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WO1991005064A1 - Procede sensible servant a mesurer les transcriptions chimeriques de l'adn contenant des translocations - Google Patents

Procede sensible servant a mesurer les transcriptions chimeriques de l'adn contenant des translocations Download PDF

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Publication number
WO1991005064A1
WO1991005064A1 PCT/US1990/005427 US9005427W WO9105064A1 WO 1991005064 A1 WO1991005064 A1 WO 1991005064A1 US 9005427 W US9005427 W US 9005427W WO 9105064 A1 WO9105064 A1 WO 9105064A1
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WO
WIPO (PCT)
Prior art keywords
mrna
measuring
level
translocation
bcl
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
PCT/US1990/005427
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English (en)
Inventor
Maryalice Stetler-Stevenson
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United States Department of Commerce
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United States Department of Commerce
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Filing date
Publication date
Application filed by United States Department of Commerce filed Critical United States Department of Commerce
Publication of WO1991005064A1 publication Critical patent/WO1991005064A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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

Definitions

  • the present invention is related generally to the measurement of mRNA present in a biological sample. More particularly, the present invention is related to a sensitive and efficient method of measuring chimeric mRNA resulting from a chromosomal translocation, especially from biopsy specimens or the like, by a novel technique combining reverse transcription and polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Follicular lymphoma is a common B-cell neoplasm which has remained largely an incurable disease. Approximately 95% of follicular lymphomas carry the t(14;18) (q32;q21) chromosomal translocation which results in joining of the putative oncogene bcl-2 on chromosome 18 to the immunoglobulin heavy chain joining region gene, JH, on chromosome 14 (Tsujimoto et al, 1984, Science 226:1097; Tsujimoto et al, 1985, Science 228:1440). This creates a unique segment of DNA composed of a joined bcl-2/JH sequence.
  • an object of the present invention to provide a sensitive, reliable and efficient method for detecting micro quantities of mRNA found in biological samples, such as biopsy specimens. It is a further object of the present invention to provide a diagnostic test for detecting and predicting the course of conditions resulting from t(14;18) translocation, such as follicular lymphoma, Hodgkins disease and the like. Other obj cts and advantages will become evident from the following detailed description of the invention.
  • Figure 1 shows different levels of expression of bcl-2.
  • RNA extracted from tumor cells (lanes 1, 2, 3, 4) and from the cell line SUDHL-6 (lanes 5 and 6) was reverse transcribed and specifically amplified using the polymerase chain reaction technique. The amplified segments are subjected to electrophoresis and Southern blotting. Southern blots were then probed with a 32-P- labeled internal oligo.
  • RNA extraction Two methods of RNA extraction are used, a microprocedure and a standard method.
  • the microprocedure involves pelleting of an aliquot of 100,00 cells in sterile Eppendorf tubes, addition of about 11 ⁇ g of yeast RNA and suspension of cells and RNA in 100 ⁇ l of quanidine isothiocyanate buffer. This is layered over 100 ⁇ l of 5.7 M CsCl and centrifuged at 75,000 rpm for two hours in the Beckman TL-100. The resulting pellet is precipitated with ammonium acetate and ethanol and resuspended in 10 ⁇ l of sterile Tris EDTA buffer.
  • the standard method uses the Fast Tract mRNA Isolation Kit (Invitrogen) and provides polyA selected mRNA.
  • the extracted RNA is reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (BRL) and one of two types of primers, " oligo dT12-18 and a mixture of a specific immunoglobulin heavy chain u constant region primer Cmu(GAGGGGGAAAAGGGTTGGGGCGGAT) and a specific immunoglobulin heavy chain gamma constant region primer C gamma (CAACTGTCTTGTCCACCTTGGTGTT) .
  • the reverse transcription reaction is terminated after production of the first strand by addition of Na-EDTA, phenol chloroform extraction and ethanol precipitation.
  • the cDNA is resuspended in 10-15 ⁇ l of tris EDTA buffer and 5 ⁇ l are amplified using the polymerase chain reaction technique (PCR) with Taq polymerase (Cetus) .
  • PCR polymerase chain reaction technique
  • the primers for PCR are the mu constant region and gamma constant region primers described above and the bcl-2 primer adjacent to the major breakpoint region (TTAGAGAGTTGCTTTACGTGGCCTG) reportedby Stetler-Stevenson et al (1988, Blood 72:1822) as well as 5' and 3' primers for a standard housekeeping gene such as beta actin.
  • PCR is performed in the Thermocycler (Perkin Elmer Cetus) using a 100 mM Tris HC1 buffer, pH8.3, containing 500 mM KC1, 15 mM MgCl, 0.1% gelatin, 2.5 units of Taq polymerase, 200 ⁇ M each of dATP, dTTP, dCTP, and dGTP as well as 1 ⁇ g of each primer.
  • the reaction mixture is heated to 91"C for 5 minutes, cooled to 55 ⁇ C for 5 minutes and brought to 74 ⁇ C for 5 minutes. Following this first round, samples are heated at 94 ⁇ C, 55°C, and 74°C for 1.5, 1.5, and 2 minutes, respectively, for a total of forty cycles.
  • PCR sample Fifty percent of the PCR sample (25 ⁇ l) is resolved by electrophoresis (100V, 4-6 hr) in either 1.5% agarose (BRL) or 3% NuSieve (FMC) and 1% SeaKemp (FMC) agarose with 0.5 ⁇ g ethidium bromide/ml in Tris-Borate buffer. Alkaline Southern transfers are performed overnight using Genescreen Plus nylon filters (DuPont) and 0.4 N NaOH as a transfer buffer.
  • BBL 1.5% agarose
  • FMC NuSieve
  • FMC SeaKemp
  • Blots are then probed with a 32P-labeled 2.8 kb EcorRI/Hindlll fragment of the bcl-2 major breakpoint region, a 32P- labeled genomic JH SauIIIA fragment, and a 32P-labeled oligo with a sequence complimentary to a region immediately 3 » to the bcl-2 primer (CAACACAGACCCACCCAGAGCCCTCCTGCCCTCCTTCCGCGGGGGC) . Amplified segments ranging between 125 and 300 base pairs are detected by autoradiography.
  • PCR amplification of contaminating genomic DNA produces a product several kilobases in length due to the nontranscribed region (intron) between JH and C mu or C gamma that is present in the rearranged gene, but not in the mRNA transcript.
  • the technique of the present invention allows easy discrimination between PCR amplified products from the mRNA and genomic levels.
  • the blots are also probed for the housekeeping gene amplified segment, such as beta actin. Comparison of the chimeric bcl-2/JH amplified segment intensity to the intensity of the beta actin amplified segment allows relative quantitation of the expression of bcl-2.
  • RNA is extracted from tissue or mononuclear cells isolated from blood and subjected to the procedures described herein above. Presence of the translocation is indicative of tumor involvement in the setting of follicular lymphoma.
  • This technique can be used to determine the level of bcl-2 expression and this may be utilized in deterr atioh of prognosis and thus in selection of proper treatment modalities.
  • This technique can be used to measure the expression of any known oncogene in tumor tissue and thus be utilized as a prognostic indicator and for selection of appropriate therapy.
  • This technique can be used to determine the presence of leukemic cells containing a translocation and thus allow early detection of relapse following therapy.
  • This technique can be used to determine the level of expression of proteolytic enzymes involved in tumor invasion and metastasis and thus provide an indicator of metastatic potential in tumors.
  • This technique is also useful in staging of Hodgkins disease in cases with known t(l4;18) translocation.
  • mRNA is isolated, reverse transcribed and the cDNA fragment amplified and quantitated by standard methodologies well known in the art; a difference from the known steady state level of mRNA being indicative of the presence of t(14;18) translocation.
  • the combined reverse transcription polymerase chain reaction is a novel technique for measurement of bcl-2 transcripts requiring only small quantities of RNA that can be easily obtained at the time of biopsy.
  • bcl-2/JH mRNA Low levels (nanogram quantities) of bcl-2/JH mRNA can still be detected because following reverse transcription the specific bcl-2/JH segment is amplified up to a million fold by the polymerase chain reaction technique (Saiki et al, 1988, Sci. 239:487). Furthermore, amplified segments are run in agarose gels without formaldehyde thus removing a significant health hazard to laboratory personnel. Detection of a segment of genomic DNA or of RNA containing the t(14;18) translocation is diagnostic for tissue or peripheral blood involvement with follicular lymphoma in patients with this primary diagnosis and in addition, determination of levels of expression allows prediction of clinical cause.
  • a diagnostic test for predicting the clinical course of a malignancy where aggressive behavior by malignant cells is associated with deregulation of an oncogene, lower expression of an antioncogene, or excessive production of proteolytic enzymes associated with metastasis comprises the step of measuring the level of target gene (oncogene, antioncogene or enzyme and the like) mRNA in tumor specimens by the method of the present invention and comparing the level of this mRNA with mRNA of normal uninvolved tissue, any statistically significant deviation from the normal levels being indicative of the clinical behavior of the tumor.
  • target gene oncogene, antioncogene or enzyme and the like

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On décrit un nouveau procédé servant à mesurer le mARN. Le procédé réunit la transcriptase reverse et la réaction en chaîne de polymérase afin d'obtenir une mesure efficace et sensible.
PCT/US1990/005427 1989-10-02 1990-09-27 Procede sensible servant a mesurer les transcriptions chimeriques de l'adn contenant des translocations Ceased WO1991005064A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41611789A 1989-10-02 1989-10-02
US416,117 1989-10-02

Publications (1)

Publication Number Publication Date
WO1991005064A1 true WO1991005064A1 (fr) 1991-04-18

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ID=23648611

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/005427 Ceased WO1991005064A1 (fr) 1989-10-02 1990-09-27 Procede sensible servant a mesurer les transcriptions chimeriques de l'adn contenant des translocations

Country Status (4)

Country Link
EP (1) EP0494968A4 (fr)
AU (1) AU630001B2 (fr)
CA (1) CA2067114A1 (fr)
WO (1) WO1991005064A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993006248A1 (fr) * 1991-09-16 1993-04-01 The United States Of America Represented By The Secretary, Department Of Health & Human Services Procede de detection de genes c-raf-1
GB2260811A (en) * 1991-10-23 1993-04-28 Yorkshire Cancer Research Camp Diagnosis of malignant tumours by mRNA detection
WO1993024655A1 (fr) * 1992-05-27 1993-12-09 Amersham International Plc Analyse d'arn
EP0597699A3 (en) * 1992-11-10 1994-09-28 Tosoh Corp Protein secreted by cancer cells.
DE4431174A1 (de) * 1994-09-01 1996-03-07 Deutsches Krebsforsch Nachweisverfahren für tumorspezifische mRNA
US5643730A (en) * 1991-09-23 1997-07-01 Pfizer Inc. Process for detecting specific mRNA and DNA in cells
EP0731806A4 (fr) * 1993-12-03 1998-12-16 St Jude Childrens Res Hospital PROTEINES ET ACIDES NUCLEIQUES SPECIFIQUES FUSIONNES PRESENTS DANS LE LYMPHOME HUMAIN t(2;5), ET LEURS METHODES DE DETECTION ET D'UTILISATION
US5869308A (en) * 1988-08-26 1999-02-09 The United States Of America As Represented By The Department Of The Health And Human Services Detection method for C-RAF-1 genes
US6696548B2 (en) 1993-12-03 2004-02-24 St. Jude Children's Research Hospital Antibodies for recognition of alk protein tyrosine/kinase receptor

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4358586A (en) * 1980-12-23 1982-11-09 Harvey Rubin Detection and isolation of endorphin mRNA using a synthetic oligodeoxynucleotide
US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009456A1 (fr) * 1989-02-16 1990-08-23 Viktor Balazs Test de malignite (test de depistage du cancer) par la reaction en chaine de la polymerase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4358586A (en) * 1980-12-23 1982-11-09 Harvey Rubin Detection and isolation of endorphin mRNA using a synthetic oligodeoxynucleotide
US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0494968A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5869308A (en) * 1988-08-26 1999-02-09 The United States Of America As Represented By The Department Of The Health And Human Services Detection method for C-RAF-1 genes
WO1993006248A1 (fr) * 1991-09-16 1993-04-01 The United States Of America Represented By The Secretary, Department Of Health & Human Services Procede de detection de genes c-raf-1
US5643730A (en) * 1991-09-23 1997-07-01 Pfizer Inc. Process for detecting specific mRNA and DNA in cells
GB2260811B (en) * 1991-10-23 1995-07-05 Yorkshire Cancer Research Camp Detection of malignant tumours
GB2260811A (en) * 1991-10-23 1993-04-28 Yorkshire Cancer Research Camp Diagnosis of malignant tumours by mRNA detection
WO1993024655A1 (fr) * 1992-05-27 1993-12-09 Amersham International Plc Analyse d'arn
US5665544A (en) * 1992-05-27 1997-09-09 Amersham International Plc RNA fingerprinting to determine RNA population differences
EP0597699A3 (en) * 1992-11-10 1994-09-28 Tosoh Corp Protein secreted by cancer cells.
EP0731806A4 (fr) * 1993-12-03 1998-12-16 St Jude Childrens Res Hospital PROTEINES ET ACIDES NUCLEIQUES SPECIFIQUES FUSIONNES PRESENTS DANS LE LYMPHOME HUMAIN t(2;5), ET LEURS METHODES DE DETECTION ET D'UTILISATION
US6174674B1 (en) 1993-12-03 2001-01-16 St. Jude Children's Research Hospital Method of detecting a chromosomal rearrangement involving a breakpoint in the ALK or NPM gene
US6451997B1 (en) 1993-12-03 2002-09-17 St. Jude Children's Research Hospital Kits for detecting chromosomal rearrangements
US6696548B2 (en) 1993-12-03 2004-02-24 St. Jude Children's Research Hospital Antibodies for recognition of alk protein tyrosine/kinase receptor
DE4431174A1 (de) * 1994-09-01 1996-03-07 Deutsches Krebsforsch Nachweisverfahren für tumorspezifische mRNA

Also Published As

Publication number Publication date
CA2067114A1 (fr) 1991-04-03
EP0494968A4 (en) 1992-11-25
AU6606190A (en) 1991-04-28
AU630001B2 (en) 1992-10-15
EP0494968A1 (fr) 1992-07-22

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