WO1999004238A2 - Reactifs, procedes de diagnostic et pronostic de troubles proliferatifs - Google Patents

Reactifs, procedes de diagnostic et pronostic de troubles proliferatifs Download PDF

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Publication number
WO1999004238A2
WO1999004238A2 PCT/US1998/014566 US9814566W WO9904238A2 WO 1999004238 A2 WO1999004238 A2 WO 1999004238A2 US 9814566 W US9814566 W US 9814566W WO 9904238 A2 WO9904238 A2 WO 9904238A2
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protein
cells
cancer
antibody
cell
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PCT/US1998/014566
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English (en)
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Michele Pagano
Giulio Draetta
Mark Rolfe
Massimo Loda
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Mitotix, Inc.
Deaconess Hospital
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Priority to AU84030/98A priority Critical patent/AU8403098A/en
Publication of WO1999004238A2 publication Critical patent/WO1999004238A2/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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • 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
    • C12Q2600/118—Prognosis of disease development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4739—Cyclin; Prad 1

Definitions

  • the cell division cycle is one of the most fundamental processes in biology which, in multicellular organisms, ensures the controlled generation of cells with specialized functions. Under normal growth conditions, cell proliferation is tightly regulated in response to diverse intra-and extracellular signals. This is achieved by a complex network of protooncogenes and tumor-suppresser genes that are components of various signal transduction pathways. Activation of a protooncogene(s) and/or a loss of a tumor suppresser gene(s) can lead to the unregulated activity of the cell cycle machinery. This, in turn, will lead to unregulated cell proliferation and to the accumulation of genetic errors which ultimately will result in the development of cancer (Pardee, Science 246:603-608. 1989).
  • CDKs cyclin-dependent kinases
  • CDK complexes are formed via the association of a regulatory cyclin subunit and a catalytic kinase subunit.
  • the combination of the kinase subunits such as cdc2, CDK2, CDK4 or CDK6
  • a variety of cyclin subunits such as cyclin A. Bl. B2, Dl, D2, D3 or E
  • the coordinated activation of these complexes drives the cells through the cell cycle and ensures the fidelity of the process (Draetta, Trends Biochem. Sci.
  • Each step in the cell cycle is regulated by a distinct and specific cyclin- dependent kinase.
  • complexes of CDK4 and D-type cyclins govern the early Gl phase ofthe cell cycle, while the activity of the CDK2/cyclin E complex is rate limiting for the Gl to S-phase transition.
  • the CDK2/cyclin A kinase is required for the progression through S- phase and the cdc2/cyclin B complex controls the entry into M-phase (Sherr, Cell 73:1059- 1065, 1993).
  • the CDK complex activity is regulated by mechanisms such as stimulatory or inhibitory phosphorylations as well as the synthesis and degradation of the kinase and cyclin subunits themselves.
  • a link has been established between the regulation of the activity of cyclin-dependent kinases and cancer by the discovery of a group of CDK inhibitors including the p ⁇ 6 INK ⁇ , p ⁇ 5 INK4b , p ⁇ 8 INK4c , pl9/p20 / ⁇ ⁇ d 5 p2 ⁇ Wafl/Cipl ; p 27Kipl and p57 ki P 2 proteins.
  • the activity of p21 is regulated transcriptionally by DNA damage through the induction of p53, senescence and quiescence (Harper et al., Cell 75:805-816, 1993).
  • the inhibitory activity of p27 is induced by the negative growth factor TGF- ⁇ and by contact inhibition (Polyak et al., Cell 78:66-69, 1994).
  • TGF- ⁇ negative growth factor
  • contact inhibition Polyyak et al., Cell 78:66-69, 1994.
  • These proteins when bound to CDK complexes, inhibit their kinase activity, thereby inhibiting progression through the cell cycle. It is generally accepted that binding of these inhibitors to the CDK/cyclin complex prevents its activation. Alternatively, these inhibitors may interfere with the interaction ofthe enzyme with its substrates or its cofactors.
  • the INK4 proteins e.g., pi 6, pi 5, pi 8 and pi 9 block exclusively the activity of the CDK4/cyclin D and CDK6/cyclin D complexes in the early Gl phase (Serrano et al., Nature 366:704-707 ', 1993), by either preventing the interaction of CDK4 and Cyclin Dl, or indirectly preventing catalysis.
  • the p21 is positively regulated by the tumor suppresser p53 which is mutated in approx. 50% of all human cancers.
  • p21 may mediate the tumor suppresser activity of p53 at the level of cyclin-dependent kinase activity
  • pi 6 is the product of a tumor suppresser gene localized to the 9p21 locus, which is frequently mutated in human cancer cells.
  • One aspect of the present invention relates to diagnostic assays for determining, in the context of cells isolated from a patient, the protein level of a CDK inhibitor, which level can be a useful diagnostic/prognostic marker for risk assessment and phenotyping cell and tissue samples.
  • the subject assay provides a method for determining if an animal is at risk for a disorder characterized by aberrant cell proliferation, differentiation and/or apoptosis, and also may be used for prognostic purposes when such aberrant cell phenotypes are known.
  • the subject method can be used for diagnosing a hyperproliferative disorder in a patient which disorder is associated with the destabilization of a CKI protein, such as p27 ki P', in cells of the patient, comprising: (i) ascertaining the level of a CKI protein in a sample of cells from the patient; and (ii) diagnosing the presence or absence of a hyperproliferative disorder utilizing, at least in part, the ascertained level of the CKI protein, wherein a reduced level of CKI protein in the sample, relative to a normal control sample of cells, correlates with the presence of a hyperproliferative disorder.
  • a CKI protein such as p27 ki P'
  • the subject method is a prognostic method for evaluating a cancer patient's risk of death and/or recurrence of a cancer, comprising (i) ascertaining the level of a CKI protein in a sample of cancer cells from the patient; and (ii) predicting the patient's risk of death and/or recurrence of a cancer utilizing, at least in part, the ascertained level of CKI protein, wherein a reduced level of CKI protein in the sample, relative to a normal control sample of cells, correlates with an increased risk of death and/or recurrence of a cancer.
  • the present invention also provides a test kit for detecting stability CKI proteins comprising an antibody, or fragment thereof, which selectively binds to a CKI protein, e.g., the antibody selectively binds to the p27 protein of SEQ ID No. 2.
  • the antibody is labeled with a detectable label, e.g., with a label selected from the group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • the test kit will also include a nucleic acid probe for detecting the amount of CKI transcript in a cell sample.
  • FIG. 1 panels A-F, illustrates the expression of p27 in normal colon mucosa.
  • Colon mucosae (x200) oriented with the luminal surface toward the top was stained with hematoxylin (a), anti-p27 monoclonal antibody (B), anti-p27 monoclonal antibody blocked by preincubation with purified recombinant p27 (C), anti Ki-67 antibody (E), or probed with p27 cDNA (D).
  • Cells expressing p27 are stained, the nonepithelial cells that appear stained represent intramucosal T lymphocytes (B).
  • Panel F shows and immunoblot of normal mucosa (lanes 1 and 3) and recombinant purified his-tagged p27 (lanes 2 and 4) with anti-p27 monoclonal antibody (lanes 1 and 2) and anti-p27 monoclonal antibody preadsorbed with purified recombinant p27 (lanes 3 and 4).
  • FIG. 2 panels A-F, illustrates p27 expression in tumors in different stages of differentiation. Immunohistochemistry with anti-p27 antibody (x200).
  • A well differentiated colon carcinoma
  • B poorly differentiated colon carcinoma
  • C colon carcinoma with well differentiated (right) and poorly differentiated (left) areas.
  • D-F moderately differentiated colon carcinoma stained with hematoxylin
  • D anti-p27 monoclonal antibody
  • E anti-p27 monoclonal antibody blocked by preincubation with purified recombinant p27
  • Figures 3A-C illustrate that the lack of p27 is associated with poor survival in patients with colorectal carcinoma.
  • A Life table analysis with expression of p27 as absent, weak (1- 50% of positive tumors cells), or strong (>50% of positive tumor cells) in total number of tumors;
  • B life table analysis with expression of p27 scored as absent or present in total number of tumors;
  • C life table analysis with expression of p27 as absent, weak (1-50% of positive tumors cells), or strong (>50% of positive tumor cells) in stage II tumors.
  • Figure 4 illustrates that p27 mRNA and protein expression do not always correlate.
  • A- C p27 mRNA detection by in situ hybridization (400X);
  • B-D immunohistochemistry with anti-p27 Mab (400X).
  • Figure 5 illustrates the kinetics of p27 degradation in colorectal carcinoma samples.
  • Purified recombinant p27 was incubated for the indicated times either alone (A) or in the presence of extracts from tumors expressing high (B), moderate (C) or low (D) p27 levels.
  • Samples in panels E and F were incubates with either proteasome-depleted (E) or proteasome reconstituted (F) extract from a tumor having low p27 protein levels.
  • the reaction products were analyzed by immunoblotting as Pagano et al. (1995) Science 269:682-685 with anti-p7 Mab.
  • Figure 6 is a table showing the correlation between p27 abundance and p27 degradation activity in human colorectal carcinomas.
  • Bank # is the number assigned to the samples at the Deaconess Hospital tumor bank. Protein levels were assessed by immunoblot: +++. detection of a very strong signal; ++, detection of a strong signal; + detection of a very weak signal; -, no signal detected even after long exposure; ND, not determined. The relative intensity of the immunoblotting signals was the average of several experiments.
  • ++ corresponds to the p27 amount found in extracts from normal human fibroblasts. Degradation activity: +++, completely degraded between 30 minutes and 3 hours incubation; ++, degraded between 6 hours and an overnight incubation; +. stable even after overnight incubation (see figure 5 for examples).
  • +++ corresponds to the p27 degradation activity found in extracts from normal human fibroblasts (Pagano et al., supra).
  • Figure 7 is a table showing univariate analysis of potentially prognostic markers.
  • Figure 8 represents a table showing clinico-pathologic features of study population in relation to p27 status.
  • Figure 9 is a table showing multivariate analysis of potential prognostic markers by survival, using Cox models.
  • Progression through the cell-cycle is marked by a series of irreversible transitions that separate discrete tasks necessary for faithful cell duplication. These transitions are negatively regulated by signals that constrain the cell-cycle until specific conditions are fulfilled. Entry into mitosis, for example, is inhibited by incompletely replicated DNA or DNA damage. These restrictions on cell-cycle progression are essential for preserving the fidelity of the genetic information during cell division.
  • the transition from G ] to S phase coordinates cell proliferation with environmental cues, after which the checks on the cell-cycle progression tend to be cell autonomous.
  • the signals that restrict cell-cycle progression during G j are extracellular proteins which inhibit cell proliferation, growth factor or amino acid depletion, and cell-cell contact. Disruption of these signaling pathways uncouples cellular responses from environmental controls and may lead to unrestrained cell proliferation.
  • Eukaryotic cells in general, require cyclin-dependent kinases (CDKs) for progression through G ] and entry into S phase.
  • CDKs cyclin-dependent kinases
  • both D-and E-type cyclins are rate limiting for the G ] to S transition, and both reduce, but do not eliminate, the cell's requirement for mitogenic growth factors.
  • cyclins and CDKs have been found to be negatively regulated by either intracellular or extracellular signals that inhibit cell proliferation.
  • various protein subunits have been identified and shown to inhibit the activity of CDKs. For this reason, these proteins have been named cyclin kinase inhibitors (CKIs).
  • the p27 protein (p27 Ki P') is a CKI protein implicated in the negative regulation of G j progression in response to a number of antiproliferative signals (Polyak et al. (1994) Cell 78:59.
  • studies in macrophages have linked cyclic AMP-induced growth arrest to an increase in the amount of p27 protein, whereas the antiproliferative drug rapamycin abrogates a small reduction in p27 abundance observed after colony-stimulating factor- 1 stimulation (Kato et al. (1994) Cell 79:487).
  • interleukin-2-induced proliferation of T cells results in a decrease in the amount of p27 protein, an effect that can be prevented by addition of rapamycin (Bunce et al., (1994) Leukemia 8:595).
  • the p27 protein was first identified as an activity present in extracts derived from Gl cells able to inhibit CDK activities in vivo.
  • the p27 gene was then cloned by the ability of its protein product to interact with CDKs and was found to share homology with p21 and block the cell cycle in Gl when overexpressed in mammalian cells.
  • the primary regulation of p27 protein level during Gl is the result of ubiquitin-mediated degradation (Pagano et al. (1995) Science 269:682).
  • the CKIs have been suggested as potential antioncogenes since their function is often missing in transformed cells.
  • pi 5 and pi 6 genes have been found mutated, deleted or inactivated by methylation in a large number of human malignancies.
  • p21 is transcriptionally induced by the tumor suppressor p53, whose function is lacking in about 50% of human tumors. It has been found that in p53 minus cells, p21 is poorly expressed and is not associated with CDKs.
  • the p27 gene analyzed by Southern blot and PCR-SSCP in a large number of human cancers and human cell lines showed no structural alterations of point mutations (Kawamat et al. (1995) Cancer Res 55:2266; Ponce-Castaneda et al. (1995) Cancer Res 55:1211 ; and Pietenpol et al. (1995) Cancer Res 55:1206).
  • the level of p27 protein can be correlated with progression of a hyperproliferative disorder.
  • the level of p27 protein can be used predictively to evaluate whether a sample of cells contains cells which are, or are predisposed towards becoming, transformed cells.
  • the subject method can be characterized as including a step of detecting, in a sample of cells from the subject, the presence (level of) or absence of a p27 protein.
  • the method ofthe present invention can be carried out using any of a large number of assay techniques for detecting destabilization of p27 protein, and importantly, provides the ability to discern between different molecular causes underlying aberrant cell growth, proliferation and/or differentiation.
  • the subject method can be used to assess the phenotype of cells which are known to be transformed, the phenotyping results being useful in planning a particular therapeutic regimen.
  • absence or low p27 protein expression is a powerful negative diagnostic and prognostic marker for a variety of cancerous diseases.
  • the loss of p27 protein can be utilized in decisions regarding, e.g., the use of more aggressive therapies.
  • Prognosis in clinical cancer is an area of great concern and interest. It is important to know the aggressiveness of the malignant cells and the likelihood of tumor recurrence in order to plan the most effective therapy.
  • Breast cancer for example, is managed by several alternative strategies. In some cases local-regional and systemic radiation therapy is utilized while in other cases mastectomy and chemotherapy or mastectomy and radiation therapy are employed.
  • treatment decisions for individual breast cancer patients can be based on, for example, the number of axillary lymph nodes involved with disease, estrogen receptor and progesterone receptor status, the size of the primary tumor, and stage of disease at diagnosis.
  • DNA aneuploidy and proliferative rate can help in predicting the course of disease (Dressier et al., (1988) Cancer 61 :420; and Clark et al., (1989) N. Engl. J. Med. 320:627).
  • the subject method provides a means for accurately predicting the course of disease for breast cancer patients.
  • the ability to detect destabilization ofthe p27 protein can facilitate separation of patients with good prognosis, e.g., who will need little to no further therapy, from those more likely to recur who might benefit from more intensive treatments. This index can be combined with other prognostic methods.
  • the subject method can be used to distinguish those node-negative patients on the basis of significantly elevated or reduced risk of mortality, and suggests that this index may be useful in determining which patients would benefit from, e.g., continued and/or more aggressive therapy (such as adjuvant therapies).
  • the subject method provides a procedure for predicting tumor recurrence in cancer patients in general, once the primary tumor is detected.
  • the present invention is a significant step in the ability to predict with some confidence the likelihood of cancer recurrence. It is clear from the extensive studies on the p27 protein that it has an important physiological role, but until now no one has been able to relate its cellular levels or presence to clinical manifestations of dysfunction. Now the survival risk to cancer patients can be better assessed and aggressive therapies applied as indicated to those in high risk groups.
  • prognosis is art recognized and, as set out above, concerns the likelihood that an individual may suffer occurrence, relapse or distant relapse of cancerous disease.
  • Relapse is the recurrence of tumor growth due to propagation of tumor cells remaining in the host after treatment, new tumor cell development, or the like.
  • Distant relapse concerns tumor dissemination such that tumor growth occurs at a site distant from the site of the original tumor.
  • the length of the relapse-free survival time is the period between either surgical removal of the tumor or the suppression or mitigation of tumor growth and the recurrence of cancerous disease.
  • Prognosis may be affected by various criteria such as histological type, tumor grade, tumor size, ploidy, and expression of certain hormone receptors such as estrogen receptor, and, according to the present invention, the stability of p27. These criteria provide some guidance in determining the need for and efficacy of subjecting the patient to various cancer therapies, such as irradiation, adjuvant therapy or surgical procedures such as mastectomy in the case of breast cancer.
  • the appended examples describe that levels of p27 protein, but not levels of p27 mRNA, are decreased in a variety of different tumor cells. For example, we have found that normal colonic mucosa, and well to moderately differentiated (less aggressive) adenocarcinomas showed a strong p27 nuclear signal. In contrast, most ofthe poorly differentiated, highly aggressive adenocarcinomas showed a very low percentage of p27 positive cells. Furthermore, a highly significant correlation between the presence of p27 and survival of the patient was found. No correlation between the mitotic index of a particular tumor and p27 expression was found. Likewise, evaluation of biopsied tissue of a series of patients illustrates that the level of p27 protein provides a diagnostic and prognostic marker for breast cancers.
  • the subject method is applicable to the diagnosis/prognosis of such breast cancer types as ductal, mucinous, lobular and the like.
  • the cancer may be detected at any stage of tumor development, including hyperplasia, in situ and the like.
  • the subject invention finds particular application for diagnosis where the patient carries an axillary lymph-node negative, ductal carcinoma ofthe breast.
  • detection of p27 stability may serve as a marker for the presence of cancerous cells and also allow for determination of the prognosis of the patient carrying the tumor.
  • the subject method can also be used to augment the detection and/or prognosis of such solid tumors as, for example, carcinomas (particularly epithelial-derived carcinomas) of such tissues as ovaries, lung, intestinal, pancreas, prostate, testis, liver, skin, stomach, renal, cervical, colorectal, and head and neck; melanomas; and sarcomas such as Kaposi's sarcoma and rhabdomyosarcoma.
  • the subject method is used to assess a malignant or pre-malignant epithelial carcinoma.
  • the diagnostic methods of the subject invention may also be employed as follow-up to treatment, e.g., quantitation ofthe level of p27 protein may be indicative of the effectiveness of current or previously employed cancer therapies as well as the effect of these therapies upon patient prognosis.
  • the present invention makes available diagnostic assays and reagents for detecting loss of p27 protein from a cell in order to aid in the diagnosis and phenotyping of proliferative disorders arising from, for example, tumorigenic transformation of cells, or other hype ⁇ lastic or neoplastic transformation processes, as well as differentiative disorders, such as degeneration of tissue, e.g. neurodegeneration.
  • the inventors understand that the inactivation of the p27 protein occurs post-transcriptionally via an increase in its degradation due to, e.g.,, deregulation ofthe ubiquitin-proteosome pathway.
  • the p27 protein is an apparent substrate for ubiquitin-mediated proteolysis.
  • the rate of degradation of the p27 protein can be increased in particular cells, e.g., the protein is destabilized, leading to a net loss of p27 protein in the cytoplasm/nucleus ofthe cell.
  • the present method there is provided a method for evaluating an individual's risk (e.g., likelihood) of having or developing a disorder marked by aberrant proliferation or dedifferentiation. Where such disorders have already been diagnosed, the present method, by facilitating careful phenotyping of transformed cells, can improve the choice of intervention strategies by clinicians. For instance, the aggressiveness of the therapy to be used may be influenced by the knowledge of whether loss of regulation of proliferation or differentiation is caused by destabilization of a CKI protein, or other genetic abnormality.
  • another aspect of the present invention relates to diagnostic assays and reagents for detecting similar destabilization of other CKI proteins.
  • another aspect ofthe present invention concerns an assay for detecting destabilization of such CIP proteins as p2lC /,l and p57 KIP2 , as well as the INK4 proteins such as p ⁇ 6 INK4a , pl 5 / ⁇ ⁇ b , P 18 / ⁇ ⁇ c , and pl9/p20 /jV ⁇ d .
  • an “inhibitor of CDK activation” refers to a naturally occurring protein which interacts with a cyclin dependent kinase and prevents activation of a kinase activity of the CDK either by, for example, inhibiting formation of CDK complexes including regulatory subunits, inhibiting interaction of the CDK subunit with activating kinases or phosphatases. inhibiting substrate binding, inhibiting ATP binding, and/or inhibiting conformational changes required for enzymatic activity. Accordingly, such inhibition may be by a direct, competitive mechanism, or by an indirect, non- or uncompetitive mechanism.
  • CKI protein refers to a protein which is an inhibitor of CDK activation.
  • CKI proteins include members of the INK4 family, such as p ⁇ 6 INK4 ⁇ - or pl5/ ⁇ «*B and members ofthe CIP/KIP family, such as p2 ⁇ IP p27 KIP1 , and p57 KIP2 .
  • CIP/KIP protein refers to members of another CKI protein family which includes p27 KIP1 (Polyak et al. (1994) Cell 78:67-74); ⁇ 2 ⁇ ClP (WAF1/SDI1/CAP20; Xiong et al. (1993) Nature 366:701-704); and p57 KIP2 (Lee et al. (1995) Genes Dev 9:639-649; and Matsuoka et al. (1995) Genes Dev. 9:650-662).
  • the CIP/KIP proteins each have a CDK inhibitory motif (a CDK-binding motif) of about 50 amino acids, referred to herein as a "p21/p27" inhibitory domain, which is conserved in members ofthe CIP/KIP family.
  • the term "INK4 protein” refers to a family of structurally related CDK inhibitors characterized by a fourfold repeated ankyrin-like sequence (Elledge et al. (1994) Curr. Opin Cell Biol. 6:874-878), and the ability to bind to CDKs, especially CDK4 and CDK6.
  • Exemplary members of this protein family include pi 6 (INK A/MTS 1 ; Serrano et al (1993) Nature 366:704-707); pl5 (INK4B; Hannon et al. (1994) Nature 371 :257-261); pl8 (INK4c; Guan et al. (1994) Genes Dev.
  • CDK cyclin dependent kinase
  • CDC2, CDK2. CDK3, CDK4, CDK5. CDK6 and CDK7 The sequence for wild-type CDK protein can be found, in GenBank.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (D ⁇ A), and, where appropriate, ribonucleic acid (R ⁇ A).
  • D ⁇ A deoxyribonucleic acid
  • R ⁇ A ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either R ⁇ A or D ⁇ A made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • the term "gene” refers to a nucleic acid comprising an open reading frame, e.g., encoding a CKI protein, including (optionally) intron sequences.
  • the nucleic acid is D ⁇ A or R ⁇ A.
  • protein polypeptide
  • peptide peptide
  • allelic modification or mutation refers to such genetic lesions as, for example, deletion, substitution or addition of nucleotides to a gene, as well as non-wild type splicing of mR ⁇ A transcribed from the gene.
  • “Mis-expression” of a gene refers to aberrant levels of transcription of the gene relative to those levels in a normal cell under similar conditions.
  • p27-minus refers to a cell phenotype wherein the cell possess a reduced cellular amount of the p27 protein relative to a normal cell of similar tissue origin.
  • a p27-minus cell may have less than 50%, 25%, 10%, or 5% ofthe p27 that a normal control cell,
  • abnormal hype ⁇ roliferation refers to proliferation of cells which is undesired, e.g., such as may arise it due to transformation and/or immortalization of the cells, e.g., neoplastic or hype ⁇ lastic, for pu ⁇ oses of wound healing, cosmetic, etc.
  • aberrant dedifferentiation refers to loss of differentiation of cells of a tissue such that the afflicted tissue losses at least a portion of the normal phenotype and function for animal at that development stage.
  • adult tissue undergoing aberrant dedifferentiation will be characterized by loss of at least a portion of the functional performance of that tissue in an adult organism.
  • apoptosis refers to unwanted cell death caused by apoptosis, e.g., as may occur in a variety of degenerative disorders, including such neurodegenerative disorders as Alzheimer's disease and Parkinson's disease.
  • patient refers to an animal, preferably a mammal, including humans as well as livestock and other veterinary subjects.
  • one aspect of the present invention relates to diagnostic assays for determining, in the context of cells isolated from a patient, if the level of a p27 protein is significantly reduced in the sample cells.
  • the assay evaluates the level of p27 protein in the test cells, and, preferably, compares the measured level with p27 protein levels detected in at least one control cell, e.g., a normal cell and/or a transformed cell.
  • the assay of the instant application detects destabilization of the p27 protein which decreases the level of protein in the cell, particularly in the nucleus.
  • the number of cells with a particular p27 phenotype may then be correlated with patient prognosis.
  • the p27 phenotype of the lesion is determined as a percentage of cells in a biopsy which are found to have abnormally low levels of p27 protein. Such expression may be detected by immunohistochemical assays, dot-blot assays, ELISA and the like.
  • a significant number of examined cells may comprise at least all the cells contained within 3 to 6 fields of a tissue sample observed at a magnification of lOOx . As the number of cells examined in a tissue sample increases, the confidence one may have in classifying a sample as p27-minus increases.
  • the presence of the p27 protein in a sample of cells can be determined by immunoassays or histochemical staining employing antibodies reactive with the p27 protein, e.g., antibodies specifically reactive with the p27 protein of SEQ ID. No. 2.
  • Antibodies can be prepared conventionally (as described below) employing, e.g., the human p27 protein or antigenic fragments thereof, as the immunogen.
  • isolated p27 protein can be generated according to the methods described in PCT application PCT US95/07361. Human p27 is not required, and antibodies raised against p27 from other vertebrates can be cross-reactive with the human protein.
  • Immunoassays are commonly used to quantitate the levels of proteins in cell samples, and many other immunoassay techniques are known in the art.
  • the invention is not limited to a particular assay procedure, and therefore is intended to include both homogeneous and heterogeneous procedures.
  • Exemplary immunoassays which can be conducted according to the invention include fluorescence polarization immunoassay (FPIA). fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).
  • FPIA fluorescence polarization immunoassay
  • FIA fluorescence immunoassay
  • EIA enzyme immunoassay
  • NIA nephelometric inhibition immunoassay
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • An indicator moiety, or label group can be attached to the anti-p27 antibody and is selected so as to meet the needs of various users ofthe method which are often dictated by the availability of assay equipment and compatible immunoassay procedures.
  • General techniques to be used in performing the various immunoassays noted above are known to those of ordinary skill in the art.
  • immunohistochemical staining may be used to determine the number of cells having the p27-minus phenotype.
  • a multiblock of tissue is taken from the biopsy or other tissue sample and subjected to proteolytic hydrolysis, employing such agents as protease K or pepsin.
  • proteolytic hydrolysis employing such agents as protease K or pepsin.
  • the tissue samples are fixed by treatment with a reagent such as formalin, gluteraldehyde, methanol, or the like.
  • the samples are then incubated with an antibody, preferably a monoclonal antibody, with binding specificity for p27.
  • This antibody may be conjugated to a label for subsequent detection of binding.
  • Samples are incubated for a time sufficient for formation of complexes comprising p27 antigen and p27-specific antibody. Binding of the anti-p27 antibody is then detected by virtue of a label conjugated to this antibody.
  • a second labeled antibody may be employed, e.g., which is specific for the isotype ofthe anti-p27 antibody.
  • the substrate for the enzyme may be added to the samples to provide a colored or fluorescent product.
  • suitable enzymes for use in conjugates include horseradish peroxidase. alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.
  • the assay is performed as a dot blot assay.
  • the dot blot assay finds particular application where tissue samples are employed as it allows determination of the average amount of p27 associated with a single cell by correlating the amount of p27 in a cell-free extract produced from a predetermined number of cells.
  • tissue sample taken from a patient, and the number of cells in each sample is determined.
  • Tissues or cells which express p27 preferably tissues or cells which express a known amount of p27, may serve as a positive control and additionally may serve as a standard for comparison of results with test samples. Tissues or cells which lack p27 may be employed as a negative control.
  • single-cell suspensions may be produced from tissue in a variety of ways (e.g. incubation with EDTA in PBS. trypsin digestion, etc.).
  • the number of cells in each sample may be determined by various conventional means, e.g. by Trypan blue or eosin staining.
  • Cell-free extracts may be prepared from the single-cell suspensions by extraction with a detergent, such as Triton X-100, sonication or other conventional means known in the art.
  • the protein concentration of each cell-free extracts may be determined by methods known in the art and dilutions from each extract employed in the assay.
  • Each sample may than be bound to a soluble support, such as nitrocellulose or the well of a microtiter plate. Following washing to remove unbound material, areas of the support which are not bound to extract components may be blocked by incubation with a blocking agent such as PBS containing bovine serum albumin (BSA) to reduce non-specific binding of subsequent reagents to the support.
  • a blocking agent such as PBS containing bovine serum albumin (BSA) to reduce non-specific binding of subsequent reagents to the support.
  • BSA bovine serum albumin
  • the samples may then be washed and subsequently incubated with an anti-p27 antibody, such as a monoclonal antibody, to allow for formation of complexes of p27 and the p27-specific antibody. Binding of the antibody may be detected and analyzed as described above.
  • the amount of p27 detected may be correlated with the number of cells in the original sample to determine the average amount of p27 protein per cell.
  • the average amount of p27 protein per cell in the test sample may then be compared to the average amount of p27 protein per cell in standard samples and patient diagnosis/prognosis determined.
  • the antibody is advantageously labeled with a detectable label such as with a fluorochrome, metal particle, radioisotope or enzyme, or alternatively can be detected by an indirect immunofluorescence technique.
  • a detectable label such as with a fluorochrome, metal particle, radioisotope or enzyme
  • the latter is accomplished by subsequent reaction of the cells previously reacted with the first antibody, with a second antibody specific for the first antibody.
  • the second antibody is ideally labeled with a detectable label as above.
  • Those antibodies not attached either directly or indirectly (i.e., by attachment through another antibody in turn attached to the antigen or antibody) to the intracellular p27 protein are removed by a washing process and the remaining cells analyzed for the presence of label. Detection of the label is in turn related to the presence ofthe intracellular p27 protein.
  • CDK proteins can be used to detect/quantitate p27 protein.
  • the level of p27 protein can be detected in cell lysates by chromatography or gel electrophoresis.
  • the rate of degradation of p27 can be assessed in the sample cells.
  • labeled p27 protein can be added to lysate from the sample cells and the rate of degradation monitored, e.g., by detecting fragments of the labeled protein or, alternatively, detecting ubiquitination of the ectopic p27.
  • proteolysis inhibitors can be added to the cells or cell lysates, and the rate of appearance of ubiquitination products of a heterologous or endogenous p27 protein can be detected. In each instance, the rate is compared to rates determined for control cells, e.g., cells of a known p27 phenotype.
  • the cells can be metabolically labeled and the rate of degradation of the endogenous p27 protein detected.
  • the detection of p27 transcript in the cell sample can facilitate the determination of the molecular basis for loss of p27 protein in a cell sample, e.g., to distinguish reduced transcription or translation from protein destabilization.
  • the subject method may include a further step of determining the level of p27 mRNA in a sample of cells, e.g., by using a probe which hybridizes under stringent conditions to the p27 nucleic acid of SEQ ID No. 1.
  • the sample cells can be isolated to include a specific subset of phenotypes of cells from a given tissue, or can include be derived to include all or a substantial portion of cells representative of the tissue. Subsets of cells can be isolated, for example, by use of cell surface markers or careful sectioning of a tissue. For instance, in certain embodiments other antigens expressed by tumor cells may be detected, e.g., to determine the percentage of p27-minus cells in a given sub-population of cells from a sample.
  • Tumor antigens of interest include breast carcinoma; prostate specific antigen (PSA); carcinoembryonic antigen (CEA); alpha- fetoprotein (AFP); CA125 antigen; soluble IL-2 receptor, progesterone receptor; estrogen receptor, and the like.
  • PSA prostate specific antigen
  • CEA carcinoembryonic antigen
  • AFP alpha- fetoprotein
  • CA-242 The tumor-associated antigen CA-242 is expressed in the vast majority of colorectal tumors and is only weakly expressed or absent in normal colonic tissue.
  • HEA125 MAb, HT29-15 Mab colonrectal carcinoma
  • BCD-B4 MAb or NCRC-1 1 Mab breast carcinoma
  • anti-PSA antibody or PD41 Mab prostate carcinoma
  • ALT-04 Mab lung carcinoma
  • B72.3 MAb, HMD4 MAb or COC183B2 ovarian carcinoma
  • HEA-125 MAb, MM46 MAb, or 9.2.27 Mab melanomas
  • MGb 2 MAb, or ZCE 025 Mab gastric carcinoma
  • BW494 Mab pancreatic carcinoma
  • CEB9 Mab uterine cervical carcinoma
  • HSAN 1.2 Mab neuroblastoma
  • HISL-19 Mab neuroendocrinomas
  • TRA-1-60 Mab salivaminoma
  • PCT application PCT/US95/07361 describes antibodies, proteins and nucleic acids for detecting p27 protein and mRNA levels. Exemplary antibodies and nucleic acid probes are also known in the art for other CKI proteins and can be readily adapted for use in the subject method. For example PCT applications PCT/US95/04636, PCT/US95/16553 and PCT/US96/01643 describes exemplary antibodies, proteins and nucleic acid probes which can be used as reagents in the subject method for embodiments directed to detection of ink4 proteins.
  • PCT applications PCT/US93/09945 and PCT/US96/04563 describes antibodies, proteins and nucleic acid probes which could be used in the present method to detect destabilization of p21 ci P 1 and p57 ki P 2 , respectively.
  • anti-p27 antisera or anti-p27 monoclonal antibodies can be made using standard methods.
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., an antigenic fragment which is capable of eliciting an antibody response).
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. For instance, a peptidyl portion of the protein represented by
  • SEQ ID No. 2 can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • anti-p27 antisera can be obtained and, if desired, polyclonal anti-p27 antibodies isolated from the serum.
  • antibody producing cells lymphocytes
  • immortalizing cells such as myeloma cells
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the p27-protein of interest and the monoclonal antibodies isolated. Synthetic antibodies, e.g., generated by combinatorial mutagenesis and phage display, are equivalents of antibodies generated by immunization.
  • the labeled antibody may be a polyclonal or monoclonal antibody.
  • the labeled antibody is a purified labeled antibody.
  • the term “antibody” includes, for example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term “antibody” includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
  • the detectable marker may be, for example, selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factor. Methods of labeling antibodies are well known in the art.
  • Antibody fragments which contain the idiotype of a p27 antigen can be generated by known techniques.
  • such fragments include, but are not limited to: the F(ab')2 fragments generated by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated by reducing disulfide bridges ofthe F(ab')2 fragments.
  • immunoglobulin fragments include, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments.
  • immunoglobulin fragments can be prepared, for example, by proteolytic enzyme digestion, for example, by pepsin or papain digestion, reductive alkylation, or recombinant techniques. The materials and methods for preparing immunoglobulin fragments are well known to those skilled in the art.
  • the immunoglobulin may be a single chain antibody ("SCA”). These can consist of single chain Fv fragments (“scFv”) in which the variable light (“V[L]”) and variable heavy (“V[H]”) domains are linked by a peptide bridge or by disulfide bonds. Also, the immunoglobulin may consist of single V[H] domains (dAbs) which possess antigen-binding activity. See, e.g., Winter and Milstein, (1991) Nature 349:295; and Glockshaber et al., (1990) Biochemistry 29:1362.
  • SCA single chain antibody
  • Synthetic antibodies e.g., generated by combinatorial mutagenesis and phage display, are equivalents of antibodies generated by immunization.
  • the subject method can include a step of determining the level of mRNA transcripts for a CKI protein, e.g., preferably to discern between loss of protein stability and decreased translation and/or transcription as a cause for low CKI protein levels.
  • a diagnostic kit of the present invention will include a nucleic acid probe/primer which can specifically detect the presence or absence of a CKI DNA or RNA sequence.
  • the cellular nucleic acid can be detected by proteins which selectively bind certain nucleic acid sequences, e.g., such as DNA binding domain from transcription factors.
  • the present invention therefore provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of SEQ ID No. 1, or naturally occurring mutants thereof.
  • the probe/primer further comprises a label group attached thereto and able to be detected, e.g. the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • oligonucleotide probes can be modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo.
  • exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775).
  • the sample nucleic which is analyzed by the subject method can be isolated from any cell or collection of cells, though preferably is obtained from the cells from the same tissue sample used to score for p27 protein levels.
  • isolating RNA from a cellular source any of which may be used to practice the present method.
  • the Chomczynski method e.g., isolation of total cellular RNA by the guanidine isothiocyanate (described in U.S. Patent No. 4,843,155) used in conjunction with, for example, oligo-dT strepavidin beads, is an exemplary mRNA isolation protocol.
  • RNA as desirable, can be converted to cDNA by reverse transcriptase, e.g., poly(dT)-primer first strand.
  • reverse transcriptase e.g., poly(dT)-primer first strand.
  • cDNA synthesis by reverse transcriptase, followed by second strand synthesis (DNA pol I).
  • kits in accordance with the subject would therefore comprise an antibody specific for p27.
  • that antibody can labeled for detection.
  • the kit can include a second labeled antibody which can detect the first antibody.
  • the kit may also include various controls and washing buffers.
  • the kit can include a swelling solution.
  • another antibody for a cell marker is provided as a means for identifying the general population of cells in which a p27 minus phenotype is to detected.
  • the kit may also include a nucleic acid probe for detecting the presence of a p27 transcript or cDNA thereof.
  • the present invention specifically contemplates any and all variations of these reagents, as well as similar kits for use in the subject assay as pertaining to other CKI proteins.
  • Cdks cyclin dependent kinases
  • CKIs cyclin dependent kinase inhibitors
  • p27 is a potential tumor suppressor gene
  • the level of p27 expression was analyzed by immunohistochemistry and in situ hybridization in archival sections from one hundred and seventy one patients with colorectal carcinomas, operated from 1965 to 1993, (median follow up was 36 months). We also determined p27 degradation activity in a subset of tumors.
  • Cdks cyclin dependent kinases
  • Several distinct cyclin-Cdk complexes have been identified and found to be active and required at various stages ofthe cell cycle.
  • the activity of Cdks is tightly regulated by both activating and inactivating phosphorylation events (reviewed by 2 ).
  • a third class of subunits has been identified and shown to inhibit the activity of Cdks.
  • Ckls cyclin kinase inhibitors
  • the first family includes p21 (also called CPI 1, Picl, Sdil and Wafl; ' 5 ' 6 - 7 - 8 ), p27 (also called d Kipl and Pic2; 9 10 ), and p57 (also called Kip2; " ⁇ I2 ).
  • the second family includes pi 6 (also called Ink4A, Mtsl, Cdkn2 and Cdk4i; ,3 - ,4 ), pi 5 (also called Ink4B, Mts2; ,4, 15 - 16 ' ,7 ), pi 8 (also called Ink4C and Ink6A; 15 ,8 ), and pl9/p20 (also called Ink4D and Ink ⁇ B; 18 - 1920 ).
  • pi 6 also called Ink4A, Mtsl, Cdkn2 and Cdk4i; ,3 - ,4
  • pi 5 also called Ink4B, Mts2; ,4, 15 - 16 ' ,7
  • pi 8 also called Ink4C and Ink6A; 15 ,8
  • pl9/p20 also called Ink4D and Ink ⁇ B; 18 - 1920 .
  • Ckis have been suggested as the products of potential antioncogenes since their function is often missing in transformed cells.
  • pi 5 and pi 6 genes have been found mutated, deleted or inactivated by methylation in a large number of human malignancies (reviewed by 21 ).
  • P21 is transcriptionally induced by the tumor suppressor p53 6- 22 whose function is lacking in about 50% of human tumors 3 . It has been found that in p53 minus cells, p21 is poorly expressed and is not in association with Cdks 24 .
  • the p27 gene analyzed by Southern plot and PCR-SSCP in a large number of human cancers and human cell, lines showed no structural alterations or point mutations 25 ' 26, 27 .
  • p27 was first identified as an activity present in extracts derived from Gl cells able to inhibit Cdk activities in vitro 28 ' 29 .
  • the p27 gene was then cloned by the ability of its protein product to interact with cdks and was found to share homology with p21 and block the cell cycle in Gl when overexpressed in mammalian cells 9 ' 10 .
  • Mammalian p27 protein accumulates in cells treated with cAMP 30 , lovastatin 3 ', TGFB 29 ' 32 , or rapamycin 33 and this increase is thought to be involved in the Gl block induced by these drugs.
  • p27 has been found to be expressed at high levels in quiescent cells, probably playing an important role in maintaining cells in GO 33 - 34 - 35 .
  • cells undergoing differentiation have elevated level of p27 36 ' 37 .
  • the downregulation of p27 protein level during Gl is the result of ubiquitin-mediated degradation
  • Paraffin blocks were recovered from one hundred and seventy one patients who were operated at the Deaconess Hospital for adenocarcinoma of the colon or rectum between 1965 and 1993. These patients, randomly chosen with respect to date of operation, site of tumor and other factors, belong to a subset of a database containing information about clinic and pathologic variables and outcomes of the Joint Center for Radiation Therapy, Boston, MA 39 . Outcome was verified and updated through the Tumor Registry and Medical Record Departments at the Deaconess Hospital as well as through contacts with municipal governments for death certificates when appropriate. Cancerous tissues from these patients had been characterized in term of clinical and pathological variables.
  • stage IV carcinomas Due to the extremely small subset of stage IV carcinomas (and less than 1% stage IV), accurate conclusions could not be drawn concerning survival; iii) Patients with negative nodes had a median survival rate of 308 months while patients 1-3 or more than 3 positive nodes had median survivals of 44 and 14 months, respectively (Wilcoxon p ⁇ 0.0001). p27 Expression. Formalin-fixed, paraffin-embedded 5 um sections were mounted on charged glass slides, deparaffinized, and rehydrated through graded alcohols. Immunohistochemistry and in-situ hybridization were performed by an automated processor (Ventana Gene II, Ventana Medical Systems, Arlington, AZ).
  • Immunohistochemistry slides were subjected to microwaving in lOmmole/L citrate buffer, pH 6.0 (BioGenex, San Ramon, CA.) in a 750 W oven inside a pressure cooker for a 30 minute period. Slides were allowed to cool at room temperature for 30 minutes. p27 mouse monoclonal antibody (mAb) (Transduction Laboratories, #K25020) was applied for 24 minutes at 37°C at a concentration of 1 :200 in the automated stainer. Steps performed by the instrument include blocking with normal horse serum, application of a secondary antibody conjugated to the avidin-biotin peroxidase complex, and visualization with 3,3 diaminobenzidine as a substrate with standardized development times. Identical reaction times permitted accurate comparison of all samples. The slides were counterstained with methyl green and coverslipped. A cocktail comprised of antibodies with no known human recognition site 40 was used as a negative control.
  • An osteosarcoma cell line MG-63 (obtained from the American Type Culture Collection) was used as a positive control. Following 48 hours of serum starvation (which is necessary to increase levels of p27, 35 cells from two confluent flasks were harvested, fixed in neutral buffered formalin for 8 hours, and paraffin-embedded. 5 micron sections of the cell block were utilized in each run as a positive control. Pre-abso ⁇ tion of the antibody with the protein used to generate it abolished p27 staining (Fig. 2).
  • RNAse ribonuclease
  • UDP cold uridine triphosphate
  • DTT dithiothreitol
  • p27 staining for both immunochemistry and in situ hybridization, was evaluated blindly (without knowledge of the clinic and pathologic parameters) or outcome and scored for degree of p27 expression as either 0 (less than 1% cells stained), 1 (1 -50% cells stained), or 2 (greater than 50% cells stained).
  • Degradation assay 1 g of each frozen human tissue sample was sectioned and quickly homogenized at 15,000 ⁇ m with a Brinkman Polytron homogenizer (PT 3000) in 1 ml of ice cold double distilled water. 46 The sample was frozen and thawed 3 times. The lysate was spun down at 15,0-0-0 rpm in a Beckman JA-20.1 rotor for 45 minutes in 4°C. The supernatant was retrieved and frozen at -80°C. This method of preparation of total extract preserves ubiquitinating enzymes. 38 ' 47
  • Purified histidine-tagged p27 (p27-his 6 , bacterially expressed and purified as in 38 ) was incubated at 37°C for different times in 30 ⁇ l of degradation mix containing 100 ⁇ g of protein human tissue homogenates, 40 mM Tris-HCl (pH 8.0), 4 mM MgCl 2 , and 1 mM DTT, 2 mM ATP, 10 mM creatine phosphokinase, 10 mM creatine phosphate and 5 ⁇ M ubiquitin. Degradation of p27 was analyzed by immunoblotting with p27 mAb.
  • protease inhibitors phenyl-methyl sulfonyl fluoride (PMSF), 0.1 mM; leupeptin, 1 ⁇ g/ml; soybean trypsin inhibitor, 10 ⁇ g/ml; L-l Chlor-3-(4-tosylamido)-4 Phenyl-2-butanon (TPCK); L-l Chlor-3-(4- tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK); 10 ⁇ g/ml; aprotinin 1 ⁇ g/ml.
  • PMSF phenyl-methyl sulfonyl fluoride
  • TPCK L-l Chlor-3-(4-tosylamido)-4 Phenyl-2-butanon
  • TLCK L-l Chlor-3-(4- tosylamido)-7-amino-2-heptanon-hydrochloride
  • 10 ⁇ g/ml aprotin
  • Immunoblotting Conditions for immunoblotting-have been previously described. 49 Proteins were transferred from gel to a nitrocellulose membrane (Novex) by wet blotting as described in 50 . Filters were subjected to immunoblotting using a chemiluminescence (DuPont-NEN) detection system according to the manufacturer's instructions.
  • DuPont-NEN chemiluminescence
  • Anti-p27 antibody detects non proliferating cells of normal colon mucosa.
  • FIG. 2 shows examples of well and poorly differentiated colon carcinomas (Fig. 2A and 2B, respectively) as well as a tumor displaying both components (Fig. 2C) stained with anti p27 antibody.
  • p27 expression is prognostic in colorectal carcinoma patients.
  • p27 is an independent prognostic variable (p ⁇ 0.0031) after controlling for age. sex. histologic grade, stage, and site. The absence of p27 increases the relative risk of dying from colorectal cancer by almost 4 fold (relative risk 3.81).
  • Fig. 4 shows two typical examples: the tumor in panels A and B was positive for both p27 mRNA and protein expression; the second (panels C and D) expressed p27 mRNA but not protein.
  • p27 degradation pathway was enhanced in tumors with low p27 levels.
  • Fig. 5 shows a typical kinetics of p27 degradation obtained by using different samples of different conditions.
  • Figure 6 shows that tumors with high levels of p27, as detected by western blot, presented low or very low p27 degradation activity (measured as described in the Material and Methods) compared to tumors with low/absent p27. These latter types of tumors, indeed, showed a high or a very high activity. This degradation activity was not due to a non-specific increase in the activity ofthe ubiquitin- proteasome pathway in some tumors. In fact, other cell cycle proteins found to be degraded by the ubiquitin-proteasome pathway, specifically, p21 and cyclin A (S.W.T. and M.P., unpublished results), were degraded in a similar fashion with all the extracts we tested ( Figure 6).
  • the Cki p21 is a transcriptional target of p53. 6 In tumor cells lacking functional p53, p21 levels are low and p21 is not found in association with cyclin-dependent kinases. 24 Immunocytochemical studies have demonstrated that there is a global decrease in p21 expression in human tumors compared to normal tissues. 36 In 75% of tumor cell lines and in a large number of human tumors, the pi 6 gene is deleted or mutated (reviewed by 2I .) Similarly, members of the pi 6 family, pi 5 and pi 8. can be the targets of genetic alterations. Thus, transformation often involves the inactivation of Cdk inhibitors.
  • stage I and IV cancer While predicting outcome in patients with stage I and IV cancer is easy, the present staging method creates broad groups of patients whose survival may range at least forty percent within stage II and III. The same is. true for predicting outcome based on the state of differentiation of the tumor. In fact, while patients with poorly differentiated, anaplastic tumors have as very poor survival rate, it is difficult to predict outcome in patients with well/moderately differentiated cancers. Improved prognostication may derive by evaluating the expression of molecular markers in tumor samples (see 59 for a review).
  • the cell cycle inhibitor p27 is an independent prognostic marker in small (Tla,b) invasive breast carcinomas
  • p27 assessment by immunohistochemistry is simple, cost- effective, reproducible and can be performed on all primary tumors. In addition, it yields prognostic information in node negative patients. Assessment of p27 could be useful to identify patients with small invasive breast carcinomas who might benefit from adjuvant therapy.
  • Cdk activation requires (i) association with an activating subunit (cyclin); (ii) de-phosphorylation by a family of dual-specificity phosphatases called Cdc25 (for a review see 9 ; and (iii) dissociation from a Cdk inhibitor subunit (Dki, review in l0 ).
  • the Cki p27 is a potential tumor suppressor.
  • p27 deficient mice develop pituitary tumors with 100% penetrance and display increased body size "' 12 ' 13 .
  • the Adenoviral El A 14 and the Human Papilloma viral E7 (P. Jansen-Duerr, pers. comm. oncoproteins inactivate p27 by dissociating it from cyclin-Cdk complexes.
  • no homozygous deletions and only rare mutations on the p27 gene have been found so far in cell lines or in human tumors 16, I7 ' 18 .
  • p27 abundance is regulated at the post-transcriptional level 19 ' 20 , we reasoned that, even in presence of a wild type gene, tumors might obtain a growth advantage from lack of p27 due to an increase in its degradation as we previously proposed in colorectal cancers 21 . Therefore, we analyzed the expression of p27 protein to assess the relationship between lack of p27 expression and aggressive behavior in small invasive breast cancers.
  • ER ⁇ PR, p53, MVD, Ki-67/MIB-l, cdc25B, or c-erb-B-2 were not significantly associated with survival by univariate analysis (Figure 7).
  • High and low p27 expression in patients was equally distributed in the positive and negative node groups (Figure 8).
  • p27 is a novel prognostic marker the expression of which is associated with a better outcome in patients with Tla,b breast carcinomas who undergo potential curative surgery.
  • Low p27 expression correlates with decreased survival, and may be utilized in Tla,b node-negative patients to predict prognosis.
  • adjuvant therapy which benefits patients with advanced breast cancer, may also be appropriate for patients with Tla,b carcinomas expressing little p27.
  • stage Tl defined as tumors less than 1 cm in greatest diameter as determined by macroscopic measurement
  • invasive breast cancers diagnosed between 1969 and April 1994 were identified.
  • Patients with distant metastasis at time of diagnosis, exclusive in-situ disease, synchronous bilateral breast carcinomas or a history or previous malignancies were excluded from this study.
  • Age at diagnosis, biochemical steroid hormone receptor levels (available in 133 patients), modalities of therapeutic interventions, time to recurrence or metastasis, length of overall survival and cause of death were recorded.
  • 53 received adjuvant therapy (51 > of which was radiation therapy alone).
  • 19 node positive patients 1 received chemotherapy 5%, 1 radiation therapy (5%), 3 hormonal therapy (16%), 7 radiation and chemotherapy (37%), 1 radiation and tamoxifen (5%) and 2 radiation, chemo and hormonal therapy (11%).
  • the median tumor size was 0.66 cm (range from 0.1 to 1.0 cm). Tumors ⁇ than 5mm in size (Tla) made up 31% of the series. Distribution of different histologic grades and presence or absence of extensive intraductal component in the patient population are outlined in Figure 7. The majority of carcinomas were ductal type (139 or 69%), while 45 (22%) were tubular carcinomas, 11 (5%) were lobular, 6 )3%) were colloid carcinomas, and 1 was a metaplastic carcinoma. Eleven patients (5%) developed local disease recurrence while 19 (9%) had radiologically or histopathologically documented distant metastases prior to death. Twenty six patients (13%) died of disease or disease-related complications. One hundred two patients underwent axillary node dissection and 19 of these had nodal metastases.
  • a representative block of formalin-fixed, paraffin-embedded carcinoma was selected for immunohistochemical (c-erb B-2, p53, factor 8, Mibl , ER and PR, p27) and in-situ hybridization (esc 25B) studies.
  • Immunohistochemistry was performed on an automated processor (Ventana ES, Ventana Medical Systems, Arlington, AZ). Formalin-fixed, paraffin embedded 5u sections mounted on charged glass slides were deparaffinized and rehydrated through graded alcohol before being placed in the automated immunostainer. Specificity of the p27 staining was assessed by : i) pre-abso ⁇ tion of the antibody with the protein used to generate it abolished p27 staining; ii) a cocktail comprised of antibodies with no known human recognition site used as a negative control; iii) positive staining of serum-starved MG-63 osteosarcoma cells (obtained from the American Type Culture Collection) in each immunoperzidase run.
  • Steps performed in the immonostainer include blocking with normal horse serum, application of the primary antibody, a biotinylated secondary antibody, and visualization with diaminobenzidine substrate, with standardized development time (allowing reproducible comparison between samples and between runs).
  • the primary antibodies used were: monoclonal an -c-erb B-2 (Ciba-Corning, Alameda, CA) dilution 1 :150, incubation time 20 minutes; monoclonal anti- factor VIII related antigen (DAKP, Ca ⁇ enteria, CA) dilution 1 :100, incubation time 16 minutes, monoclonal anti-hormone receptors (ER and PK), (Ventura Medical Systems, Arlington, AZ); incubation time 32 minutes; monoclonal anti-p53 clone Ab-6 (Oncogene Science) dilution 1 :500, incubation time 32 minutes; monoclonal anti-MIB-1, (Immunotech, Westbrook, ME) dilution 1
  • a prior modified antigen retrieval procedure was employed by microwaving deparaffmized slides in citrate buffer (pH 6.1) (Biogenex, San Ramon, CA) within a pressure cooker at power 10 (750W oven) for 20 minutes (ER and PR), power 5 for 16 minutes (p53 and MIBl, a power 7 for 16 minutes (p27).
  • In-situ hybridization ln-situ hybridization for cdc 25B was performed on the automated processor (Ventura Gen II, Ventana Medical Systems, Arlington, AZ) on 154 cases in which there was adequate preservation of mRNA. Digoxigenin-labeled probes were utilized for in situ hybridization as previously described 23 .
  • Stained tumor sections were evaluated by estimating the percentage of invasive carcinoma cells positively stained by a primary antibody on the entire tissue section p27 staining, for both immunochemistry and in situ hybridization, was evaluated in a coded manner (without knowledge of the clinic and pathologic parameters or outcome) and scored independently for degree of p27 expression by two pathologist (M.L. and P.T.) At least 10 high power fields were counted. For c-erb B-2, a distinct brown membranous staining was scored as positive. For factor VIII, a distinct brown cytoplasmic staining in endothelial cells was considered positive. Areas with the highest vessel density were chosen at low magnification for counting.
  • Microvessel density was assessed by counting up to 3 fields at X200 field as previously described 7 . The range was 5 to 262 microvessels per X200 microscopic field, with a median value of 50. This value was used as a cutoff point. For all the other markers, namely p53, MIB-1, p27, ER and PR, a distinct brown nuclear staining was scored as positive. A cutoff value of ⁇ 10%> was used for p53, MIB-1. c-erb-B-2, and ER/PR, and ⁇ 50% for p27, 50% of p27 positive tumor cells represented the median of this series.
  • Sytosolic estrogen receptor assays were performed as previously described 24 cdc25B expression was assessed by in situ hybridization. Because expression of cdc25B, when present, was found throughout the tumor, sections were evaluated for intensity of staining which was scored as 0, 1 , 3 to 3+. Only 3+ were considered overexpressors and this group was compared to 0, 1+, and 2+ combined.
  • the primary study outcome was survival, which was measured from the date of surgery to the date of last follow up or death. Survival was censored of the patient was still alive or died from other causes. Survival curves were constructed using the method of Kaplan-Meier 23 . Univariate survival curves were compared using a Wilcoxon procedure 26 and differences between prognostic factors were tested for statistical significance with the log rank analysis 27 (Figure 9).
  • a Cox proportional hazards model 28 for the risk ratio was used to assess the simultaneous contribution of the following baseline covariates; age, grade, nodal status, presence or absence of EIC (Cox Model 1 ; Figure 9), and expression of the various markers (Cox Model 2; Figure 9) or all parameters combined (not shown).
  • Ki-67/MIB-l was excluded from Model 2 and the combined Cox because of 1) the scarcity of cases >10% positive cells (6/199) and 2) all deaths were clustered in the ⁇ 10% group. All other covariates were retained in the model to illustrate lack of effect in the presence of other significant factors.
  • the distribution of p27 was compared to the distribution of each baseline covariate using the Jonckheer-Te ⁇ stra Test2P for doubly ordered data, Kruskai-WallisJO was used for single ordered data, and Fishers' exact test for categorical data. A p-value ⁇ 0.05 was required for significance. Two-sided tests were performed throughout all analyses.
  • GGT TCC CCA AAT GCC
  • CCC AAG AAG CCT GGC 576 Gly Ser Pro Asn Ala Gly Ser Val Glu Gin Thr Pro Lys Lys Pro Gly 180 185 190

Abstract

La présente invention concerne des méthodes diagnostique et pronostique qui évaluent le phénotype et l'agressivité d'un trouble par détection de la stabilité d'une protéine inhibitrice de cycline kinase (CKI).
PCT/US1998/014566 1997-07-15 1998-07-14 Reactifs, procedes de diagnostic et pronostic de troubles proliferatifs WO1999004238A2 (fr)

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Cited By (16)

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Publication number Priority date Publication date Assignee Title
EP1023459A1 (fr) * 1997-05-15 2000-08-02 Thomas Jefferson University Determination de niveaux de p27 inhibiteur de kinase dependant de la cycline comme facteur de pronostic chez des patients atteints de cancer
WO2001011361A2 (fr) * 1999-08-04 2001-02-15 Dako A/S Trousse diagnostique et son utilisation
WO2001040271A2 (fr) * 1999-12-01 2001-06-07 Ludwig Institute For Cancer Research Antigenes associes au cancer et utilisations correspondantes
EP1208232A1 (fr) * 1999-06-10 2002-05-29 Sloan-Kettering Institute For Cancer Research Marqueurs du cancer de la prostate
EP1333278A1 (fr) * 2002-02-01 2003-08-06 MTM molecular tools in medicine Procédé prognostic des tumeurs gastro-intestinales
EP1388734A1 (fr) * 2002-08-01 2004-02-11 MTM Laboratories AG Méthode de diagnostic en utilisant une solution d'échantillon
WO2003092719A3 (fr) * 2002-04-29 2004-03-18 Yissum Res Dev Co Procede et composition pour moduler la phosphorylation de la beta-catenine
US6972170B1 (en) 1997-12-01 2005-12-06 Sloan-Kettering Institute For Cancer Research Markers for prostate cancer
US7306926B2 (en) 1998-07-01 2007-12-11 Mtm Laboratories Ag Method for detecting carcinomas in a solubilized cervical body sample
US7932047B2 (en) 2003-08-25 2011-04-26 Mtm Laboratories, Ag Method for detecting neoplastic disorders in a solubilized body sample
US8012945B2 (en) 2001-11-09 2011-09-06 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
US9340581B2 (en) 2001-10-03 2016-05-17 Washington University Ligands to radiation-induced molecules
US9738725B2 (en) 2011-07-29 2017-08-22 Washington University Antibodies to TIP-1
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer
US10449261B2 (en) 2014-07-24 2019-10-22 Washington University Compositions targeting radiation-induced molecules and methods of use thereof
US11352436B2 (en) 2017-02-10 2022-06-07 Washington University Antibodies to TIP1 and methods of use thereof

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EP1023459A4 (fr) * 1997-05-15 2002-01-09 Univ Jefferson Determination de niveaux de p27 inhibiteur de kinase dependant de la cycline comme facteur de pronostic chez des patients atteints de cancer
EP1023459A1 (fr) * 1997-05-15 2000-08-02 Thomas Jefferson University Determination de niveaux de p27 inhibiteur de kinase dependant de la cycline comme facteur de pronostic chez des patients atteints de cancer
US6972170B1 (en) 1997-12-01 2005-12-06 Sloan-Kettering Institute For Cancer Research Markers for prostate cancer
US7306926B2 (en) 1998-07-01 2007-12-11 Mtm Laboratories Ag Method for detecting carcinomas in a solubilized cervical body sample
EP1208232A4 (fr) * 1999-06-10 2003-01-15 Sloan Kettering Inst Cancer Marqueurs du cancer de la prostate
EP1208232A1 (fr) * 1999-06-10 2002-05-29 Sloan-Kettering Institute For Cancer Research Marqueurs du cancer de la prostate
WO2001011361A2 (fr) * 1999-08-04 2001-02-15 Dako A/S Trousse diagnostique et son utilisation
WO2001011361A3 (fr) * 1999-08-04 2001-11-15 Monotec Gmbh Trousse diagnostique et son utilisation
WO2001040271A2 (fr) * 1999-12-01 2001-06-07 Ludwig Institute For Cancer Research Antigenes associes au cancer et utilisations correspondantes
WO2001040271A3 (fr) * 1999-12-01 2002-04-18 Ludwig Inst Cancer Res Antigenes associes au cancer et utilisations correspondantes
US10086073B2 (en) 2001-10-03 2018-10-02 Washington University Ligands to radiation-induced molecules
US9340581B2 (en) 2001-10-03 2016-05-17 Washington University Ligands to radiation-induced molecules
US8012945B2 (en) 2001-11-09 2011-09-06 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
US8927288B2 (en) 2001-11-09 2015-01-06 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
US8617521B2 (en) 2001-11-09 2013-12-31 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
WO2003065035A1 (fr) * 2002-02-01 2003-08-07 Mtm Molecular Tools In Medicine Methode permettant de mesurer et de pronostiquer l'evolution de tumeurs gastro-intestinales
EP1333278A1 (fr) * 2002-02-01 2003-08-06 MTM molecular tools in medicine Procédé prognostic des tumeurs gastro-intestinales
US7358060B2 (en) 2002-02-01 2008-04-15 Mtm Laboratories, Ag Method for monitoring and prognosis of disease course of gastrointestinal tumors
WO2003092719A3 (fr) * 2002-04-29 2004-03-18 Yissum Res Dev Co Procede et composition pour moduler la phosphorylation de la beta-catenine
WO2004013632A1 (fr) * 2002-08-01 2004-02-12 Mtm Laboratories Ag Procede de diagnostic a base de solution
US7517662B2 (en) 2002-08-01 2009-04-14 Mtm Laboratories, Ag Method for solution based diagnosis
AU2003262554B2 (en) * 2002-08-01 2009-02-19 Ventana Medical Systems, Inc. Method for solution based diagnosis.
JP2005534313A (ja) * 2002-08-01 2005-11-17 エムティーエム ラボラトリーズ アクチェンゲゼルシャフト 溶液ベースの診断のための方法
EP1388734A1 (fr) * 2002-08-01 2004-02-11 MTM Laboratories AG Méthode de diagnostic en utilisant une solution d'échantillon
US10697966B2 (en) 2002-08-01 2020-06-30 Ventana Medical Systems, Inc. Method for detecting cervical dysplasia
US7932047B2 (en) 2003-08-25 2011-04-26 Mtm Laboratories, Ag Method for detecting neoplastic disorders in a solubilized body sample
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer
US9738725B2 (en) 2011-07-29 2017-08-22 Washington University Antibodies to TIP-1
US10259884B2 (en) 2011-07-29 2019-04-16 Washington University Antibodies to GRP78
US10449261B2 (en) 2014-07-24 2019-10-22 Washington University Compositions targeting radiation-induced molecules and methods of use thereof
US11352436B2 (en) 2017-02-10 2022-06-07 Washington University Antibodies to TIP1 and methods of use thereof

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