoforms which may arise from alternative splicing of follistatin mRNA or mutant or polymorphic forms of follistatin. It should still further be understood to extend to any protein encoded by the follistatin gene, any subunit polypeptide, such as precursor forms which may be generated, and any follistatin protein, whether existing as a monomer, multimer or fusion protein.
  • mammal as used herein includes humans, primates, livestock animals (eg. horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg. mice, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, deer, foxes).
  • livestock animals eg. horses, cattle, sheep, pigs, donkeys
  • laboratory test animals eg. mice, rats, guinea pigs
  • companion animals eg. dogs, cats
  • captive wild animals eg. kangaroos, deer, foxes.
  • the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human.
  • the present invention is predicated on the finding that levels of two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin, and in particular inhibin- ⁇ , activin- ⁇ A , activin- ⁇ c or follistatin, are modulated in neoplastic tissue as compared to normal tissue or non-malignant neoplastic tissue.
  • inhibin- ⁇ , activin- ⁇ A , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin are modulated in neoplastic tissue as compared to normal tissue or non-malignant neoplastic tissue.
  • the "normal” level is the level of protein or encoding nucleic acid molecule, in a biological sample corresponding to the sample being analysed, of an individual who has not developed a. neoplasm nor is predisposed to developing a neoplasm.
  • the "normal” level also includes reference to the level of these molecules in non- neoplastic regions of the tissue which is the subject of analysis. This latter method of analysis is a relative form of analysis in terms of the normal and test levels being determined from non-neoplastic and test tissues, respectively, derived from a single individual.
  • the method of the present invention should also be understood to encompass non-relative analysis means such as tlxe analysis of test results relative to a standard result which reflects individual or collec ⁇ ve results obtained from healthy individuals, other than the patient in issue.
  • Said "normal level” may be a discrete level or a range of levels. In this regard, it should be understood that levels may be assessed or momtored by either quantitative or qualitative rea-douts.
  • the reference level may also vary between individual forms (such as differently pro essed forms) of these molecules.
  • Reference to the marker levels of "corresponding grade" neoplastic cells should be understood as a reference to the levels which one observes or detects in any other neoplastic cell of the same grade as the cell which-, is under analysis, whether that be a cell or cells which are present in the tissue which is the subject of analysis or cells which are found in a corresponding but separate biological sample harvested from either the same individual or a different individual. It should be understood that this form of analysis may be relevant due to the fact that not all cells of a defined grade will necessarily progress in the same manner. For example, not all grade 4 cells may necessarily progress to metastises. Accordingly, differences between similar populations of cells in terms of the levels of the panel of markers defined herein may provide extremely valuable information. One may also seek to compare levels of these markers in cells of different grades. It should also be understood that the discussion, above, in relation to relative versus non- relative analyses, standard results and discrete versus ranges of levels applies equally in this context.
  • the terms “increase”, “decrease” and “modulation” refer to increases and decreases in inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E and follistatin levels relative either to a control level or control level range) or to an earlier result determined from the patient in issue, this la-tter reference point being particularly relevant in the context of the ongoing monitoring of a patient, as hereinafter described.
  • inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E and/or follistatin exhibit dual roles as an indicator of carcinogenesis in the context of the development of both low grade and high neoplasms.
  • Reference to the "onset" of an advanced stage cancer, in particular an advanced stage prostate cancer, should be understood as a reference to one or more cells of that individual exhibiting an advanced stage growth characteristic In this regard, the advanced stage cancer may be well developed in that a mass of proliferating cells has developed.
  • the advanced stage cancer may be a-t a very early stage in that only relatively few divisions of the cells characterising the cancer have occurred at the time of diagnosis.
  • the method of the present invention facilitates the identification of increased expression of two or more of inhibin- ⁇ , activin- ⁇ ,_v, activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin in these cells and, therefore, their detection.
  • the present invention also extends to the assessment of an individual's predisposition to the development of certain classes of high grade neoplasm, such as metastatic cancer. It should be understood that a corresponding definition applies with respect to the onset or predisposition to the onset of an early stage cancer.
  • the preferred method is to detect an increase in two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin levels in order to diagnose the onset of or predisposition to the onset of an ad ⁇ vanced stage cancer, or a decrease in two or more of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin to diagnose the onset or a predisposition to the onset of an early stage cancer
  • the detection of decreases in inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , a_ctivin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin levels in the context of advanced stage cancers or increase in these molecules in the context of early stage cancers may be desired under certain circumstances.
  • a radical prostatectomy is not performed one may seek to monitor for improvements in the disease state of the prostate (characterised by a decrease in marker levels) during the course of prophylactic or therapeutic treatment of the patient.
  • patients presenting with early stage prostate cancer in the form of very early symptoms of prostate disease or a genetic or environmental predisposition to the development of prostate disease one may monitor for elevation of low levels of the marker molecules back to normal levels during the course of treatment.
  • inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin in the context of early stage cancers, in general, provides a means of detecting normalisation of tumour suppression function in the tissue in issue. This may be indicative of an effective treatment regime. It should " be understood that in accordance with this aspect of the present invention, inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin levels will likely be assessed relative to one or more previously obtained results, as hereinbefore described.
  • mapping the modulation of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin in any one or more classes of biological samples is a highly sensitive and accurate indicator of the status of an individual or the effectiveness of a therapeutic or prophylactic regime which is currently in use.
  • the method of the present invention should therefore be understood to extend to monitoring for increases or decreases in two or more of inhibin- ⁇ , activin- ⁇ A , actrvin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin levels in an individual relative to a control level (as hereinbefore defined) or relative to one or more earlier levels determined from said individual.
  • another aspect of the present invention is directed to a method of monitoring for the onset or progression of a neoplasm in a mammal said method comprising screening for the modulation in the level of two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D , activin- ⁇ E or follistatin in said mammal wherein the level of said inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin relative to the normal level of inhibin is indicative of the onset or progression of said neoplasm.
  • markers are three or four of inhibin- ⁇ , activin- ⁇ A, activin- ⁇ c or follistatin.
  • said neoplasm is a malignant breast neoplasm.
  • said neoplasm is a malignant ovarian neoplasm.
  • said neoplasm is a malignant neoplasm of the thyroid, testis or adrenal gland.
  • said neoplasm is a malignant neoplasm of the prostate, skin, lymph node, lung, salivary gland, liver, gall bladder, pancreas. , oesophagus, stomach, colon, rectum, kidney, bladder, small intestine, large intestine, larynx, nasal cavity, throat, neural tissue or endometrium or testis.
  • said neoplasm is a malignant neoplasm of the prostate.
  • the method of the present invention has widespread applications including, out not limited to, the diagnostic or prognostic analysis of cancer, in particular prostate cancer, or any condition characterised by the presence of cancer, for example, the conditions associated with advanced prostate cancer such as urine retention, haematuria, urinary incontinence, kidney failure, bone pain, bone fragility, spinal cord damage, osteoarthritis, lethargy, loss of appetite, nausea, diarrhea, constipation or cachexia.
  • the diagnostic or prognostic analysis of cancer in particular prostate cancer
  • any condition characterised by the presence of cancer for example, the conditions associated with advanced prostate cancer such as urine retention, haematuria, urinary incontinence, kidney failure, bone pain, bone fragility, spinal cord damage, osteoarthritis, lethargy, loss of appetite, nausea, diarrhea, constipation or cachexia.
  • Means of screening for changes in inhibin- ⁇ , activin- ⁇ A, activin- ⁇ , activin- ⁇ c, actrvin- ⁇ o, activin- ⁇ E or follistatin (herein referred to as "the markers") levels in an individual, or biological sample derived therefrom, can be achieved by any suitable method, which would be well known to the person of skill in the art, such as but not limited to:
  • Molecular imaging [Moore et al, BBA, 1402:239-249, 1988; Weissleder etal. Nature Medicine, ⁇ ->:351-355, 2000] is the in vivo imaging of molecular expression that correlates with the macro-features currently visualized using "classical" diagnostic imaging techniques such as X-Ray, computed tomography (CT), MU, Positron Emission Tomography (PET) or endoscopy.
  • CT computed tomography
  • PET Positron Emission Tomography
  • endoscopy Historically, detection- of malignant tumor cells in a background of normal or hyperplastic benign tissue is often based on differences in physical properties between tissues, which are frequently minimal, resulting in low contrast resolution.
  • Application of expression profiling will define the differences in "molecular properties" between cancer and normal tissues that arise as a result of malignant transformation.
  • FISH Fluorescent In Situ Hybridization
  • QRTPCR Quantitative Reverse Transcriptase Polymerase Chain Reaction
  • Flow cytometric qualification of competitive RT-PCR products [Wedemeyer et al., W. Clinical Chemistry 48:9 1398-1405, 2002] or array technologies.
  • a labelled polynucleotide encoding the markers may be utilized, as a probe in a Northern blot of an RNA extract obtained from a tissue.
  • a nucleic acid extract from the animal is utilized in concert with oligonucleotide primers corresponding to sense and antisense sequences of a polynucleotide encoding inhibin- ⁇ , or flanking sequences thereof, in a nucleic acid amplification reaction such as RT PCR, real time PCR or SAGE.
  • NLSIPSTM very large scale immobilized primer arrays
  • R ⁇ A is isolated from a cellular sample suspected of containing the marker R ⁇ A, e.g. total R ⁇ A isolated from human prostate cancer tissue.
  • R ⁇ A can be isolated by methods known in the art, e.g. using TRIZOLTM reagent (GIBCO-BRL/Life Technologies, Gaithersburg, Md.).
  • Oligo-dT, or random-sequence oligonucleotides, as well as sequence- specific oligonucleotides can be employed as a primer in a reverse transcriptase reaction to prepare first-strand cD ⁇ As from the isolated R ⁇ A.
  • Resultant first- strand cD ⁇ As are then amplified with sequence-specific oligonucleotides in PCR reactions to yield an amplified product.
  • PCR Polymerase chain reaction
  • R ⁇ A and/or D ⁇ A a preselected fragment of nucleic acid
  • oligonucleotide primers sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR can be used to amplify specific RNA sequences and cDNA transcribed from total cellular RNA. See generally Mullis et al., 1987; Erlich, 1989.
  • amplification of specific nucleic acid sequences by PCR relies upon oligonucleotides or "primers" having conserved nucleotide sequences wherein the conserved sequences are deduced from alignments of related gene or protein sequences, e.g. a sequence comparison of mammalian inhibin- ⁇ genes.
  • one primer is prepared which is predicted to anneal to the antisense strand and another primer prepared which is predicted to anneal to the sense strand of a cDNA molecule which encodes inhibin- ⁇ .
  • the reaction mixture is typically subjected to agarose gel electrophoresis or other convenient separation technique and the relative presence of the marker specific amplified DNA detected.
  • marker amplified DNA may be detected using Southern hybridization with a specific oligonucleotide probe or comparing is electrophoretic mobility with DNA standards of known molecular weight. Isolation, purification and characterization of the amplified DNA may be accomplished by excising or eluting the fragment from the gel (for example, see references Lawn et ah, 1981; Goeddel et ah, 1980), cloning the amplified product into a cloning site of a suitable vector, such as the pCRII vector (Invitrogen), sequencing the cloned insert and comparing the DNA sequence to the known sequence of the marker. The relative amounts of marker mRNA and cDNA can then be determined.
  • an antibody according to the invention having a reporter molecule associated therewith, may be utilized in immunoassays.
  • immunoassays include but are not limited to radioimmunoassays (RIAs), enzyme- linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs), Western blotting which are well known to those of skill in the art.
  • RIAs radioimmunoassays
  • ELISAs enzyme- linked immunosorbent assays
  • ICTs immunochromatographic techniques
  • Western blotting which are well known to those of skill in the art.
  • Immunoassays may include competitive assays. It will be understood that the present invention encompasses qualitative and quantitative immunoassays.
  • Suitable immunoassay techniques are described, for example, in U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labelled antigen-binding molecule to a target antigen.
  • Two-site assays are particularly favoured for use in the present invention.
  • an unlabelled antigen-binding molecule such as an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • another antigen-binding molecule suitably a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
  • any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
  • the results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including minor variations as will be readily apparent.
  • a first antibody having specificity for the antigen or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient and under suitable conditions to allow binding of any antigen present to the antibody.
  • the antigen-antibody complex is washed and dried and incubated with a second antibody specific for a portion of the antigen.
  • the second antibody has generally a reporter molecule associated therewith that is used to indicate the binding of the second antibody to the antigen.
  • the amount of labelled antibody that binds, as determined by the associated reporter molecule, is proportional to the amount of antigen bound to the immobilized first antibody.
  • An alternative method involves immobilizing the antigen in the biological sample and then exposing the immobilized antigen to specific antibody that may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound antigen may be detectable by direct labelling with the antibody. Alternatively, 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 reporter molecule associated with the antigen-binding molecule may include the following:- (a) direct attachment of the reporter molecule to the antibody;
  • the reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a paramagnetic ion, a lanthanide ion such as Europium (Eu 34 ), a radioisotope including other nuclear tags and a direct visual label.
  • a colloidal metallic or non- metallic particle a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Suitable enzymes suitable for use as reporter molecules is disclosed in U.S. Patent Nos. U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338.
  • Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ -galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzymes may be used alone or in combination with a second enzyme that is in solution.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by Dower et ah, International Publication No. WO 93/06121. Reference also may be made to the fluorochromes described in U.S. Patent Nos. 5,573,909 [Singer et al], 5,326,692
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • the substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody- antigen complex, allowed to bind, and then the excess reagent washed away.
  • a solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • fluorescent compounds such as fluorescein, rhodamine and the lanthanide, europium (EU) may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent- labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest.
  • Immunofluorometric assays IFMA
  • IFMA Immunofluorometric assays
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.
  • any suitable technique may be utilised to detect inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ o, activin- ⁇ E or follistatin or its encoding nucleic acid molecule.
  • the nature of the technique which is selected for use will largely determine the type of biological sample which is required for analysis.
  • tissue section such as a prostate tissue section, in terms of both a morphological analysis of the cell types which are present and the levels of two or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ and/or follistatin protein or expressed gene levels.
  • Means of screening for the presence of neoplastic cells in an organ, if necessary, can be achieved by any suitable method which would be well known to the person of skill in the art.
  • tissue sections for morphological analysis may be prepared in any suitable manner such as in the form of frozen sections or wax embedded sections.
  • tissue biopsy for example, a needle biopsy
  • tissue sections may be prepared in any suitable manner such as in the form of frozen sections or wax embedded sections.
  • needle biopsies are utilised, one may nevertheless harvest tissue of sufficient size to enable analysis of the tissue's architecture.
  • cellular aspirates it may be necessary to render the cells a single cell suspension and analyse the morphology of a population of cells derived therefrom. This is a less ideal method but may nevertheless achieve the objective of identifying the existence of particular grade neoplastic cells.
  • any suitable histological technique will enable the grading of the cells.
  • tissues can be harvested and stored in the form of formalin fixed tissue, frozen sections, gluteraldehyde fixed or bouins fixed tissue.
  • histological techniques for achieving morphological analysis one could utilise, ter alia, haemotoxylin and eosin, immunoperoxidase, electronmicroscopy, in situ staining or PCR in situ.
  • biological sample should be understood as a reference to any sample of cells or tissue which is derived from an organism.
  • the cells may be single cells, cultured cells or part of a tissue.
  • the biological sample may be derivable from any human or non-human mammal, as detailed above. It should be further understood that reference to “organism” includes reference to embryos and foetuses.
  • the biological sample may be any sample of material derived from the organism. This includes reference to both samples which are naturally present in the organism, such as tissue and body fluids in a mammal (for example biopsy specimens such as lymphoid specimens, resected tissue, tissue extracts, blood, lymph fluid, faeces, bronchial secretions or cell culture medium) and samples which are introduced into the body of the organism and subsequently removed, such as, for example, the saline solution extracted from the lung following a lung lavage or from the colon following an enema. It also includes reference to cells which originated from an organism but have been maintained in vitro, for example cell lines, or which have been manipulated or treated subsequently to removal from the organism, for example immortalised or genetically modified cells or tissues.
  • tissue and body fluids in a mammal for example biopsy specimens such as lymphoid specimens, resected tissue, tissue extracts, blood, lymph fluid, faeces, bronchial secretions or cell culture medium
  • the biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing.
  • a biopsy sample may require homogenisation prior to testing.
  • the sample comprises cellular material
  • the sample may be partially purified or otherwise enriched prior to analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to select out a sub-population of particular interest.
  • neoplastic condition is a lymphoma
  • a lymph node biopsy or a blood or marrow sample would likely provide a suitable source of tissue for testing. Consideration would also be required as to whether one is monitoring the original source of the neoplastic cells or whether the presence of metastases or other forms of spreading of the neoplasia from the point of origin is to be monitored. In this regard, it may be desirable to harvest and test a number of different samples from any one organism.
  • Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting two or more of inhibin- ⁇ , activin- ⁇ A , activin- ⁇ , activin- ⁇ c, activin- ⁇ D, activin- ⁇ E or follistatin protein or encoding nucleic acid molecule and reagents useful for facilitating the detection by the agent in the first compartment. Further means may also be included, for example, to receive a biological sample.
  • the agent may be any suitable detecting molecule.
  • Immunohistochemistry was performed on radical prostatectomy tissue from 28 prostate cancer patients with Gleason Grade sum of greater than or equal to 7. After being deparaffinated the tissue underwent a pretreatment step of microwave heating in 0.1 M glycine (pH 4.5) for activin- ⁇ c, and 0.01M citrate (pH 6) for inhibin- ⁇ , follistatin 315 and activin- ⁇ A. The sections were immunostained for activin- ⁇ c subunit, inhibin- ⁇ , follistatin 315 and activin- ⁇ A protein using the DAKO Autostainer (DAKO, Carpinteria, USA).
  • DAKO DAKO Autostainer
  • the antibody was detected by incubation with Envision polymer-antimouse-horse radish peroxidase (DAKO, Carpinteria, USA) for 15 minutes and visualized by reaction with diaminobenzidine (DAB) (DAKO, Carpinteria, USA) for 5 minutes.
  • DAKO Envision polymer-antimouse-horse radish peroxidase
  • DAB diaminobenzidine
  • Inhibin- ⁇ , activin- ⁇ c and follistatin 315 immunostaining was significantly increased in high grade cancers of Gleason grade 4 or 5 compared to adjacent benign secretory epithelium (p ⁇ 0.05; figure 1).
  • immunostaining for activin- ⁇ c, follistatin 315 and activin- ⁇ A significantly increased in Gleason grade 4 or 5 prostate cancer compared to Gleason grade 3 prostate cancer (p ⁇ 0.05; figure 7).
  • Inhibin- ⁇ , activin- ⁇ c and follistatin 315 immunostaining were significantly increased in cribriform cancers compared to benign epithelium (p ⁇ 0.05; figure 2).
  • immunostaining for inhibin- ⁇ , activin- ⁇ c and follistatin 315 significantly increased in cribriform pattern prostate cancer compared to Gleason grade 3 prostate cancers (p ⁇ 0.05; figure 8).
  • Activin- ⁇ A was not significantly different in cribriform cancers compared to either benign epithelium or Gleason grade 3 cancer (figure 2 and 8).
  • Cribriform cancers are a histopathological sub-type of Gleason grade 3 or 4 prostate cancers based on cellular morphology. Patients diagnosed with cribriform pathology have poor outcome and survival compared to other grade 3/4 prostate cancers [McNeal and Yemoto, 1 96, Am J Surg Pathol, 20: 802-14; Rubin et al., Am JSurg Pathol, 22: 840-8, 1998; Wilcox et al., Hum Pathol, 29: 1119-23, 1 998; Cohen et al., 2000, Prostate, 43:11-9]. Therefore, these data support the hypothesis that the expression of the panel of markers is increased during progression of prostate cancers and is associated with poor patient prognosis.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS heat- inactivated foetal calf serum
  • LNCaP cells were plated at a density of 240,000 cells per well, in DMEM + 5% FCS into 12-well plates (Falcon) for 48hrs. LNCaP cells were transiently transfected with pRK5- ⁇ c (6 ⁇ g) at a ratio of 1 :2 DNA ( ⁇ g) to Lipofectamine PLUS ( ⁇ l) and 1 :1 ratio of DNA ( ⁇ g) to Lipofectamine reagent ( ⁇ l) according to the manufacturer's instructions.
  • PC3 cells were plated at 200,000 cells per well in DMEM + 10°/o FCS into 12 well plates (70-80% confluence) for 24 hrs. PC3 cells were transiently transfected with pRK5- ⁇ c or pRK5 control (3 ⁇ g) using
  • the human INHA cDNA subcloned into pcDNA3.1 was obtained from Invitrogen, Carisbad, CA, USA.
  • LNCaP cells seeded at 6.24-xlO 5 cells/well in a 6-well plate were cultured (50-80% confluence) for 24 hours.
  • Lipofectamine plus (Invitrogen, Carisbad, CA, USA) was then used for transfections according to the manufacturer's instructions. Briefly, the cells were transfected with 3.84 ⁇ g high quality, linearised plasmid DNA using 4 ⁇ l Lipofectamine and 19.2 ⁇ l Lipofectamine plus.
  • the transfection media was replaced with DMEM + 10%FCS, and after 48hrs, media was replaced with selection media (DMEM supplemented with 10%FCS and 360 ⁇ g/ml zeocin).
  • PC3 cells seeded at 4.8xl0 5 cells/well in a 6-well plate were cultured (50-80% confluence) for 24 hours.
  • Superfect Qiagen Pty Ltd, Clifton Hill, Victoria, Australia
  • the cells were transfected with 8.64 ⁇ g plasmid DNA using 14.4 ⁇ l of Superfect.
  • the transfection media was replaced with DMEM + 10%FCS, and after 48hrs, media was replaced with selection media (DMEM supplemented with 10%FCS and 360 ⁇ g/ml zeocin). Individual colonies surviving after 2-3 weeks selection were picked and propagated in DMEM supplemented with 10%FCS and 360 ⁇ g/ml zeocin. Integration of the plasmids was confirmed by genomic PCR. Expression of inhibin- ⁇ from the plasmids was confirmed by RT-PCR and Western blot with RI monoclonal antibody raised to the mature ⁇ C region of the inhibin- ⁇ subunit.
  • LNCaP cells overexpressing either activin- ⁇ c or inhibin- ⁇ demonstrated a significant reduction in proportion of cells in S-phase compared to LNCaP cells not transfected with activin- ⁇ c or inhibin- ⁇ cDNA (p ⁇ 0.05; grey bars; figure 3).
  • PC3 cells overexpressing either activin- ⁇ c or inhibin- ⁇ demonstrated a significant increase in proportion of cells in S-phase compared to PC3 cells not transfected with activin- ⁇ c or inhibin- ⁇ cDNA (p ⁇ 0.05; grey bars; figure 4).
  • LNCaP and PC3 prostate cancer cells represent early and advanced stages of human prostate cancer progression, respectively.
  • LNCaP cells are dependant on androgens and fail to produce tumours in vivo [Fisher et al., Cell Tissue Res, 307: 337-45, 2002].
  • PC3 cells are not dependant on androgens for growth -and are highly metastatic in vivo (Fisher et al., 2002, supra). Therefore, these data support the hypothesis that activin- ⁇ c and inhibin- ⁇ are tumour suppressive in early stage prostate cancer but become growth- promoting or pro-metastatic in advanced prostate cancer.
  • Tissue samples from patients with metastatic prostate cancel to the lymph node were obtained from Melbourne Pathology.
  • Activin- ⁇ A immunolocalization was investigated using monoclonal E4 antibody
  • follistatin immunolocalisation was investigated using the monoclonal H10 antibody
  • activin- ⁇ c was investigated using the monoclonal 25/4 antibody which also detects the activin- ⁇ E subunit peptide.
  • Sections were de-paraffinated and underwent antigen retrieval. Sections to be stained were -heated in the microwave in antigen retrieval solutions as follows: H10 in 0.1M glycine (pH 4.5), E4 in 0.01 mol/L Tris buffer (pH 9.7) and 25/4 in 0.01M citrate buffer (pH 6).
  • E4 antibody was diluted 1:750
  • H10 was diluted 1:75
  • 25/4 antibody was diluted 1:100.
  • the mouse IgG antibody was diluted 1:20 and incubated for 60 minutes at room temperature. Sections were washed with PBS and incubated for 50 min with biotinylated horse antimouse secondary antibody (DAKO Corp., Botany, Australia) at a dilution of 1:200 in PBS. Sections were "washed with PBS and incubated with ABC reagent from the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Peterborough, UK) for 40 min.
  • Peroxidase activity was detected using 3,39- diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc.). The reaction was terminated by immersion in distilled water, and the sections were counterstained with Mayers' hematoxylin (Sigma), washed with tap water, dehydrated, and permanently mounted with DPX (BDH, Poole, UK).
  • the parental LNCaP, empty vector clones (L16, L17, L18) and the inhibin ⁇ transfected clones (LI, L5, L8) were plated in triplicates in 6 well plates in DMEM plus 10%-FCS and grown until approximately 70-80% confluence.
  • the cell monolayer was then wou ⁇ ided by scraping the surface with a blue 1ml plastic pipette tip to leave an approximately 1mm wide clearing.
  • the cultures were washed several times with media to remove cells liberated during the wounding process and then cultured in fresh DIv EM plus 10%FCS.
  • the same fields were photographed at two or more day intervals using a digital camera. The images were analyzed, wound width measured and plotted as percentage of wound closure relative to day 0. This experiment was repeated twice.
  • mice 6-8 weeks of age were obtained from ARC. They were housed in microisolator cages with free access to sterilized food and water. The experiments were performed a week after the arrival of the animals. All the protocols were approved, by the Animal Ethics Committee of Monash Medical Centre, Melbourne, Australia.
  • the parental LNCaP, empty vector clones (L16, L17, L18) and the inhibin ⁇ transfected clones (LI, L5, L8) were used.
  • a total of 5 x 10 cells were inoculated subcutaneously in the presence of Matrigel into SCID mice. Control animals were inoculated with Matrigel alone. Mice were maintained in their microisolator cages and were monitored for tumor growth and tumor size weekly until the tumors reached 1cm when the mice were euthanasized and the tumors excised.
  • mice A total of 5 x 10 cells in 0.01ml were injected orthotopically into the ventral lobe of th-e prostate gland after the mice was surgically opened at the lower abdomen.
  • the abdominal wall was sutured using absorbable sutures, and the skin closed with a skin-staple.
  • Mice were weighed twice weekly to ensure close monitoring of health. After 10-12 weeks, tit e mice were euthanasized and surgically opened for determination of tumor weights.
  • In vitro wound healing assays can be used to analyze migration.
  • the extent of cell migration was estimated by the rate of the wound closure over a period of time.
  • Wound closure was slower in LNCaP clones overexpressing inhibin ⁇ (LI, L5, L8) compared to empty vector clones (L16, L17, L18) and the parental LNCaP cells.
  • inhibin ⁇ In order to study the effects of inhibin ⁇ on tumor growth, an in vivo model of tumor growth in SCID mice was used.
  • LNCaP clones overexpressing inhibin ⁇ demonstrated significant reduction in tumor size (*** p ⁇ 0.001) compared to the parental LNCaP cells.
  • the prostate tumors formed after orthotopical injection of the LNCaP clones overexpressing inhibin ⁇ into the ventral pro state demonstrated significant decrease in tumor weights (* p 0.01 - 0.05; *** p ⁇ 0. OOl) compared to the parental LNCaP cells (figures 9-11).
  • Cipriano S. C Chen, L., Kumar, T. R. and Matzuk, M. M. (2000) 'Follistatin is a modulator of gonadal tumor progression and the activin- induced wasting syndrome in inhibin-deficient mice', Endocrinology, 141, 2319-27

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Abstract

L'invention concerne, de façon générale, un procédé de diagnostic, de prédiction ou de régulation du développement ou de la progression d'un cancer, et plus particulièrement, un procédé de diagnostic, de prédiction ou de régulation du développement ou du progrès d'un cancer de la prostate chez un mammifère. Cette invention concerne, plus spécifiquement, un procédé destiné à repérer les cancers en stade précoce et en stade avancé, ou des prédispositions aux cancers, par criblage de changements dans le niveau de deux ou plusieurs expressions d'inhibine-α, d'activine-βA, d'activine-βB, d'activine-βC, d'activine-βD, d'activine-βE ou de follistatine chez un mammifère. Cette invention concerne également un procédé destiné à diagnostiquer ou à réguler des états associés ou caractérisés par l'apparition d'un cancer.
PCT/AU2005/000542 2004-04-16 2005-04-15 Procede destine a reguler la progression d'un cancer WO2005100593A1 (fr)

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US11/578,711 US20080038729A1 (en) 2004-04-16 2005-04-15 Method For Monitoring The Progress Of Cancer
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8026060B2 (en) 2006-01-11 2011-09-27 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US10179936B2 (en) 2009-05-01 2019-01-15 Genomic Health, Inc. Gene expression profile algorithm and test for likelihood of recurrence of colorectal cancer and response to chemotherapy
CN114075281A (zh) * 2021-11-16 2022-02-22 福州迈新生物技术开发有限公司 抗Inhibin-α蛋白单克隆抗体、细胞系及其制备方法和应用
CN116358954A (zh) * 2023-02-22 2023-06-30 深圳市昭蓝生物科技有限公司 复合质控品及其制备方法和产前筛查试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IN2013MN00441A (fr) * 2010-10-07 2015-05-29 Astute Medical Inc

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030108896A1 (en) * 2001-06-28 2003-06-12 Thomas Vogt Method for detection of melanoma
WO2004053487A1 (fr) * 2002-12-12 2004-06-24 Monash University Procede de diagnostic et de traitement, et agents utilises pour des etats caracterises par une modulation du niveau de l'activine $g(b)c
WO2004086048A1 (fr) * 2003-03-28 2004-10-07 Prostate Diagnostics Pty. Ltd. Procede et agents diagnostiques et therapeutiques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005083438A1 (fr) * 2004-02-27 2005-09-09 Monash University Procede de determination d'un pronostic pour des patients atteints d'un cancer a degre modere

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030108896A1 (en) * 2001-06-28 2003-06-12 Thomas Vogt Method for detection of melanoma
WO2004053487A1 (fr) * 2002-12-12 2004-06-24 Monash University Procede de diagnostic et de traitement, et agents utilises pour des etats caracterises par une modulation du niveau de l'activine $g(b)c
WO2004086048A1 (fr) * 2003-03-28 2004-10-07 Prostate Diagnostics Pty. Ltd. Procede et agents diagnostiques et therapeutiques

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
COBELLIS L. ET AL: "Gonadal malignant germ cell tumours express immunoreactive inhibin/activin subunits.", EUROPEAN JOURNAL OF ENDOCRINOLOGY., vol. 145, 2001, pages 779 - 784, XP008075383 *
LA ROSA S. ET AL: "Localization of inhibits and activins in normal endocrine cells and endocrine tumors of the gut and pancreas: an immunohistochemical and in situ hybridization study.", JOURNAL OF HISTOCHEMISRY AND CYTOCHEMISTRY., vol. 52, no. 2, February 2004 (2004-02-01), pages 217 - 225, XP008092210 *
MCPHERSON S.J. ET AL: "Expression of activin A and follistatin core proteins by human prostate tumor cell lines.", ENDOCRINOLOGY., vol. 140, no. 11, 1999, pages 5303 - 5309, XP008075300 *
PETRAGLIA F. ET AL: "Expression and secretion of inhibits and activin in normal and neoplastic uterine tissues. High levels of serum antivin A in women with endometrial and cervical carcinoma.", JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM., vol. 83, no. 4, 1998, pages 1194 - 1200, XP008075301 *
RISBRIDGER G.P. ET AL: "Elevated expression of inhibin alpha in prostate cancer.", JOURNAL OF UROLOGY., vol. 171, January 2004 (2004-01-01), pages 192 - 196, XP005368718 *
RISBRIDGER G.P. ET AL: "The contribution of inhibits and activins to malignant prostate disease.", MOLECULAR AND CELLULAR ENDOCRINOLOGY., vol. 180, 2001, pages 149 - 153, XP003000861 *
ROBERTSON D.M. ET AL: "Inhibin/activin and ovarian cancer.", ENDOCRINE RELATED CANCER., vol. 11, March 2004 (2004-03-01), pages 35 - 49, XP008075338 *
See also references of EP1756301A4 *
WILDI S. ET AL: "Overexpression of activin A in stage IV colorectal cancer.", GUT., vol. 49, 2001, pages 409 - 417, XP008075384 *
YOSHINAGA K. ET AL: "Clinical significance of the expression of activin A in esophageal carcinoma.", INTERNATIONAL JOURNAL OF ONCOLOGY., vol. 22, 2003, pages 75 - 80, XP008075386 *
ZHENG W. ET AL: "Imbalanced expression of inhibin and activin subunits in primary epithelial ovarian cancer.", GYNECOLOGIC ONCOLOGY., vol. 69, 1998, pages 23 - 31, XP008075299 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8026060B2 (en) 2006-01-11 2011-09-27 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8029995B2 (en) 2006-01-11 2011-10-04 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8153378B2 (en) 2006-01-11 2012-04-10 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8153380B2 (en) 2006-01-11 2012-04-10 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8153379B2 (en) 2006-01-11 2012-04-10 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8198024B2 (en) 2006-01-11 2012-06-12 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8273537B2 (en) 2006-01-11 2012-09-25 Genomic Health, Inc. Gene expression markers for colorectal cancer prognosis
US8367345B2 (en) 2006-01-11 2013-02-05 Genomic Health Inc. Gene expression markers for colorectal cancer prognosis
US10179936B2 (en) 2009-05-01 2019-01-15 Genomic Health, Inc. Gene expression profile algorithm and test for likelihood of recurrence of colorectal cancer and response to chemotherapy
CN114075281A (zh) * 2021-11-16 2022-02-22 福州迈新生物技术开发有限公司 抗Inhibin-α蛋白单克隆抗体、细胞系及其制备方法和应用
CN116358954A (zh) * 2023-02-22 2023-06-30 深圳市昭蓝生物科技有限公司 复合质控品及其制备方法和产前筛查试剂盒

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JP2007532889A (ja) 2007-11-15

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