EP0960214A4 - DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER - Google Patents

DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER

Info

Publication number
EP0960214A4
EP0960214A4 EP97951529A EP97951529A EP0960214A4 EP 0960214 A4 EP0960214 A4 EP 0960214A4 EP 97951529 A EP97951529 A EP 97951529A EP 97951529 A EP97951529 A EP 97951529A EP 0960214 A4 EP0960214 A4 EP 0960214A4
Authority
EP
European Patent Office
Prior art keywords
seq
marker
pcr
prostate
mrna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP97951529A
Other languages
German (de)
English (en)
Other versions
EP0960214A1 (fr
Inventor
David Ralph
Gang An
Mark O'hara
Robert Veltri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Urocor Inc
Original Assignee
Urocor Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Urocor Inc filed Critical Urocor Inc
Publication of EP0960214A1 publication Critical patent/EP0960214A1/fr
Publication of EP0960214A4 publication Critical patent/EP0960214A4/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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

Definitions

  • primers to detect the message of IL 8 using the transcribed portions of the marker sequence as set forth in the listing in Genebank Accession # M28130 may hybridize to nucleotides 1482 to 1503 and the complement of nucleotides 1626-1647. These particular primers would amplify a segment of message of the marker gene 166 base pairs in length.
  • Certain metastatic marker genes disclosed herein do not have reading frames for translation disclosed. However, one of ordinary skill in the art may translate the identified sequences or segments thereof in the three potential reading frames to obtain peptides or proteins for use in generating antibodies to these marker genes. Such antibodies may be used to purify the proteins of the marker genes, and the identity of protein being detected is confirmed by peptide sequencing of the protein. Once confirmed as binding the translation products of the marker genes corresponding to SEQ ID NO: 1 and Genebank accession # T03013, and/or SEQ ID NO:2, the antibodies that bind the marker gene protein would be useful in detecting, diagnosis, or prognosis of metastatic cancer.
  • the invention also broadly comprises methods for identifying biomarkers for use in prognostic or diagnostic assays of a disease state, using the technique of RNA fingerprinting to identify RNAs that are differentially expressed between individuals with the disease state versus normal individuals.
  • RNA fingerprinting to identify RNAs that are differentially expressed between individuals with the disease state versus normal individuals.
  • FIG. 5 Ability of UC325 (IL-8) and t-PSA combined to distinguish BPH and Stages A,
  • hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions.
  • Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature.
  • a medium stringency condition may be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition may be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • hybridization conditions may be readily manipulated depending on the desired results.
  • the following codon chart may be used, in a site-directed mutagenic scheme, to produce nucleic acids encoding the same or slightly different amino acid sequences of a given nucleic acid:
  • Substitutional variants typically exchange one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with another of similar shape and charge.
  • prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli ,
  • the coding sequences may be ligated to an adenovirus transcription/ translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) results in a recombinant virus that is viable and capable of expressing proteins in infected hosts.
  • the detection of an antigen encoded by a disease state marker nucleic acid, or an increase in the levels of such an antigen, in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with the disease state.
  • the basis for such diagnostic methods lies, in part, with the finding that the nucleic acid disease state markers identified in the present disclosure are overexpressed in peripheral blood samples from individuals with the disease state (see Examples 1 through 4 below). By extension, it may be inferred that at least some of these markers produce elevated levels of encoded proteins, that may also be used as disease state markers.
  • the encoded proteins or peptides of the disclosure have utility as immunogens, e.g., in connection with vaccine development, in immunohistochemistry and in ELISA assays.
  • One evident utility of the encoded antigens and corresponding antibodies is in immunoassays for the detection of disease state marker proteins, as needed in diagnosis and prognostic monitoring.
  • Under conditions effective to allow immunecomplex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • the "suitable” conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C, or may be overnight at about 4°C or so.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a spectrophotometer.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependentDNA polymerase).
  • RNA-dependentDNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art and may be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding.
  • kits This generally comprises preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • kits generally comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.
  • nucleic acids would be extracted from these samples and amplified as described above. Some embodiments would utilize kits containing pre-selected primer pairs or hybridization probes. The amplified nucleic acids would be tested for the markers by, for example, gel electrophoresis and ethidium bromide staining, or Southern blotting, or a solid-phase detection means as described above. These methods are well known within the art. The levels of selected markers detected would be compared with statistically valid groups of individuals with metastatic, non-metastatic malignant, or benign tumors or normal individuals. The diagnosis and prognosis of the individual patient would be determined by comparison with such groups. Another embodiment of the present disclosure involves application of RT-PCR techniques to detect a disease state using probes and primers selected from sequences comprising Genebank
  • the (TOSOH) total PSA assay reacted equally to the free and bound (PSA-ACT) forms of PSA.
  • the (lmmulite) free PSA assay system was unable to detect the bound fraction of PSA (PSA- ACT) below a concentration of 20 ng/ml.
  • Antibodies for detecting both total and free PSA were unable to detect PSA covalently linked to ⁇ -2 macroglobulin (PSA-MG or occult PSA).
  • RNA fingerprinting by PCR, primed with oligonucleotides of arbitrary sequence was performed on RNAs isolated from peripheral human blood. Bands which appeared to be differentially expressed were cloned.
  • RNAs and proteins genes and gene products for the above described markers of metastatic prostate cancer are included within the scope of the disclosure herein described. It will also be recognized that the diagnosis and prognosis of metastatic prostatic cancer by detection of the nucleic acid products of these genes are included within the scope of the present invention. Serological and other assays to detect these mRNA species or their translation products are also indicated. It is obvious that these assays are of utility in diagnosing metastatic cancers derived from prostate and other tissues.
  • Table 8 clearly demonstrates a relationship between tumor burden and serum UC-325 gene product measured by IL-8 assay. Note that as biopsy-confirmed clinical stage of the cancer increases, so does the IL-8 serum marker concentration, whereas the same relationship did not occur with [t-PSA] or f/t PSA ratio.
  • ACAATCCGGAGGCATCAGAAACT 3' (SEQ ID NO:32). These oligonucleotides direct the amplification of a 277 nucleotide long PCRTM product that is specific for UC331.
  • the oligonucleotides used in the relative quantitative RT-PCRTM studies that independently confirmed the differential expression of UC332 were designed using the sequences of the cDNA with the GenBank accession number D87451.
  • These UC332 specific oligonucleotides had the sequences 5' AGCCCCGGCCTCCTCGTCCTC 3' (SEQ ID NO:33) and 5' GGCGGCGGCAGCGGTTCTC 3' (SEQ ID NO:34).
  • These oligonucleotides direct the amplification of a 140 nucleotide long PCRTM product that is specific for UC332.
  • AATTCATAAA AAAATTCATT CTCTGTGGTA TCCAAGAATC AGTGAAGATG CCAGTGAAAC 540

Abstract

Cette invention, qui a trait à des méthodes diagnostiques permettant de déceler des états pathologiques chez l'homme, décrit également les sondes génétiques et les techniques afférentes utilisées pour déceler des états pathologiques et surveiller leur évolution. Elle concerne, plus particulièrement, des sondes et des techniques permettant d'évaluer la présence d'espèces d'ARN dont l'expression dans le sang périphérique de personnes souffrant desdits états pathologiques diffère de celle ayant lieu chez des individus en bonne santé. L'invention porte, en outre, sur un modèle de diagnostic à plusieurs variables s'appliquant au cancer de la cancer de la prostate chez des sujets dont le taux plasmatique total d'antigène prostatique spécifique (PSA) est modérément élevé (≥ 2,0 ng/ml). Les résultats de dosages sériques concernant le produit génique UC325 [Interleukine-8(IL-8)], le taux plasmatique total d'antigène prostatique spécifique (t-PSA) ainsi que les rapports relatifs à l'absence de PSA/PSA total (f/t PSA) ont été combinés pour renforcer la sensibilité du diagnostic du cancer de la prostate dans une population relevant de l'urologie diagnostiquée comme étant atteinte, soit d'un cancer de la prostate limité à l'organe proprement dit (stade clinique A et B), soit d'un cancer de la prostate non limité à l'organe (stade clinique C ou D) ou encore d'une hyperplasie prostatique bénigne (BPH). L'invention traite également des possibilités supplémentaires qu'offre la connaissance des taux sériques en produit génique UC325 de définir avec exactitude les stades d'évolution du cancer de la prostate, indépendamment des taux de PSA, qu'il s'agisse de t-PSA ou de f/t PSA.
EP97951529A 1996-12-06 1997-12-05 DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER Ceased EP0960214A4 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US3261996P 1996-12-06 1996-12-06
US32619P 1996-12-06
US3270196P 1996-12-12 1996-12-12
US32701P 1996-12-12
US4157697P 1997-03-24 1997-03-24
US41576P 1997-03-24
PCT/US1997/022105 WO1998024935A1 (fr) 1996-12-06 1997-12-05 DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER

Publications (2)

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EP0960214A1 EP0960214A1 (fr) 1999-12-01
EP0960214A4 true EP0960214A4 (fr) 2004-08-04

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Country Link
EP (1) EP0960214A4 (fr)
AU (1) AU722819B2 (fr)
CA (1) CA2273847C (fr)
WO (1) WO1998024935A1 (fr)

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AU5515198A (en) 1998-06-29
AU722819B2 (en) 2000-08-10
WO1998024935A1 (fr) 1998-06-11
CA2273847C (fr) 2013-08-13
EP0960214A1 (fr) 1999-12-01

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