WO2005083438A1 - Procede de determination d'un pronostic pour des patients atteints d'un cancer a degre modere - Google Patents

Procede de determination d'un pronostic pour des patients atteints d'un cancer a degre modere Download PDF

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WO2005083438A1
WO2005083438A1 PCT/AU2005/000283 AU2005000283W WO2005083438A1 WO 2005083438 A1 WO2005083438 A1 WO 2005083438A1 AU 2005000283 W AU2005000283 W AU 2005000283W WO 2005083438 A1 WO2005083438 A1 WO 2005083438A1
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neoplasm
grade
activin
prostate
inhibin
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PCT/AU2005/000283
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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 claimed from AU2004902162A external-priority patent/AU2004902162A0/en
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Publication of WO2005083438A1 publication Critical patent/WO2005083438A1/fr

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    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates generally to a method of prognosing or monitoring the development or progress of a moderate grade cancer to a higher grade cancer in a mammal. More particularly, the present invention contemplates a method of prognosing or monitoring the development or progress of a moderate grade prostate cancer to a higher grade prostate cancer in a mammal.
  • the method of the present invention is predicated on the analysis of one or both of protein/gene expression levels or cellular morphological characteristics. Also provided are diagnostic agents for use in the method of the invention.
  • a related aspect of the present invention is directed to a method of classifying moderate grade cancers, in particular prostate cancers, as high level or low level cancers on the basis of unique cellular morphology and/or protein or gene expression levels.
  • Prostate cancer is a disease that occurs in men mostly over the age of 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 of prostatic cancer is high and similarly prevalent rates occur in most countries. Globally, prostate cancer now represents the third highest incidence of cancer after 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. Further, the incidence of prostate cancer appears to be increasing. This may be partly due to a 'real' increased risk, but is certainly related to the increased likelihood of detection, via PSA tests, and the increased number of biopsy operations or TURPs. 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.
  • 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 majority of prostrate 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 and signals a significantly less hopeful prognosis.
  • PSA levels do not always correlate with prostate cancers.
  • Certain limitations also exist with some aspect of the Gleason score. Briefly, prostate pathologies are graded with a Gleason grading of from 1 to 5, in order of increasing malignancy.
  • Grade 1 very well differentiated adenocarcinoma.
  • Grade 2 well differentiated adenocarcinoma.
  • Grade 3 moderately differentiated adenocarcinoma.
  • Grade 4 poorly differentiated adenocarcinoma.
  • Grade 5 undifferentiated adenocarcinoma.
  • Gleason grade 1 and 2 pathologies are regarded as low grade, Gleason grade 3 as a moderate grade and Gleason grade 4 and 5 as high grade.
  • patients with predominantly grade 1 or 2 pathologies are usually assumed to have rumours which are not aggressive.
  • patients with grade 4 and 5 pathologies are generally regarded as having very aggressive tumours which may have spread outside the gland.
  • the most difficult pathology for a clinician to formulate a prognostic opinion in respect of is the Gleason grade 3 pathology. Since this is the most common grade of tumour observed by pathologists, the inability to currently predict whether the tumour is likely to spread and become aggressive is a significant limitation.
  • TGF transforming growth factor
  • Activins regulate cell growth or differentiation by binding activin receptors and initiating a signalling cascade [Pangas, S. A. and Woodruff, T. K. (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 trans genie mice bearing a targeted deletion of the inhibin ⁇ subunit [Matzuk, M. M., Finegold, M. J., Su, J. G., Hsueh, A. J. and Bradley, A. (1992), Nature, 360, 313-9; Matzuk, M. M. and Bradley, A. (1994), Semr ⁇ Cancer Biol, 5, 37-45; Matzuk, M. M., Finegold, M. J., Mather, J. P., Krumrnen, L., Lu, H. and Bradley, A.
  • Inhibin ⁇ and activin ⁇ subunits, and follistatins are synthesized in the human prostate.
  • Activin ⁇ A - and ⁇ s-subunits are also localized to the epithelium of benign tissues, but their expression is unchanged and present in poorly differentiated adenocarcinomas of the prostate [Thomas, T. Z., Wang, H., Niclasen, P., O'Bryan, M. K., Evans, L. W., Groome, N. P., Pedersen, J. and Risbridger, G. P. (1997), JClin Endocrinol Metab, 82, 3851-8; Thomas, T. Z., Chapman, S. M., Hong, W., Gurusingfhe, C, Mellor, S. L., Fletcher, R., Pedersen, J.
  • moderate grade cancers can be further categorised into those which are likely to become aggressive versus those which are not likely to become aggressive (i.e. high level grade 3 vs. low level grade 3, respectively) based either on unique morphological characteristics of the grade 3 cells or based on differences in cellular or systemic levels of one or more of inhibin ⁇ , activin ⁇ A , activin ⁇ c or follistatin.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
  • One aspect of the present invention provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the progression of said neoplasm to a higher grade, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said neoplasm progressing to a higher grade.
  • Another aspect of the present invention provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the progression of said prostate neoplasm to a higher grade, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said prostate neoplasm progressing to a higher grade.
  • Yet another aspect of the present invention provides a method of detecting or monitoring a predisposition, in a mammal with a Gleason grade 3 prostate neoplasm, to the progression of said prostate neoplasm to a higher grade, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said Gleason grade 3 prostate neoplasm progressing to a higher grade.
  • Still another aspect of the present invention preferably provides a method of detecting or monitoring a predisposition, in a mammal with a Gleason grade 3 prostate neoplasm, to the progression of said prostate neoplasm to a Gleason grade 4 or 5, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said Gleason grade 3 prostate neoplasm progressing to a higher grade.
  • Yet still another aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the onset of a condition characterised by said higher grade neoplasm, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of the onset of said condition.
  • Still yet another aspect of the present invention provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the onset of a condition characterised by a higher grade prostate neoplasm, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of the onset of said condition.
  • a related aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the progression of said neoplasm to a higher grade said method comprising morphologically assessing said neoplasm wherein: (i) the presence of cells characterised by an optically non-clear cytoplasm, enlarged nucleus and/or enlarged nucleoli is indicative of said neoplasm progressing to a higher grade; and
  • a further aspect of the present invention therefore provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the progression of said neoplasm to a higher grade said method comprising morphologically assessing said prostate neoplasm wherein:
  • Yet another aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the onset of a condition characterised by a higher grade neoplasm, said method comprising morphologically assessing said neoplasm wherein:
  • Yet another aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the onset of a condition characterised by a higher grade neoplasm, said method comprising morphologically assessing said neoplasm wherein:
  • activin ⁇ A increased levels of protein and/or gene expression of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin correlates to a high level neoplasm.
  • a method of classifying a moderate grade prostate neoplasm comprising morphologically assessing said neoplasm wherein: (i) the presence of cells characterised by an optically non-clear cytoplasm, enlarged nucleus and/or enarlged nucleoli correlates to a high level neoplasm;
  • activin ⁇ A increased levels of protein and/or gene expression of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin correlates to a high level neoplasm.
  • Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting the marker proteins or encoding nucleic acid molecules and reagents useful for facilitating the detection by the agent in the first compartment.
  • Figure 1 shows immunolocalisation of inhibin ⁇ (A), activin ⁇ c (B), follistatin 315 (C) and activin ⁇ A (D) protein in serial sections of human prostate cancer patient tissue.
  • "Low” grade 3 cells are identified morphologically as cells with nuclei that are a similar size or slightly larger than a nucleus in a normal benign cell; an absent or a small nucleolus and optically clear cytoplasm. In these "low” grade 3 cells the nucleus is placed towards the base of the cell.
  • "High” grade 3 prostate cancer cells have enlarged nuclei and nucleolus compared to normal/ benign or “low” grade 3 cells. The cytoplasm is a pink-blue color (arrowhead) and the nuclei are placed more centrally when compared to the "low” grade 3 prostate tumour cells.
  • A "Low” grade 3 prostate tumour cells do not display inhibin ⁇ subunit protein staining, in contrast to "high” grade 3 prostate tumour cells which immunolocalise this protein to the cytoplasm.
  • B Activin ⁇ c subunit protein imunolocalises to the smooth muscle cells and the cytoplasm of "high” grade 3 prostate tumour cells. In contrast, “low” grade 3 prostate tumour cells do not display any activin ⁇ c subunit protein staining.
  • C Follistatin 315 protein positively immunolocalises to the cytoplasm of "high” grade 3 prostate tumour cells; however no staining is present in the "low” grade 3 prostate tumour cells.
  • D "Low” grade 3 prostate tumour cells do not display activin ⁇ subunit protein staining, in contrast to "high” grade 3 prostate tumour cells which display cytoplasmic staining of this protein.
  • Figure 2 shows the percentage of activin ⁇ c protein staining in either "low” and “high” grade 3 prostate tumour cells in radical prostatectomy tissue sections.
  • Open square symbols represent the percentage of activin ⁇ c protein staining in areas of "low” grade 3 tumour cells.
  • Closed triangular symbols represent the percentage of activin ⁇ c protein staining in areas of "high” grade 3 tumour cells.
  • Areas of "low” grade 3 tumour cells predominantly displayed a lower percent of cells with activin ⁇ c protein staining (between 0-40%), while "high” grade 3 tumour cells displayed between 90-100% of cells with activin ⁇ c protein staining.
  • Figure 3 shows visually the separation of these protein markers (inhibin ⁇ (A), activin ⁇ c (B), follistatin 315 (C) and activin ⁇ A (D)) with predominantly higher intensity of staining observed in "high” grade 3 prostate tumour cells and predominantly less intensity of staining observed in "low” grade 3 prostate tumour cells.
  • Figure 4 shows visually the predominantly high intensity of staining of these protein markers (inhibin ⁇ (A), activin ⁇ c (B), follistatin 315 (C) and activin ⁇ A (D)) in cribriform patterns (star symbols).
  • Figure 5 shows the expression of dimer proteins that contain the activin ⁇ c subunit in nom al human serum and prostate cancer patient serum using Western blot analysis. Proteins were separated by 15% SDS-PAGE gel. Lane 1 and 2: normal human patient #1; Lane 3 and 4: prostate cancer patient serum with a Gleason score of 6, Lane 5 and 6: prostate cancer patient serum with a Gleason score of 6, Lane 7 and 8: prostate cancerpatient serum with unknown Gleason score; Lane 9: normal human patient #2. In lanes 1 and 2, normal serum displayed a single band of approximately 20 kDa (arrow) was detected indicating the presence of either activin C ( ⁇ c- ⁇ c) or activin BC ( ⁇ rr ⁇ c) which have approximate sizes of 20 and 21 kDa, respectively.
  • activin C ⁇ c- ⁇ c
  • activin BC ⁇ rr ⁇ c
  • the approximately 23 kDa sized band may be indicative of the presence of activin AC ( ⁇ A- ⁇ c) that has an approximate size of 23 kDa.
  • the approximately 25-28 kDa sized band may also be a dimer of the activin ⁇ c subunit protein.
  • Lane 9 normal serum displayed a single band of approximately 20 kDa band (arrow) was detected indicating the presence of either activin C ( ⁇ c- ⁇ c) or activin BC ( ⁇ - ⁇ c) which have approximate sizes of 20 and 21 kDa, respectively, as well as a faint band at approximately 23 kDa sized band may be indicative of the presence of activin AC ( ⁇ A - ⁇ c) that has an approximate size of 23 kDa.
  • Figure 6 shows the expression of activin ⁇ c subunit protein dimers in normal human serum and prostate cancer patient serum using Western blot analysis. Proteins were separated by 15% SDS-PAGE gel. Lane 1: prostate cancer patient (#1) serum; Lane 2: prostate cancer patient (#2) serum. In lanes 1 and 2 multiple bands were detected in patients with prostate cancer at approximately 20 - 21 kDa (arrow), and 23kDa (arrow) and 25-26 kDa (arrow).
  • the 20 - 21 kDa band may be indicative of activins C or BC
  • the 23 kDa sized band may be indicative of the presence of activin AC ( ⁇ A - ⁇ c)-
  • the approximately 25-26 kDa sized band may be a dimer of the activin ⁇ c subunit which was not present in normal serum.
  • Lane 4 male normal (#1) serum
  • Lane 2 male normal (#2) serum.
  • normal serum displayed one bands of approximately 20-21 kDa band (arrow) indicating the presence of either activin C ( ⁇ c - ⁇ c) or activin BC ( ⁇ - ⁇ c) which have approximate sizes of 20 and 21 kDa respectively.
  • a band of 23 kDa (arrow) was detected in normal serum, indicating the presence of activin AC, although this was at lower levels than prostate cancer patient serum in lanes 1 and 2.
  • Figure 7 shows the expression of inhibin ⁇ subunit protein dimers in normal human serum and prostate cancer patient serum using Western blot analysis. Proteins were separated by 15% SDS-PAGE gel. Lane 1: normal male serum (#1). Lane 3: prostate cancer patient serum (#1). Lane 3: prostate cancer patient serum (#3). Lane 4: prostate cancer patient serum (#4). Inhibin ⁇ subunit protein was detected at a size of approximately 26 kDa in prostate cancer patient serum samples in Lane 2 and Lane 4. Inhibin ⁇ subunit protein was undetectable normal serum (Lane 1) and prostate cancer patient serum (Lane 4) demonstrating that inhibin ⁇ subunit protein is expressed differentially in prostate cancer serum.
  • Figure 8 shows western immunoblot analysis by double labelling for activin ⁇ A subunit and activin ⁇ C subunit.
  • A Single channel image labelled by E4 antibody (activin ⁇ A, channel 800).
  • B Single channel image labelled by ⁇ C clone 1 antibody (activin ⁇ C, channel 700).
  • C Overlay two channels image of activin ⁇ A (green) and activin ⁇ C (red), with activin ⁇ A and activin ⁇ C dimer (yellow).
  • Lane 1 to 4 serum from normal males; Lane 5: prostate cancer patient with Gleason grade sum 6 (3+3) whose cancer did not metastasise; Lane 6: prostate cancer patient; Lane 7: prostate cancer patient; Line 8: prostate cancer patient; Line 9: prostate cancer patient.
  • the present invention is predicated, in part, on the finding that moderate grade cancers can be differentiated into those likely to become aggressive (high level grade 3) versus those not likely to become aggressive (low level grade 3) based on differences in cellular morphology and/or expression of one or more of inhibin ⁇ , activin ⁇ A , activin ⁇ c or follistatin. This is despite the common knowledge that moderate grade cancers are extremely difficult and unpredictable to differentiate in terms of their likely aggression. Accordingly, the present determination has now facilitated the development of highly sensitive and informative means of predicting moderate grade cancer progression.
  • one aspect of the present invention provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the progression of said neoplasm to a higher grade, said method comprising screening for the level of one or more of activin ⁇ A, activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said neoplasm progressing to a higher grade.
  • 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.
  • 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.
  • 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.
  • 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.
  • the neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell.
  • neoplasms include, but are not limited to, those of the pancreas, brain and neural tissue, adrenal gland, thyroid gland, stomach, colon, urinary bladder, endometrium, breast, lymph node, skin, salivary gland, bone, nasal cavity, duodenum, gallbladder, uterine cervix, thymus, spleen, seminal vesicle, larynx, tongue, small intestine, rectum, oesophagus, or soft tissue, fallopian tube, testis, ovary, kidney, pituitary, lung, uterus, tonsil, appendix or myometrium.
  • said neoplasm is a prostate cell neoplasm.
  • the present invention therefore more particularly provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the progression of said prostate neoplasm to a higher grade, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said prostate neoplasm progressing to a higher grade.
  • neoplasm "grade” 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. Without limiting the present invention to any one theory or mode of action, the
  • Gleason system is based on the architectural pattern of the glands of the prostate tumour.
  • tumours 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: (i) Gleason Grades 1 and 2
  • 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. A tabulated representation of the Gleason grading system is provided in Table 3.
  • 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.
  • a “moderate grade” neoplasm should be understood as a reference to a neoplasm which comprises regions of Gleason grade 3 neoplastic cells (herein referred to as a "grade 3 prostate neoplasm"). More preferably, said moderate grade neoplasm is one in which the grade 3 classification forms part of the Gleason score. Accordingly, in a most preferred embodiment, said moderate grade prostate neoplasms are those with a Gleason score of 3+1, 3+2, 3+3 or 3+4.
  • the present invention therefore preferably provides a method of detecting or monitoring a predisposition, in a mammal with a Gleason grade 3 prostate neoplasm, to the progression of said prostate neoplasm to a higher grade, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said Gleason grade 3 prostate neoplasm progressing to a higher grade.
  • said grade 3 regions form part of the Gleason score of the prostate neoplasm in issue and, even more preferably, said neoplasm corresponds to a Gleason score of 3+1, 3+2, 3+3 or 3+4.
  • the method of the present invention provides a means of predicting whether or not a moderate grade neoplasm will progress to a higher grade.
  • 'higher grade should be understood a Gleason grade of 4 or 5, or equivalent grade thereof. In this regard, it should therefore be understood that progression to a higher grade is a reference to the progression of the grade 3 cells of the neoplasm to a higher grade.
  • the Gleason score of the tissue may or may not progress to a score which is usually associated with aggressive growth.
  • the grade 3 cells of a tissue with an initial score of 3+2 undergoes transition to a higher grade
  • the grade 3 cell component of the neoplasm has progressed to a higher Gleason grade
  • the neoplasm is regarded as having progressed to a higher grade within the definition of the present invention.
  • the moderate grade prostate neoplasm is one which has progressed to a neoplasm exhibiting the equivalent of a Gleason score of 8, 9 or 10, this correlating to an aggressive neoplastic state.
  • the present invention therefore preferably provided a method of detecting or monitoring a predisposition, in a mammal with a Gleason grade 3 prostate neoplasm, to the progression of said prostate neoplasm to a Gleason grade 4 or 5, said method comprising screening for the level of one or more of activin ⁇ A, activin ⁇ c, inhibin or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of said Gleason grade 3 prostate neoplasm progressing to a higher grade. Still more preferably, said higher grade neoplasm correlates to a prostate neoplasm of Gleason score 8, 9 or 10.
  • neoplasms as defined herein should be understood to correlate to neoplasms which are also routinely termed “advanced cancers", “aggressive cancers” and “metastatic cancers". It should also be understood that 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.
  • prostate cancer may develop subsequently to removal of the prostate.
  • Reference to the terms “malignant neoplasm” and “cancer” herein should be understood to be interchangeable. This is particularly relevant in the context of the present invention where any delay in removal of a prostate comprising Gleason grade 3 cells which are predisposed to progressing to a higher grade cancer may result in progression occurring during the period of delay, thereby potentially resulting in the appearance of metastases subsequently to removal of the prostate. Nevertheless, not every higher grade cancer falling within the definition provided herein will necessarily correlate to a metastatic cancer, although many will.
  • the progression of the grade 3 cellular component of a neoplasm in accordance with the present invention to a higher grade may or may not occur concomitantly with the progression of one or more of the other non-grade 3 cellular components to a higher grade or the appearance of cribriform patterns.
  • the method of the present invention is directed to differentiating moderate/grade 3 neoplastic cells into those which may ultimately progress and potentially lead to a poor outcome for a patient (i.e. progress to a higher grade/become aggressive) and those which may not.
  • the present invention is therefore directed to detecting the predisposition of a given grade 3 neoplastic cell to progress aggressively or not.
  • the present invention provides a useful means for performing ongoing testing of a patient in order to monitor for adverse changes to the nature of the grade 3 neoplasm, which changes, in accordance with the findings disclosed herein, predict a poor outcome due to likely progression to a higher grade neoplasm.
  • the more commonly used method is likely to be directed to the detection of increased levels of activin ⁇ A , activin ⁇ c, inhibin ⁇ and/or follistatin in order to classify moderate grade 3 neoplasms into those likely to progress to a higher grade versus those not likely to progress, one may nevertheless seek to monitor for the converse. For example, where a radical prostatectomy is not performed, one may seek to monitor for changes in the grade 3 classification of the prostate, and its prognostic implications in relation to the development of higher grade cancers, during the course of prophylactic or therapeutic treatment of the patient. Alternatively, patients presenting with symptoms of prostate disease or a genetic or environmental predisposition to the development of prostate disease may be monitored for the appearance of a grade 3 neoplasm and may be thereafter classified according to the predisposition classification of the present invention.
  • activin ⁇ A increases in the level of expression of activin ⁇ A , activin ⁇ c, inhibin ⁇ and/or follistatin in the context of moderate grade neoplasms are indicative of a predisposition to the progression of the moderate grade neoplasm to a higher grade.
  • 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”.
  • 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- ⁇ o),activin CE ( ⁇ c- ⁇ ) or inhibin C ( ⁇ - ⁇ c ) proteins. Accordingly, it should be understood that one may screen for activin ⁇ c in its monomeric, homodimeric or heterodimeric form. A corresponding definition applies with respect to "activin ⁇ A M .
  • activin ⁇ c and activin (3 A have a structure similar to other activins and other members of the TGF ⁇ superfamily.
  • the structure of activins 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).
  • 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).
  • 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, multimer or fusion protein.
  • Multimeric protein forms of inhibin ⁇ include for example the heterodimeric ⁇ polypeptide (for example ⁇ A , ⁇ , ⁇ c 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.
  • follistatin 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 one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ and follistatin expression are modulated in moderate grade prostate neoplasms with a poor prognosis as compared to normal prostate tissue.
  • activin ⁇ A activin ⁇ A
  • activin ⁇ c activin ⁇ c
  • inhibin ⁇ follistatin expression
  • one may screen for changes to the levels of those molecules at either the protein or the encoding nucleic acid molecule level.
  • reference herein to screening for the level of one or more of "activin ⁇ A, activin ⁇ c, inhibin ⁇ and follistatin should be understood to include reference to screening for either the protein or its encoding primary RNA transcript or mRNA.
  • Control levels may be either “normal” levels or the levels of corresponding moderate grade neoplastic cells.
  • 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 grade 3 neoplasm nor is predisposed to developing a grade 3 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 the analysis of test results relative to a standard result which reflects individual or collective 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 monitored by either quantitative or qualitative readouts.
  • the reference level may also vary between individual forms (such as differently processed forms) of these molecules.
  • references to the levels of "corresponding moderate grade neoplastic cells” should be understood as a reference to the levels which one observes or detects in any other moderate grade prostate neoplastic cell, 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 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” and “modulation” refer to increases and decreases in levels relative either to a control reference level (or control reference level range) or to an earlier result determined from the patient in issue, this latter reference point being particularly relevant in the context of the ongoing monitoring of a patient.
  • the present invention is predicated on differentiating the predisposition of a moderate grade neoplasm, in particular a prostate neoplasm, to progress to a higher grade (or not), in addition to detecting the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin one must also establish the existence of a grade 3 neoplasm, relative to which these levels are analysed. This may be achieved by any suitable method which could be designed and implemented by the person of skill in the art.
  • tissue section such as a prostate tissue section
  • a tissue section such as a prostate tissue section
  • a morphological analysis of the cell types which are present and the levels of activin ⁇ A , activin ⁇ c, inhibin ⁇ and/or follistatin protein or expressed gene levels.
  • Means of screening for the presence of moderate grade neoplastic cells can be achieved by any suitable method which would be well known to the person of skill in the art. For example one may harvest a population of cells for analysis via organ biopsy (for example, a needle biopsy), organ removal or aspiration. Where a section of intact organ is available, one may prepare tissue sections for morphological analysis - such tissue sections may be prepared in any suitable manner such as in the form of frozen sections or wax embedded sections. To the extent that needle biopsies are utilised, one may nevertheless harvest tissue of sufficient size to enable analysis of the tissue's architecture. To the extent that cellular aspirates are harvested, it may be necessary to render the cells a single cell suspension and analyse the morphology of a population of cells derived therefrom.
  • organ biopsy for example, a needle biopsy
  • tissue removal or aspiration Where a section of intact organ is available, one may prepare tissue sections for morphological analysis - such tissue sections may be prepared in any suitable manner such as in the form of frozen sections or wax embedded sections.
  • 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, inter 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 biological material derived from an individual such as, but not limited to, mucus, stool, urine, blood, serum, cell extract, biopsy specimens and fluid which has been introduced into the body of an individual and subsequently removed such as, for example, the saline solution extracted from the lung following lung lavage or the solution retrieved from an enema wash.
  • 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. For example, a biopsy sample may require homogenisation or sectioning prior to testing.
  • Means of screening for changes in activin ⁇ A , activin ⁇ c, inhibin ⁇ and/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: (i) In vivo detection of the markers. Molecular Imaging may be used following administration of imaging probes or reagents capable of disclosing altered expression levels of the markers mRNA or protein expression product in the prostate tissues.
  • Molecular imaging (Moore, A., Basilion, J., Chiocca, E., and Weissleder, R., BBA, 1402:239-249, 1988; Weissleder, R., Moore, A., Ph.D., Mahmood-Bhorade, U., Benveniste, H., Chiocca, E.A., Basilion, J.P. Nature Medicine, 6:351-355, 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), MRI, Positron Emission Tomography (PET) or endoscopy.
  • CT computed tomography
  • PET Positron Emission Tomography
  • FISH Fluorescent In Situ Hybridization
  • QRTPCR Quantitative Reverse Transcriptase Polymerase Chain Reaction
  • Flow cytometric qualification of competitive RT-PCR products Wedemeyer, N., Potter, T., Wetzlich, S. and Gohde, 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 the prostate.
  • a nucleic acid extract from the animal is utilized in concert with oligonucleotide primers corresponding to sense and antisense sequences of a polynucleotide encoding the markers, or flanking sequences thereof, in a nucleic acid amplification reaction such as RT PCR, real time PCR or SAGE.
  • RT PCR real time PCR
  • SAGE nucleic acid amplification reaction
  • NLSIPSTM very large scale immobilized primer arrays
  • R ⁇ A is isolated from a cellular sample suspected of containing the markers 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 the markers 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 the markers.
  • the reaction mixture is typically subjected to agarose gel electrophoresis or other convenient separation technique and the relative presence of the markers specific amplified DNA detected.
  • the markers 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 the markers DNA may be accomplished by excising or eluting the fragment from the gel (for example, see references Lawn et ah, 1981; Goeddel et al., 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 markers. The relative amounts of the markers 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
  • 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. The antigen in this case is the markers or a fragment thereof.
  • 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.
  • 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 :-
  • the reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemilumine scent 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 Other exemplary fluorochromes include those discussed by Dower et al., 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 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896,
  • 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.
  • Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting the marker proteins or encoding nucleic acid molecules 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.
  • the method of the present invention has widespread applications including, but not limited to, the diagnosis, in an individual with a moderate grade neoplasm, or a predisposition to the progression of this neoplasm to a higher grade and, further, the predisposition to the development of a condition characterised by said higher grade neoplasm such as, in the context of a prostate cancer for example, urine retention, haematuria, urinary incontinence, kidney failure, bone pain, bone fragility, spinal cord damage, osteoarthritis, lethargy, loss of appetite, nausea, diarrhoea, constipation or cachexia.
  • a condition characterised by said higher grade neoplasm such as, in the context of a prostate cancer for example, urine retention, haematuria, urinary incontinence, kidney failure, bone pain, bone fragility, spinal cord damage, osteoarthritis, lethargy, loss of appetite, nausea, diarrhoea, constipation or cachexia.
  • yet another aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the onset of a condition characterised by said higher grade neoplasm, said method comprising screening for the level of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of the onset of said condition.
  • the present invention provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the onset of a condition characterised by a higher grade prostate neoplasm, said method comprising screening for the level of one or more of activin ⁇ A, activin ⁇ c, inhibin ⁇ or follistatin protein and/or gene expression in said mammal wherein an increase in the level of said protein and/or gene expression is indicative of the onset of said condition.
  • said moderate grade neoplasm is a Gleason grade 3 neoplasm and even more preferably a neoplasm which corresponds to a Gleason score of 3+1, 3+2, 3+3 or 3+4. Still more preferably, said higher grade neoplasm is a Gleason grade 4 or 5 neoplasm which, most preferably, correspond to a Gleason score of 8, 9 or 10.
  • said neoplasm is a prostate neoplasm and said condition is urine retention, haematuria, urinary incontinence, kidney failure, bone pain, bone fragility, spinal cord damage, osteoarthritis, lethargy, loss of appetite, nausea, diarrhoea, constipation or cachexia.
  • the inventors have also determined that the differentiation of moderate grade cancers into those which exhibit a predisposition to progressing to a higher grade and those which do not can also be determined via morphological analysis of a target cell population. Accordingly, this provides either an alternative means of characterising the moderate grade neoplastic cells in issue or an additional test to be performed in concert with the marker analysis hereinbefore described.
  • This morphological method is based on the determination that moderate grade prostate neoplastic cells which exhibit a predisposition to progressing to a higher grade exhibit morphological features which are distinct from those that do not exhibit this predisposition.
  • a related aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the progression of said neoplasm to a higher grade said method comprising morphologically assessing said neoplasm wherein: (i) the presence of cells characterised by an optically non-clear cytoplasm, enlarged nucleus and/or enlarged nucleoli is indicative of said neoplasm progressing to a higher grade; and
  • the morphological analysis upon which the Gleason grading system is based is directed to applying a standard set of criteria to categorise the cells of a tissue. These criteria are based on the pattern of growth of the cells, in particular the pattern of infiltration of the cells through the tissue.
  • the Gleason sub- grouping system which divides the Grade 3 patterns into 3A, 3B and 3C, is based on the structure and architecture of the gland. For example, under the Gleason system, Grade 3 A is defined by large acini, 3B is defined by small acini and 3C is defined by cribriform.
  • the Gleason system is based on analysing acini architecture and not morphological characteristics.
  • Bostwick and Gleason described the cytoplasm of the cells comprising these three Grade 3 subdivisions as "more basophilic than patterns 1 or 2". This indicates that they regard the cytoplasm of all three grades as blue in colour and make no reference to the existence of any change between them. However, merely examining these characteristics does not enable one to differentiate between the potential differentiative capacity of the moderate grade cancers in terms of their progression to higher grade cancers.
  • the present invention therefore more particularly provides a method of detecting or monitoring a predisposition, in a mammal with a moderate grade prostate neoplasm, to the progression of said neoplasm to a higher grade said method comprising morphologically assessing said prostate neoplasm wherein:
  • references to an "enlarged” or “non-enlarged” nucleus should be understood as a reference to the size of the subject nucleus relative either to a corresponding normal cell or to other moderate grade cells present in the tissue sample. That is, a nucleus that is substantially larger than that observed in a corresponding non-neoplastic cell is defined as "enlarged”. Cells which are defined as “non-enlarged” are those which exhibit a nucleus size similar to that of a corresponding non-neoplastic cell. In this regard, the nucleus of the Grade 3 cell under examination may be very slightly larger than that of a corresponding non-neoplastic cell but, for the purpose of the present invention, fall within the scope of the term "non- enlarged”.
  • any given tissue sample will generally comprise populations of both low level moderate grade neoplastic cells and high level moderate grade neoplastic cells, although this will not always be the case (the terms "low level” and "high level” in the context of the moderate grade neoplastic cells is defined hereinafter but, briefly, correspond to the moderate grade neoplastic cells which are not likely to progress to higher grade versus those which are likely to progress to a higher grade, respectively).
  • the low level moderate grade neoplastic cells comprise a nucleus which is positioned towards the base of the cell whereas the high level moderate grade neoplastic cells comprise a nucleus which is generally placed more centrally than that observed in respect of the lower level moderate grade neoplastic cells.
  • the nucleolus is a small, dense body, one or more of which can be present in the nucleus of eukaryotic cells.
  • Nucleoli are rich in RNA and protein. They are not bounded by membrane and are not observed during mitosis.
  • the presence of enlarged nucleoli in moderate grade neoplastic cells is indicative of the predisposition of those cells to progressing to a higher grade neoplasm.
  • the total absence of nucleoli or the presence of small nucleoli is indicative of a predisposition not to progress to a higher grade neoplasm.
  • nucleoli can be performed either relative to normal cells or, more preferably, relative to other moderate grade neoplastic cells which are present in the tissue which is the subject of examination.
  • the nucleoli of the moderate grade neoplastic cells in any given tissue will generally clearly fall into either a larger sized category or a smaller sized category when analysed relative to one another. Still further, even if analysed relative to the nucleoli of normal or benign cells, the nucleoli of the high level moderate grade neoplastic cells will be larger in size. It should also be understood that in the low level moderate grade neoplastic cells, it may occur that there are no nucleoli present, although it should nevertheless be understood that some cells may exhibit one or more small nucleoli.
  • cells which are undergoing a high turnover rate are characterised by the presence of RNA and proteins in the cytoplasm. Accordingly, histological staining techniques will usually enable visualisation of the presence of RNA and/or protein in the cytoplasm.
  • Reference to "optically clear” and “optically non-clear” should therefore be understood as a reference to whether or not the cytoplasm of a cell appears clear subsequently to a haemotoxylin and eosin staining procedure, or equivalent staining procedure.
  • haemotoxylin binds to basophilic proteins in the nucleus (and therefore nuclear proteins which have leaked into the cytoplasm will also be bound by haemotoxylin) while eosin generally binds to cytoplasmic proteins. Accordingly, in high level moderate grade neoplastic cells, the nucleus will appear blue/pink and possibly grainy in appearance due to the presence of RNA and protein in the cytoplasm. However, the low level moderate grade neoplastic cells will comprise either no cytoplasmic content when analysed in accordance with a haemotoxylin and eosin-like staining procedure or will exhibit a very minimal cytoplasmic content.
  • haemotoxylin and eosin like staining procedure should be understood as a reference to any staining procedure which achieves the same functional outcome as haemotoxylin and eosin in terms of binding to and enabling visualisation of the RNA and protein content which may be present in a cytoplasm. Accordingly, although the haemotoxylin and eosin staining technique is the preferred staining technique, the person skilled in the art may substitute any other suitable staining technique which achieves the same functional objective. It will also be appreciated that in performing this type of histological stain, the subject tissue sections have normally been fixed.
  • any given histological characteristic may be made with reference to a normal cell or, more preferably, the analysis is made relative to other corresponding moderate grade neoplastic cells since the relative comparison of these neoplastic cells results in clear identification of the sub-group to which any given neoplastic cell belongs.
  • tissue sections are treated with a haematoxylin-eosin stain.
  • Alternative stains include, but are not limited to Masson Trichrome, PAS (periodic acid schif), alcian blue.
  • Suitable fixation methods include, but are not limited to formalin, glutaraldehyde fixation or frozen section.
  • said moderate grade neoplasm is a Gleason grade 3 neoplasm and even more preferably a neoplasm which corresponds to a Gleason score of 3+1, 3+2, 3+3 or 3+4. Still more preferably, said higher grade neoplasm is a Gleason grade 4 or 5 neoplasm which, most preferably, corresponds to a Gleason score of 8, 9 or 10.
  • said neoplasm is a prostate neoplasm.
  • Yet another aspect of the present invention is directed to a method of detecting or monitoring a predisposition, in a mammal with a moderate grade neoplasm, to the onset of a condition characterised by a higher grade prostate neoplasm, said method comprising morphologically assessing said neoplasm wherein:
  • said moderate grade neoplasm is a Gleason grade 3 neoplasm and even more preferably a neoplasm which corresponds to a Gleason score of 3+1, 3+2, 3+3 or 3+4. Still more preferably, said higher grade neoplasm is a Gleason grade 4 or 5 neoplasm which, most preferably, corresponds to a Gleason score of 8, 9 or 10.
  • said neoplasm is a prostate neoplasm and said condition is urine retention, haematuria, urinary incontinence, kidney failure, bone pain, bone fragility, spinal cored damage, osteoarthritis, lethargy, loss of appetite, nausea, diarrhoea, constipation or cachexia.
  • activin ⁇ A increased levels of protein and/or gene expression of one or more of activin ⁇ A , activin ⁇ c, inhibin ⁇ or follistatin correlates to a high level neoplasm.
  • references to "high level” should be understood as a reference to a “higher grade” as hereinbefore defined.
  • Reference to “low level” is a reference to a neoplasm which does not exhibit a predisposition to progress to a higher grade.
  • said moderate grade neoplasm is a Gleason grade 3 neoplasm and even more preferably a neoplasm which corresponds to a Gleason score of 3+1, 3+2, 3+3 or 3+4.
  • said high level neoplasm is a Gleason grade 4 or 5 neoplasm which, most preferably, corresponds to a Gleason score of 8, 9 or 10.
  • said neoplasm is a prostate neoplasm.
  • Immunohistochemistry was performed on radical prostatectomy tissue from 28 prostate cancer patients with Gleason Grade Score of greater than or equal to 7. After being de- paraffinated the tissue underwent a pretreatment step of microwave heating in 0.1M 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-anti- mouse-horse radish peroxidase (DAKO, Carpinteria, USA) for 15 minutes and visualised by reaction with diaminobenzidine (DAB) (DAKO, Carpinteria, USA) for 5 minutes.
  • DAKO Envision polymer-anti- mouse-horse radish peroxidase
  • DAB diaminobenzidine
  • Activin ⁇ c, activin ⁇ A, follistatin 315, and inhibin ⁇ staining intensity was recorded by histopathologists in "low” grade 3 tumour cells, "high” grade 3 tumour cells and cribriform pathologies.
  • Inhibin ⁇ (A), activin ⁇ c (B), follistatin 315 (C) and activin ⁇ A (D) protein localisation was investigated in radical prostatectomy tissue from patients with prostate cancer.
  • Figure 1 is representative of observations that "low" grade 3 prostate tumour cells display little or no staining of inhibin ⁇ , activin ⁇ c, follistatin 315 and activin ⁇ A proteins (arrow) in contrast to "high" grade 3 prostate tumour cells which have stronger cytoplasmic staining (arrowhead).
  • EXAMPLE 8 Method SDS-page was performed under non reducing conditions using 15% polyacrylamide gel. ⁇ Human normal serum and prostate cancer patient serum samples were diluted 1 :3 with 4% SDS, boiled at 100°C in heat block for 5 minutes and supernatants were collected. Inhibin ⁇ PO#12 antibody was added at 1:6000 in 5% milk in lxTBS overnight at 4°C. The serum samples, treated with 4% SDS, were diluted in 1:2 in sample buffer (Tris, P/o SDS, glycerol, and 0.01% bromophenol blue, pH 7.2).
  • the membrane was blocked (5% Non-fat milk powder, 0.05% Tween in lx TBS) for 90 minutes.
  • Activin ⁇ c clone 1 antibody was added at 1:6000 in 5% milk in lxTBS overnight at 4°C.
  • the membrane was incubated with goat anti-mouse HRP 1: 10,000 in 5% milk in TBS for 45 minutes at RT.
  • ECL plus substrate was added according to manufacturer's instructions.
  • the membrane was placed in an x- ray cassette and exposed to X-Omat film (Kodak).
  • Serum from three prostate cancer patients with a Gleason score of 6 (with one patient with unknown Gleason), displayed multiple bands at approximately 20-21 kDa, 23 kDa and 25-28 kDa (Lanes 3-8). It could be predicted that the approximately 20-21 kDa bands are activin C or BC, 23 kDa band is activin AC and the 25-28 kDa sized band also may be a dimer of the activin ⁇ c subunit.
  • activin ⁇ C subunit dimers are present in serum from men with prostate cancer. Additionally, these dimers are larger and more intense than the activin ⁇ C subunit dimers present in serum
  • FIG. 6 demonstrates that activin ⁇ C dimer proteins have different forms and are at a higher concentration in serum from men with prostate cancer compared to serum from normal males.
  • Prostate cancer patient serum (Lane 1 and 2) displayed three bands at sizes of approximately 20-21 kDa, predicted to be activins C or BC; 23kDa; 23kDa predicted to be activin AC and a larger 25-28kDa activin ⁇ C subunit dimer.
  • normal male serum (Lane 3 and 4) contained activin ⁇ C bands at 20 kD and a weaker 23kDa band, however no activin ⁇ C subunit dimer band at 25-28 kDa was undetectable.
  • inhibin subunit protein is expressed at different levels in serum from patients with prostate cancer as compared to normal serum.
  • Inhibin subunit protein at a size of approximately 26kD, was detected in serum from two patients with prostate cancer (Lane 2 and 4).
  • inhibin ⁇ subunit protein was undetectable in normal male serum (Lane 1) and another prostate cancer patient (Lane 3).
  • Cipriano S. C Chen, L., Kumar, T. R. and Matzuk, M. M. (2000), Endocrinology, 141, 2319-27
  • Table 1 corresponds to Figure 3 describing the mean intensity of staining of inhibin ⁇ (A), activin ⁇ c (B), follistatin 315 (C) and activin ⁇ A (D) in "low” or "high” grade 3 areas of prostate cancer cells.
  • Table 2 corresponds to Figure 4 describing the mean intensity of staining of inhibin ⁇ (A), activin ⁇ c (B), follistatin 315 (C) and activin ⁇ A (D) in Grade 3/4 cribriform patterns observed in seven patients. High intensity of staining was observed for inhibin ⁇ protein (3.455), activin ⁇ c protein (3.95), follistatin 315 protein (4.0) and activin ⁇ A protein (3.85) markers.

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Abstract

L'invention concerne de manière générale un procédé destiné à pronostiquer ou à réguler le développement ou la progression d'un cancer à degré modéré vers un cancer à degré supérieur chez un mammifère. Plus particulièrement, l'invention concerne un procédé destiné à pronostiquer ou à réguler le développement ou la progression d'un cancer de la prostate à degré modéré vers un cancer de la prostate à degré plus élevé chez un mammifère. Le procédé de l'invention est basé sur l'analyse des niveaux d'expression génique/protéique et/ou des caractéristiques morphologiques cellulaires. L'invention concerne également des agents de diagnostic utilisés dans le procédé de l'invention. Un des aspects de cette invention concerne un procédé de classification des cancers à degré modéré, notamment des cancers de la prostate, par cancers à niveau élevé ou à niveau faible sur la base de la morphologie cellulaire unique et/ou des niveaux d'expression génique ou protéique.
PCT/AU2005/000283 2004-02-27 2005-02-28 Procede de determination d'un pronostic pour des patients atteints d'un cancer a degre modere WO2005083438A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1756301A1 (fr) * 2004-04-16 2007-02-28 Monash University Procede destine a reguler la progression d'un cancer
EP1756301A4 (fr) * 2004-04-16 2008-01-23 Univ Monash Procede destine a reguler la progression d'un cancer

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