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WO2016070230A1 - Détection de mutations dans les séquences de gènes chimères leucémiques - Google Patents

Détection de mutations dans les séquences de gènes chimères leucémiques Download PDF

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Publication number
WO2016070230A1
WO2016070230A1 PCT/AU2015/000667 AU2015000667W WO2016070230A1 WO 2016070230 A1 WO2016070230 A1 WO 2016070230A1 AU 2015000667 W AU2015000667 W AU 2015000667W WO 2016070230 A1 WO2016070230 A1 WO 2016070230A1
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Prior art keywords
sequence
umid
mutations
bcr
molecules
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Inventor
Wendy Tara PARKER
David Tak On YEUNG
Susan Branford
Hamish Steele Scott
Joel Micah GEOGHEGAN
Andreas Wolfgang SCHREIBER
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Central Adelaide Local Health Network Inc
Adelaide University
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University of South Australia
Central Adelaide Local Health Network Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods of detecting mutations, particularly rare sequence mutations, in polynucleotide molecules. These methods involve the use of a novel technique, Single Molecule Consensus Sequencing (SMCS).
  • SMCS Single Molecule Consensus Sequencing
  • Chronic myeloid leukaemia otherwise known as chronic granulocytic leukaemia (CGL)
  • CML chronic myeloid leukaemia
  • CGL chronic granulocytic leukaemia
  • the disease is invariably fatal within 3 to 5 years.
  • the diagnosis is made during a relatively benign chronic phase (the "chronic phase”).
  • the disease progresses through an "accelerated phase” to a terminal “blastic phase” or “blast crisis” phase that is generally refractory to therapy (Goldman el ah, 2003).
  • the disease is characterised by the overproduction of granulocytes (also referred to as blasts or leukaemic blasts) in the blood marrow.
  • CML occurs when a bone marrow stem cell develops a new and abnormal chromosome referred to as the Philadelphia (Ph) chromosome. What causes this chromosome to appear in some people is unknown (although in some cases it may have arisen due to radiotherapy for other cancers), and it is not familial nor can it be passed to offspring.
  • the Ph chromosome comprises the fusion gene, BCR-ABL1, which represents the central molecular pathology of CML.
  • This gene encodes the BCR-ABLl fusion protein, which is a constitutively-activated tyrosine kinase that aberrantly activates a series of molecular pathways causing deregulated cell proliferation, differentiation, DNA repair and apoptosis (Melo et ah, 2007).
  • TKI BCR-ABL l tyrosine kinase inhibitor
  • Glivec® imatinib
  • TKI-resistant mutations More than 100 TKI-resistant mutations have now been described in CML patients (Branford et al., 2009). This has driven the development of more potent TKIs. Now available are the "second- generation" TKIs, nilotinib (Tasigna®; Novartis Pharmaceuticals Pty Ltd) and dasatinib (Sprycel®; Bristol-Myers Squibb Company, Princeton, NJ, United States of America) which, between them, are active against most imatinib-resistant BCR-ABLl protein mutants. A notable exception however, is the most commonly detected mutation, T3151, which confers resistance to imatinib, nilotinib and dasatinib.
  • Ponatinib (IclusigTM; ARIAD Pharmaceuticals lnc, Cambridge, MA, United States of America) is a "third-generation" TKI that inhibits all known BCR-ABL l protein mutants, including T3151, at clinically achievable doses (O'Hare et al., 2009).
  • ponatinib therapy has been associated with a relatively high incidence of side-effects, especially arterial thrombotic events such as coronary and peripheral vascular disease (Cortes et al., 2013).
  • in vitro studies have demonstrated that certain compound mutants are still likely to cause resistance (O'Hare et al., 2009; Zabriskie et al., 2014).
  • ABLOO l Novartis AG, Basel, Switzerland
  • the BCR-ABLl fusion gene as derived from the Ph chromosome is also central to the pathogenesis of other haematological neoplasms.
  • BCR-ABLl is not uncommonly found in a subtype of adult acute lymphoblastic leukaemia (ALL), associated with the older patient, and adverse prognosis.
  • ALL acute lymphoblastic leukaemia
  • TKIs are also increasingly recognised as a valuable addition to this standard of care (Chalandon et al, 2015).
  • the increased use of TKI for ALL is also associated with KD mutations as an evolving cause of disease resistance.
  • KD mutations in Ph+ ALL more commonly found at treatment failure, and more frequently confer resistance to both first and second generation TKIs, BCR-ABLl mutants that confer high level resistance, such as T3 151, E255K and Y253H, are more common in Ph+ ALL as compared to CML, as are compound mutations (Zabriskie et ah, 2014; Soverini et al, 2014).
  • Ph chromosome and acute myeloid leukaemia have been described, and the use of imatinib in these cases has also been reported to be associated with KD mutations as a pathway of treatment resistance (Reboursiere et al , 2015).
  • Ph+ leukaemias It is widely accepted that techniques used to interrogate the BCR-ABLl KD in the ascertainment of treatment resistant mutations in CML are also applicable and transferable to Ph+ ALL and Ph+ AML without, or with minimal, further modification. These diseases are referred to hereinafter as Ph+ leukaemias.
  • the present invention provides a method for identifying and/or enumerating sequence mutations within the kinase domain (KD) of a fusion gene comprising ABLI or a portion thereof encoding all or substantially all of the KD, wherein the method of the present invention comprises the steps of:
  • the present invention provides a kit for use in the method of the present invention.
  • a kit may comprise, for example, appropriate primer molecules, and/or buffer solutions, preparations of deoxyribonucleotide triphosphates (dNTPs) etc.
  • dNTPs deoxyribonucleotide triphosphates
  • Figure 1 provides: (A) a schematic diagram outlining an embodiment of the method according to the present invention, employing the use of a novel technique, Single Molecule Consensus Sequencing (SMCS).
  • SMCS Single Molecule Consensus Sequencing
  • the diagram depicts how, by uniquely tagging individual BCR-ABLl transcripts prior to PCR amplification, the method enables the identification and/or enumeration of those individual transcripts and hence differentiation between compound and polyclonal mutations; and
  • (B) provides tabulated results comparing compound mutations detected by the method depicted in (A) and amplicon next generation sequencing (NGS) using Ion Torrent. Only mutations within the region examined by the SMCS are summarised (nb. four (4) mutations were outside of this region).
  • the present invention provides a novel method which allows for the accurate and sensitive discrimination between compound mutants and multiple single mutants in patients with ABU fusion gene-driven diseases such as Ph+ leukaemias.
  • This method involves a technique herein termed as Single Molecule Consensus Sequencing (SMCS).
  • SMCS Single Molecule Consensus Sequencing
  • SMCS enables the identification and enumeration of sequence mutations (eg point mutations) present in RNA, cDNA or gDNA that may be associated with, for example, disease or treatment outcomes (eg TK1 drug resistance).
  • sequence mutations eg point mutations
  • the technique is particularly suitable for rare sequence mutations, however the technique may also be applied to the identification and enumeration of more commonly arising sequence mutations (eg mutations that may be present in all cells of a particular cancer).
  • SMCS enables one to distinguish rare mutations that may only be present in, for example, some cells from a particular cancer from mutations that may be caused by polynucleotide molecule amplification and/or sequencing reactions (ie artefacts).
  • the SMCS technique also enables compound mutations to be distinguished from instances where there are multiple versions of the relevant polynucleotide molecule (eg within a sample of a particular cancer) each with one different mutation within a particular cancer; as will be appreciated from the above, the problem with detecting compound mutations is that artefacts introduced during the amplification and/or sequencing reactions used in present methods can make it appear that two different mutations in the same gene are present in the same cell, when in fact they are present in separate cells.
  • the SMCS technique involves the use of "unique molecular ID tags" (UMIDs) or molecular "barcode” sequences to tag all of the polynucleotide molecules of interest within a sample.
  • UIDs unique molecular ID tags
  • barcode molecular "barcode”
  • the UMlD-tagged molecules are then copied and the sequence of the copies (ie amplicons) is obtained by, preferably, NGS.
  • the sequence of the original polynucleotide molecule is inferred from the consensus of the copies with the same UMID-tag sequence to thereby overcome misleading results caused by artefact mutations introduced during the amplification and/or sequencing reactions.
  • the present invention is particularly described with reference to the BCR-ABLl gene and chronic myeloid leukaemia (CML).
  • CML chronic myeloid leukaemia
  • the invention is considered to be more broadly applicable to other disease associated ABLl fusion genes, including but not limited to the BCR-ABLl- e fusion gene associated with some forms of acute lymphoblastic leukaemia (ALL), namely ETV6 (TEL)-ABLl which, in one described example, is a fusion of sequences from exon 5 of the ETV6 (TEL) gene (ie the ETS variant 6 gene) and exon 2 (also known as a2) of the ⁇ gene (Yeung et al., 2015).
  • ALL acute lymphoblastic leukaemia
  • TEL ETV6
  • TEL ETV6
  • TEL ETS variant 6 gene
  • exon 2 also known as a2
  • the present invention provides a method for identifying and/or enumerating sequence mutations (such as rare sequence mutations) within the kinase domain (KD) of a fusion gene comprising ABLJ or a portion thereof encoding all or substantially all of the KD.
  • the fusion gene may be associated with a disease (eg BCR-ABLl associated with CML, Ph+ ALL or Ph+ AML, or a fusion gene characteristic of another ABLJ translocation-associated neoplasm; for example the ETV6 (TEL)-ABLl fusion gene associated with some forms of ALL).
  • the method of the present invention therefore comprises the steps of:
  • RNA transcripts ie mRNA
  • ABLl a fusion gene comprising ABLl or cDN A molecules produced from transcripts of a fusion gene comprising ABLl;
  • a primer extension reaction eg 2-6 cycles
  • a first primer pair comprising forward and reverse primer molecules targeted so as to generate polynucleotide molecules comprising a polynucleotide sequence corresponding to all of the fusion gene or a portion thereof comprising a target region spanning a fusion gene breakpoint, an adjacent upstream sequence (eg of at least 20 nucleotides in length) and a downstream KD-encoding portion of the ABLl sequence (eg encoding exons 2 to 10, or 4 to 7), wherein one of said primer molecules (preferably the reverse primer or the primer closest to the KD- encoding portion) comprises a short sequence of random nucleotides providing an individual unique molecular ID tag (UMID), along with a fusion gene-specific sequence and a universal 5' tail sequence, to thereby tag each of the generated polynucleotide molecules with an individual UMID;
  • UID individual unique molecular ID tag
  • the method enables the identification (ie detection) of, for example, mutations (including rare sequence mutations and compound mutations) present in the fusion gene transcripts, which may be associated with disease (eg a Ph+ leukaemia such CML), or otherwise, of some disease- or treatment- associated characteristic (eg disease stage or drug resistance, particularly T I resistance).
  • the method may also enable, for example, rare sequence mutations to be distinguished from artefact mutations arising from amplification and/or sequencing reactions. As will be apparent from the above, avoiding such misleading results can be of considerable clinical significance.
  • rare sequence mutation is to be understood as referring to a "low level” mutation that may show a frequency of ⁇ 10% (eg a mutation that is present in less than 10% of individuals with an ABL1 fusion gene). Such rare sequence mutations may, for example, be present in all tumour cells in a sample or only in a portion or sub-population of the tumour cells in a sample. Further, a rare sequence mutation may consist of, for example, a point mutation (eg a single nucleotide variant; SNV), or an insertion or deletion mutation. Moreover, rare sequence mutations may represent polyclonal mutations or compound mutations.
  • the term "compound mutation” will be well understood by those skilled in the art and refers to one of two or more mutations present on the same polynucleotide mol ecule (ie one of multiple mutations present on the same polynucleotide molecule). These mutations may "compound” to cause, for example, an altered activity of an encoded protein, polypeptide or protein domain, which may in turn, be the cause of disease or, otherwise, of some disease- or treatment-associated characteristic (eg disease stage or drug resistance).
  • a "polyclonal mutation” will be understood by those skilled in the art as referring to one of two or more mutations present on different copies of a
  • polynucleotide molecule ie one of multiple single mutations found on different copies of a particular polynucleotide molecule.
  • the sample used in step (i) of the method comprises mRNA or complementary DNA (cDNA) prepared from any suitable body sample obtained from, for example, blood, serum, plasma, or the like (eg tumour tissue sample).
  • cDNA complementary DNA
  • the sample used in step (i) comprises cDNA.
  • cDNA refers to a DNA molecule that has a nucleotide sequence that is complementary to a molecule of messenger RNA (mRNA) which may be synthesised with reverse transcriptase using the mRNA as template.
  • mRNA messenger RNA
  • the cDNA does not contain intron sequences.
  • the sample for use in the method of the present invention may comprise cDNA as prepared by any of the methods well known to those skilled in the art.
  • the cDNA molecules present in the sample may be tagged with an individual unique molecular ID tag (UMID) in step (ii) by conducting a primer extension reaction (eg using a high-fidelity DNA polymerase enzyme) with a first primer pair comprising forward and reverse primer molecules, wherein one of said primer molecules (preferably the reverse primer) comprises a short sequence of random nucleotides providing the UMID, along with a fusion gene-specific sequence (ie a first fusion gene- specific sequence) and a universal 5' tail sequence.
  • the other primer molecules will also comprise a fusion gene-specific sequence (ie a second fusion gene-specific sequence).
  • the forward and reverse primer molecules are selected so that the primer extension reaction generates polynucleotide molecules comprising a polynucleotide sequence corresponding to at least the target region of the fusion gene.
  • the first fusion gene-specific sequence of the reverse primer may bind at the 3' end of the ABLl sequence within exon 7 or 9, while the second fusion gene-specific sequence of the forward primer may bind within the adjacent upstream sequence of the other gene member of the fusion gene (eg within exon el or el 3 of BCR or within exon 4 of ETV6 (TEL)).
  • the fusion gene-specific sequences of the forward and reverse primers may allow for code degeneracy (ie the primer molecules may be degenerate primers), or otherwise, the primer extension reaction may include multiple primers as required, to ensure tagging of all of the cDNA molecules.
  • the mRNA molecules may be tagged with an individual unique molecular ID tag (UMID) in step (ii) by conducting a primer extension reaction with a first primer pair comprising forward and reverse primer molecules (eg using a reverse transcriptase enzyme followed by synthesis of the second strand with a high-fidelity DNA polymerase enzyme), wherein one of said primer molecules (preferably the reverse primer) comprises a short sequence of random nucleotides providing the UMID, along with a fusion gene-specific sequence (ie a first fusion gene-specific sequence) and a universal 5' tail sequence.
  • a primer pair comprising forward and reverse primer molecules
  • a reverse transcriptase enzyme followed by synthesis of the second strand with a high-fidelity DNA polymerase enzyme
  • the other primer molecules will also comprise a fusion gene-specific sequence (ie a second fusion gene-specific sequence).
  • the forward and reverse primer molecules are selected so that polynucleotide molecules are generated which comprise a polynucleotide sequence corresponding to at least the target region of the fusion gene.
  • the first fusion gene-specific sequence of the reverse primer may bind at the 3' end of the ABLl sequence within exon 7 or 9, while the second fusion gene-specific sequence of the forward primer may bind within the adjacent upstream sequence of the other gene member of the fusion gene (eg within exon el or el 3 of BCR or within exon 4 of ETV6 (TEL)).
  • the primer extension reaction may include multiple primers as required to ensure tagging of all of the mRNA molecules.
  • the primer extension reaction may be conducted using any one of the suitable methodologies well known to those skilled in the art.
  • the primer extension reaction will comprise a two (2) cycle reaction.
  • the UMID sequences may be provided by generating random nucleotide sequences of, for example, 10-25 nucleotides in length (preferably, 15-20 nucleotides in length, and more preferably, 18 nucleotides in length).
  • the primer molecules comprising the UMID bind to the cDNA/mRNA (through the complementary fusion gene-specific sequence) in a simple manner forming a regular duplex structure devoid of any significant loop structure and, as such, those skilled in the art will understand that the method of the present invention does not employ molecules such as inversion probes (eg single molecule Molecular Inversion Probes (smMIP) described by Hiatt el ai, 2013).
  • the product of step (ii) is a reaction mixture wherein each one of the cDNA/mRNA molecules present in the sample is tagged with an individual UMID.
  • excess primers comprising the UMID are preferably degraded using any suitable methodology (eg by incubation with 60U of Exonuclease I at 37°C for 60 mins).
  • step (iii) the amplification of the UMID-tagged polynucleotide molecules to generate UMID- tagged amplicons comprising a polynucleotide sequence corresponding to all or substantially all of the said D-encoding portion, may be achieved using any of the suitable methodologies well known to those skilled in the art.
  • the amplification is performed using a standard polymerase chain reaction (PCR) amplification method (preferably a non-linear amplification method) using a pair of primers (ie forward and reverse primer molecules) defining the 5' and 3' ends of the desired polynucleotide sequence of the KD-encoding portion.
  • PCR polymerase chain reaction
  • the respective primer sequences hybridise to the 3' end of one strand (ie to thereby “define” the 3' end) and the 3' end of a complementary strand (ie to thereby “define” the 5' end) of the particular sequence so as to enable that sequence to be amplified.
  • this may be achieved by using a primer pair comprising a first primer molecule that comprises a nucleotide sequence that is complementary to the sequence at one end of the desired polynucleotide sequence and a second primer molecule that comprises a nucleotide sequence that targets a standard universal 5' tail sequence.
  • a primer pair comprising a first primer molecule that comprises a nucleotide sequence that is complementary to the sequence at one end of the desired polynucleotide sequence and a second primer molecule that comprises a nucleotide sequence that targets a standard universal 5' tail sequence.
  • each of the 20 nucleotides is perfectly complementary to the corresponding nucleotide of the particular target nucleotide sequence
  • a lesser degree of complementarity eg 95% complementary; wherein for a primer sequence of 20 nucleotides in length there may be one "mismatch" nucleotide and 19 nucleotides that are perfectly complementary with the corresponding nucleotide of the particular target nucleotide sequence.
  • step (iii) may employ primer molecules that allow for code degeneracy (ie the primer molecules may be degenerate primers), or otherwise, the amplification may include multiple primers as required, to ensure amplification of all of the tagged polynucleotide molecules.
  • the method will be conducted using a standard PCR amplification, in some circumstances, it may be preferred to perform the amplification step using a "nested" PCR amplification method using a further, "outside", pair of primers. Nested PCR amplification methods are well known to those skilled in the art.
  • primer molecules suitable for use in primer extension reactions and amplification reactions may be in accordance with techniques and guidelines well known to those skilled in the art (eg as described in Sambrook, J. and D. W. Russell, Molecular Cloning: a laboratory manual, Cold Spring Harbor Press, Third Edition (2001) at Chapter 8 (particularly Table 8-3), the entire disclosure of which is hereby incorporated by reference).
  • the amplicons produced in step (iii) may be no more than about 1 kb in length (although longer amplicons may also be suitable) and, perhaps preferably, will be about 650-750 nucleotides in length nucleotides in length comprising all or substantially all of the KD-encoding portion of the ABU sequence.
  • substantially all it is to be understood that the amplicons comprise at least two and, more preferably, at least three exons of the KD-encoding portion of the ABU sequence.
  • the amplicons comprise at least four exons of the KD-encoding portion (eg exons 2 to 10, exons 4 to 10 or exons 4 to 7).
  • each of the UMID-tagged amplicons is sequenced, conveniently by using a next generation sequencing (NGS) platform such as, for example, 454 pyrosequencing (Roche Diagnostics Corporation, Branford, CT, United States of America), Illumina (Solexa) sequencing (lllumina Inc, San Diego, CA, United States of America), SOLiD sequencing (Life Technologies, Carlsbad, CA, United States of America) or, alternatively, Ion Torrent semiconductor sequencing (Life Technologies).
  • NGS next generation sequencing
  • the technique is also amenable for use with new and emerging sequencing technologies such as PacBio (Pacific Biosciences, Menlo Park, CA, United States of America), Oxford Nanopore (Oxford Science Park, Oxford, United Kingdom) or Qiagen GeneReader (Qiagen, Hilden, Germany).
  • step (v) bioinformatic analysis is conducted on the sequences (or "reads") obtained in step (vi) to identify a consensus sequence for all sequenced amplicons comprising a common UMID (eg comprising a "Read Group” or read “family”).
  • the consensus sequence information may then reveal any sequence mutations that were present in the KD-encoding portion of the polynucleotide molecules present in the sample, since mutations arising from the polynucleotide molecule amplification and/or sequencing reactions (ie artefact mutations) will only be present in a small numbers of the sequences (ie in a few- reads only).
  • the bioinformatic analysis identifies reads derived from a single initial fusion gene transcript by virtue of the common UMID.
  • the consensus sequence of reads with a common UMID may be determined using automated variant calling and filtering algorithms, and represents the sequence of an initial cDNA/mRNA molecule present in the sample, thereby overcoming artefact mutations arising from the polynucleotide molecule amplification and/or sequencing reactions.
  • Steps (iv) and (v) may be conducted concurrently.
  • the term "consensus sequence” refers to the order of the most frequent nucleotides found in the sequences (ie reads) of the UMID-tagged amplicons (eg as produced in step (iv) of the method of the first aspect of the present invention) comprising a common UM1D.
  • the consensus sequence for a given group of amplicons comprising a common UMID may reveal any sequence mutations that were present in the
  • polynucleotide molecules present in the sample since mutations arising from the polynucleotide molecule amplification and/or sequencing reactions (ie artefact mutations) will only be present in a small numbers of the sequences (ie in a few reads only) and will therefore not be represented in the consensus sequence. Moreover, since the consensus sequence is produced from amplicons ultimately generated from a single polynucleotide molecule in the sample, the presence of two or more mutations in the consensus sequence may identify compound mutations (ie two or more mutations present on the same polynucleotide molecule present in the sample).
  • Mutations present in the consensus sequence may be recognised by sequence comparison against an appropriate reference sequence (eg the "wild-type” (WT) sequence), using standard methodologies and tools well known to those skilled in the art (eg Burrows-Wheeler sequence aligner (BWA); Li et al., 2010 and Genome Analysis Toolkit (GATK) such as that available from Appistry Inc, St Louis, MO, United States of America).
  • WT wild-type sequence
  • BWA Burrows-Wheeler sequence aligner
  • GATK Genome Analysis Toolkit
  • the number of amplicons present with a common UMID can be readily ascertained. This may be done by bioinformatic analysis of the NGS reads. For example, reads comprising a common UMID are grouped into Read Groups (or "families") and aligned to a reference sequence using BWA. The variants within each Read Group are called using GATK. Variants passing bioinformatic filtering that are present in the majority of reads within each Read Group represent those in the initial amplified molecule, whereas those present in only a few reads are artefacts.
  • the ratio of the number of Read Groups with a consensus sequence bearing a WT or reference sequence versus groups bearing variants may then be expressed as a percentage or ratio to allow description of prevalence of each mutant present.
  • Artificial, simulated samples created from known mixtures of WT/reference and mutant sequences may be used to empirically determine, and periodically calibrate, the sensitivity of the assay, or otherwise act as quality assurance. This may be particularly helpful when samples are of poor quality and/or quantity (eg some clinical samples such as formalin-fixed paraffin embedded sections, circulating tumour DNA samples and samples taken to assess minimal residual disease following treatment).
  • the present invention provides a method for identifying and/or enumerating sequence mutations (such as rare sequence mutations) within the kinase domain (KD) of the BCR-ABLl fusion gene associated with Ph+ leukaemias.
  • sequence mutations such as rare sequence mutations
  • the structure of BCR-ABLl (encompassing numerous variants) has been well described in, for example, Melo and Chuah, 2007, the content of which is hereby incorporated by reference in its entirety.
  • the KD-encoding portion of BCR-ABLl comprises six exons of the ABL1 portion of the fusion gene denoted exon 4 to 10 (also a4 to al O).
  • the chromosomal translocation point of BCR-ABLl that is the breakpoint or junction at which the two genes are fused, commonly arises from the major breakpoint cluster region (M-bcr) between exons e l 2 and e l6 of BCR and breakpoints of ABL within exon a2, and numerous fusion gene variants have been identified.
  • M-bcr major breakpoint cluster region
  • the "typical" transcripts of BCR-ABLl include transcripts denoted as e l a2 (a transcript produced from a fusion between exon e 1 of BCR and exon a2 of ABU), e 13a2 and e 14a2 (which can also arise from alternative splicing), but other BCR-ABLl transcripts are also well known including rare or "atypical” e l a2 (arising from the minor breakpoint cluster region (m-bcr) of BCR), e l a3, e2a2, e6a2, e l 3a3, e l4a3 and e l 9a2 variant forms.
  • m-bcr minor breakpoint cluster region
  • the e l 3a2 and e l2a2 transcripts encode a BCR-ABL l protein of 210 kDa, while the e l a2 transcript encodes for a BCR-ABL l protein of 190 kDa and the el 9a2 transcript encodes for a BCR-ABL l protein of 230 kDa.
  • the method of the present invention may be conducted in a manner to identify and/or enumerate sequence mutations (such as rare sequence mutations) within the kinase domain (KD) of one or more of the BCR-ABLl fusion gene and/or transcript variants, including but not limited to those mentioned above.
  • the method of the present invention therefore comprises the steps of:
  • a primer extension reaction eg 2-6 cycles
  • a first primer pair comprising forward and reverse primer molecules targeted so as to generate polynucleotide molecules comprising a polynucleotide sequence corresponding to all of the BCR-ABLl fusion gene or a portion thereof comprising a target region spanning a fusion gene breakpoint, an adjacent upstream 5CR-derived sequence (eg of at least 20 nucleotides in length) and a downstream KD-encoding portion of the ABL1 sequence (eg encoding exons 4 to 10 or exons 4 to 7), wherein one of said primer molecules (preferably the reverse primer or the primer closest to the KD-encoding portion) comprises a short sequence of random nucleotides providing an individual unique molecular ID tag (UMID), along with a BCR-ABLl - specific sequence (3') and a universal 5' tail sequence, to thereby tag each of the generated polynucleotide molecules with an individual UMID;
  • UID individual unique molecular ID
  • the sample used in step (i) of the method of this embodiment comprises BCR-ABLl transcripts or cDNA (produced from BCR-ABLl transcripts) prepared from any suitable body sample obtained from, for example, blood, serum, plasma, or the like.
  • the cDNA/mRNA sample used in step (i) of the method of this embodiment is prepared from a white blood cell pellet.
  • the sample used in step (i) of the method of this embodiment comprises cDNA.
  • the cDNA molecules may be tagged with an individual unique molecular ID tag (UMID) in step (ii) by conducting a primer extension reaction with a first primer pair comprising forward and reverse primer molecules, wherein one of said primer molecules (preferably the reverse primer) comprises a short sequence of random nucleotides providing the UMID, along with a BCR-ABLl -specific sequence (ie a first BCR-ABL 1 -specific sequence) and a universal 5' tail sequence.
  • the other primer molecules will also comprise a BCR-ABLl -specific sequence (ie a second BCR-ABL 1 -specific sequence).
  • the forward and reverse primer molecules are selected so that the primer extension reaction generates polynucleotide molecules comprising a polynucleotide sequence corresponding to at least the target region of the BCR- ABLl fusion gene.
  • the first BCR-ABL 1 -specific sequence of the reverse primer may bind at the 3' end of the ABLl sequence within exon 7 or 9, while the second BCR-ABLl -specific sequence of the forward primer may bind within the BCR sequence (eg within exon e l or e l3).
  • the forward and reverse primers may allow for code degeneracy, or otherwise, the primer extension reaction may include multiple primers as required, to ensure tagging of all of the cDNA molecules.
  • the primer extension reaction may preferably comprise a two (2) cycle reaction.
  • the UMID sequences may be provided by generating random nucleotide sequences of, for example, 10-25 nucleotides in length (preferably, 15-20 nucleotides in length, and more preferably, 18 nucleotides in length).
  • the primer molecules comprising the UMID bind to the cDNA (through the complementary BCR-ABLl -specific sequence) in a simple manner forming a regular duplex structure devoid of any significant loop structure (as such, the method of this embodiment does not employ molecules such as inversion probes).
  • the product of step (ii) is a reaction mixture wherein each one of the cDNA molecules present in the sample is tagged with an individual UMID. Following the UMID tagging, excess primers comprising the UMID are preferably degraded using any suitable methodology.
  • the tagged molecules produced in step (ii) are polynucleotide molecules encoding a "typical" transcript BCR-ABLl (eg el a2, e l 3a2 and e l4a2).
  • a "typical" transcript BCR-ABLl eg el a2, e l 3a2 and e l4a2
  • the method is also applicable for polynucleotide molecules encoding an "atypical" transcript such as those mentioned above.
  • the target region may comprise a portion of the BCR-ABLl fusion gene of, for example, no more than 2.5 kb (excluding intron sequences), but shorter sequences may be preferable such as a portion of no more than about 1.5 kb (excluding intron sequences) or a portion of no more than about 1 kb (excluding intron sequences).
  • step (iii) the amplification of the UMID-tagged polynucleotide molecules to generate UMID- tagged amplicons which comprise a polynucleotide sequence to all or substantially all of the said KD- encoding portion, may be achieved with any of the suitable methodologies well known to those skilled in the art.
  • the amplification may be preferably performed using a standard polymerase chain reaction (PCR) amplification method (preferably a non-linear amplification method) using a pair of primers (ie forward and reverse primer molecules) defining the 5' and 3' ends of the desired
  • PCR polymerase chain reaction
  • primers ie forward and reverse primer molecules
  • the amplicons produced in step (iii) may be no more than about 1 kb in length (although longer amplicons may also be suitable) and, perhaps preferably, will about 650-750 nucleotides in length nucleotides in length comprising all or substantially all of the KD-encoding portion of the ABL1 sequence.
  • the amplicons comprise at least two and, more preferably, at least three exons of the KD-encoding portion of the ABL1 sequence.
  • the amplicons comprise at least four exons of the KD-encoding portion (eg exons 4 to 7).
  • each of the UMID-tagged amplicons is sequenced, conveniently by using a next generation sequencing (NGS) platform.
  • NGS next generation sequencing
  • bioinformatic analysis is conducted on the sequences (or "reads") obtained in step (vi) to identify a consensus sequence for all sequenced amplicons comprising a common UMID (eg comprising a "Read Group”).
  • the consensus sequence information may then reveal any sequence mutations that were present in the KD-encoding portion of the polynucleotide molecules present in the sample.
  • the bioinformatic analysis identifies reads derived from a single initial BCR-ABLl molecul e by virtue of the common UMID.
  • the consensus sequence of reads with a common UMID may be determined using automated variant calling and filtering algorithms, and represents the sequence of an initial BCR-ABLl cDNA molecule present in the sample, thereby overcoming artefact mutations arising from the polynucleotide molecule amplification and/or sequencing reactions. Steps (iv) and (v) may be conducted concurrently.
  • the method of this embodiment of the present invention may thereby enable, for example, rare sequence mutations to be distinguished from artefact mutations arising from amplification and/or sequencing reactions.
  • the method of this embodiment of the present invention may enable the identification of the presence of compound mutations, present in the BCR-ABLl fusion gene, associated with advanced Ph+ leukaemia (eg advanced CML disease) and inferior therapeutic outcomes (Parker et al., 2012) and/or drug resistance.
  • the method of the present invention may be varied for the detection of minimal residual disease (MRD) by enabling the accurate and sensitive detection of sequence mutations associated with the particular disease.
  • MRD minimal residual disease
  • the term "minimal residual disease” will be well understood by those skilled in the art and refers to any small amount of remnant diseased tissue or cells in a subject during or after disease therapy. MRD is the major cause of relapse in leukaemia and cancer. More particularly, in this embodiment, the method may enable the assessment of whether a subject who has been treated (eg treated for Ph+ leukaemia) is free of the disease (eg the treatment has eradicated the diseased cells) or whether remnant diseased tissue or cells remain.
  • the method of this embodiment may enable the assessment of whether the treatment is or is not being effective.
  • the method may be used so as to allow comparison of the efficacy of different treatments, as well as monitoring the subject's remission status and recurrence of the disease. As such, an informed decision may be made on whether the patient may benefit from further treatment (perhaps with an alternative drug or drug regimen).
  • Ph+ leukaemia and other ABL1 translocation associated neoplasms the continual absence of residual leukaemia could identify patients where TKI treatment can be stopped wi th a limited probability of relapse.
  • the method further comprises the step of:
  • step (vi) where at least one disease-associated sequence mutation is identified within the consensus sequences, the method indicates the presence of minimal residual disease. Conversely, if no disease-associated sequence mutation is identified within the consensus sequences, the method indicates that the subject may be free of disease.
  • minimal residual disease may be detected by identifying and/or enumerating all sequenced amplicons comprising a common UMID.
  • the amplicons may be representative of the presence in the sample of BCR-ABL1 cDNA molecules.
  • the cDNA sample would preferably be prepared from mRNA isolated from a white blood cell pellet and, as such, the BCR-ABL1 cDNA molecules would represent transcripts of the BCR-ABL1 fusion gene.
  • the method of this further embodiment enables the detection and/or quantification of minimal residual disease.
  • the present invention provides a kit for use in a method of the present invention.
  • a kit may comprise, for example, appropriate primer molecules, and/or buffer solutions, preparations of deoxyribonucleotide triphosphates (dNTPs) etc.
  • SMCS Single Molecule Consensus Sequencing
  • Individual BCR-ABLl cDNA molecules were tagged with a unique molecular identifier (UMID) sequence using a two (2) cycle primer extension reaction performed using the polymerase chain reaction (PCR) technique and a robust high-fidelity DNA polymerase enzyme, and a set of BCR-ABLl -specific primers (nb. the forward primer is dependent on the type of BCR-ABLl transcript being targeted); namely:
  • the forward primer binds to BCR at nucleotides c.2645 to c.2668 (NM 021574) within exon el 3.
  • the reverse primer consists of sequence complementary to ABLl (NM_()05157.5) at the 3' end (at nucleotides c.1224 to c.1243 within exon 7), flanked by the UMID and a universal sequence (ie a portion of the Illumina sequencing adaptor, underlined) at the 5' end to allow amplification using a universal primer in subsequent steps.
  • the UMID consisted of 15 or 18 randomised nucleotides, generating >1 billion or >60 billion distinct sequences, respectively.
  • the forward primer in this case binds to BCR at nucleotides c.1 1 16 to c.1 137 within exon e 1.
  • Tagging with UMID was performed using BCR-ABLl cDNA generated from -0.5 ⁇ g of the total RN A and 400 nM of each primer in a 25 reaction. NEBNext High-Fidelity 2X PCR master mix (New England BioLabs Inc, Ipswich, MA, United States of America) was used for all PCRs. The UMID-tagged cDNA molecules are about 1.5 kb in length. Following UMID tagging, excess UM ID-containing primers were degraded by incubation with 60U of Exonuclease I at 37°C for 60 mins. The Exonuclease I was then heat inactivated (95°C for 5 min).
  • the uniquely-tagged BCR-ABLl molecules were amplified using 18-28 cycles of PCR with the BCR forward primer and a reverse primer complementary to the universal sequence in the UMID-containing primer (5' ACACTCTTTCCCTACACGACGCTC; SEQ ID NO: 4) in a 50 ⁇ xL reaction.
  • the products were purified with 0.6X AMPure® XP beads (Agilent
  • Excess primers were degraded using Exonuclease I, and then a final five (5) cycle PCR was performed to incorporate sample indexes ([i7] and [i5J to allow sample pooling for sequencing; lllumina Inc.) and sequences for binding the lllumina flow cell:
  • haplotype within the original samples, the consensus sequences were collated into unique sequences.
  • G ⁇ T nucleotide changes The most frequent error was G ⁇ T nucleotide changes, which is consistent with mis-incorporation of an A opposite an 8-oxo-guanine during the first round of PCR, resulting in G ⁇ T errors.
  • 8-oxo-guanine is prevalent in ancient DNA and is caused by nucleic acid oxidation during sample storage.
  • G ⁇ T nucleotide changes were most frequent in samples which had been stored for long periods of time (nb. most RNA samples had been stored for over 5 years) and were collected into PAXgene® tubes (Qiagen).
  • the second most prevalent nucleotide changes were G ⁇ A, which are caused by cytosine deamination to uracil causing mis-incorporation of an A opposite the uracil during the first round of PCR.
  • This nucleotide change was commonly observed at specific genomic positions, suggesting that it is likely due to in vivo cytosine deamination by RNA editing enzymes, such as those of the APOBEC family
  • Parent nodes were therefore interpreted as representing the clonal diversity of interest within the biological sample, with children nodes representing uninteresting changes of parent haplotypes.
  • mock samples were created by mixing compound mutant plasmids or patient samples (cDNA or RNA) with different BCR-ABL 1 mutations (up to 5 replicates each of a total of 14 mock samples were examined). Examination of the raw sequencing reads of the mock samples revealed a complex spectrum of mutants, similar to previous clinical reports (Soverini et ah, 2013; Khorashad et al., 2103). Using SMCS, however, enabled bioinformatic filtering of these artefacts, largely eliminating PCR amplification and sequencing errors, and exclusively reported the compound and polyclonal mutants known to be present in the mock samples.
  • a mock sample was generated by mixing 5 plasmids containing different compound mutations at various ratios with a plasmid containing unmutated BCR-ABLl.
  • the mock sample contained 65% unmutated BCR-ABL and 35%, 1 %, 0. 1 %, 0.05% and 0.01 % of each of the 5 compound mutations.
  • Six replicates of the mock sample (between 1 ,741 and 3,721 BCR-ABLl molecules were examined per replicate) were examined and it was found that it was possible to detect compound mutants present at a frequency of 0.1 % or greater (ie reproducible detection of 3 of the 5 plasmids containing compound mutations).
  • the amplicon NGS method detected 36 compound mutants within the 25 patients. Of the 32/36 mutations that were present within the region examined by SMCS, only eight (8) mutations were detected. Based on observations previously published in Parker et al., 2014, 16 of the 24 compound mutants that were not detected by SMCS were considered to likely represent PCR
  • the SMCS method was further evaluated by examining samples of 91 imatinib-resistant CML patients for which extensive examination of the BCR-ABLl kinase domain had been previously performed using Sanger sequencing (detection limit - 10%) and a mass-spectrometry based mutation assay (detection limit ⁇ ().2%)(Parker et al., 201 1). Neither Sanger sequencing nor the mass-spectrometry assay is able to distinguish compound mutations from polyclonal mutations.
  • the samples examined were collected immediately before starting (ie "baseline”) second-line TKl treatment with a second-line TKl (nilotinib or dasatinib), and clinical and molecular follow-up data was available.
  • the method By using samples of BCR-ABLl cDNA molecules (eg generated from patient mPvNA), the method not only allows examination of multiple exons of sequence using a current, clinically applicable, sequencing platform, but also abrogates the need for patient-specific primers to isolate their unique BCR-ABLl gene fusion molecules, a necessary step to enable cost-effective sequencing of the fusion molecules which may be scarce within a clinical sample.

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Abstract

L'invention concerne des procédés de détection de mutations, en particulier des mutations rares dans des séquences, dans le domaine kinase (KD) d'un gène chimère notamment ABL1. Ces procédés impliquent l'utilisation d'une nouvelle technique, appelée séquençage consensus à molécule unique (SMCS). Les procédés permettent plus particulièrement de détecter une combinaison de mutations présentes sur la même molécule polynucléotidique BCR-ABL1, qui peut être à l'origine d'une activité altérée d'une protéine, d'un polypeptide ou d'un domaine de protéine codé(e), qui peut à son tour être la cause d'une leucémie myéloïde chronique (LMC) ou d'une leucémie lymphoblastique aiguë (LLA) ou alors de certaines caractéristiques associées à une maladie ou à un traitement (par exemple, le stade de la maladie ou une résistance aux médicaments).
PCT/AU2015/000667 2014-11-05 2015-11-05 Détection de mutations dans les séquences de gènes chimères leucémiques Ceased WO2016070230A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107723353A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种白血病驱动基因的高通量检测方法
CN112687341A (zh) * 2021-03-12 2021-04-20 上海思路迪医学检验所有限公司 一种以断点为中心的染色体结构变异鉴定方法
CN112687341B (zh) * 2021-03-12 2021-06-04 上海思路迪医学检验所有限公司 一种以断点为中心的染色体结构变异鉴定方法
CN115896038A (zh) * 2023-02-02 2023-04-04 陕西师范大学 一种bcr-abl突变工程细胞及其构建方法与应用
CN116287162A (zh) * 2023-02-14 2023-06-23 赣南医学院 检测bcr-abl1融合基因及其酪氨酸激酶区突变和启动子甲基化的试剂盒及使用方法
CN120432016A (zh) * 2025-07-08 2025-08-05 四川大学 一种药物耐药性预测方法、装置、电子设备及存储介质

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