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WO1992009702A1 - Procedes et compositions de detection et de quantification de tumeurs hematopoietiques - Google Patents

Procedes et compositions de detection et de quantification de tumeurs hematopoietiques Download PDF

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WO1992009702A1
WO1992009702A1 PCT/US1991/008707 US9108707W WO9209702A1 WO 1992009702 A1 WO1992009702 A1 WO 1992009702A1 US 9108707 W US9108707 W US 9108707W WO 9209702 A1 WO9209702 A1 WO 9209702A1
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tal
dna
chromosome
probe
patient
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PCT/US1991/008707
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R. Graham Smith
Richard J. Baer
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University Of Texas System Board Of Regents
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • 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
    • C12Q1/6841—In situ hybridisation
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Definitions

  • the present invention relates to methods and compositions for diagnosis and prognosis of hematopoietic tumors in a human, and for detecting and quantitating minimal residual disease in hematopoietic tumors in a human.
  • the present invention relates to methods and compositions for detecting alteration of tal-l locus on chromosome 1 of human cells for the diagnosis and prognosis of T-cell acute lymphoblastic leukemia, and also relates to using oligonucleotide probes to monitor residual T-cell acute lymphoblastic leukemia in the blood, bone marrow and other body fluids from patients who are being treated for this leukemia.
  • T-cell acute lymphoblastic leukemia comprises about 157. of all cases of acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • features that distinguish this disease from B-lineage ALL include higher incidence in males, older mean age at diagnosis, high mean blood leukocyte count and the frequent presence of a mediastinal mass.
  • significant improvements in long-term disease-free survival for both children and adults have resulted from contemporary therapeutic programs, about 40% of children and at least 607. of adults with T-ALL still relapse and die of drug-resistant disease. Such failure is presumably due to residual leukemic cells which resist standard therapy.
  • Chromosome 1 harbors a genetic locus (designated ⁇ al. for T-cell acute leukemia) involved in leukemogenesis.
  • the tal-l gene was identified upon analysis of t(l: 14) (p34;qll) .
  • the breakpoint regions derived from one recurrent cytogenetic defect in T-ALL, namely the t(l; 14) (p34; ll) translocation were isolated and sequenced (Chen, Q., et al. (1990) EMBO J. 9:415-424; Chen, Q. , et al. (1990) J. Exp. Med. 172:1403-1408).
  • the breakpoints on chromosome 1 clustered within a 1 kb region. This translocation cleaves the tal-l gene on chromosome 1, separating its 5' end from the rest of the gene which is transposed into the T cell receptor ⁇ /y locus on chromosome 14.
  • the tal-l gene potentially encodes a protein containing a helix-loop-helix domain, which is found in a growing number of highly conserved DNA binding proteins involved in the regulation of growth and development.
  • Several genes in this family are known to be disrupted in subsets of ALL (Leder, P., et al. (1983) Science 222:765-771; Mellentin, J.D., et al.
  • site-specific rearrangements at the tal-l locus characterize nearly 30% of T-ALLs: about 3% are due to translocation (tal 1 alleles), while 26% result from an interstitial deletion which is too small to be detected cytogenetically (tal 11 alleles).
  • T-ALL T-ALL
  • a sensitive assay using a specific marker, for detecting and monitoring minimal residual leukemia cells in patients under treatment to develop strategies for the prevention of the recurrence of the disease.
  • an object of the present invention is to provide a novel method for the diagnosis and prognosis of hematopoietic tumors in a human patient by detecting an alteration, such as deletion and rearrangement, of tal-l locus on chromosome 1 in cells of a human patient.
  • Another object of the present invention is to provide a nucleic acid hybridization probe for the detection of altered tal-l locus on chromosome 1 in cells of a human patient.
  • Still another object of the present invention is to detect deletion of the tal-l gene in human cells by fluorescent in situ chromosome hybridization.
  • an object of the present invention is to provide a novel, sensitive and specific assay method for detecting minimal residual tumor, such as T-ALL, cells in patients.
  • Another object of the present invention is to provide a specific oligonucleotide probe to detect and monitor residual leukemic cells in the blood, bone marrow and other body fluids from patients.
  • Yet another object of the present invention is to provide a sensitive diagnostic test to select the best therapy for each individual leukemic patient through an understanding of how the tumor responds to each phase of treatment.
  • Another object of the present invention is to exploit both immune receptor gene and oncogene rearrangements as clonal markers of tumor populations in a patient.
  • Another object of the present invention is to provide a method and composition for the quantitation of minimal residual hematopoietic tumor cells in a patient in remission.
  • Still another object of the present invention is to detect unique DNA rearrangements by sensitive polymerase chain reaction methodology to provide clonal assays for ALL.
  • Yet another object of the present invention is to provide a test kit for detecting and monitoring of hematopoietic tumor cells in a patient.
  • Still another object of the present invention is to provide a test kit for quantitating minimal residual hematopoietic tumor cells in remission cells of a patient. Briefly, it is disclosed a method and composition to detect deletion and rearrangement of tal-l locus on chromosome 1 from human cells to confirm T-ALL in a patient.
  • PCR polymerase chain reaction
  • TdT terminal deoxynucleotidyl transferase
  • TCR T cell receptor
  • FIG. 1 illustrates rearrangement of the tal-l gene in T-ALL cells.
  • FIG. 2 shows that tal-l gene rearrangements in cell lines from unrelated T-ALL patients.
  • FIG. 3 illustrates that deleted tal-l (tal° rearrangements are tumor-specific.
  • FIG. 4 shows the structure of the tal d rearrangement.
  • FIG. 5 shows the normal structure of the AA-0.6 locus.
  • FIG. 6 illustrates the tal rearrangement generates a 90 kilobasepair deletion.
  • FIG. 7 shows nucleotide sequence encompassing the tal rearrangement of RPMI 8402.
  • FIG. 8 illustrates the deletion junction of tal d from RPMI8402 cells.
  • FIG. 9 shows that ______ deletion junctions resemble the coding joints of assembled immunoglobulin genes.
  • FIG. 10 (A) Sequence of the deleted tal-l (tal d ) allele derived from the cultured T-ALL cell line RPMI 8402. The germline sequences of the centromeric (top line) and telomeric (bottom line) sides of the rearrangement are aligned with the tal" allele (middle line) . About 90 kb of the normal sequence is deleted due to the rearrangement. The distal end of this deletion lies in the first intron of the tal-l gene. Junctional nucleotides not found in the germline sequences are in lower case letters. Oligonucleotides PI and P2 are used to amplify ______ alleles.
  • Oligonucleotides PI and F amplify a 251 bp fragment on the 5' (centromeric) side of normal tal-l alleles and serve as control primers in PCR assays.
  • Oligonucleotide H is used to detect amplified tal d alleles in hybridization assays. Dots covered in the top line signify nucleotides omitted from the figure.
  • the tal d sequences are aligned with germline centromeric (top line) and telomeric (bottom line) sequences, and non-germline nucleotides are shown in lower case letters.
  • the sizes of fragments amplified with oligonucleotides PI and P2 are shown at the right.
  • FIG. 11 Sequence of the translocated tal-l (tal ⁇ ) allele in T- ALL from patient L-23. Chromosome lp (top line) and 14q (bottom line) sequences are aligned with the tal* allele (middle line) .
  • the breakpoint on chromosome lp is in the first known intron of the tal-l gene, 2.5 kb 3' of the distal side of the tal d rearrangements.
  • the translocation creates a der lp chromosome, which joins the first exon of the tal-l gene to a rearranged D ⁇ 2-J ⁇ l gene on chromosome 14qll.
  • Non-germline junctional nucleotides are shown in lower case letters.
  • D 2 and J 1 segments are overlined.
  • Heptamer-nonamer recombination sequences surrounding the germline D 2 segment are highlighted with overlying dots.
  • Oligonucleotides XI and X2 are used to amplify the L23 tal* allele; oligonucleotide H2 is used to detect this amplified product in a hybridization assay. Dots covered in the top and middle lines signify nucleotides omitted from the figure.
  • FIG. 13 Detection of tal. 1 alleles by amplification/- hybridization assay.
  • A Ethidium bromide- stained 10% polyacrylamide gel of PCR products.
  • B Southern hybridization of PCR products with oligonucleotide H2 (FIG. 11).
  • FIG. 14 Quantitation of tal d alleles using an internal standard in PCR assay.
  • A Quantitation of L14D ______ alleles. Varying amounts of RPMI 8402 DNA, shown as genome copies above the lanes, were added to PCR mixtures containing either 50 or 250 genome copies of L14D DNA. The products were analyzed on a 10% polyacrylamide gel and stained with ethidium bromide. The L14D and RPMI 8402 products are marked on the left side of the figure; a size marker (194 bp) is shown on the right. The band intensities were quantitated densitometrically and the ratios of these intensities are shown below the lanes.
  • B Quantitation of L54F1 tal d alleles. Varying amounts of MOLT 16 DNA were added to PCR mixtures containing 1.9 X 10 5 genome copies of L54F1 DNA. The results are displayed as described in (A) .
  • novel methods and compositions are provided for the diagnosis and prognosis of hematopoietic tumors in a human host suspected of having such tumors. Novel methods and compositions are also provided for the monitoring and quantitation of residual tumor cells in a patient undergoing treatment. It has now been demonstrated in the present invention that a locus (designated tal) on chromosome 1 is altered in the tumor cells of a significant proportion (about 25%) of patients with T-ALL. Chromosomal rearrangements or alteration can be of deletion or translocation type.
  • the DNA was then added to a polymerase chain reaction mixture which contained a first and a second oligonucleotide probe, each of which was substantially concordant with regions of chromosome 1, and which regions spanned the site of rearrangements in the tal-l gene.
  • a polymerase chain reaction mixture which contained a first and a second oligonucleotide probe, each of which was substantially concordant with regions of chromosome 1, and which regions spanned the site of rearrangements in the tal-l gene.
  • the separated products were further analyzed by molecular hybridization with a third oligonucleotide probe which was concordant with a region of chromosome 1, and which region was in between the regions of concordance to the first and the second oligonucleotide probes. If rearrangements of the tal-l gene were detected and confirmed in a patient, these rearrangements were quantitated as follows: An "unknown" DNA from the patient was added to different quantities, precisely measured, of a DNA standard which contained the tal-l rearrangements. The standard was generally derived from T-ALL cells and was chosen to yield an amplified product which would differ in size from the amplified product derived from the "unknown" DNA.
  • the quantitating method comprised the steps of:
  • the t(l; 14) (p34;qll) translocation is found in 3% of T-ALL.
  • the breakpoint on chromosome 1 interrupts the tal-l gene, which potentially encodes a protein with a helix-loop-helix DNA binding motif.
  • a remarkably site-specific deletion interrupts the same gene in an additional 26% of T-ALL.
  • nearly one-third of these leukemias contain clustered rearrangements of the tal-l locus which were exploited as markers for residual disease.
  • Four (4) patients with T-ALL were monitored; 3 of the leukemias contained a deleted (tal d ) and one a translocated (tal*) tal-l allele.
  • tal-l alleles were recognized by an amplification/hybridization assay which could detect 10 rearranged tal-l alleles per 10 6 copies of the normal genome.
  • Rearranged tal-l alleles were not identified in normal peripheral blood mononuclear cells, thymocytes or bone marrow cells. Blood and marrow cells were collected from patients from the 4th through 20th month of antileuke ic treatment. tal d alleles were found in the blood of one patient during the 4th month of treatment but not thereafter. Using a quantitative assay to measure the fraction of tal d alleles in DNA extracts, it was estimated that this month 4 sample contained 150 tal d copies per 10 6 genome copies.
  • t(l; 14) p34;qll
  • tal 1 The patient with t(l; 14) (p34;qll) (tal 1 ) leukemia developed a positive assay during the 20th month of treatment. By standard criteria, all 4 patients remain in complete remission 11-20 months into treatment. Thus, genetic lesions at the tal-l locus provide clonal markers for monitoring minimal residual disease in approximately 30% of patients with T-ALL.
  • An increasing variety of therapeutic modalities has appeared, including novel doses and schedules of existing drugs, newer agents such as pentostatin (deoxycoformycin) , biologic response modifiers such as cc-interferon, immunotoxins, and allogeneic or autologous bone marrow transplantation.
  • a locus (designated tal) on chromosome 1 is altered in the tumor cells of a significant proportion (about 25%) of patients with T-ALL.
  • the tal-l locus alterations on chromosome 1 can be readily detected by Southern hybridization analysis or by the polymerase chain reaction. The uses of this invention are fourfold: First, the tal-l locus alterations on chromosome 1 can be used to facilitate the diagnosis of T-ALL.
  • the tal-l locus alterations on chromosome 1 can be used prognostically to identify T-ALL patients that are likely to suffer a relapse of leukemia after the initial therapy.
  • the tal—1 locus alterations on chromosome 1 can also be used prognostically to track minimal levels of residual disease in T-ALL patients during treatment and during remission.
  • different oligonucleotides can be used to quantitate and measure 10 rearranged tal-l alleles per one million copies of normal genome.
  • tal-l locus on chromosome 1 observed in different patients are identical, i.e., they all arose from a precise 90 kilobasepair ("kb") deletion that disrupts the coding region of tal-l in a manner analogous to the t(l; 14) (p34;qll) translocation.
  • kb kilobasepair
  • the extraordinary precision of these deletions suggests that they are mediated by a site-specific DNA recombinase.
  • analysis of the deletion junctions indicates that ______ rearrangement is engendered by aberrant activity of the same recombinase that controls immunoglobulin and T cell receptor gene assembly.
  • tal-l locus denotes a region of DNA approximately 200 kb upstream and approximately 200 kb downstream of tal—1 transcription unit.
  • the DNA extract containing chromosome 1 can be isolated from a human tissue such as blood or bone marrow.
  • Site-specific DNA rearrangements in ALL cells provide clonal markers for the detection of residual disease. These rearrangements are of two sorts: physiologic immune receptor gene rearrangements and pathologic recombinational events such as chromosomal translocations, deletions and insertions. Examples of the former process are TCR - VDJ segment rearrangements which have been exploited as clonal markers for T—ALL populations. Potential disadvantages of this approach include the requirement for specific probes for each clone, the dominance of new rearrangements during clonal progression and doubts regarding true tumor specificity of the particular rearrangement being monitored. Use of the latter, pathological kinds of rearrangements as clonal markers may overcome these limitations, especially if a site-specific abnormality were found in a large fraction of T-ALLs.
  • the tal d rearrangement was observed in at least 25% of T-ALL samples, including leukemic specimens obtained directly from T-ALL patients and leukemic cell lines established from these patients. No similar lesions in other hematopoietic tumors, including pre-B-ALL and the other forms of T cell neoplasia, were observed.
  • alteration of the tal-l gene either by tal d rearrangement or t(1; 14) (pl3; qll) translocation, appears to be predominantly, if not exclusively, associated with T-ALL.
  • type A mRNA includes the three coding exons provisionally designated la, II and III, while type B mRNA includes exons lb, II and III (see FIG. 1A discussed below).
  • type B mRNA includes exons lb, II and III (see FIG. 1A discussed below).
  • Three of the four t(1; 14) (p32;qll) translocations analyzed feature breakage within a 1 kb region of the tal-l locus. It is noteworthy that the translocation breakpoint region and the site of tal d rearrangement both fall between exons la and lb.
  • tal d deletion and t(1; 14) (p32; qll) translocation are structurally equivalent alterations of tal-l in that each removes exon la from the remainder of the locus and thereby precludes the production of type A mRNA.
  • the effect of these lesions on expression of type B mRNA cannot be evaluated until its transcription start site is defined.
  • a remarkable feature of the tal d rearrangement is its apparent site-specificity, especially in view of the substantial size of the deletion that it engenders.
  • the only site-specific DNA rearrangements observed in vertebrates are those involved in the assembly of the immunoglobulin (Ig) and T cell receptor (TCR) genes during lymphoid development. Since tal rearrangements arise in T-lineage cells, these may also be mediated by the same recombination system. Rearrangements within the Ig/TCR loci are directed by signals that flank the rearranging gene segments and presumably serve as recognition sites for the Ig/TCR recombinase.
  • CACAGTG consensus heptamer of Ig/TCR recombination signals
  • heptamers should be relatively inefficient at directing recombination since they are not associated with conserved nonamer elements and they bear sequence deviations from the consensus heptamer that are likely to reduce, but not eliminate, the rate of recombination.
  • the role of the Ig/TCR recombinase in tal d rearrangement can be further evaluated by examination of tal d deletion junctions from T-ALL patients.
  • a "coding joint” which constitutes the fusion of two gene segments of the rearranging locus
  • a "signal joint” comprised of the two recombination signals that had previously flanked the rearranged gene segments.
  • Signal joints are usually formed in a conservative fashion without the loss or gain of nucleotides at the recombination junction.
  • coding joints are diversified as a result of both the random trimming and random addition of nucleotides at the junction.
  • the ______ deletion junctions bear a striking resemblance to the coding joints of assembled Ig/TCR genes. For example, if cleavage within germline AA-
  • 0.6 occurs adjacent to the proposed heptamer (see FIG. 9 discussed below), then a variable trimming of nucleotides (0 to 22 residues) clearly takes place before relegation of the AA-0.6 end to form tal d : exonucleolytic trimming of the B2EE-2.0 sequence (0 to 8 residues) is also evident. Moreover, 21 of the 22 tal d junctions bear random nucleotides (0 to 13 residues) that are not derived from germline sequences, and thus may have been generated in a manner similar to the
  • N-region nucleotides of Ig/TCR coding joints acquire nonrandom insertions of defined mono- and di-nucleotides (designated P nucleotides). Although the complete rules for their identification are complex, P nucleotides are only found appended to coding ends that have not suffered exonucleolytic trimming. Notably, a thymidine residue (underlined in FIG. 9 discussed below) that fulfills the criteria of P nucleotides can be seen in each of the six tal d junctions that bears an untrimmed AA-0.6 sequence (CCRF-HSB-2,
  • tal junctions and the coding joints of assembled Ig/TCR genes implies that tal d deletions are mediated by the Ig/TCR recombinase.
  • Aberrant activity of the recombinase has also been implicated in the formation of chromosome translocations involving the Ig/TCR loci. Nevertheless, at least one of the two recombining elements responsible for these translocations corresponds to an Ig/TCR sequence that normally serves as a recombination signal.
  • the site of this common rearrangement within the first known intron of the tal-l locus is approximately 1 kb 5' of a cluster of breakpoints found in the t(1; 14) (p34;qll) translocations in T-ALL.
  • the remarkably focused nature of the rearrangements provides an opportunity to monitor the leukemic clones with straightforward genomic PCR assays. A single pair of amplimers sufficed for all tal d alleles, while a few additional pairs should be adequate to detect the tal* cases.
  • T-ALLs should thus be amenable to disease monitoring based upon these tal-l rearrangements.
  • the success of this approach depends upon the absence of tal-l rearrangements in normal hematopoietic cells. The site-specific nature of these rearrangements raises the question whether they could play a role in normal T cell development or function.
  • aberrant trans-rearrangements involving immune receptor genes have been demonstrated by PCR of normal thymocyte DNA. Since both kinds of tal-l rearrangements studied herein may originate from misdirected action of the immune receptor recombinase, cell populations containing mature and developing T lymphocytes were examined for evidence of ______ alleles.
  • the modified assay relies on an internal standard which contains a second tal d allele distinguishable from the first based on the size of the amplified product. This assay accurately measured 50 to 250 copies of ______ alleles present in a large excess of normal DNA. Thirty copies of the L54 _____ allele were found in the L54F1 sample, during the 4th month of treatment.
  • a test kit which permits both the detection, or diagnosis, and the monitoring of the hematopoietic tumor cells in a patient. Further, a test kit is provided which permits quantitating the residual hematopoietic tumor cells in remissions cells of a patient.
  • the detection and monitoring test kit comprises: A first and a second oligonucleotide probe, each of which is substantially concordant with first and second regions of chromosome 1, and which regions span the site of rearrangements of tal-l gene on chromosome 1 found in a DNA sample isolated from a patient having hematopoietic tumor cells.
  • the kit may further comprise a third oligonucleotide probe which is substantially concordant with a third region of chromosome 1, which region lies in between the first and second regions, which are sites of concordance with the first and the second oligonucleotide probes.
  • the detection and monitoring test kit comprises: A first and a second oligonucleotide probe, both of which are substantially concordant with regions of chromosome 1, and which regions span the site of rearrangements of tal-l locus on chromosome 1 of a first DNA sample isolated from a patient having T- ALL.
  • These first two oligonucleotide probes shall be used to amplify a product, the presence of which indicates a rearrangement of the tal-l gene found in patient with T-ALL.
  • test kit may contain a second DNA sample having such rearrangements, and a third DNA sample without having such rearrangements. These second and third DNA samples can be used to confirm that the detection system is working properly.
  • the quantitating test kit comprises: A first and a second oligonucleotide probe, both of which are substantially concordant with regions of chromosome 1, and which regions span the site of rearrangements of tal-l gene on chromosome 1 of a first DNA sample isolated from a patient having residual hematopoietic tumor cells; a third oligonucleotide probe which is substantially concordant with a region of chromosome 1, which regions lies in between the sites of concordance with the first and the second oligonucleotide probes; a first series of internal standards of measured dilutions of a second DNA sample from a second T-ALL patient having tal-l rearrangements, this first series is provided which, after amplification, yields a large-sized product; and a second series of internal standards of measured dilutions of a third DNA sample from a third T-ALL patient, the second series is provided which, after amplification, yields a small-sized product.
  • the methods and compositions of the present invention utilize the following materials and general methods: Tumor Specimens and Cell Lines Leukemic specimens were provided by the Pediatric Oncology Group
  • Genomic DNA libraries of BamHI-digested patient DNAs were constructed in phage vector ⁇ 2001 (Karn, J. et al. (1984) Gene 32:217-224) .
  • a cDNA library of poly(A)-selected RNA from CCRF-CEM cells (Foley, G.E. et al. (1965) Cancer 18:522-529) prepared in the phage vector ⁇ ZAP II (Short, J.M.
  • the hybridization of B2EE-2.0 was perfectly concordant with chromosome 1 and randomly associated (18-65% discordancy) with every other human chromosome (Table 1).
  • Table 1 Concordancy analysis of each human chromosome with B2EE-2.0 in the 17 hybrids of the hybrid clone human mapping panel.
  • -+/+ have the chromosome and B2EE, +/- have the chromosome but not B2EE, -/+ do not have the chromosome but have B2EE, -/- have neither the chromosome nor B2EE.
  • AA-0.6 DNA fragment by somatic cell hybrid analysis was conducted exactly as described for the B2EE-2.0 fragment (Chen, Q. et al. (1990) EMBO J. 9:415-424).
  • Pulsed-field gel electrophoresis (Schwartz, D.C. and Cantor, C.R. (1984) Cell 37:67-75) was conducted on a transverse alternating-field (Gardiner, K. et al. (1986) Som. Cell Mol. Genet.
  • Amplification of tal d deletion junctions was conducted by the polymerase chain reaction (Saiki, R.K. et al. (1988) Science 239:487- 491) using oligonucleotide primers C (AGGGGAGCTCGTGGG AGAAATTAAG) and D (TCACAATCCCACCGCATGCACA).
  • the reaction conditions were similar to those described (Cheng, J.-T. et al. (1990) J. Exp. Med. 171:489-501).
  • the amplification products were fractionated fay electrophoresis on 10% polyacrylamide gels and visualized by ethidium bromide staining.
  • the amplification products were phosphorylated with polynucleotide kinase (New England Biolabs) and cloned into the S al site of M13mpl8 for nucleotide sequence analysis.
  • FIG. 1A shows a restriction map of the tal-l gene in its normal configuration.
  • the small arrows designate the sites of chromosome breakage due to t(1; 14) (p32; qll) translocations from patients 4 and 5 (Chen, Q. et al. (1990) EMBO J. 9:415-424), DU.528 (Begley, C.G. et al. (1989) Proc. Natl.
  • the (1; 14) (p32;qll) chromosome translocation in leukemic cells is only observed in 3% of T-ALL patients. If this DNA rearrangement represents a junction of the t(1; 14) (p32; qll) translocation, then sequences upstream of the divergence point should be derived from chromosome 1. Therefore a 2.0 kb EcoRI fragment from this region was isolated and subcloned into a plasmid vector. This clone (B2EE-2.0) was then used as a probe in Southern filter hybridizations with DNAs extracted from a panel of 17 human/hamster somatic cell hybrids with randomly segregated human chromosomes (Thompson, L.E. et al.
  • FIG. 1 shows Southern analyses of BamHI-digested DNAs hybridized with B2EE-2.0, a probe representing sequences from the translocation breakpoint region of tal-l.
  • FIG. IB shows the rearrangement of the tal-l gene in T-ALL cell lines.
  • a Southern filter of BamHI-digested DNAs was hybridized with tal-l probe B2EE-2.0.
  • the DNAs were derived from T-ALL cell lines Jurkat (lane 1), RPMI8402 (2), CCRF-CEM (3), MOLT-3 (4), M0LT-13 (5), MOLT-16 (6), PEER (7), and CCRF-HSB-2 (8).
  • Hindlll ⁇ DNA marker fragments are indicated in kb.
  • FIG. 1C shows the rearrangement of the tal-l gene in primary T-ALL cells.
  • a Southern filter of BamHI-digested DNAs was hybridized with B2EE-2.0. The DNAs were derived from peripheral blood obtained from T-ALL patients before treatment.
  • FIG. 2A shows the Southern analysis of genomic DNAs digested with any of six different restriction endonucleases and hybridized with the B2EE-2.0 probe. Genomic DNAs were derived from the non-leukemic B cell line RPMI83902 (lanes 1) and the T-ALL cell line RPMI8402 (lanes 2). Thus, FIG.
  • FIG. 2A shows the hybridization pattern obtained for DNA from the T-ALL cell line RPMI8402; in each digest the B2EE-2.0 probe detected an equimolar ratio of the normal DNA fragment and a rearranged DNA fragment, indicating that one allele of tal-l had undergone a structural rearrangement in RPMI8402 cells.
  • FIG. 2B shows the Southern analysis of DNAs hybridized with the B2EE-2.0 probe. The DNAs of CCRF-CEM, a leukemic cell line derived from an unrelated T-ALL patient are shown by lanes 2; DNA' s derived from non-leukemic control cells are shown by lanes 1.
  • the B2EE-2.0 probe detects DNA rearrangement of one of the two alleles of tal-l.
  • the rearranged DNA fragment in each restriction digest of CCRF-CEM DNA is similar in size to that observed in RPMI8402 DNA, indicating that both cell lines bear an identical rearrangement of the tal-l locus.
  • an identical pattern of rearranged DNA fragments was obtained upon analysis of each of the other T-ALL samples with tal-l gene alterations. Therefore, a high proportion of T-ALL patients exhibit a common rearrangement of the tal-l locus (designated tal") that, at least at the level of Southern analysis, appears to be the same in each patient.
  • the tal d Rearrangement is Tumor-Specific.
  • FIG. 3 shows tal d rearrangements are tumor specific. Southern hybridization analysis of EcoRI-digested DNAs hybridized with the B2EE-2.0 tal-l probe; tal d gene rearrangements are seen in DNAs of T-ALL cell lines (RPMI8402 and CCRF-HSB-2) but not non-leukemic B cell lines (RPMI8392 and CCRF-SB, respectively) from the same patients.
  • tal d rearrangements can be detected in DNA from peripheral blood obtained from T-ALL patients before treatment (lanes L) but not after remission induction (lanes R) .
  • tal d rearrangement is tumor- specific.
  • tal is apparent in T lymphoblastoid lines from two T-ALL patients (RPMI8402 and CCRF-HSB-2), but not in non-leukemic B cell lines derived from the same patients (RPMI8392 and CCRF-SB, respectively) (Hayata, I. et al. (1975) In Vitro 11:361-368). Similar results were obtained upon analysis of fresh specimens from T-ALL patients.
  • tal d is not a genetic polymorphism of the tal-l gene, but instead represents an acquired alteration that appears to be restricted to the leukemic cells of T-ALL patients.
  • D. The ______ Rearrangement is Generated by Local DNA Recombination.
  • a bacteriophage ⁇ library of genomic DNA from RPMI8402 cells was screened with B2EE-2.0, and several clones with inserts spanning the rearrangement were obtained (e.g., ⁇ BLI and ⁇ BL3).
  • FIG. 4A it is shown ⁇ BLI and ⁇ BL3 clones isolated by screening a library of genomic DNA
  • DNA from the tal d -positive RPMI8402 cell line with probe B2EE-2.0 was compiled from those of ⁇ BLI and ⁇ BL3, and is compared to that of the normal tal-l locus.
  • the downstream endpoint of the _____ deletion is denoted with a large arrow.
  • the B2BE-0.9 and B2EE-2.0 probes are derived from normal tal-l sequences, and the AA-0.6 probe is derived from novel sequences engendered by the tal d rearrangement.
  • the major breakpoint region of t(l; 14) (p32; ll) is bracketed and the position of the HLH-encoding exon III is indicated.
  • FIG. 4B depicts a Southern filter identical to that shown in FIG. 2A as being hybridized with the B2BE-0.9 probe. Genomic DNAs in lanes 1 were derived from the non-leukemic B cell line RPMI83 2 (lanes 1) and the T-ALL cell line RPMI8402 (lanes 2).
  • a restriction map encompassing the tal" rearrangement was compiled by analysis of these clones, and in FIG. 4 this map is compared to that of the normal tal—1 locus. As illustrated, the maps diverge within a 0.25 kb EcoRI-SphI fragment of the normal tal-l locus, at a position approximately one kb upstream of the t(l; 14) (p32;qll) breakpoint region. As a consequence of the rearrangement, novel DNA sequences are juxtaposed with the tal-l locus (FIG ' . 4).
  • AA-0.6 was perfectly concordant with chromosome 1 and randomly associated (18-65% discordancy) with every other human chromosome.
  • the regional localization of AA-0.6 was determined by analysis with a panel of hybrids containing broken derivatives of chromosome 1 (Stallings, R.L. 0 et al. (1988) Am. J. Hum. Genet. 43:144-151). Since the AA-0.6 probe showed low discordance with short arm markers (17-24%) and high discordance with long arm markers (44-75%) , it is likely to be derived from the short arm of chromosome 1. Moreover, the same hybrid panel had been analyzed previously with the B2EE-2.0 probe (Chen, Q. et al. 5 (1990) EMBO J.
  • FIG. 5 shows the normal structure of AA-0.6 locus.
  • the ⁇ BH3 and ⁇ SP3 clones were obtained by screening a library of genomic DNA from the tal d -neeative 5 cell line SUP-T1 with probe AA-0.6.
  • the restriction map of the unrearranged AA-0.6 locus was compiled from those of ⁇ BH3 and ⁇ SP3.
  • the upstream endpoint of the tal d deletion is denoted with an arrow. Restriction sites are marked as in FIG. 1A.
  • the ______ Rearrangement Represents a DNA Deletion of about 90 kb. °
  • the tal d rearrangement might conceivably arise by any of a number of distinct processes, including local DNA inversion, duplication, insertion or deletion.
  • both normal and leukemic DNAs were hybridized with B2BE-0.9, a probe located immediately upstream of B2EE-2.0 in normal DNA, but on the 5 opposite flank of the rearrangement site (see FIG. 4A) . If tal rearrangement occurs without loss of genetic material, then Southern analyses with B2BE-0.9 should reveal rearranged DNA fragments upon digestion of RPMI8402 DNA with restriction enzymes that recognize sites flanking both B2BE-0.9 and B2EE-2.0 (e.g., BamHI, Hindlll, Bglll, PstI) . Nevertheless, as shown in FIG. 4B, only normal DNA fragments are detected with B2BE-0.9.
  • B2BE-0.9 exhibits the same pattern of hybridization with genomic DNA from each of the other T-ALL samples that bear tal d .
  • _____ is generated by local DNA deletion, with concomitant loss of sequences between the two recombining elements (represented by AA-0.6 and B2EE- 2.0).
  • AA-0.6 and B2EE-2.0 originate from the same region of chromosome 1, the restriction maps of normal genomic DNA around these markers do not overlap. Indeed, direct comparison of these maps indicates that AA-0.6 and B2EE-2.0 are separated by at least 35 kb (FIGS. 1A and 5). Consequently, if tal d is generated by simple deletion, then the segment of DNA deleted is likely to be substantial.
  • FIG. 6A shows that Notl-digested genomic DNAs from the non-leukemic B cell line RPMI8392 (lanes 1, 3, and 5) and the T-ALL cell line RPMI8402 (lanes 2, 4, and 6) were fractionated by pulsed-field gel electrophoresis and transferred to a membrane filter. The filter was hybridized, stripped of radioactivity and rehybridized successively with probes B2EE-2.0 (FIG. 4A) , AA-0.6 (FIG. 4A), and WI (FIG. 6B) . Size markers are concatamers of ⁇ DNA spaced at about 50 kb intervals.
  • FIG. 6B shows the NotI restriction map of the normal and rearranged alleles of tal-l.
  • the positions of the AA-0.6 (AA), WI, and B2EE-2.0 (B2) probes are indicated.
  • the WI probe represents sequences located 12 kb upstream of B2EE-2.0 in the normal tal-l allele.
  • the sizes of NotI restriction fragments that co—hybridize with AA-0.6 and B2EE-2.0 are indicated in kb.
  • the asterisk denotes the NotI site that exhibits variable resistance to NotI digestion.
  • the AA-0.6 and B2EE-2.0 markers reside within a 190 kb NotI fragment; the 220 kb species is likely to arise in certain cell lines as a consequence of partial cytosine methylation at one of the flanking NotI sites.
  • the relative position of AA-0.6 within the 190 kb fragment can be deduced by Southern analysis of Notl-digested DNA from cells bearing tal d .
  • the tal d rearrangement generates a 130 kb NotI fragment that co-hybridizes with the AA-0.6 and B2EE-2.0 probes (FIG. 6A, lanes 2 and 4).
  • these probes co-hybridize with either a single rearranged 130 kb fragment (e.g., CCRF-CEM) or with two rearranged fragments of 130 and 100 kb (e.g., CCRF-HSB-2).
  • B2EE-2.0 sequences that contain a NotI recognition site As depicted schematically in FIG. 6B, this in turn localizes AA-0.6 to a position approximately 90 kb upstream of B2EE-2.0 in normal genomic DNA. It is noteworthy that tal-l DNA probes located between AA-0.6 and B2EE-2.0 (e.g., WI; FIG. 6B) hybridize with the normal 220/190 kb NotI fragment(s) but not with the rearranged 130/100 kb species (FIG. 6A, lanes 5 and 6).
  • the deletion junction from RPMI8402 cells can be localized to a 0.7 kb BamHI-SphI fragment, the sequence of which is presented in FIG. 7, which shows the nucleotide sequence encompassing the tal rearrangement of RPMI8402.
  • the sequence includes the 0.7 kb BamHI-SphI fragment of the tal d allele of RPMI8402 cells.
  • the rearrangement site was determined by comparative analysis of normal and rearranged tal-l sequences (see FIG. 8). The positions of synthetic oligonucleotide primers are indicated; the oligonucleotide C sequence is as shown, whereas the oligonucleotide D sequence is the reverse complement of that shown.
  • nucleotide sequences of corresponding germline DNA in the vicinity of AA-0.6 and B2EE-2.0 were determined. In FIG. 8, these sequences are aligned so as to illustrate the deletion junction. Interestingly, the RPMI8402 junction contains a stretch of nine nucleotides which are not derived from germline sequences in the region of either AA-0.6 or B2EE-2.0.
  • FIG. 8 shows the deletion junction of tal d from RPMI8402 cells.
  • the tal d deletion junction of RPMI8402 (B) is identified by comparison with germline sequences of the AA-0.6 region (A) and the B2EE-2.0 region (C) .
  • the nine nucleotide residues at the junction are not derived from germline sequences of AA-0.6 or B2EE-2.0. Heptamer sequences of the putative recombination signals are marked with asterisks.
  • each product is comprised of AA-0.6 sequences juxtaposed with B2EE-2.0 sequences in a fashion similar to that of the RPMI8402 junction.
  • FIG. 9 each product is comprised of AA-0.6 sequences juxtaposed with B2EE-2.0 sequences in a fashion similar to that of the RPMI8402 junction.
  • FIG. 9 depicts tal d deletion junctions as resembling the coding joints of assembled immunoglobulin genes.
  • the tal junctions of three additional tal d -positive cell lines and 18 tal d -positive primary T-ALL specimens were isolated by PCR amplification using oligonucleotides C and D (FIG. 7).
  • the tal d junctions are aligned with germline sequences from AA-0.6 and B2EE-2.0.
  • Heptamer sequences of the putative recombination signals within AA-0.6 and B2EE-2.0 are marked with asterisks, and the proposed sites of recombination are denoted with arrows.
  • junctional nucleotides in lowercase letters are not derived from the germline sequences of AA- 0.6 or B2EE-2.0.
  • the underlined thymidine residues at the AA-0.6 junctions of CCRF-HSB-2, MOLT16, patients 60, 80, and 83 are proposed to be P nucleotides (Lafaille, J.J. et al. (1990) Cell 59:859-870). Nevertheless, the deletion junction from each patient is unique due to sequence variation at the recombination site (FIG. 9) .
  • the junctional diversity generated by the tal rearrangement is reminiscent of that engendered during site-specific rearrangement of the immunoglobulin and T cell receptor genes.
  • Leukemic cells that harbor the tal-l gene deletion can also be readily identified by fluorescent in situ chromosome hybridization (FISH). Within a given leukemic cell, the tal-l deletion only involves one of the two homologs of chromosome 1. In these cells, DNA probes that recognize tal-l sequences located within the 90 kb deletion region
  • deletion probes will hybridize to one homolog (i.e., the normal homolog) of chromosome 1, whereas DNA probes that recognize other sequences on chromosome 1 (referred to as “control probes”) will hybridize to both homologs.
  • control probes DNA probes that recognize other sequences on chromosome 1
  • the hybridization patterns of the deletion probe and the control probe can be distinguished by a two-color FISH analysis in which each probe is marked with a distinct fluorochrome such as Texas red or fluorescein isothiocyanate (FITC) .
  • FITC fluorescein isothiocyanate
  • a typical general protocol for detection of the tal-l deletion by FISH is as follows:
  • the deletion probe green, e.g., ⁇ SU25, see below
  • the deletion probe is nick- translated (Bethesda Research Laboratories Nick-Translation System) with digoxigenin-11-dUPT (deoxyuridine 5'-triphosphate) (Boehringer Mannheim Biochemicals) and the control probe (red, e.g., L-myc genomic DNA) is similarly nick-translated with biotin-11-dUTP (Enzo Diagnostic).
  • These probes are then hybridized to either i ⁇ terphase nuclei or metaphase spreads of the cells to be tested under conditions essentially described by Tkachuk et al. (Tkachuk, D.C.
  • the digoxigenin-labelled deletion probe is then detected by incubation with sheep—antibody to digoxigenin followed by FITC—conjugated rabbit-antibody to sheep—antibody.
  • the biotin-labelled control probe is detected by incubation with Texas red-avidin.
  • the hybridization signals of both probes are then visualized simultaneously with a fluorescent microscope equipped with a double band-pass filter set. Two-color FISH with the deletion (green) and control (red) probes should result in two green and two red hybridization signals in the nuclei of normal cells. In contrast, a single green and two red hybridization signals should be observed in leukemic cells that harbor the tumor-specific tal-l gene deletion.
  • DNA from the phage clones ⁇ SU25 and ⁇ WIll can be used as
  • ⁇ SU25 and ⁇ WIll each contain a genomic DNA fragment
  • control probe any other sequence derived from chromosome 1 can serve as the "control probe".
  • control probe it may be preferable to use a control probe that is not closely linked to the tal-l locus at lp32-34. Specifically, detection of the tal-l gene deletion by FISH can be carried out as follows:
  • Test cells are to be hybridized by a published procedure (Pinkel, D. et al. (1988) Proc. Natl. Acad. Sci. USA 85:9138). Cells are thermally denatured at 72 ° C for about 5 min, dehydrated in an ethanol series, air-dried, and then can be placed at 37°C.
  • a hybridization mixture (10 ⁇ l) containing each probe (2 ng/ ⁇ l), 50% formamide/2x standard saline citrate (SSC), 10% dextran sulfate, and human genomic DNA (1 mg/ml, sonicated to 200 to 600 bp) is then heated to about 70 ° C for about 5 min, incubated for about 30 min at 37 ° C, placed on the warmed slides, covered with a 20 mm by 20 mm cover slip, sealed with rubber cement, and incubated overnight at 37 ° C. Slides are washed three times in 50% formamide/2x SSC for about 20 min each at 42 ° C, twice in 2x SSC at 42 C for about 20 min each, and rinsed at room temperature in 4x SSC.
  • the signal is amplified by applying biotinylated goat antibody to avidin ⁇ Vector Laboratories Inc., 5 ⁇ g/ml in PNM [0.1 M NaH 2 PO 4 /0.1 M Na 2 HP0 4 , pH 8 (PN) containing 5% nonfat dry milk and 0.02% sodium azide and centrifuged to remove solids] ⁇ , washed twice in PN for about 5 min, followed by another layer of Texas red-avidin in PNM.
  • biotinylated goat antibody to avidin ⁇ Vector Laboratories Inc., 5 ⁇ g/ml in PNM [0.1 M NaH 2 PO 4 /0.1 M Na 2 HP0 4 , pH 8 (PN) containing 5% nonfat dry milk and 0.02% sodium azide and centrifuged to remove solids] ⁇ , washed twice in PN for about 5 min, followed by another layer of Texas red-avidin in PNM.
  • the digoxigenin-labeled deletion probe is detected by incubation with sheep antibody to digoxigenin (Boehringer Mannheim Biochemicals, Indianapolis, IN; 15.4 ⁇ g/ml in PNM) for about 30 min, washed twice in PN for about 5 min, followed by a rabbit-antibody to sheep conjugated with FITC (Organon Teknika-Cappel, 1:50 in PNM). After washing twice for about 5 min in PN, the signal can be amplified by applying a sheep antibody to rabbit immunoglobulin G (IgG) conjugated to FITC (Organon Teknika-Cappel, 1:50 in PNM). The slides are then rinsed in PN. Slides are mounted in 10 ⁇ l of fluorescence antifade solution [Johnson,
  • PCR Polymerase chain reactions
  • Oligonucleotide primers PI and P2 used to detect tal d alleles, were constructed from the nucleotide sequences on either side of the common deletion (FIG. 10A) .
  • Another set of oligonucleotides, XI and X2 was prepared for the detection of the tal* translocation in leukemia L23 (FIG. 11). High molecular weight DNA (0.1-10 ⁇ g) was added and the corresponding number of genome copies was calculated assuming that 1.5 X 10 diploid cells contain I ⁇ g DNA.
  • Amplification was accomplished in a Perkin Elmer Cetus thermal cycler in 60 cycles. The first cycle consisted of 3 min at 94 degrees, 1 min at 61 degrees and 2 min at 72 degrees; in subsequent cycles the melting step lasted 1 min. Control PCR mixtures were run with every experiment and included normal liver or thymus DNA and approximately 1 ng tal d DNA. Oligonucleotides PI and F (FIG. 10A) were used to confirm the integrity of the DNA samples; all samples contain at least one normal germline chromosome lp, which yields a 250 bp fragment upon PCR containing these primers. Standard precautions were taken to avoid contamination of PCR reaction mixtures with PCR products (Kwok, S. , and R. Higuchi (1989) Nature.339: 237-238) . Hybridization.
  • oligonucleotide H was an 18-mer which detected all tal d alleles (FIG. 10A) .
  • the second were 4 oligonucleotides which detected specific rearrangements in the tal d leukemias L14, L54 and L81 (FIG. 10B) or the tal* leukemia L23 (oligonucleotide H2, FIG. 11). End-labeling of oligonucleotides, hybridization and stringent washing was carried out as described (Jonsson, O.G., et al. (1990) Blood. 76:2072-2079).
  • Oligonucleotide primers for PCR and hybridization were constructed using an Applied Biosyste s Model 380B DNA Synthesizer
  • Oligonucleotides F, H, PI and P2 are shown in FIG. 10A; oligonucleotides XI, X2 and H2 are shown in FIG. 11. Quantitative PCR Assay for tal d alleles.
  • PCR was carried out as described above, except that internal standard DNAs were added to each reaction (Sambrook, J. , et al. (1989) "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, 14.30-14.33).
  • the standards were chosen to yield an amplified product which differed in size from the target product by approximately 10 bp.
  • RPMI 8402 DNA was used as a standard in assays for L14 alleles
  • MOLT-16 DNA was used in assays for L54 alleles (FIG. 10B) .
  • DNA standards were serially diluted into normal liver or thymus DNA (8 ⁇ g) .
  • the dilutions of standard DNA were added to PCR mixtures containing target DNAs whose tal d alleles were to be quantitated.
  • the products were separated by electrophoresis through 10% polyacrylamide gels and visualized by staining with ethidium bromide.
  • the original number of target tal d alleles was estimated by densitometric comparison of staining intensities of the standard and target products.
  • Equal numbers of standard and target alleles present at the start of amplification were shown to generate equal yields of products. Refined estimates of target allele number were obtained by repeating assays in the presence of a narrow range of 2-fold dilutions of standard DNAs.
  • each tal d allele differs by several nucleotides at the proximal and distal ends of the deletion and in the sequence of the extra non-germline nucleotides which replace the deletion. These features enable the specific detection of these alleles by PCR and hybridization assays, as described above. A similar strategy was used to detect the t(l;14) (p32;qll) (tal*) allele in material from patient L23 (FIG. 11). Distribution of tal d alleles.
  • tal d -bearing cells should not be found in normal hematopoietic tissues. Using the PCR assay, no evidence of such cells in normal peripheral blood mononuclear cells
  • DNA was extracted from diagnostic (D) or followup (F) peripheral blood or bone marrow ( ⁇ ) leukocytes and assayed by the amplification/hybridization procedure for tal d (*) or tal* ( ⁇ ) alleles. (+), alleles detected; (-) , alleles not detected. Both blood and bone marrow from patient L54 were assayed at month 10. tal d alleles in sample L54F1 were quantitated as described in Methods. Characteristic bands representing tal fragments were amplified in PCRs containing the diagnostic bone marrow samples from each of these 3 patients (FIG. 12, lanes 1, 6 and 10).
  • FIG. 12 Examples of these negative assays are shown in FIG. 12, lanes 5 and 11.
  • tal d alleles were detected in a blood sample drawn from patient L54 during the 4th month of therapy (FIG. 12, lane 7).

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Abstract

Procédés et compositions de diagnostic et de pronostic de tumeurs hématopoïétiques, telles que la leucémie lymphoblastique aiguë des lymphocytes T, chez l'homme. L'invention consiste à utiliser des sondes d'hybridation d'acide nucléique pour détecter, par hybridation chromosomique in situ par fluorescence, la délétion du locus tal-1 sur le chromosome 1 des cellules humaines, afin de confirmer la leucémie lymphoblastique aiguë chez le patient. On décrit également des procédés et des compositions pour contrôler la présence de cellules tumorales hématopoïétiques minimales résiduelles, telles que les cellules affectées par la leucémie lymphoblastique aiguë dans les cellules de rémission d'un patient. L'invention consiste à utiliser des sondes d'hybridation d'acide nucléique pour détecter l'altération du locus tal-1 sur le chromosome 1 d'un extrait d'ADN afin de confirmer la présence de cellules affectées par la leucémie lymphoblastique aiguë chez le patient. On décrit également des procédés de quantification des cellules hématopoïétiques, ainsi que des trousses de détection et de contrôle des cellules tumorales hématopoïétiques chez un patient, de même que des trousses de quantification des cellules tumorales hématopoïétiques chez un patient en période de rémission.
PCT/US1991/008707 1990-11-28 1991-11-21 Procedes et compositions de detection et de quantification de tumeurs hematopoietiques WO1992009702A1 (fr)

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WO1999014366A2 (fr) * 1997-09-18 1999-03-25 Erasmus Universiteit Rotterdam Detection d'une pathologie residuelle minime dans des tumeurs malignes lymphoides
WO2004108144A1 (fr) * 2003-06-06 2004-12-16 Kringle Pharma Inc. Préparations contenant des cellules

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EMBO JOURNAL. vol. 9, no. 10, October 1990, EYNSHAM, OXFORD GB pages 3343 - 3351; L.BROWN ET AL.: 'Site-specific recombination of the tal-1 gene is a common occurence in human T-cell leukemia' cited in the application *
EMBO JOURNAL. vol. 9, no. 2, February 1990, EYNSHAM, OXFORD GB pages 415 - 424; QI CHEN ET AL.: 'The tal gene undergoes chromosome translocation in T cell leukemia and potentially encodes a helix-loop-helix protein' cited in the application *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014366A2 (fr) * 1997-09-18 1999-03-25 Erasmus Universiteit Rotterdam Detection d'une pathologie residuelle minime dans des tumeurs malignes lymphoides
WO1999014366A3 (fr) * 1997-09-18 1999-05-06 Univ Erasmus Detection d'une pathologie residuelle minime dans des tumeurs malignes lymphoides
WO2004108144A1 (fr) * 2003-06-06 2004-12-16 Kringle Pharma Inc. Préparations contenant des cellules
JP4563937B2 (ja) * 2003-06-06 2010-10-20 敏一 中村 細胞含有製剤

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