EP0943013A2 - Detection d'anomalies chromosomiques - Google Patents

Detection d'anomalies chromosomiques

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Publication number
EP0943013A2
EP0943013A2 EP97946747A EP97946747A EP0943013A2 EP 0943013 A2 EP0943013 A2 EP 0943013A2 EP 97946747 A EP97946747 A EP 97946747A EP 97946747 A EP97946747 A EP 97946747A EP 0943013 A2 EP0943013 A2 EP 0943013A2
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Prior art keywords
seq
nucleic acid
primers
type
strandedness
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EP97946747A
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German (de)
English (en)
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Niels Pallisgaard
Peter Hokland
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Individual
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Individual
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to methods for detection of the presence or absence of chromosomal abnormalities associated with a condition, notably a malignant neoplastic disease, in a subject and defined by at least one characteristic nucleic acid sequence.
  • the invention further relates to DNA fragments having specific nucleic acid sequences and their use as either cDNA primers or primers in molecular amplification reactions leading to the detection of chromosomal abnormalities.
  • the invention also relates to a kit comprising selected primers for use of detection according to the invention.
  • Chromosomal translocations appear to be important events in the development of tumours (especially haematopoietic tumours) and more than 50 different consistently occurring translocations have been described (Rabbitts, T. H. Nature 372:143 (1994)). Many of the chromosomal aberrations have been found to be specific to particular subtypes of leukaemia or lymphoma .
  • the identification of translocations in haematopoietic malignancies is therefore of great diagnostic and prognostic value.
  • the diagnosis of acute leukaemia is multidisciplinary with standard pathology, immunology and cytogenetics as the most often used methodologies.
  • immunopheno- typing using flow cytometry and monoclonal antibodies provide a speedy and accurate differentiation between lymphoid and myeloid lineages, while a bone marrow biopsy delineates the degree of malignant infiltration simultaneously with revealing the extent of remaining normal haematopoiesis .
  • Neither immunophenotyping nor histology seem to be able to provide satisfactory tools for prognosticating the patients.
  • cytogenetic evaluation while being time consuming, has been shown to delineate both patient groups with favourable as well as poor prognosis.
  • the basis for the value of cytogenetics as a prognostic tool is the existence of a number of balanced chromosomal translocations, where unique genetic sequences are created (for review see Rabbitts, 1995) . Cloning of the translocation break points have indicated that these genes can be altered at the level of their expression or in the properties of the encoded proteins. These alterations appear to play an integral role in the development and possibly in the progression of the disease.
  • the translocations may alter the function or activities of cellular proto-oncogenes located at or near the breakpoint. These proto-oncogenes are normally involved in control of cellular growth, differentiation or apoptosis.
  • the oncogenic conversion may occur by two general mechanisms, either (i) by juxtaposition of a cellular proto-oncogene to the regulatory element of a tissue specific gene, e.g. the immunoglobulin and T-cell receptor genes in leukaemia, leading to inappropriate expression of the oncogene (Leder, P. et al .
  • Translocational breakpoints are highly conserved and ge- nerally within the introns of the affected genes. This is properly due to constrains on the reading frame and on protein (mal) function, but also intron size and the presence of repeated (e.g. Alu) sequences or sequences homologous to B- and T-cell specific recombinase recognition sites within the introns may target and influence the frequency of translocations.
  • repeated (e.g. Alu) sequences or sequences homologous to B- and T-cell specific recombinase recognition sites within the introns may target and influence the frequency of translocations.
  • fusion-genes have been found in several variant sizes. Sequence analysis has revealed that the reading frame of the fusion protein variants is preserved, and shows that there may be some freedom in the joining of protein domains in the generation of the oncogene.
  • one of the genes involved in the fusion is most often a transcription factor which appears to have a direct role in haematopoiesis and which, following the translocation, is frequently fused to a second gene not normally active in haematopoietic cells. In some instances the same gene is involved in fusion with more that one chromosomal partner.
  • a translocational breakpoint gene may have several fusion partners, the most promiscuous example is the MLL gene at chromosome band llq23, where 10 different fusion partners together with an internal duplication has been described.
  • the MLL/AF4 fusion gene, detected in t(4;ll) (q21;q23) translocations, is only observed in paediatric ALL, whereas the MLL/AF6 fusion gene detected in t(6;ll) (q27;q23) translocations is seen in a subgroup of AML patients (Prasad, R, et al., Cancer Res. 53:5624 (1993)).
  • the t (10; 11) (pl4;q23) translocation where the MLL is fused to the AF10 gene, has been described in both paediatric ALL and AML patients. Thus depending on fusion partner the MLL gene can contribute to the pathogenesis of either lymphoid or myeloid malignancies or both.
  • a number of breakpoint genes have been found fused to various partners in different translocations. e.g. E2A/PBX and E2A/HLF in t(l;19) (q23;pl3) and t(17;19) (q22;pl3) or PML/RAR ⁇ .
  • chromosome preparation from clinical samples is often not feasible and the cytogenetic based diagnoses are not sensitive to a small fraction of abnormal cell, i.e. are not helpful for monitoring for relapse. Cytogenetic analysis may detect gross aberrations, but not submicroscopic alterations. Some of these problems may be overcome by using PCR based techniques. However, only four different chromosomal abnormalities have been identified by multiplex- PCR, Repp, R. et al; Detection of Four Different llq23 Chromosomal Abnormalities by Multi- plex-PCR and Flourescence-Based Automatic DNA-Fragment Analysis : Leukemia (1995) 9:210-215
  • a translocation specific fusion gene can be detected by PCR techniques, indicating that a second chromosomal rearrange- ment has occurred restoring the normal karyotype.
  • a PCR analysis is rapid and very sensitive, but will generally only detect one specific fusion-gene.
  • a major drawback of the PCR method is that it is time consuming and that false negatives are difficult to detect.
  • the high number of diversified translocations which have hitherto been demonstrated in acute leukaemia, has precluded its use as a screening tool.
  • the inventors of the present invention have provided a method for detection of the presence or absence of chromosomal abnormalities which are associated with a condition in a subject and are each defined by at least one characteristic nucleic acid sequence.
  • the method comprises subjecting a sample of nucleic acids to a multiplex molecular amplification procedure using multiple and mutually distinct primers in one single reaction mixture, wherein each of the primers defines an end of at least one characteristic nucleic acid sequence.
  • the method according to the invention makes it possible, with a minor amount of work, to screen a sample of nucleic acids for a very high number of chromosomal abnormalities which may occur in a subject. By employing the methods of the invention it has become possible to detect about 50 chromosomal rearrangements with more than 80 subtypes, all performed in one single experimental round.
  • One distinguishing feature of the invention is the use of amplification primers which constitute the one half of more than one pair of primers used in the amplification reaction, thereby reducing the number of primers necessary to carry out the amplification of all sequences of interest.
  • translocations fall within families, wherein one half of one translocation product is present in at least one other translocation product.
  • two such translocation products should be detected, it would only be necessary to use a total of three primers.
  • a total number of 6 primers could ideally be used; this is in strong contrast to the 10 primers which would be necessary when performing a multiplex amplification utilizing the prior art primer systems.
  • the inventor has utilized the existence of families of translocations so as to reduce the total number of primers necessary to detect virtually all known chromosomal abnormal - ities associated with malignant diseases of haematopoietic origin.
  • it has been achieved to perform multiplex PCR reactions wherein the number of primers is no less than 7, i.e. an unprecedented high number of primers present in a multiplex PCR for this purpose.
  • the invention relates to a method for detection of the presence or absence of chromosomal abnormalities, each of these chromosomal abnormalities being associated with a condition in a subject and each of these chromosomal abnormalities being defined by at least one characteristic nucleic acid sequence, the method comprising
  • step b) retrieving the product (s) from step b) , and detecting the presence and/or absence of amplified characteristic nucleic acid sequences and thereby the presence or absence of corresponding chromosomal abnormalities
  • the multiplex molecular amplification procedure comprises the use of at least 7 mutually distinct primers in one single reaction mixture, each of the at least 7 mutually distinct primers defining an end of at least one characteristic nucleic acid sequence, and wherein at least one of the at least 7 mutually distinct primers defines the first ends of at least two characteristic nucleic acid sequences, said at least two characteristic nucleic acid sequences each being defined in their opposite ends by mutually distinct primers selected from the remainder of the at least 7 mutually distinct primers, whereby the number of amplified characteris- tic nucleic acid sequences which can be detected upon conclusion of the amplification reaction is at least ⁇ xn+1, wherein n is the number of the at least 7 mutually distinct primers .
  • the invention pertains to a method for detection of the presence or absence of chromosomal abnormalities, each chromosomal abnormality being associated with a condition in a subject and each chromosomal " abnormality being defined by at least one characteristic nucleic acid sequence, the method comprising
  • step b) retrieving the product (s) from step b) , and detecting the presence and/or absence of amplified characteristic nucleic acid sequences and thereby the presence or absence of corresponding chromosomal abnormalities
  • the multiplex molecular amplification reaction comprises
  • n a number of mutually distinct primers each defining an end of a characteristic nucleic acid sequence
  • n mutually distinct primers defines first ends of at least two mutually distinct characteristic nucleic acid sequences, said at least two mutually distinct characteristic nucleic acid sequences being defined in their opposite ends by at least two mutually distinct primers selected from the remainder of the n mutually dis- tinct primers, whereby the number of amplified characteristic nucleic acid sequences which can be detected upon conclusion of the amplification procedure is at least xn+1.
  • the number of primers used in one single reaction mixture is at least 7, but higher numbers are preferred such as at least 8, 10, 12, 14, 16, 20, 26, or at least 30.
  • the number of primers in one single reaction mixture will be at most 50, but in certain situations the number will be at most 40 or even 35.
  • chromosomal abnormality and “chromosomal abnormalities” denote chromosomal sequences of nucleic acids which are usually not detectable in normal healthy subjects whereas these sequences are typically found in subjects suffering from diseases, having an increased risk of developing said diseases, or having well-defined chromosomal defects. Typically, such chromosomal abnormalities are translocations, inversions, deletions, duplications .
  • chromosomal abnormality will normally be associated with a condition such as a disease (often malignant) or a chromosomal defect, or in other words, the chromosomal abnormality will be present in a significantly higher percent of subjects having the condition than in the average population.
  • a "characteristic nucleic acid sequence” is a consecutive stretch of nucleotides which is comprised in the genome of a subject having a chromosomal abnormality and usually not in the "average” healthy subject. Further, the characteristic nucleic acid sequence is one the nucleotide sequence of which is uniquely tied to the chromosomal abnormality, i.e. it will not be found in any substantial number of nucleic acid samples from subjects which do not harbour the chromosomal abnormality.
  • molecular amplification procedure a in vi tro procedure in which a nucleic acid sequence is multiplied by use of priming sequences ("primers”) which anneal to a target sequence (the “template”) and means for initiating and sustaining amplification of the extension products of the primers or complements thereof.
  • priming sequences primers
  • template target sequence
  • methods are well-known in the art, but as exemplary can be mentioned the methods described in EP-0 200 362, EP-0 201 184, EP-A-0 368 906, EP- A-0 379 368, EP-A-0 540 693.
  • multiplex molecular amplification procedures When referring to a "multiplex" molecular amplification procedure is, as well-known in the art, meant a molecular amplification procedure which comprises the use of at least three primers and which results in the amplification of at least two target sequences.
  • multiplex molecular amplification procedures are described in a number of patent publications, cf. e.g. EP-A-0364 255.
  • the preferred molecular amplification method according to the invention is multiplex PCR.
  • the primers used in the amplification procedure must be "distinct", by which term is meant nucleic acid primers which are not 100% identical in sequence and which furthermore will not, under the chosen amplification conditions, exhibit substantial mutual competition for annealing to a given target sequence.
  • Important embodiments of the ' methods of the invention are those wherein the sample of nucleic acids is derived from subjects in the form of cDNA. It will be understood that this requires the use of preceding method steps wherein cDNA is provided by employing reverse transcription of mRNA derived from the subjects and such a procedure thus limits the number of characteristic sequences to be detected to those which are actually transcribed in at least some of the subject's cells.
  • the procedures for retrieving mRNA from cells are well-established in the art and involve relatively few problems in a standard setup.
  • the methods of the invention are in no way restricted to use of cDNA as template molecules in the amplification procedures.
  • One interesting possibility will be to extract chromosomal DNA from the subject's cells and perform the multiplex molecular amplification either directly thereon or restriction fragments thereof. In this way it will be possible to detect chromosomal abnormalities which do not give rise to an appa- rent phenotype at the time of extraction but which nevertheless may be an important marker for the condition of interest.
  • the molecular amplification procedure performed in the method of the invention utilises cDNA obtained by use of specific or non-specific cDNA primers in a separate molecular amplification procedure wherein the templates in the procedure are in the form of mRNA derived from the subject.
  • the cDNA primers used are normally non-specific, and the mRNA extraction is therefore "randomly" primed.
  • the present inventor has discovered that markedly superior results are obtained in the detection phase when a mixture of specific cDNA primers are used for synthesis of cDNA from total RNA.
  • the use of speci- fic cDNA primers has given rise to an approximately 25 to 125 fold increase in sensitivity depending on the system in question.
  • the cDNA primers are specific and in fact, the use of specific cDNA primers when coupled to subsequent multiplex molecular amplification procedures is in itself believed to be a novel approach which leads to a substantially higher sensitivity in the multiplex PCR reaction.
  • another part of the invention is the combination of such specifically primed cDNA production with subsequent multiplex molecular amplification.
  • telomere sequences By the term “specific" when used in conjunction with cDNA primers is herein meant that the cDNA primers are predesigned to anneal to target RNA sequences which predominantly exist in RNA transcribed from the above-defined characteristic nucleic acid sequences.
  • the number of cDNA primers is preferably at least 20, such as at least 25, such as at least 30, such as at least 50, at least 100, at least 150, or at least 200.
  • cDNA or any other source of template nucleic acids
  • This can, according to the invention be done by ensuring that the conditions for obtaining cDNA (or other template nucleic acids) derived from the subject are compatible with the conditions of the molecular amplification procedure.
  • the chemical composition of the medium for the molecular amplification procedure in the inventive method should be substantially the same as that of e.g.
  • any residual cDNA primers should be different from the melting point of the primers used in the multiplex molecular amplification procedure. In this way, it will be possible to restrict the manipulation of test tubes to a minimum and thereby avoid contamination of the samples prior to the multiplex molecular amplification procedure.
  • a preferred embodiment of the invention is a method of multi- plex molecular amplification, wherein said multiplex molecular amplification is a nested molecular amplification procedure such as a nested polymerase chain reaction.
  • nested PCR enhances the specificity of any PCR reaction by excluding a large number of artefactual amplification products resulting from the initial round of PCR.
  • Suitable nested PCR methods to be used according to the present invention are those described in USP 4,683,195, and especially the procedures described in EP-A-0 519 338, because these further ensures that no change of media or reactants between the individual steps in the nested molecular amplification procedure need be performed.
  • An important embodiment of the methods of the invention is a method wherein the chromosomal abnormality is the presence of a transcribed fusion gene.
  • a number of expressed fusion genes have been identified which are related in a highly significant manner to various malignant diseases of haematopoietic origin and therefore the detection of the presence of such fusion genes provides important and useful information of the prognosis of the subject, since certain of the malignancies are known to be susceptible to specific regimens of treatment.
  • the presence of such a transcribed fusion gene is typically the result of an inversion, a deletion, a duplication, or activation of a proto-oncogene.
  • Said activated proto-oncogene is typically selected from the group consisting of Hox-11 and evi-l and others as presented in
  • any genetic variant which is predominantly seen in e.g. malignant cells may be detected according to the invention, when the material subjected to the methods of the invention is not cDNA but for instance nucleic acid fragments derived from a chromosomal source.
  • a gene like c-myc which is often overexpressed, may be detected and used as an indication of illness.
  • preferred embodiments of the methods of the invention are those wherein at least one of the chromosomal abnormalities is associated with a malignant neoplastic condition, especially a systemic neoplastic malignancy, since a relatively large number of these have been shown to be associated with e.g. expressed fusion genes.
  • such systemic neoplastic malignancies are selected from the group consisting of leukaemia such as acute leukaemia (AL) , chronic leukaemia (CL) , T-cell acute leukaemia (T-ALL) , B-cell acute leukaemia (B-ALL) , T- cell chronic leukaemia (T-CLL) , B-cell chronic leukaemia (B- CLL) , prolymphocytic leukaemia (PLL) , acute undifferentiated leukaemia (AUL) , acute myelogenous leukaemia (AML) , chronic myelogenous leukaemia (CML) , chronic myelomonocytic leukaemia (CMML) , acute promyelocytic leukaemia (APL) , pre-B-ALL, and pro-B-ALL; lymphoma such as Burkitt's lymphoma (BL) , non-Hodgkins lymphoma
  • dup(llq23) (dup exon 5-9/2); dup(llq23) (dup exon 5-9/4); inv(16) (pl3;q22) ; t(l;ll) (p32;q23) ; t (1;19) (q23;pl3) ; t (10;11) (pl4;q23) t(10;ll) (pl4;q23) t (10;11) (pl4;q23) t(10;14) (q24;qll) t (11;17) (q23;q21) 11; 19) (q23;pl3.1) ; 11; 19) (q23;pl3.3) ; 12; 21) (pl3;q22) 12; 22) (pl3;qll) 15; 17) (q21;q22) 15; 17) (q21;q22) 15; 17) (q21;q22) 15; 17) (q21;q22) (q21;q22) inv
  • malignancies are non- systemic neoplastic malignancies selected from the group consisting of carcinoma, adenocarcinoma, liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, glioma, neuroblastoma, medullablastoma, malignant melanoma, neurofibrosarcoma, heamangiosarcoma, lymphangiosarcoma, malignant teratoma, dysgerminoma, seminoma, and choriocarcinoma.
  • the neoplastic disease is carcinoma it is preferably selected from the group consisting of carcinoma of the breast, bronchus, colorectum, stomach, prostate, ovary, lymphoid tissue, lymphoid marrow, uterus, pancreas, oesophagus, urinary bladder, kidney, or skin.
  • Especially interesting malignant neoplastic conditions are selected from the group consisting of papillary thyroid carcinoma, Ewing's sarcoma, liposarcoma, rhabdomyosarcoma, synovial sarcoma, and melanoma of soft parts, since all of these are positively associated with genomic rearrangements, cf. Rabbits 1994.
  • the sample of nucleic acids " used in the methods of the invention is typically derived from cells of the bone marrow in the subject or from peripheral blood cells in the subject. This is especially interesting in the cases wherein the disease to detect is a malignant disease of the haematopoietic system, but also conditions characterized by chromosomal defects (e.g. Downs syndrome) may be detected this way.
  • the sample may be derived from any other source in the subject, but interesting origins are placental cells, foetal cells, and amniotic fluid.
  • a sample of 5 million mononuclear cells will normally be sufficient to deliver an amount of nucleic acid of 5 ⁇ g (between 3 and 8 ⁇ gr) RNA, however it depends on the growth rate of the cells.
  • a 20 ml sample from the bone marrow generally corresponds to 5 ⁇ g (between 3 and 8 ⁇ g) RNA. Accordingly, as little as 10 ml of the sample may be sufficient for the method according to the present invention
  • At least one of the primers used in the multiplex molecular amplification procedure may according to the invention be labelled.
  • the label can be a radioactive label, a coloured label, a fluorescent label, a biotinyl group, an enzymatic group, a phosphate, an amin a tiol, or any other moiety which can be detected directly or indirectly.
  • a biotinyl group may in itself be labelled, but it is also possible to detect the presence of the biotinylated nucleic acid fragment by reacting the mixture with labelled avidin or streptavidin.
  • the primers are labelled with a fluorescent label or a coloured label.
  • a primer exists in two versions which are different labelled e.g., a labelled and an unlabelled, and is used in differendt known amounts the amplification products will exhibit an average degree of labelling which correspons with the initial defined ratios of label due to the stochastic distribution of the labels in the amplified product.
  • relatively few fluorescent labels of primers are known, it is possible to detect a large number of different amplified fragments when the above- indicated technique is used.
  • primers which pairwise gives rise to amplified products of different lengths.
  • the presence or absence of amplified products is then detected by use of various methods which are able to detect the amplified fragments on basis of their size/sequence, methods such as gel electrophoresis, sequence analysis, HPLC, FPLC, flouresence spectofotometri and other suitable chromatograhphic methods.
  • a labelled means for detecting the amplified products may be used, such as other nucleic acid fragments which will hybridize to the product and thereafter be detect- able by virtue of the label.
  • Such methods are well-known in the art .
  • the sample of nucleic acids is subjected to at least two multiplex molecular amplification as defined herein, i.e. the sample (s) derived from the subject is split into several aliquots which each are subjected to a multiplex molecular amplification procedure according to the invention.
  • the at least two multiplex molecular amplification procedures are carried out in parallel, and it is especially preferred that the at least two multiplex molecu- lar amplification procedures are carried out under substan- tially the same conditions with respect to physical parameters and timing; the latter preferred embodiment has the advantage of allowing the use of e.g. the same reaction buffers (with the exception of primers) and the same thermo- " cycling scheme for all aliquots. In essence, all reactions are thus performed in the same thermocycler.
  • the exact number of different amplification procedures the sample is subjected to may vary, but is preferably at least 3, such as at least 4, 5, 6, 7, 8, 9, 10, 11, 12, or at least 15. Higher numbers may be necessary, depending on the number of families of chromosomal abnormalities need be detected.
  • the internal standard used in one of the methods of the invention is preferably a cDNA molecule derived from the subject and most preferably said cDNA molecule is obtained by use of specific or non-specific cDNA primers in a molecular amplification procedure wherein the templates in the procedure are in the form of mRNA derived from the subject.
  • the internal standard is also cDNA which has been obtained in parallel to the other cDNAs, including the molecular amplification procedure leading to the provision of the other cDNAs .
  • RNA fragment of known sequence is added to the total RNA mixture from which mRNA is extracted, whereafter the subsequent products of the known RNA are obtained from reverse transcription PCR; in this way, all steps but the total RNA extraction are confirmed;
  • a cDNA molecule constituting the internal standard corresponds to a constitutively expressed RNA fragment.
  • a sequence from the constitutively expressed gene E2A is used as target.
  • any "normal" gene sequence may be employed as internal standard.
  • primers used in the multiplex molecular amplification procedure are designed using standard software known to the skilled person, and a number of criteria must be met by the primers in the reaction mixture:
  • primers must hybridize to their respective target sequences at or below substantially the same temperature, preferable within a temperature diffenrence of 5°C (they should have the same melting point) ; in the setup reported in the following examples, the melting point has been chosen to be approximately 70°C,
  • primers must be substantially specific for their respec- tive target sequences, meaning that they will not initiate polymerization from other template sequences than "their own” and that they are not capable of hybridizing with each other. This specificity is obtained by the fact that the primers are completely complementary to the target sequence, however up to 3 point differencies (mutations) may still result in a specific priming.
  • primers should exhibit substantially no intramolecular hybridization, or in other words, there must be a minimum of secondary structure in each primer, that is normally the case when the delta G is above -1 within the primer,
  • primers must have a higher melting point in the 5' -end than in the 3' -end, i.e. they have a high internal stability in the 5' end and a relatively low stability in the 3' end, the difference in melting point is preferable above 1°C, such as above 2°C, preferable above 3°C, such as above 4°C and more preferred above 5°C, such as above 6°C, howver, the exact difference may also depends on other desired properties of the primers .
  • no two primers are, in the molecular amplification pro- cedure, capable of together initiating and sustaining amplification of nucleic acid fragments in the sample which correspond to normally occurring sequences not associated with a condition in the subject,
  • no primer should preferably contain more than 5 consecu- tive guanidyl residues, such as not more than 3 guanidyl residues . 7) they should exhibit substantially no intermolecular hybridization, which may be obtained for the primer dimer having a delta G being above -10.
  • a further restraint on the choice of primers is that they should pairwise give rise to fragments of different lengths when the molecular amplification procedure is the one resulting in the amplified products to be detected (i.e. the last molecular amplification procedure, e.g. the second PCR in a nested PCR) and the procedure is one wherein the amplified products are distinguished by their length/sequence.
  • the amplified fragment should typically have a length of between 100-400 bp.
  • primers can be varied in an almost indefinite number of ways as long as they conform with the sequences of e.g. the fusion genes to be detected and the above criteria are met.
  • PCR primers the primers having any of SEQ ID NOs 33-177 are currently being especially preferred.
  • cDNA primers the primers having any of SEQ ID NOs 1-32 and 178-182 are cur- rently being preferred.
  • the invention also relates to a kit comprising 7 mutually distinct primers.
  • the kit may comprise any disired combination of primers for the methods for detection of the presence or absence of chromosomal abnormalities according to the present invention.
  • the kit may comprise primers selected from the group of cDNA primers consisting of SEQ ID NO: 1 through SEQ ID NO: 32 and SEQ ID NO: 178 through SEQ ID NO: 182 and of PCR primers selected from SEQ ID NO: 33 trough SEQ ID NO: 177.
  • the kit according to the present invention may also comprise additives such as buffers, enzymes, and stabilizing agents known in the art.
  • the primers are attached to a surface of a device such as a well, e.g of a multiplate, a cappilary tube, a stick, or a bead (such as a magnetic bead) .
  • a device such as a well
  • the primers may be dried or in other any suitable form including being contained in a polymer vehicle.
  • the primers in the kit are in a liquid form contained in e.g. a tube or well.
  • FIG. 1 Setup of the multiplex PCR amplification reaction.
  • the figure shows representative results of gel electrophores- es of nucleic acid samples from one patient subjected to 8 multiplex nested PCR amplifications, each multiplex nested PCR using the primer mixes 1-8.
  • the upper lane (lane 1) represents molecular weight markers.
  • the band which is present in lanes 1-8 is the internal standard, while the second band in lane 5 (primer mix 4) is a detected chromosomal abnormality as highlighted in bold.
  • FIG. 2A and 2B The figure shows gel electrophoreses of nucleic acid samples from 18 individual patients, subjected to multiplex nested PCR amplification using the primer mixes 1-8. In each patient one or more chromosomal abnormalities has been detected by the PCR giving rise to a typical and readily identifiable pattern of bands. Above each panel, representing one patient, the actual chromosomal abnormality has been specified.
  • Figure 3 The figure shows samples positive in the multiplex analysis reanalyses with individual primer sets.
  • the upper three panels each represent gel electrophoreses of multiplex nested PCR performed on nucleic acid samples from one patient. From each of these gels one lane shows that the individual harbours a chromosomal abnormality, the precise nature and variant of which cannot be readily determined.
  • nucleic acids from each patient were subjected to another round of PCR (lower three panels) this time using individual primer sets able to determine the nature of the fusion genes involved.
  • Bone marrow or peripheral blood samples were fractionated on a Ficoll gradient and cryopreserved before use.
  • the cell lines Karpas-299, ML-2, Mono-Mac-6, NB-4, 697, JOSK-I, NALM-6 and RPMI-8402 were obtained from DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) - (DSM accession numbers 31, 15, 124, 207, 42, 155, 128 and 290 respectively).
  • the cell lines RS4;11 and MV-4-11 were obtained from the American Type Culture Collection (Rock- ville, MD) (ATCC accession numbers CRL 1873 and CRL 9591, respectively) .
  • the cell line HAL- 01 is described in Ohyashiki et al . (1991), Leukaemia 5: pp. 322-331.
  • the cell lines were all cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum.
  • the medium for the cell line Mono-Mac-6 was supplemented with 9 ⁇ g/mL bovine insulin.
  • RNA was prepared either by the guanidinium thiocyanate- phenol chloroform method [Chomczynski, Anal. Biochem 162:156, 1987] or by using a RNeasy Kit (Quiagen) according to the manufacturer's instructions.
  • the RNA solution was subsequently treated with 0.1 unit/ ⁇ L RNase-free DNase (Boeh- ringer) in 50 mmol/L Tris-HCl, pH 8.0 , 10 mmol/L MgCl 2 at
  • RNA was pelleted in an eppendorf centrifuge at 13.000 rpm for 30 minutes and washed with 80% ethanol. The RNA was resuspended in 25 ⁇ L DEP ddH 2 0 and 5 ⁇ L withdrawn for quantification on a Genequant (Pharmacia) . Subsequently the RNA was diluted to 0.1 ⁇ g/ ⁇ L and stored until use at -80°C in 10 ⁇ L aliquots.
  • RNA was incubated for 5 minutes with a mixture of translocation-specific-cDNA-primers (2.5 pmol of each) and then reverse transcribed to cDNA by incubation at 37°C for 45 minutes in a total volume 25 mL containing 20 units RNase inhibitor (Boehringer) , 1 mmol/L of each dNTP, 10 mmol/L dithiothreitol , 1XRT buffer (50 mmol/L Tris-HCl pH 8.3, 75 mmol/L KC1 , 3 mmol/L MgCl 2 ) , and 400 units Moloney murine leukaemia virus reverse transcriptase (BRL, Bethesda, MD) .
  • RNase inhibitor Boehringer
  • the cDNA reaction mixture was diluted with ddH 2 0 to 50 ⁇ L.
  • the PCR amplification was performed as 8 parallel nested (two round) multiplex reactions in a Perkin Elmer 9600 thermocycler.
  • Five ⁇ L of diluted cDNA reaction was added to each of eight 20 ⁇ L multiplex mixtures which contained 1.1 X PCR buffer (10 mmol/L Tris-HCl pH 8.3, 50 mmol/L KC1 , 1.5 mmol/L MgCl 2 ) , 0.2 mmol/L of each dNTP, 12,5 pmol of each primer and 1.5 unit AmpliTaq-Gold polymerase (Perkin Elmer) .
  • the first PCR reaction time consisted of an initial activation of the polymerase at 95°C for 15 minutes, followed by 25 cycles of PCR amplification (annealing at 58°C for 30 seconds, elongation at 72°C for 1 minute, and denaturation at 95°C for 30 seconds) .
  • a 1 ⁇ L aliquot from each of the 8 PCR reactions was transferred to eight 24 ⁇ L second round multiplex mixtures which contained 1 X PCR buffer (10 mmol/L Tris- HCl pH 8.3, 50 mmol/L KC1 , 1.5 mmol/L MgCl 2 ) , 0.2 mmol/L of each dNTP, 5-12.5 pmol of each primer and 1.5 unit AmpliTaq- Gold polymerase.
  • the second PCR reaction time consisted of an initial activation of the polymerase at 95°C for 15 minutes, followed by 20 cycles of PCR amplification (annealing at 58°C for 30 seconds, elongation at 72 °C for 1 minute, and denaturation at 95°C for 30 seconds) followed by a 10 minutes extension at 72°C. Fifteen ⁇ L of the PCR reactions were electrophoresed in a 1.5% agarose gel for 60 minutes at 100V and stained with ethidiumbromide .
  • Positive samples were reanalyzed to verify/determine the translocation (s) by performing cDNA and nested PCR with the individual primer sets using the same conditions as for the multiplex PCR except that only 0.75 unit/reaction of AmpliTaq-Gold polymerase was used. Positive samples with limiting amount of RNA were reanalyzed by performing only the second round of PCR with the individual primer sets using 1 ⁇ L from the first round multiplex PCR as template. This analysis was performed with and without the internal control primers and translocations were confirmed by DNA sequence analysis. Negative controls without DNA template were included for all PCR reactions mixtures.
  • filtertips were used in all steps and four different laboratories with indigenous pipettes were used for the preparation of stock solutions, the RNA preparation and cDNA synthesis/setup of first PCR, the first to second PCR transfer, and the gel electrophoresis .
  • PCR oligonucleotide primers were designed using the Windows primer analysis software OLIGO version 5.0 (National Biosciences Inc., Plymouth, MN) using data from the EMBL DNA database. Oligonucleotide primers were supplied HPLC purified from DNA Technology, Science Park, DK-8000 Aarhus . DNA sequencing was performed on agarose gel purified PCR fragments using a Taq DyeDeoxy Terminator Sequencing kit (Perkin Elmer) on an automated 373A DNA sequencer (Applied Biosysterns, Foster City, CA) . Both strands of the PCR fragments were sequenced.
  • the cDNA primers were: 1. 12-mer: CBFBMYHA: 1752L12 ,
  • AMLIEVI 1897U21 60 5 AML1MGT8:1895U20 128 12.5
  • ALLl(llq23) AL 1:3955U24 36 A L1:3996U24 88 to rn (NP: AL 1AF1:4048 22 65 ALL1AF1:4031L21 130 301 bp
  • E2Aexl2/HLFex4 (II) 33 R3B 207 ALL t ⁇ 12;2i ⁇ pl3;q22) TEL(12pl3) TEUAML1 44 R3C 293 ALL
  • TLS/ERG 32 R4C 413 ALL t(7;10Xq35;q32) Activation of HOXll ⁇ 48 R4D 212 RPMI8402 T-ALL, t(10;14Xq24;qll) HOXll (10q32) AML, ALL, CML
  • RARA ⁇ (17q21) PLZF/RAR ⁇ (B:1452) 23 R8A 402 APL t(15;17Xq21;q22) PML(15q21) PMLex3/RAR ⁇ ex2 30 R8C 393 NB4 APL RAR ⁇ (17q21) S-form ( BCR3)
  • AML acute myelogenous leukemia
  • ALL acute lymphoblastic leukemia
  • CML (-BC) chronic myeloid leukemia (in blast crisis); MDS, myelodysplastic syndrome
  • APL acute promyelocytic leukemia
  • AUL acute undifferenti- ated leukemia
  • CMML chronic myelomonocytic leukemia
  • ALCL anaplastic large cell lymphoma
  • AMMOL acute myelomonocytic leukemia.
  • # Numbers in parentheses indicates the size of the co-amplified PCR fragment resulting from the MLLl exon 5 primer. ⁇ No fusion mRNA is generated, only expression of the gene is tested for.
  • MLLex9/MLLex2 M49 MIA 408 (653) AML dupMLL (llq23) MLL (l lq23) MLLex5/MLLex4 M101 M1B 193 T
  • MLLex9/AF-lp M5 M2A 331 (576) T t(ll;17 ⁇ q23;q21) MLLl (llq23) MLLex5/AF17 M22 M2B 282
  • AML AF17 (17q21) t(10;l l)(pl2;q23) MLLl (Ilq23) MLLex5/AF10 (A:2222) M21A M2C 203
  • E2Aexl2/HLFex4 (II) M33 M3B 207 ALL t(12;2i ⁇ pl3;q22) TEL(12pl3) TELAML1 M44 M3C 242 ALL
  • AML acute myelogenous leukemia
  • ALL acute lymphoblastic leukemia
  • CML (-BC) chronic myeloid leukemia (in blast crisis); MDS, myelodysplastic syndrome
  • APL acute promyelocytic leukemia
  • AUL acute undifferenti- ated leukemia
  • CMML chronic myelomonocytic leukemia
  • ALCL anaplastic large cell lymphoma
  • AMMOL acute myelomonocytic leukemia.
  • # Numbers in parentheses indicates the size of the co-amplified PCR fragment resulting from the MLLl exon 5 primer.
  • AMLIEVI 1897U21 60 AML1EVI:2345L21 128 Cell line: m AMLl(21q22) AML1EVI:2376L24 97 SKffl o m M102 t(3;21)(q26;q22) EVI-l(3q26) AMLIEVI: 1897U21 60 AML1MGT8:1895U20 128 m AMLl(21q22) AML1EVI:2776L22 155 AML1EVI:2720L22 158
  • McDNA-mix (12.5 ⁇ l) :
  • Figs. 1-3 Exemplary results of the inventive methods appear from Figs. 1-3.
  • the internal positive control cf. e.g. lanes 1-3 and 5-8 in Fig. 1 .
  • a positive sample will manifest itself as a band deviating from the position of the internal standard (cf. lane 4 in Fig. 1) .
  • the precise location in the gel can then identify precisely the kind of rearrangement (in this case t(8;21) which results in the positive reaction. Absence of the internal positive control indicates that the method should be repeated since false negatives might be present.
  • Figs. 2A and B shows essentially the same picture as Fig. 1, although for different chromosomal rearrangements; the gene identified is indicated over each panel.
  • the sample is subjected to individual (non-multiplex) PCR reactions using specific primers selected from the NP mixes listed above. Thereby, the precise variant of the chromosomal rearrangement can be determined (cf. Fig. 4).

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Abstract

L'invention concerne une méthode de détection de l'absence ou de la présence d'anomalies chromosomiques associées à un état chez un individu, chacune d'elles étant définie par au moins une séquence nucléotidique caractéristique. En général, ladite méthode consiste à soumettre un échantillon d'acides nucléiques à une procédure d'amplification moléculaire multiplex, laquelle consiste à utiliser au moins 7 amorces différentes dans un seul mélange réactionnel. Chacune des amorces différentes au nombre minimum de 7 définit une extrémité d'au moins une séquence nucléotidique caractéristique, et au moins une des amorces définit les premières extrémités d'au moins deux séquences nucléotidiques caractéristiques, lesquelles sont définies dans leurs extrémités opposées par des amorces distinctes choisies parmi les amorces restantes. Le nombre de séquences nucléotidiques caractéristiques amplifiées pouvant être détectées à la fin de la réaction d'amplification est d'au moins 1/2xn+1, n étant le nombre correspondant aux amorces distinctes, soit au moins 7. Dans un mode de réalisation, l'utilisation d'un étalon positif contenant I) un fragment d'acide nucléique présent dans l'échantillon, et (II) des amorces pour l'amplification d'une séquence nucléotidique dudit fragment d'acide nucléique fait partie de la procédure.
EP97946747A 1996-12-06 1997-12-08 Detection d'anomalies chromosomiques Withdrawn EP0943013A2 (fr)

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US7598226B2 (en) 1998-12-28 2009-10-06 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
US6969518B2 (en) 1998-12-28 2005-11-29 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
EP1272673B1 (fr) * 2000-03-24 2007-03-14 Olympus Corporation Analyse multiplex specifique d'acides nucleiques
ES2281416T3 (es) * 2000-04-03 2007-10-01 Corixa Corporation Metodos, composiciones y sistemas para la deteccion y monitorizacion del cancer de mama.
US7087414B2 (en) 2000-06-06 2006-08-08 Applera Corporation Methods and devices for multiplexing amplification reactions
US7179598B2 (en) 2001-11-20 2007-02-20 The Regents Of The University Of California Early leukemia diagnostics using microsphere arrays
US7301017B2 (en) * 2002-05-30 2007-11-27 Kolesnick Richard N Kinase suppressor of Ras inactivation for therapy of Ras mediated tumorigenesis
FR2842534B1 (fr) * 2002-07-19 2006-01-20 Inst Nat Sante Rech Med Amplification multiplex quantitative a l'echelle d'un genome, et applications a la detection de remaniements genomiques
WO2004051218A2 (fr) 2002-12-04 2004-06-17 Applera Corporation Amplification multiplex de polynucleotides
DK2972353T3 (da) 2013-03-11 2023-03-27 Meso Scale Technologies Llc Forbedrede fremgangsmåder til udførelse af multipleksanalyser
CN105838793B (zh) * 2016-04-22 2019-07-12 上海荻硕贝肯生物科技有限公司 用于定性检测白血病融合基因的引物、试剂盒及方法

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EP0515573A1 (fr) * 1990-02-23 1992-12-02 The Board Of Trustees Of The Leland Stanford Junior University Facteurs de transcription jouant un role pathogene dans des neoplasmes humains
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