[go: up one dir, main page]

WO1997008339A1 - Dosage diagnostic pour leucemie myelogene chronique - Google Patents

Dosage diagnostic pour leucemie myelogene chronique Download PDF

Info

Publication number
WO1997008339A1
WO1997008339A1 PCT/US1995/010919 US9510919W WO9708339A1 WO 1997008339 A1 WO1997008339 A1 WO 1997008339A1 US 9510919 W US9510919 W US 9510919W WO 9708339 A1 WO9708339 A1 WO 9708339A1
Authority
WO
WIPO (PCT)
Prior art keywords
abl2
rna
oligonucleotide
detector
solid phase
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/US1995/010919
Other languages
English (en)
Inventor
Janice Brown
Connie Lockhart-Bruce
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.)
Dade International Inc
Original Assignee
Dade International 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 Dade International Inc filed Critical Dade International Inc
Priority to AU34178/95A priority Critical patent/AU3417895A/en
Publication of WO1997008339A1 publication Critical patent/WO1997008339A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates generally to the field of methods for conducting a chronic myelogenous leukemia (CML) assay to detect or monitor CML cells in a human patient.
  • CML chronic myelogenous leukemia
  • Chronic myelogenous leukemia is a hematological disorder that is the result of neoplastic transformation of pluripotent stem cells. Symptoms include fatigue, anemia, leukocytosis and splenomegaly.
  • the first phase is called the chronic phase and is associated with an increased production of committed myeloid and lymphoid progenitor cells. Terminal differentiation is maintained in this phase and an elevated granulocyte count is produced.
  • the chronic phase progresses to the second phase, which is also called the blast crisis phase. In the second phase, terminal differentiation is lost and unregulated proliferation of immature lymphoid and myeloid blast cells occurs.
  • the Philadelphia (Ph) chromosome was first described in 1960 as an abbreviated chromosome found in the bone marrow of patients with CML.
  • the Ph chromosome is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;qll).
  • the potential breakpoints on chromosome 22 band qll occur in a small 5.8 kb region called the breakpoint cluster region (ber).
  • the breakpoint cluster region is part of a large ber gene which contains four exons.
  • Potential breakpoints on chromosome 9 are scattered over a distance of at least 100 kb, but are all located 5' to the c-abl proto-oncogene.
  • the Ph translocation transfers the c-abl gene from its position on chromosome 9 band q34 to the Ph chromosome.
  • FIGURE 1 is a schematic drawing of the translocation region of the Philadelphia chromosome consisting of either bcrl-abll or bcr3-abl2.
  • FIGURE 2 gives the DNA sequence for the human chimeric bcr/c-abl fusion protein gene, exons 2-5.
  • the chimeric DNA produces a hybrid transcript containing either ber exon 2 to c-abl exon 2 or ber exon 3 to c-abl exon 2.
  • the resultant chimeric gene transcribes an 8.5 kb mRNA that contains 5' ber and 3' c-abl in a head-to-tail tandem sequence.
  • This hybrid protein encoded by the chimeric mRNA has elevated tyrosine kinase activity and its appearance parallels the onset of clinical symptoms of CML and ALL.
  • ALL the translocations of c-abl to ber have been mapped to the intron between exons pl and p2 of the ber gene.
  • This gene transcribes a 7.0 kb mRNA that also contains 5' ber and 3' c-abl in a head-to tail tandem sequence.
  • Current methods for diagnosing CML include karyotype determination and Southern blot analysis. Karyotype analysis requires expertise in cytogenetics and takes weeks to obtain the results. Southern blot analysis normally takes several days to perform and requires extensive laboratory equipment.
  • the present invention includes a method of detecting or monitoring human chronic myelogenous leukemia (CML) cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA.
  • CML chronic myelogenous leukemia
  • RNA from hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions.
  • RNA/capture agent complex comprising a binding ligand and a capture oligonucleotide specific for the bcr2-abl2 and bcr3-abl2 translocation regions is hybridized to the amplified RNA to form an RNA/capture agent complex.
  • This binding may be carried out at about 42°C.
  • Oligonucleotides specific for a target region are defined as oligonucleotides that hybridize specifically to the appropriate target under appropriate hybridization conditions.
  • the RNA/capture agent complex is coupled to a solid phase having a receptor specific for the binding ligand coupled to the capture agent. The solid phase is washed and a detector agent is added to the solid phase.
  • the detector agent comprises a detector label and a detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions.
  • the addition of the detector agent may be carried out at room temperature.
  • the amount of the labeled detector oligonucleotide bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
  • the present invention also includes a method for detecting or monitoring CML cells in a human patient wherein a capture agent and a detector agent are simultaneously added to RNA that has been amplified using a pair of primers capable of amplifying both the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the capture agent comprises a binding ligand and a capture oligonucleotide specific for the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the detector agent comprises a detector label and a detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions.
  • the target complex made up of the amplified RNA, bound capture agent and bound detector agent is then applied to a solid phase having a receptor specific for the binding ligand coupled to it.
  • the solid phase is then washed and the amount of detector agent bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
  • the present invention further includes the following method for detecting or monitoring human CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA.
  • the RNA from a patient's hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions, and the amplification product is applied to a solid phase having a capture agent specific for the bcr2-abl2 and bcr3-abl2 translocation regions coupled to it.
  • the solid phase to which the amplified RNA is bound is then washed.
  • a detector agent comprising a detector label and an oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions is applied to the washed solid phase.
  • the amount of the detector agent bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
  • the present invention also includes the following method for detecting or monitoring human CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal
  • RNA from a patient's hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the amplification product is then incubated with a labeled detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions to form a reaction product/ detector complex.
  • the reaction product/ detector complex is applied to a solid phase having a capture agent coupled to it, where the capture agent is specific for the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the primer pairs used in the methods of the present invention may comprise a sense-RNA oligonucleotide containing a polymerase binding site and an antisense-RNA oligonucleotide lacking a polymerase binding site.
  • a sense-RNA oligonucleotide is a primer that generates a sense RNA in the amplification reaction and an antisense-RNA oligonucleotide is a primer that generates an antisense RNA in the amplification reaction.
  • These primers may be BB323/BB329, BB326/BB313 or BB325/BB329.
  • the RNA-based nucleic acid amplification may be self sustained sequence replication (3SR).
  • the binding ligand may be biotin and the receptor specific for the binding ligand may be either avidin or streptavidin.
  • the capture oligonucleotide may be CAP6, CAP7, CAP8, CAP10, CAP14 or CAP14T.
  • the detector oligonucleotide may be DET1, DET350, DET351, DET352, DET1-46 or DET1-3 and the detector label may be horseradish peroxidase, fluorescein, alkaline phosphatase or other appropriate detector molecule.
  • the solid phase may be a microwell plate, plastic beads, glass beads, magnetic particles or other solid phase materials known to those of ordinary skill in the diagnostic arts.
  • FIG. 1 is a schematic drawing of the bcr2-abl2 and bcr3-abl2 translocation region of the Philadelphia chromosome.
  • Fig. 2 gives the DNA sequence for the human chimeric bcr/c-abl fusion protein gene, exons 2-5.
  • Fig. 3 depicts the hybridization regions for capture agents CAP6, CAP7, CAP8, CAP10 and CAP14.
  • Fig. 4 depicts the hybridization regions for detector agents DET350, DET351, DET352, DET618, DET643 and DET646 (DET646 is also called DET1).
  • Fig. 5 depicts the location of the regions of hybridization for the primer pairs BB170/BB332 and BB170/BB333.
  • Fig. 6 depicts the location of the regions of hybridization for the primer pairs BB172/BB173, BB172/BB174, BB172/BB175 and BB172/BB176.
  • Fig. 7 depicts the location of the regions of hybridization for the primer pairs BB189/BB329, BB189/BB330, BB189/BB331, BB189/BB332, BB189/BB333 and BB189/BB188.
  • Fig. 8 depicts the location of the regions of hybridization for the primer pairs BB316/BB173, BB316/BB174, BB316/BB175, BB316/BB176 and BB316/BB309.
  • Fig. 9 depicts the location of the regions of hybridization for the primer pairs BB300/BB320, BB300/BB329, BB300/BB173, BB300/BB174, BB300/BB175 and BB300/BB176; BB300b/BB320, BB300b/BB173, BB300b/BB174, BB300b/BB175 and BB300b/BB176.
  • BB300 and BB300b hybridize to the same sequence region;
  • BB300 differs from BB300b in that it has a polymerase binding site sequence attached to it.
  • Fig. 10 depicts the location of the regions of hybridization for the primer pairs BB322/BB329 and BB322/BB331; BB323/BB329, BB323/BB330,
  • BB323/BB331 and BB323/BB333 BB325/BB313, BB325/BB328, BB325/BB329, BB325/BB330, BB325/BB331 and BB325/BB333; BB326/BB313, BB326/BB329 and BB326/BB330; and BB334/BB332 and BB334/BB333.
  • Fig. 11 depicts the hybridization regions for primer pair BB325/BB329, capture agent CAP14T and detector agent DET1.
  • Fig. 12 is a slot blot of 3SR products probed with oligo BB302, which is specific for the bcr2-abl2 translocation.
  • Fig. 13 is a slot blot of 3SR products probed with BB303, which is specific for the bcr3-abl2 translocation.
  • Fig. 14 is a summary of a portion of a clinical test. Karyotype results are given as the fraction of cells containing the Ph chromosome out of the total number of cells observed. Abbreviations are as follows: hydroxy urea (HU), interferon (IFN), true positive (TP), true negative (TN), false negative (FN). The CML EPA was scored as positive if the specimen absorbance value was greater than the positive cutoff value of 0.438 (i.e. 0.200 + OD value of the amplification blank).
  • the assays detect both the bcr2-abl2 and the bcr3-abl2 translocation associated with CML. These assays do not detect CML in the absence of the Ph chromosome, nor do they detect ALL even if the ALL patient has the Ph chromosome.
  • a blood sample is obtained from the CML patient and total RNA is extracted from the peripheral blood cells. The extracted RNA is contacted with primers that are capable of hybridizing at positions corresponding to sequences flanking the bcr2-abl2 and bcr3-abl2 translocation region of the Philadelphia chromosome. The RNA is then subjected to an amplification procedure such as self-sustained sequence replication (3SR) or nucleic acid sequence-based amplification (NASBA).
  • 3SR self-sustained sequence replication
  • NASBA nucleic acid sequence-based amplification
  • the reaction product if any, is captured onto a solid phase such as a microwell by means of a capture agent.
  • a solid phase such as a microwell by means of a capture agent.
  • a 96-well microwell plate facilitates automation, as these plates can be read with an ELISA plate reader, a fluorogenic reader, or any other reader capable of detecting the selected detector agent. Plates other than 96-well plates may be used such as 6, 24 or 48 cluster plates. These plates are available in the form of strip wells or as conventional culture plates.
  • Other suitable solid phases include, without limitation, glass, plastic or magnetic beads or other acceptable surfaces.
  • the detector agent can be an oligonucleotide probe to which a label, such as horseradish peroxidase (HRP), has been coupled.
  • a solution-based hybridization can be performed.
  • the amplified sample is simultaneously mixed with a capture agent and a detector agent to form a target/capture agent/ detector agent complex.
  • the complex is then applied to a microwell.
  • the amount of label bound to the solid phase is measured and correlated with the amount of amplification.
  • the readings are analyzed to determine the presence, progression or regression of CML in a semi- quantitative manner.
  • the amplified sample is placed in a microwell plate coated with a capture agent. After the amplified sample is coupled to the bound capture agent, any unbound material is rinsed away. A detector agent is then applied under conditions that allow it to hybridize to the captured target, and the amount of label bound to the solid phase is measured.
  • the amplified sample can be incubated with the detector agent to form a sample /detector agent complex. The complex is then added to a microwell coated with a capture agent.
  • a microwell format is used to detect specific sequences derived from the bcr-abl chimeric mRNA. Transcripts are amplified using the self sustained sequence replication (3SR) reaction.
  • the assay comprises two fundamental steps. The first step involves isolation and 3SR amplification of bcr-abl mRNA from leukemic cells. The second step involves the capture and detection of the amplified product in a microwell.
  • the amplified sample is mixed with a biotinylated oligonucleotide that has a base sequence complementary to the abl region of the amplified bcr-abl product.
  • a biotinylated oligonucleotide that has a base sequence complementary to the abl region of the amplified bcr-abl product.
  • the sample is then added to a streptavidin coated microwell.
  • the biotin portion of the oligonucleotide binds with the streptavidin, thereby immobilizing any biotinylated capture oligonucleotides that are hybridized to the amplified bcr-abl product. Unbound products are subsequently washed out of the microwell.
  • Detection of the captured bcr-abl product is achieved with a peroxidase-labelled oligonucleotide that hybridizes to a sequence complementary to the ber region of the amplified product. Following the incubation, unhybridized peroxidase-labelled oligonucleotide is washed away. Substrate is added and color is produced if the capture- bcr- flfo/-peroxidase complex is present in the microwell. The color production is quantified and compared to negative and positive controls to determine the presence of bcr-abl RNA.
  • EXAMPLE 1 Oligonucleotides All oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer (Millipore, Marlborough, MA) using standard phosphoramidite chemistries as indicated by the manufacturer's directions. Oligonucleotides that contained a biotin molecule at the 5' end of the DNA were synthesized using biotin phosphoramidite (Glenn Research, Sterling, VA). Oligonucleotides that contained an amine at the 3' end of the DNA were made using an Amino-On column (catalogue number 20-2957-42, Glenn Research).
  • the primers that were tested for use in 3SR amplifications of the bcr2-abl2 and bcr3-abl2 translocation regions of the Philadelphia chromosome in the assay of the present invention are listed in Table 1 below and are identified in the Sequence Listing as SEQ. ID. Nos. 1-30 and 44. All primers listed below are oriented in the 5' to 3' direction from left to right.
  • sequence AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-A represents the T7 polymerase binding site with the final four bases (GGGA) being the preferred transcription start site.
  • Capture agents were oligonucleotide sequences coupled to a binding ligand, such as biotin. Capture agents that were tested in the present experiments are listed in Table 2 and are identified in the Sequence Listing as SEQ. ID. Nos. 31-36.
  • FIGURE 3 depicts the locations of the regions of hybridization for the capture agents. These capture oligonucleotide sequences were immobilized on streptavidin-coated plates as described in Example 5 below.
  • Detector agents were oligonucleotide sequences coupled to a label such as horseradish peroxidase (HRP), fluorescein, or alkaline phosphatase. Detector oligonucleotide sequences (amine-derivatized) that were tested in the present experiments are listed in Table 3 below and are identified in the Sequence Listing as SEQ. ID. Nos. 37-42.
  • FIGURE 4 depicts the location of the regions of hybridization for the capture agents.
  • K562 is a line of pleuroeffusion cells derived from a 53 year old woman suffering from chronic myelogenous leukemia (CML) in blast crisis.
  • CML chronic myelogenous leukemia
  • the K562 cell line contains the bcr2-abl2 translocation.
  • the cells were grown in RPMI with 10% fetal bovine serum and maintained by standard culture methods.
  • K562 cells were removed from culture vessels and then collected by centrifugation following the methods of Shtivelman, et al., Nature, 315: 550-554 (1985).
  • the cells were solubilized by vortexing at room temperature in 450 ⁇ l of lysing buffer comprising 0.5% SDS, 50 mM Tris pH 7.4, 0.1 M dithiothreitol, and 5 mM vanadyl ribonucleoside complex.
  • An equal volume of the salt solution, 1.6 M KCl and 50 mM MgCl 2 was added to each sample and detergent-protein complexes removed by centrifugation. The supernatant was precipitated with isopropanol or an equal volume of 5% w/v cetyltrimethylammonium bromide (CTAB).
  • CTCAB cetyltrimethylammonium bromide
  • the proteins can be extracted by using standard acid phenol extraction procedures.
  • the nucleic acids were collected by centrifugation and washed twice with 70% ethanol.
  • the various oligonucleotide primers were synthesized both with and without the T7 polymerase binding site and the amplification levels were determined. Surprisingly, it was found that the bcr-abl target, unlike most other targets, was amplified to higher levels when the polymerase binding site was on the primer that generated the sense RNA. All the primers set forth in Table 1 hybridized to total RNA isolated from the K562 cell line and functioned in amplification of the translocation region. FIGURES 5-10 depict the location of the regions of hybridization for the primers.
  • the preferred primer concentration for 3SR was generally about 15 pmole of each primer per reaction.
  • the preferred primer pairs were BB323/BB329, BB326/BB313 and BB325/BB329. The most preferred primer pair was BB325/BB329.
  • Primer pairs were tested in 3SR reaction mixes with and without dimethyl sulfoxide (DMSP) and sorbitol as additives for amplification.
  • DMSP dimethyl sulfoxide
  • a 5 ⁇ l aliquot of each nucleic acid sample was used as a template in a 3SR amplification reaction in an RNase-free 1.5 ml microcentrifuge tube.
  • the reaction mixture contained 20 ⁇ l of a 5X buffer (200 mM Tris HCl, pH 8.1, 150 mM MgCl 2 , 100 mM KCl, 50 mM dithiothreitol, 20 mM spermidine), 5 ⁇ l (15 pmol) of each of the priming oligonucleotides, 20 ⁇ l of a 5X nucleoside triphosphate mix (35 mM rNTP's, 5 mM dNTP's), and 45 ⁇ l of DEPC-treated H 2 P.
  • a 5X buffer 200 mM Tris HCl, pH 8.1, 150 mM MgCl 2 , 100 mM KCl, 50 mM dithiothreitol, 20 mM spermidine
  • 5 ⁇ l 15 pmol
  • BB189/BB329 BB189/BB330, BB189/BB331, BB189/BB332, BB189/BB333 BB316/BB173, BB316/BB174, BB316/BB175, BB316/BB176
  • BB300/BB320 BB300/BB329, BB300/BB173, BB300/BB174, BB300/BB175, BB300/BB176
  • BB300b/BB320 BB300b/BB173, BB300b/BB174, BB300b/BB175, BB300b/BB176
  • Assay Plate Design Micro wells were coated with streptavidin as follows. A solution containing 10 ⁇ g/ml streptavidin in 50 mM sodium borate, pH 9.0 was incubated in a covered microwell plate at 37°C for four hours. The plates were then washed five times with 0.05% Tween-20, 10 mM Tris, pH 7.4. The coated plates were then packed with a desiccant in an aluminum foil pouch and stored at 4°C until ready for use.
  • CAP14, CAP14T and CAP8 were the preferred capturing agents, giving the highest OD values at an absorbance wavelength of 450 nm (A 450 ) using a plate reader system sold by Baxter Healthcare
  • the concentration of the capture agent during in-solution hybridization with target also was an important factor in optimizing the assay. Excessive levels of capture agent inhibited specific binding to the streptavidin microwell.
  • Table 6 gives the signal-to-noise ratio of different concentrations of capture agents tested in the assay. The signal-to-noise ratio was calculated by comparing the OD (A 450 ) of wells containing target, detector agent and various concentrations of capture agent to wells containing only a labelled capture agent.
  • Optimal (highest) signals from capture agent CAP14T were obtained at 42°C.
  • Kinetics of hybridization of capture agents to the 3SR amplified target were optimized at 30 minutes in 0.1% polyvinylpyrrolidone (PVP), 2X SSC (0.3 M NaCl, 0.03 M sodium citrate).
  • the CML detection assay can be performed in a format in which the amplified target is incubated with the capture agent and the detector agent simultaneously in the microwell.
  • the amplified target sequence first is incubated with the capture agent to allow the capture agent to bind the target sequence.
  • the capture agent/target complex is applied to the microwells and unbound material is washed away.
  • the detector agent is applied to the microwell. The preferred procedure allows the detector agent to hybridize only to the captured 3SR target without the dilution effect seen in the simultaneous format, where detector agent can hybridize to free capture oligonucleotides in solution.
  • the preferred basic hybridization solution contained 0.1% PVP and 2X SSC.
  • the long term stability of the detector agent was also evaluated. It was found that by adding 1% (w/v) BSA and 30% (v/v) glycerol to the basic hybridization solution (0.1% PVP, 2X SSC), the life span of the detector agent was increased. The presence of glycerol had the added benefit of decreasing the background signal. A final concentration of 30% glycerol gave the maximum signal with the minimum background.
  • Figure 11 depicts the hybridization regions for the preferred primer pair BB325/BB329, preferred capture agent CAP14T and preferred detector agent DET1.
  • EXAMPLE 6 Isolation of Total RNA from Clinical Samples Three-milliliter blood samples were collected from patients and placed into citrate or EDTA treated tubes. Heparinized tubes were not used as they were found to inhibit the RNA amplification reaction. Samples were stored at room temperature and processed within 24 hours of specimen collection.
  • the tube containing the blood sample was mixed by inversion 5 times.
  • the entire 3 ml of blood was transferred into a 15 ml polypropylene conical tube.
  • Three milliliters of phosphate buffered saline (PBS) were added and mixed with the blood by inversion.
  • a Histopaque gradient was prepared by pipetting 6 ml Histopaque 1119 (Sigma Co., St. Louis, MO) into a 15 ml polypropylene conical tube.
  • Six milliliters of the diluted blood were slowly pipetted over the Histopaque, avoiding mixing.
  • the tube was placed into a clinical centrifuge (swinging bucket type) and spun at 900 x g for 30 minutes at room temperature.
  • the white blood cells (WBCs) from the serum/Histopaque interface were collected using a plugged pasteur pipette and transferred to another 15 ml sterile conical tube. The WBCs were counted using a hemocytometer. After counting the cells, 7 x IO 6 WBCs were transferred to another tube and the volume was raised to 10 mis with PBS. The cells were mixed by inversion and pelleted by centrifugation at 900 x g for 15 minutes at room temperature. The PBS was carefully decanted without disturbing the cell pellet. Another 10 ml of PBS was added, and the cells were spun at 900 x g for 15 minutes at room temperature. Again, the PBS was carefully decanted without disturbing the cell pellet.
  • RNAzol BTM (Teltest, Friendswood, TX) was added to the cell pellet and vortexed for 30 seconds or until the cell pellet was homogenized. Alternatively, the cell pellet was homogenized by repeatedly pipetting the solution. The homogenate was transferred to a sterile RNAse- free microcentrifuge tube. One hundred microliters of chloroform were added and the tube was vortexed for 30 seconds, stored on ice for 5 minutes and then centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous phase was carefully collected, avoiding the interphase, and transferred to another RNAse-free microcentrifuge tube.
  • RNA pellet was washed twice by adding 1.0 ml of 75% ethanol, vortexing for 5 seconds and centrifuging at 7,500 rpm for 5 minutes at 2-25 °C.
  • RNA pellet was stored on ice until used in the amplification process.
  • RNAse-free microcentrifuge tubes were labeled with the proper specimen or control number for the enzyme probe assay (EPA).
  • One positive amplification control and one amplification blank were run each time an amplification was performed.
  • the reagents in Module 1 i.e. amplification reagent, positive amplification control template, enzyme mix, capture buffer and detection reagent
  • All the reagents except the amplification reagent were then placed on ice.
  • the amplification reagent was warmed to 42°C. Once a reagent was used, it was returned to -70°C.
  • Reagent A amplification reagent composed of buffer (40 mM Tris HCl, pH 8.1, 30 mM MgCl 2 , 20 mM KCl, 10 mM dithiothreitol, 4 mM spermidine), 7 mM rNTP's, 1 mM dNTP's, and 15 pmol of each of the priming oligonucleotides BB325 and BB329) and 5 ⁇ l of Reagent B (positive amplification control template composed of 0.2 attamole/ ⁇ l in vitro generated RNA transcript of the bcr-abl region in 10 mM Tris, 1 mM EDTA, pH 7.4 ) were added to the microcentrifuge tube labelled "positive amplification control.”
  • Reagent A were added to the microcentrifuge tube labelled "amplification blank.” No template was added to the amplification blank.
  • Reamplification reagent A amplification reagent composed of
  • the tubes containing the specimens and controls were then transferred to a 65°C (+ 1°C) water bath for 2 minutes and then to a 41 °C ( ⁇ 1°C) water bath for 5 minutes.
  • Five microliters of Reagent C (enzyme mix containing 30 units AMV Reverse Transcriptase, 2 units RNAse H and 100 units T7 RNA polymerase) were added to each tube and the mixture was gently vortexed. The tube was observed to ensure that the RNA pellet was not attached to the upper walls of the microcentrifuge tube.
  • the mixture was incubated in a 41 °C ( ⁇ 1°C) water bath for 1 hour.
  • the number of EPA microwells needed for the assay was determined.
  • One negative control (amplification blank), two positive controls (one positive amplification control and one positive hybridization control), and one substrate control (empty well) were run each time the assay was performed.
  • the number of wells required for specimens and controls was determined.
  • An appropriate number of commercially available strips of pre-coated wells were removed from the packaging and placed in a 96-well frame provided by the manufacturer. Care was taken so as to not scratch the bottom surface of the wells as scratches could interfere with the reading of the test results.
  • Reagent D capture buffer composed of 8 pmol biotinylated oligonucleotide in 4X SSC, 0.2% PVP
  • Reagent D capture buffer composed of 8 pmol biotinylated oligonucleotide in 4X SSC, 0.2% PVP
  • microwell plate was covered with a plate lid and incubated at room temperature for 20 minutes. After the incubation, the plate lid was removed and each well was washed 3 times with 300 ⁇ l wash solution (2X SSC, 0.05% Tween-20, 0.01% thimerosal) using a microwell plate washer or wash bottle. Following the last wash, the plate was inverted and tapped on a paper towel to remove excess wash solution. Two drops of Reagent 2 (detection reagent composed of 1 pmol peroxidase labelled detector oligonucleotide in 30% glycerol, 5X SSC, 0.1% PVP, and 1% BSA) was promptly added to the center of each well except the empty substrate control well.
  • Reagent 2 detection reagent composed of 1 pmol peroxidase labelled detector oligonucleotide in 30% glycerol, 5X SSC, 0.1% PVP, and 1% BSA
  • microwell plate was covered with the lid and incubated at room temperature for 30 minutes. Again the plate lid was removed and each well washed 3 times with 300 ⁇ l wash solution using a microwell plate washer or wash bottle. Following the last wash, the plate was inverted and tapped on a paper towel to remove excess wash solution. Two drops of Reagent 3 (peroxidase substrate containing 0.04% (w/v) 3,3',5,5' tetramethylbenzidine
  • TMB Tetrahydrofurane
  • 0.02% hydrogen peroxide solution manufactured by Kirkegaard & Perry, Gaithersburg, MD
  • the microwell plate was covered with the plate lid and incubated at room temperature for 15 ⁇ 2 minutes.
  • the plate reader e.g. Baxter PRIMA or
  • Figure 14 summarizes additional clinical test results in which the results using the CML EPA can be compared to results using other tests.
  • the data in Figure 14 reflect a sensitivity of 91% and a specificity of 100%.
  • Sensitivity is defined as the proportion of people who truly have a specific disease and are so identified by a test.
  • Specificity is defined as the proportion of people who are truly free of a specific disease and are so identified by a test.
  • OD correlate with what would be expected of a valid clinical assay for CML. This test can also be used prognostically to assess cytogenetic remission in patients with chronic myelogenous leukemia.
  • the filters were pre-wetted with hybridization solution (6X SSC, 10X Denhardts, 10 mM Tris pH 7.4, 0.2 mg/ml sheared salmon sperm DNA and 1% SDS) and then hybridized at 68°C for 45 minutes with a 32p. labeled oligonucleotide probe that was complementary to the junction sequence of bcr2-abl2 (BB302) or bcr3-abl2 (BB303).
  • hybridization solution 6X SSC, 10X Denhardts, 10 mM Tris pH 7.4, 0.2 mg/ml sheared salmon sperm DNA and 1% SDS
  • the filters were washed three times at room temperature for 5 minutes each using 1 ml buffer /cm 2 filter, where the buffer was 2X SSC, 0. 1% SDS.
  • the filters were exposed to X-ray film at -70°C with one intensifying screen.
  • the results of the 3SR amplification using clinical specimens are shown in Figure 12 and 13.
  • nucleic acid specific for the bcr-abl translocation and isolated from clinical specimens can be amplified and detected. Further, it can be distinguished whether the cells contain the bcr2-abl2 or the bcr3-abl2 translocation.

Landscapes

  • 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

Cette invention concerne des procédés permettant d'effectuer des dosages pour leucémie myélogène chronique (LMC) qui permettent de détecter ou de contrôler les cellules LMC chez un patient humain. On procède tout d'abord au prélèvement d'un échantillon chez un patient avant d'extraire la totalité de l'ARN. L'ARN extrait est ensuite mis en contact avec les primères adéquats qui entourent les zones de translocation bcr2-abl2 ou bcr3-abl2 du chromosome de Philadelphie, et les zones interprimères sont amplifiées. Après amplification, l'éventuel produit de réaction est piégé sur une phase solide à l'aide d'un agent de piégeage, puis détecté à l'aide d'un agent de détection marqué. La quantité d'agent de détection marqué est corrélée avec la présence ou la quantité de cellules LMC chez le patient.
PCT/US1995/010919 1995-08-25 1995-08-28 Dosage diagnostic pour leucemie myelogene chronique Ceased WO1997008339A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU34178/95A AU3417895A (en) 1995-08-25 1995-08-28 Chronic myelogenous leukemia diagnostic assay

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US29625895A 1995-08-25 1995-08-25
US08/296,258 1995-08-25

Publications (1)

Publication Number Publication Date
WO1997008339A1 true WO1997008339A1 (fr) 1997-03-06

Family

ID=23141269

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/010919 Ceased WO1997008339A1 (fr) 1995-08-25 1995-08-28 Dosage diagnostic pour leucemie myelogene chronique

Country Status (2)

Country Link
AU (1) AU3417895A (fr)
WO (1) WO1997008339A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998041652A3 (fr) * 1997-03-15 1998-12-17 Deutsches Krebsforsch Adn utilise pour detecter des modifications du chromosome 7
WO2000005418A1 (fr) * 1998-07-23 2000-02-03 Gen-Probe Incorporated Procedes pour detecter et mesurer des acides nucleiques episses
WO2002090563A1 (fr) 2001-05-07 2002-11-14 Applera Corporation Methodes de reduction d'artefacts de pcr dans l'amplification de microsatellites au moyen de sorbitol
RU2241989C2 (ru) * 2002-11-20 2004-12-10 Ульяновский государственный университет Способ прогнозирования течения хронического миелоидного лейкоза
US6849400B1 (en) 1997-07-23 2005-02-01 Gen-Probe Incorporated Methods for detecting and measuring spliced nucleic acids
JP2016077221A (ja) * 2014-10-17 2016-05-16 東洋鋼鈑株式会社 Ｂｃｒ−ａｂｌ阻害剤耐性関連変異の検出方法及びこれを用いたｂｃｒ−ａｂｌ阻害剤耐性を予測するためのデータ取得方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009285A1 (fr) * 1988-03-31 1989-10-05 Board Of Regents, The University Of Texas System Detection d'anormalites genomiques au moyen de transcripts uniques de genes aberrants
EP0338713A1 (fr) * 1988-04-18 1989-10-25 City Of Hope Diagnostic de leucémie chronique de myélogène
EP0430402A2 (fr) * 1989-12-01 1991-06-05 The Regents Of The University Of California Méthodes et compositions pour la coloration de chromosomes particuliers
US5066792A (en) * 1987-09-04 1991-11-19 Board Of Regents University Of Texas Gene probe for detection of specific human leukemias
WO1993012136A1 (fr) * 1991-12-11 1993-06-24 Thomas Jefferson University Detection et traitement de leucemies aigues resultant d'anomalies chromosomiques dans la region all-1

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5066792A (en) * 1987-09-04 1991-11-19 Board Of Regents University Of Texas Gene probe for detection of specific human leukemias
WO1989009285A1 (fr) * 1988-03-31 1989-10-05 Board Of Regents, The University Of Texas System Detection d'anormalites genomiques au moyen de transcripts uniques de genes aberrants
EP0338713A1 (fr) * 1988-04-18 1989-10-25 City Of Hope Diagnostic de leucémie chronique de myélogène
EP0430402A2 (fr) * 1989-12-01 1991-06-05 The Regents Of The University Of California Méthodes et compositions pour la coloration de chromosomes particuliers
WO1993012136A1 (fr) * 1991-12-11 1993-06-24 Thomas Jefferson University Detection et traitement de leucemies aigues resultant d'anomalies chromosomiques dans la region all-1

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998041652A3 (fr) * 1997-03-15 1998-12-17 Deutsches Krebsforsch Adn utilise pour detecter des modifications du chromosome 7
US6849400B1 (en) 1997-07-23 2005-02-01 Gen-Probe Incorporated Methods for detecting and measuring spliced nucleic acids
WO2000005418A1 (fr) * 1998-07-23 2000-02-03 Gen-Probe Incorporated Procedes pour detecter et mesurer des acides nucleiques episses
JP2002521037A (ja) * 1998-07-23 2002-07-16 ジェン−プローブ・インコーポレーテッド スプライシングされた核酸を検出および測定する方法
WO2002090563A1 (fr) 2001-05-07 2002-11-14 Applera Corporation Methodes de reduction d'artefacts de pcr dans l'amplification de microsatellites au moyen de sorbitol
EP1385976A4 (fr) * 2001-05-07 2005-03-09 Applera Corp Methodes de reduction d'artefacts de pcr dans l'amplification de microsatellites au moyen de sorbitol
RU2241989C2 (ru) * 2002-11-20 2004-12-10 Ульяновский государственный университет Способ прогнозирования течения хронического миелоидного лейкоза
JP2016077221A (ja) * 2014-10-17 2016-05-16 東洋鋼鈑株式会社 Ｂｃｒ−ａｂｌ阻害剤耐性関連変異の検出方法及びこれを用いたｂｃｒ−ａｂｌ阻害剤耐性を予測するためのデータ取得方法

Also Published As

Publication number Publication date
AU3417895A (en) 1997-03-19

Similar Documents

Publication Publication Date Title
Beà et al. Genetic imbalances in progressed B-cell chronic lymphocytic leukemia and transformed large-cell lymphoma (Richter's syndrome)
EP3211086B1 (fr) Procedes et compositions destines a produire des sondes d'adn a sequence unique, etiquetage de sonde d'adn et utilisation de ces sondes
AU702726B2 (en) Detection of hypermutable nucleic acid sequence in tissue
Aster et al. Detection of BCL2 rearrangements in follicular lymphoma
EP0672181A4 (fr) Procedes de detection d'acides nucleiques de mammifere isoles a partir d'un echantillon de selles, et reactifs utilises a cet effet.
JP4495349B2 (ja) 癌の遺伝マーカーを生物学的サンプルにおいて検出するための核酸配列
IE902727A1 (en) Methods and compositions for chromosome-specific staining
EP0633944A4 (fr) Procede de reaction de ligase en chaine multiplex.
JP2012505655A (ja) 無細胞体液中のｎｐｍ１核酸の検出
WO1994019492A1 (fr) Instabilite genomique specifique a une tumeur constituant un indicateur pronostique
Franks et al. Hypersecretion of androgens by polycystic ovaries: the role of genetic factors in the regulation of cytochrome P450c17α
US5220004A (en) Methods and reagents for G -65 -globin typing
US6849400B1 (en) Methods for detecting and measuring spliced nucleic acids
WO1997008339A1 (fr) Dosage diagnostic pour leucemie myelogene chronique
WO2005108611A2 (fr) Procedes et compositions pour l'amplification de mutations du diagnostic de la mucovicidose
IE920547A1 (en) Chromosome-specific staining to detect genetic¹rearrangements
EP0872561B1 (fr) Oligonucléotides spécifiques de Neisseria gonorrhoae
US5273883A (en) Methods and reagents for γ-globin typing
US5643724A (en) Methods and reagents for Glycophorin A typing
US20030082644A1 (en) Diagnostic detection of nucleic acids
US20050079496A1 (en) Methods for diagnosing and treating neoplasias using nf-at transcriptions factors
CA2255146A1 (fr) Detection d'un carcinome dans la vessie a partir d'un echantillon d'urine
EP1749025A2 (fr) Nouveaux nucleotides et sequences d'acides amines, et leurs dosages et procedes d'utilisation pour le diagnostic du cancer du colon
Zhang et al. Detection of leukemic blasts in peripheral blood specimens by reverse transcriptase polymerase chain reaction assay for myeloperoxidase mRNA
JPWO2006112483A1 (ja) びまん性大細胞型ｂ細胞リンパ腫の病型の診断方法及び予後診断の方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

WA Withdrawal of international application
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA