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WO2010048691A1 - Test de génotypage multiplex permettant la détection de mutations dans le gène k-ras - Google Patents

Test de génotypage multiplex permettant la détection de mutations dans le gène k-ras Download PDF

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WO2010048691A1
WO2010048691A1 PCT/CA2009/001226 CA2009001226W WO2010048691A1 WO 2010048691 A1 WO2010048691 A1 WO 2010048691A1 CA 2009001226 W CA2009001226 W CA 2009001226W WO 2010048691 A1 WO2010048691 A1 WO 2010048691A1
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seq
primers
ras
primer
nucleic acid
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John D. Brunstein
Amy Miu Geu Lum
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British Columbia Cancer Agency BCCA
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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • TITLE Multiplex Genotyping Assay for Detecting Mutations in K-Ras
  • the present invention relates to primers and assays for molecular genotyping, and in particular to ARMS primers suitable for multiplex-PCR for the detection of activating mutations in the K-ras gene.
  • K-ras gene mutation status may be associated with K-ras gene mutation status and in many cases this determines whether certain classes of very expensive chemotherapy will be effective.
  • the drugs Panitumumab and Cetuximab are monoclonal human antibodies used in the treatment of certain classes of colorectal cancer and potentially some lung cancers and other cancer types where involvement (i.e. activation) of the epidermal growth factor receptor (EGFR) pathway is involved.
  • EGFR epidermal growth factor receptor
  • the drug is not generally effective. Approximately 40% of potential treatment subjects would thus be excluded from expensive and ineffective treatment with this class of drug by the use of a K-ras mutation screening assay.
  • a commercially available assay from DxS Ltd. utilizes real-time PCR to target each of seven known common K-ras activating mutations. Positivity for each of the mutations is scored on the basis of differential quantitation of each potential mutant in comparison to wild-type sequence in a set of single-mutation querying ARMS (Amplification Refractory Mutation System) assays with scorpion probe detection.
  • ARMS Amplification Refractory Mutation System
  • ARMS-PCR for detecting seven known mutations in codons 12 and 13 of K- ras.
  • the ARMS primers were designed to bind to different strands of the target nucleic acid molecule and were not shown to be useful in multiplex reactions in a single tube.
  • the inventors have identified novel primers that allow for the detection of specific mutations in the human K-ras gene.
  • the primers are allele-specific and enable the simultaneous detection of mutations using Amplification Refractory Mutation System (ARMS).
  • ARMS primers permit the extension or amplification of a specific nucleotide target sequence but are refractory to other sequences that differ from the target sequence by as little as one single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • the primers disclosed herein may be used in a multiplexed genotyping assay for the detection of K-ras mutations in a single tube as opposed to the use of separate tubes for each mutation or SNP. Information regarding whether a subject has a K-ras mutation is useful for the diagnosis and treatment of cancer and for determining whether a patient with cancer would be likely to benefit from EGFR-targeted therapy.
  • the primers and methods disclosed herein allow for the determination of the mutational status for all 7 major mutations in codons 12 and 13 of K-ras
  • the mutational status of all 7 codon 12/13 SNPs or all 3 codon 61 SNPs can be determined in a single tube, multiplexed ARMS- PCR reaction.
  • the mutational status of all 10 codon 12/13 and codon 61 SNPs may be determined in a single tube, multiplexed ARMS-PCR reaction.
  • the assays described herein offer significant advantages, including, but not limited to, higher throughput, lower cost per sample, reduced complexity, simpler interpretation of results and ability to perform measurements on problematic specimens/templates such as formalin-fixed paraffin-embedded (FFPE) tissue, in addition to the more commonly encountered specimen formats.
  • FFPE formalin-fixed paraffin-embedded
  • one or more forward primers for detecting mutations in K-ras comprising nucleic acid molecules selected from: a) a nucleic acid molecule comprising any one of SEQ ID NO:1 to SEQ ID NO:11 ; b) the nucleic acid molecule of (a), wherein the 3'-portion is destabilized; and c) a nucleic acid molecule comprising any one of SEQ ID NO: 12 to SEQ ID NO:23.
  • the primers described herein further comprise a detectable label including, but not limited to, a nucleic acid sequence, peptide, luminescent compound, fluorescent compound, radiomolecule, redox label or antibody.
  • a detectable label including, but not limited to, a nucleic acid sequence, peptide, luminescent compound, fluorescent compound, radiomolecule, redox label or antibody.
  • Some embodiments described herein include primer pools for detecting mutations in K-ras.
  • One embodiment comprises a primer pool for detecting mutations in codons 12 and 13 of k-ras comprising two or more forward primers comprising nucleic acid sequences selected from the group
  • the primer pool is for detecting mutations in codons 12 and 13 of K-ras and includes primers comprising a set consisting of nucleic acid molecules with the sequences SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:
  • a further embodiment comprises a primer pool for detecting mutations in codon 61 of k-ras comprising two or more forward primers comprising nucleic acid sequences selected from SEQ ID NO:21 , SEQ ID NO:22, and SEQ ID NO:23.
  • the primer pool includes a set consisting of nucleic acid molecules with the sequences SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58.
  • the primer pools include one or more reverse primers suitable for use with the forward primers.
  • a method for detecting K-ras mutations in a sample from a subject comprising: a) providing a DNA sample from the subject; b) amplifying the sample using the forward primers described herein and one or more reverse primers in a PCR reaction wherein each forward primer is specific for a single K-ras mutation or for wild-type K-ras; and c) detecting a K-ras mutation by identifying the amplification products of step b).
  • the step of amplifying the sample is a multiplex ARMS-PCR reaction carried out in a single tube.
  • the PCR reaction optionally uses one of the primer pools described herein.
  • the forward primers are at a reaction concentration of between 50 nm and 500 nM and the reverse primers are present at a reaction concentration between 500 and 1500 nM.
  • the amplification products may be identified using Luminex TM technology.
  • Another embodiment includes a method of screening a subject with cancer for treatment with EGFR targeted therapy comprising detecting K-ras mutations in a sample from the subject using one of the methods described herein, wherein subjects with a K-ras mutation are excluded from treatment with EGFR targeted therapy.
  • kits that contain reagents necessary for carrying out the K-ras genotyping assays described herein.
  • Figure 1 is an amplicon diagram illustrating the relative position of the 7 forward mutant K-ras ARMS primers, WT forward primer and a common biotinylated reverse primer.
  • Figure 2 is an amplicon diagram illustrating the relative position of the 3 forward mutant K-ras codon 61 ARMS primers and a common biotinylated codon 61 reverse primer.
  • FIG 3 is a schematic illustration of the hybridization and capture of WT and mutant K-ras PCR products incorporating biotin and an anti-tag sequence to FlexMap tagged LuminexTM beads.
  • Figure 4 is a schematic illustration of the incubation of LuminexTM beads (including those with captured and biotinylated WT and mutant K-ras PCR products) with streptavidin-PE.
  • Figure 5 is a schematic illustration of the differential LuminexTM detection of bead classes with captured WT K-ras and mutant K-ras PCR products.
  • Figure 6 shows the detection of WT K-ras signal demonstrating good signal to noise ratios and clear interpretability of results.
  • Figure 7 shows detection of the K-ras mutant/SNP G12V in a positive control homozygous G12V cell line.
  • Figure 8 shows a cross-table (results grid) validation of the K-ras mutant multiplex assay for multiple targets and the detection of codon 12 and codon 13 K-ras mutants and WT K-ras in sample consisting of 50% K-ras mutant in WT background.
  • Figure 9 shows a cross-table (results grid) validation of the K-ras mutant triplex assay for multiple targets and the detection of codon 61 mutants and WT K-ras in sample consisting of 50% K-ras mutant in WT background.
  • Figures 10A through 10G show the determination of the limit of detection (LOD) for each of the different K-ras mutations in the codon 12/13 multiplex assay. Each plot shows observed signal intensity versus input % mutant sample in a K-ras WT background.
  • LOD limit of detection
  • Figure 11 A through 11 C show the determination of the limit of detection
  • Figure 12 shows the detection of K-ras mutations G13D and G12V in patient tissue samples previously reported as positive for these mutations when independently analyzed using a commercially available (DxS) assay.
  • DNA (20 ng) was isolated from FFPE samples.
  • Figure 13 shows the detection of K-ras mutations G13D and G 12V in patient tissue samples previously reported as positive for these mutations when independently analyzed using a commercially available (DxS) assay.
  • DNA (10 ng) was isolated from FFPE samples.
  • the present inventors disclose novel primers and methods for the detection of mutations in the human K-ras gene.
  • the primers have been shown to selectively bind to target mutations in K-ras and may be combined in a single tube, multiplex PCR reaction.
  • the primers disclosed herein are primers that selectively hybridize to and allow the amplification of a specific target sequence in the human K-ras gene.
  • the primers are Amplification Refractory Mutation System (ARMS) primers.
  • ARMS Amplification Refractory Mutation System
  • ARMS primers discriminate among nucleic acid sequences that differ by as little as a single nucleotide residue.
  • ARMS-PCR also known as allele- specific PCR
  • ARMS-PCR can be used to amplify a specific target allele while remaining refractory to the amplification of alleles that differ from the target allele.
  • ARMS-PCR therefore combines the amplification and detection of a target mutation into a single reaction.
  • Primers and assays for ARMS-PCR are often difficult to design and optimize due to the spurious amplification of wild-type (WT) alleles. Identifying ARMS primers suitable for use in single-tube multiplex PCR reactions presents an even greater challenge.
  • the forward primers disclosed herein minimize the cross-hybridization and corresponding amplification of sequences in the human genome other than the K-ras mutant targets, including WT K-ras.
  • Figure 1 shows the relative position of the ARMS primers for detecting mutations in codons 12/13 of K-ras including G12A, G12C, G12D, G12R,
  • Figure 2 shows the relative position of the ARMS primers for detecting mutations in codon 61 of K-ras including Q61 R, Q61 H and Q61 L.
  • Reverse ARMS primers suitable for use with the codon 12/13 forward ARMS primers and the codon 61 forward ARMS primers are also disclosed.
  • ARMS primers that selectively hybridize to and amplify wild-type K-ras sequences are also disclosed.
  • the sequence and length of the WT K-ras forward ARMS primer is designed so that it will not out-compete or falsely amplify a mutant K-ras target/sequence.
  • the position of the WT forward primer is optionally such that amplification of at least 6 of the 7 common Codon 12/13 K-ras mutants (SNPs) would be blocked or the efficiency of amplification of the SNPs greatly reduced if the template contains a mutation.
  • SNPs common Codon 12/13 K-ras mutants
  • one or more primers suitable for detecting mutations in K-ras as shown in Table 1.
  • Table 1 Native mutation-specific ARMS forward primers for detecting mutations in K-ras.
  • the native ARMS primers shown in Table 1 occasionally have the unwanted capacity to amplify WT K-ras. Additional primers that incorporate destabilizing mutations at the 3'-portion of the primers were therefore designed and tested for use in the K-ras genotyping assays.
  • destabilized refers to changes in primer sequence which effectively reduce the spurious amplification of WT K-ras, while retaining binding activity for a target mutation sequence.
  • 3'-portion refers to the last 5 nucleotides at the 3' end of a primer.
  • the primers are destabilized following the rules described in Ye et al. "An efficient procedure for genotyping single nucleotide polymorphisms" (Nucleic Acids Research, 2001 VoI 29:17). In other embodiments, the primers are destabilized by sequence modifications according to thermodynamic analysis.
  • primers originally identified using the rules set out in Ye et al. or primers with similar Tm values to those primers were not necessarily the primers that performed best when tested in vitro.
  • one embodiment includes primers suitable for detecting mutations in K-ras listed in Table 1 , wherein the 3'-portion of the primer is destabilized.
  • Another embodiment includes the destabilized ARMS primers suitable for detecting mutations in K-ras shown in Table 2.
  • Table 2 Destabilized mutation specific ARMS-PCR forward primers for detecting mutations in K-ras.
  • the ARMS primers directed to the various K-ras mutants as well as to WT K-ras provided herein may be varied in length, composition and/or sequence to provide modified and/or derivative primers that are also useful as ARMS-primers for the detection of K-ras mutants.
  • some embodiments described herein include primers between 10 and 35 nucleotides in length that are co-terminal to the 3'-end of the native forward ARMS primers disclosed in Table 1.
  • Another embodiment includes the primers of Tables 1 or 2 comprised in whole or in part of non-natural nucleotide analogs such as locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) or Iso-C containing nucleotides.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • Iso-C containing nucleotides Iso-C containing nucleotides.
  • said modified and/or derivative primers are of a different length, and in a preferred embodiment are shorter in length at the 5'- end of the primer compared to the parent primers shown in Tables 1 or 2.
  • the shorter modified and/or derivative primers retain or have improved their specificity to the given K-ras target as well as retain their thermal characteristics (such as Tm) compared to the parent primers shown in Tables 1 or 2.
  • Another embodiment disclosed herein includes primers and associated multiplexed single tube assays capable of detecting the 5 K-ras codon 13 activating mutations designated as G13S, G13R, G13C, G13A,
  • G13S, G13R, G13C, G13A, G13V as well as a common biotinylated reverse primer are designed and optimized using methods as described herein.
  • primers disclosed herein may be used to selectively bind to a target sequence comprising a specific K-ras mutation.
  • primers corresponding to a specific K-ras mutation are used to selectively bind to and amplify a target sequence.
  • the mutational status of the target sequence can then be determined by identifying the primer incorporated into the amplified sequence.
  • the primers disclosed herein comprise a detectable label.
  • detectable label refers to any molecule attached to a primer which facilitates the detection and/or isolation of the primer or a nucleic acid molecule comprising the primer.
  • the detectable label is covalently attached to the primer.
  • the detectable labels include a nucleic acid sequence, peptide, luminescent compound, fluorescent compound, radiomolecule, redox label or antibody.
  • more than one detectable label is attached to a primer.
  • the detectable label permits the isolation or purification of molecules that comprise the label.
  • the label may comprise a nucleic acid sequence which binds to a complementary nucleic acid sequence conjugated to a fixed support. Accordingly, the detectable label can be used to isolate or purify nucleic acid molecules that are the product of
  • the detectable label may be bound to a secondary label which is itself detectable.
  • a detectable label is attached to the native forward ARMS primers listed in Table 1. In another embodiment, a detectable label is attached to the destabilized forward ARMS primers listed in Table 2.
  • the detectable label is a flanking nucleic acid sequence that is attached to the 5' end of the primer resulting in a continuous sequence of nucleic acids. Flanking sequences may be used for product capture and to distinguish between the ARMS-PCR products described herein.
  • the detectable label is a unique 5' flanking sequence selected from the LuminexTM FlexMap anti-tag list that enables for the capture and characterization of associated PCR products.
  • anti-tag sequences for the primers are chosen sequentially based on their associated LuminexTM bead region in accordance with the manufacturers' suggested order of bead selection (Luminex Corporation, Austin, Texas).
  • mutation specific ARMS primers are labeled on their 5' end with the anti-tag sequences listed in Table 3.
  • Table 4 provides sequences for the native mutation-specific ARMS primers that include a LuminexTM bead anti-tag sequence detectable label while Table 5 provides the sequences for the 3'-destabilized mutation-specific ARMS primers that include a LuminexTM bead anti-tag sequence detectable label.
  • Table 4 Mutation-specific native ARMS primers labeled with LuminexTM anti- tag sequences.
  • Table 5 Mutation-specific 3' destabilized ARMS primers labeled with LuminexTM anti-tag sequences.
  • the forward ARMS primers described herein are suitable for use with reverse primers that permit the amplification of suitable fragments when amplified using PCR.
  • the codon 12/13 forward ARMS primers described herein are paired with a single reverse primer.
  • the reverse primer for use with the codon 12/13 forward ARMS primers has an annealing position on K-ras such that the resulting amplified target region is less than 350 base pairs in length, or preferably between 100-200 base pairs in length and most preferably between 150 and 160 base pairs in length.
  • shorter amplicons have the advantage of maximizing functionality of the K-ras multiplex assay with fragmented template samples (i.e. DNA from FFPE tissue).
  • the codon 61 forward ARMS primers described herein are paired with a single reverse primer.
  • the reverse primer for use with the codon 61 forward ARMS primers has an annealing position on K-ras such that the resulting amplified target region is less than 300 base pairs in length, less than 150 bp in length, and preferably between 100-200 base pairs
  • the reverse primer TTTATCTGTATCAAAGAATGGTCCTG results in the ARMS-PCR products with the sizes given in Table 6.
  • the forward ARMS primers are those listed for codon 61 in Table 5, the reverse primer is ATTTATGGCAAATACACAAAGAAAGC (SEQ ID NO:60) and the codon 61 ARMS-PCR products are the sizes given in Table 6.
  • Table 6 Product lengths for ARMS-PCR reactions with reverse primers SEQ ID NO:59 and SEQ ID NO:60.
  • the reverse ARMS primers are labeled with a detectable label. In one embodiment, the reverse ARMS primers are biotinylated on the 5' end of the primer. In some embodiments the reverse ARMS primers are biotinylated in order to allow detection using LuminexTM technology as described herein.
  • multiplex PCR refers to a PCR reaction that includes more than one pair of primers that can potentially result in the formation of an amplification product.
  • Multiplex PCR includes the use of more than one forward primer and a single reverse primer.
  • primer pool refers to a mixture of 2 or more primers that differ in nucleic acid sequence.
  • the primer pool comprises primers with the nucleic acid sequences listed in Table 1 or Table 2.
  • the primer pool comprises primers that include the nucleic acid sequence listed in Table 4 or Table 5.
  • the primer pool includes only forward primers, only reverse primers or a mixture of forward and reverse primers.
  • Embodiments provided herein include optimized multiplex assay concentrations of the WT primer, mutation specific K-ras primers and biotinylated K-ras reverse primer such that squelching of the mutant-specific signals by the WT primer in a WT background is limited. These primer concentrations result in high uniformity of assay results and inherent asymmetry of the PCR reactions with asymmetric PCR product yield.
  • the resulting asymmetric product have an excess of biotinylated or otherwise labeled product strands which enables the detection assay as shown in Example 1 to skip the customary denaturation (95 degrees C for 1-3 minutes prior to step 4) and washing steps (adding and removing buffer solutions by vacuum filtration between steps 5 and 6).
  • the differential relative concentrations of the primers used in this multiplex assay ensures that all mutations (SNPs) are consistently and simultaneously detected with WT K- ras.
  • the forward ARMS primers (WT and mutant specific) are present in the multiplex assay at concentrations between 50 nm and 500 nM and the reverse primers are present at a concentration between 500 and 1500 nM.
  • the K-ras WT primer is present at an assay concentration of 125 nM, each of the mutation-specific ARMS forward primers at a concentration of 250 nM and the biotinylated reverse primer at 125O nM.
  • the ARMS-PCR forward primer for the G12C mutation is at an assay concentration of 62.5 nM and the other forward ARMS primers for codons 12/13 and 61 are at an assay concentration of 250 nM.
  • the codon 61 forward ARMS primers are at an assay concentration of 250 nM.
  • the forward ARMS primers include primers for codons 12/13 and codon 61 , and the codon 12/13 and codon 61 reverse primers are both at a concentration of 1250 nM.
  • some embodiments include methods for detecting K-ras mutations in a sample from a subject comprising: a) providing a sample comprising nucleic acids from the subject; b) amplifying in an PCR reaction the sample using the forward primers of any one of claims 1 to 6 and one or more reverse primers wherein each forward primer is selective for a specific K-ras mutation or for wild-type K-ras; and c) detecting a K-ras mutation by identifying the amplification products of step b).
  • the term "subject” refers to any member of the animal kingdom, and includes mammals such as humans. The term also includes subjects having cancer or suspected of having cancer.
  • sample comprising nucleic acids refers to any sample of cells or tissue from any a subject that contains nucleic acids such as DNA or RNA.
  • the sample may be comprise a biological fluid such as blood, serum, saliva, cerebrospinal fluid, plasma, or lymphatic fluid, a tissue sample or tissue biopsy.
  • amplifying in a PCR reaction refers to the process of repeatedly denaturing and annealing forward and reverse primers to a nucleotide template in the presence of a polymerase enzyme in order to extend the primers with a sequence complementary to the template in a thermocycling reaction to produce additional copies of a nucleic acid sequence as is generally known in the art (See Dieffenbach CW and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N. Y.).
  • PCR polymerase chain reaction
  • Amplification using PCR typically requires reagents necessary to carry out amplification such as a nucleic acid precursors (dCTP, dTTP etc.) a polymerase, primers, template, and buffer.
  • a nucleic acid precursors dCTP, dTTP etc.
  • primers a polymerase
  • template a polymerase
  • buffer a buffer
  • primer refers to an oligonucleotide, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the term "selective for a specific K-ras mutation” refers to a primer which will preferentially hybridize to and amplify a target sequence containing a specific K-ras mutation.
  • the primers listed in Tables 1 , 2, 4 and 5 are each selective for given K-ras mutation.
  • the primers disclosed herein are each selective for a specific K-ras mutation under the conditions listed in Example 1.
  • the annealing temperature of the ARMS PCR reactions described herein is between about 52 and 56 degrees Celsius. In one embodiment, the annealing temperature is about 54 degrees Celsius.
  • each forward primer is specific for a given mutation
  • identifying which primer is contained in an amplification product identifies the mutation contained in the amplification product and accordingly in the sample.
  • the primer further comprises a detectable label which is unique to that primer and facilitates the capture and/or identification of an amplification product containing the primer.
  • the detectable label can be a nucleic acid sequence, peptide, luminescent compound, fluorescent compound, radiomolecule or an antibody.
  • the primers disclosed herein can be used in a multiplex PCR reaction in a single tube.
  • the multiplex reaction single tube reaction includes primers that are selective for mutations
  • the multiplex reaction single tube reaction includes primers that are selective for mutations Q61 R, Q61 H and Q61 L in codon 61 of K-ras.
  • DNA is extracted from a sample tissue of interest from a subject and is introduced into a polymerase chain reaction (PCR) containing the novel ARMS primers of the invention.
  • PCR reaction includes the 10 forward primers specific for each of the K-ras mutations (SNPs) in codons 12/13 and codon 61 as well as an ARMS forward primer for the wild-type (WT) K-ras gene and two biotinylated reverse primers common to either the target amplicons of codons 12/13 or 61.
  • each of the ARMS primers also comprises a unique 5' sequence (known as an anti-tag sequence) selected from the LuminexTM bead anti-tag list in accordance with the manufacturers' recommendations and a standard PCR master mix containing a non-proofreading thermostable polymerase.
  • the master mix is the Qiagen HotStarTaq master mix.
  • the native ARMS primers are used for the G12A and G12R K-ras mutations and destabilized ARMS primers are for the G12S, G12C, G12D, G12V and G13D mutations.
  • the destabilized mutant ARMS primers of the invention suppress spurious amplification of WT K-ras and in one embodiment of the invention are used for detection of other K-ras mutations in codons 12 and 13. In other embodiments, destabilized primers are used for each of the mutations to be detected.
  • thermocycling of the reaction leads to amplification
  • PCR of products between any annealing forward primers (K-ras WT or K-ras mutant-specific ARMS primers) and the common reverse primer as shown in Figures 1 and 2. Since mutant as well as normal cells are expected to contain WT sequence (K-ras mutations are normally heterozygous when present), there will be a WT priming site in all samples.
  • the WT amplicon therefore optionally serves as an internal control for the assay processes/variables such as DNA extraction, amplification, and the subsequent detection/classification of PCR products.
  • thermocycling conditions employed in some embodiments of the assay described in Example 1 have been experimentally optimized.
  • PCR endpoint products are detected using Luminex-based (liquid-array microsphere) technology as outlined in Figures 3-5. Briefly, the aforementioned PCR products are mixed with a number of classes of FlexMap Luminex beads corresponding to the number of assayed mutations at, for example, a concentration of 5000 of each bead class per assay. These consist of colour- coded beads with each colour code having a unique surface conjugated oligonucleotide tag. Each of these tags corresponds to (complementary to) one of the forward primer anti-tag sequences.
  • Annealing of the beads with the PCR product allows any anti-tag containing PCR products to hybridize to their complimentary bead class and thereby associates a biotin tag (contained on a reverse primer) with that bead class ( Figure 3).
  • Subsequent incubation with streptavidin-phycoerythrin (strep-PE) conjugate couples the PE fluorescent reporter to the bead class ( Figure 4).
  • the amplification products can optionally be detected by LuminexTM technology. Briefly, the incubated bead mixture is sampled by a Luminex instrument which acts similar to a flow cytometer by passing aspirated beads single file through a dual laser excited optical detection window.
  • the first laser enables readout of the passing bead colour code, while the second laser interrogates the passing bead for surface associated PE fluorescence ( Figure 5).
  • a minimum number of each bead class (for example 50 or 100 as set by operating software) is interrogated in each sample, and a Mean Fluorescence Intensity (MFI) for each bead class is calculated and displayed.
  • MFI Mean Fluorescence Intensity
  • a bead set associated with a particular K-ras mutation is scored as positive if the MFI exceeds a statistically determined cutoff criterion.
  • this criterion is set based on analysis of known negative reactions and, in one embodiment of the invention, at the (average + 5 standard deviations) of a negative sample signal. For example, in one embodiment, on a BioPlex instrument calibrated to a High RP1 setting, the positivity cutoff is 300 MFI). In trials, positive samples (i.e. WT K-ras) have been shown to yield signals approaching 17,000 MFI or nearly 57 fold above the positivity cutoff ( Figure 6), making determination of sample genotype unambiguous and easily interpreted.
  • embodiments include, but are not limited to, techniques such as primer extension, classical microarrays or line probes.
  • Methods of PCR product endpoint detection including, but not limited to, fluorescence, chemiluminescence, colourimetric techniques or measurement of redox potential may also be used with the embodiments described herein for detecting K-ras mutations.
  • the methods described herein can be used to detect the presence of K-ras activating mutations in subjects with cancer whereby the cancer is at least partially due to inappropriate activation of the cellular epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • Clinical responsiveness of said patients to EGFR targeted therapy is significantly reduced when the patients simultaneously harbour downstream activating K-ras mutations (40- 50 % of these patients have K-ras mutations).
  • application of the detection assays described herein to patients prior to anti-EGFR therapy will allow identification of those patients unlikely to respond to this treatment and enable improved disease management as well as reduced drug expenditures.
  • the assay is used to detect the presence of K-ras activating mutations in patients with colon cancer whereby the colon cancer is at least partially due to inappropriate activation of the cellular epidermal growth factor receptor (EGFR).
  • the assay is used to detect the presence of K-ras activating mutations in patients with non-small cell lung cancer (NSCLC) whereby the NSCLC is at least partially due to inappropriate activation of the cellular epidermal growth factor receptor (EGFR).
  • NSCLC non-small cell lung cancer
  • Clinical responsiveness of said patients to EGFR targeted therapy is significantly reduced when the patients simultaneously harbour downstream activating K-ras mutations (40-50 % of these patients have K-ras mutations).
  • EGFR targeted therapies include, but are not limited to, the use of panitumimab, gefitinib, erlotinib, or cetuximab.
  • some embodiments include a method of screening a subject with cancer for treatment with EGFR targeted therapy comprising detecting K-ras mutations in a sample from the subject, wherein subjects with a K-ras mutation are excluded from treatment with EGFR targeted therapy.
  • additional cancers which are at least partially caused by inappropriate activation of the cellular epidermal growth factor receptor (EGFR) and/or EGFR pathway as a whole, and would therefore be expected to benefit from EGFR targeted therapy include, but are not limited to, colon cancer, non-small cell lung cancer, head and neck cancer, and ovarian cancer.
  • EGFR cellular epidermal growth factor receptor
  • the K-ras mutation multiplex assay can be used as a screening or diagnostic tool for early detection of cancer or preneoplastic lesions and, in a particular aspect, to detect pre-cancerous lesions in the lower gastrointestinal tract.
  • the K-ras multiplex genotyping described herein is useful as a theranostic or companion diagnostic assay/tool applicable to multiple cancer types and multiple therapeutic regimens.
  • kits comprising the necessary reagents to perform any of the methods of the invention.
  • the kits may include at least one nucleic acid primer or primer pool described herein, which may be conveniently used, e.g., in a clinical or laboratory setting, to screen and identify those individuals with mutations in the K-ras gene.
  • the kits may also include nucleotides, enzymes and buffers useful for PCR as well as reagents useful for detecting the PCR products described herein.
  • the kit may also include suitable packaging and/or containers such as plastic resealable tubes or the like for holding primers or other regents, detailed instructions for carrying out the methods of the invention.
  • kits for detecting mutations in K-ras comprising (i) reagents for conducting PCR with the primers described herein and (ii) instructions for use thereof.
  • the kits include primer pools.
  • the kits optionally include reagents for detecting the PCR products using LuminexTM technology such as color-coded beads, detection buffer, streptavidin-phycoerythrin (Strep-PE), and stopping buffer.
  • the kits optionally include reagents for suitable for detecting PCR products or labeled PCR products using other technologies such as redox potential or immunodetection.
  • Sample Preparation Input material for the described assay is sample DNA extract of suitable quality for routine PCR, such as that provided by any of a number of commercial kits optimized for different specific specimen types.
  • isolation of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue (such as might be a common specimen type for this assay, and which has been used in the development of the assay to date) has been performed using the QIAamp DNA FFPE Tissue Extraction Kit (Qiagen, Hilden, Germany) with minor modification from the supplied protocol including two xylene treatments, an overnight incubation with proteinase K, and an elution volume of 30 ⁇ l.
  • DNA quantitation of the sample eluate was performed using the Nanodrop ND-1000 spectrophotometer and samples were diluted to a concentration of 10 ng/ ⁇ l.
  • Primer Design 'Native 1 primers were generated using PrimerPlex software with the exception of G12R and Q61 R, which were generated manually. Destabilized primers were manually generated using a combination of rules described by Ye et al. "An efficient procedure for genotyping single nucleotide polymorphisms" (Nucleic Acids Research, 2001 VoI 29:17) and thermodynamic analysis. Ye et al. suggest a "thermodynamic best" sequence based on their observations applied in the context of the 'strength' of the ARMS mismatch, such that a 'strong' ARMS mismatch gets a weaker destabilization and vice-versa.
  • the oligonucleotide sequence that provided the better performance for G12C was determined to be ACTTGTGGTAGTTGGGGCTT (SEQ ID NO 13), and for G12S ACTTGTGGTAGTTGGAGGTA (SEQ ID NO: 17) as found in Table 2.
  • the preferred primer for G12C exhibited a much smaller ⁇ Tm when compared to the primer originally identified using the Ye et al. rules, while the preferred primer for G12S also exhibited a smaller ⁇ Tm
  • Table 7 Thermodynamic calculations for G12C and G12S oligonucleotides. Oligonucleotides marked as (++) correspond to primers identified using the rules in Ye et al. Oligonucleotides marked with an ( * ) were selected for synthesis and tested in vitro. The reverse primers were also generated using PrimerPlex software. The specific beads and associated XMap bead anti-tag sequences chosen were taken from suggestions from MiraiBio (San Francisco, California) protocols.
  • PCR is performed in a 25 ⁇ l reaction containing 12.5 ⁇ l of Qiagen HotStarTaq Master Mix, 125 nM of K-ras codon 12/13 wild-type control primer, 250 nM each of the ARMS forward primers except for G12C at 62.5 nM, 1250 nM of the biotinylated reverse primer, 9.5 ⁇ l nuclease free water, and 2 ⁇ l (ie. 20ng DNA) of diluted template from above Sample Preparation. Cycling conditions were as follows: 95C for 10 minutes, 50 cycles of (95C for 30 sec, 54C for 30 sec, and 72C for 30 sec), 72C for 2 minutes and final hold at 4C.
  • Reading by Luminex Raw data is obtained by analysis on a Luminex instrument, running under the following parameters: (DD gate setting, high RP1 setting, etc, flow rate, timeout, 100 beads/target). This corresponds to the standard default instrument settings on a BioRad Bio-Plex instrument. For other versions of the Luminex platform, similar settings should be employed. Note that while the High RP1 calibration used here is a unique option on the Bio-Plex version of the instrument, the lower photomultiplier gain settings on other versions of the Luminex platform can be used equally well as long as equivalent positivity cutoff criteria (as described below) are employed.
  • the positivity cutoff value is set at the (average + 5 standard deviations) of the uncorrected Mean Fluorescence Intensity (MFI) of all bead sets in a set of negative samples.
  • MFI Mean Fluorescence Intensity
  • Samples that are double-mutants for K-ras and do not contain at least one wild-type allele are considered to be extremely rare. Accordingly, amplification of wild-type K-ras serves as an internal control when validating samples that also have a mutation. Samples that fail to show a positive Kras wild-type signal should be considered uninterpretable.
  • Example 10 ng of template DNA from a SW480 homozygous G 12V cell line was amplified according to Example 1 using native forward ARMS primers for G12A and G12R as found in Table 4, destabilized forward ARMS primers for G12C (SEQ ID NO:49), G12D, G12S (SEQ ID NO:53), G12V, G13D as found in Table 5, WT K-ras forward primer (SEQ ID NO:36) and a reverse primer (SEQ ID NO:59) at 1250 nM with 50 PCR cycles.
  • the cutoff analyzed under these conditions was similar to that in Example 2 (300 MFI).
  • Samples corresponding to heterozygous G12A, G12C, G12D, G12R, G12S, G12V, G13D and WT K-ras were each assayed according to Example 1 using the destabilized primers found in Table 5 for G12A, G12C (SEQ ID NO:48), G12D, G12R, G12S (SEQ ID NO:52), G12V and G13D, a WT K-ras control primer (SEQ ID NO:36) and a reverse primer (SEQ ID NO:59).
  • Samples were generated using 10 ng Genome Equivalents (GEQ) of control plasmid DNA for each mutation and 10 ng of human wild-type DNA. Accordingly, each sample contained the plasmid at a copy number such that contains the same number of mutant K-Ras genes as would be in 10 ng of mutant human DNA.
  • GEQ Genome Equivalents
  • FIG 8 shows the results for the detection of genotypes for codon 12 and codon 13 K-ras mutants and WT K-ras. Each sample genotype was readily detected.
  • Samples corresponding to Q61 H, Q61 L and Q61 R heterozygotes were genotyped according to the methods of Example 1 with the codon 61 destabilized primers listed in Table 5 and a reverse primer (SEQ ID NO:60).
  • Figure 9 shows the results for the detection of the Q61 H, Q61 L and Q61 R genotypes.
  • the triplex assay correctly identified the genotype.
  • the observed signals corresponding to the sample genotype were at least 10X above the positivity cutoff.
  • LOD Limit of Detection
  • G12A, G12C, G12D, G12R, G12S, G12V, G13D, and WT using the destabilized primers found in Table 5 for G12A, G12C (SEQ ID NO:48), G12D, G12R, G12S (SEQ ID NO:52), G12V and G13D, as well as WT K-ras (SEQ ID NO:36) and a reverse primer (SEQ ID NO:59) in an 8-plex reaction using plasmid controls for each mutant and 20 ng GEQ in WT human background.
  • Each sample was independently tested in triplicate at each of the three concentrations (5%, 25% and 50%).
  • Each sample contained DNA corresponding to one mutation plus the WT human background as described. Average MFI data for each mutant template per concentration point were obtained and used to generate this data.
  • the LOD values for the codon 12/13 mutations ranged between 3.5 and 19.7%.
  • These plasmid derived LOD values are not as sensitive as those observed using real cell lines, possibly due to supercoiling on plasmid templates making them less kinetically productive PCR templates or due to less 'bulk' DNA in the reaction.
  • all targets tested were detected to at least 5%.
  • Plasmid derived LOD values however reflect relative sensitivities with respect to real specimens with G12S being the least sensitive.
  • LOD Limit of Detection
  • Table 8 Detection of Q61 H, Q61 L and Q61 R mutations in a triplex reaction at different levels of mutant DNA in a WT human background.
  • Figures 11A through 11C show that the LOD values calculated from the above data for each mutation were each less than 5%.
  • Example 8 Detection of G12V and G13D in FFPE Tissue Samples FFPE tissue samples previously tested using a commercially available assay (DxS) were tested for mutations using the single-tube multiple assay for mutations in codons 12/13 as described in Example 1 with either 20 ng or 10 ng of DNA.
  • the primers used included native forward ARMS primers for G12A and G12R as found in Table 4, destabilized forward ARMS primers for G12C (SEQ ID NO:49), G12D, G12S (SEQ ID NO:53), G12V, G13D as found in Table 5, wild-type forward control primer(SEQ ID NO:36) and reverse primer (SEQ ID NO:59).
  • Table 9 11-plex amplification of samples containing 50% WT and 50% mutant DNA.

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Abstract

L'invention concerne un nouveau test de génotypage multiplex permettant la détection de mutations dans le gène K-ras. Le test peut être utilisé pour prédire la sensibilité de patients cancéreux à divers traitements tels qu'une thérapie ciblant le récepteur du facteur de croissance épidermique (EGFR). Le test peut être effectué dans une réaction de PCR en un seul tube et présente un certain nombre d'avantages y compris un faible coût, une vitesse et un rendement élevés, une complexité réduite, la facilité d'interprétation des résultats et sa disposition vis-à-vis de modèles d'échantillons représentant un défi tels qu'un tissu incorporé dans la paraffine et fixé par du formol (FFPE).
PCT/CA2009/001226 2008-10-28 2009-09-09 Test de génotypage multiplex permettant la détection de mutations dans le gène k-ras Ceased WO2010048691A1 (fr)

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US20130071844A1 (en) * 2010-03-24 2013-03-21 Toppan Printing Co., Ltd. Method for detecting target base sequence using competitive primer
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EP2607495A1 (fr) * 2011-12-23 2013-06-26 Genomica S.A.U. Procédé de détection des mutations KRAS
EP2898093B1 (fr) * 2012-09-21 2018-12-05 Genomica S.A.U. Procédé de détection des mutations de braf et pi3k

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130071844A1 (en) * 2010-03-24 2013-03-21 Toppan Printing Co., Ltd. Method for detecting target base sequence using competitive primer
US10093987B2 (en) 2011-10-18 2018-10-09 Exact Sciences Development Company, Llc Multiplexed KRAS mutation detection assay
WO2013058868A2 (fr) 2011-10-18 2013-04-25 Exact Sciences Corporation Dosage multiplex pour la détection de mutations kras
WO2013058868A3 (fr) * 2011-10-18 2014-05-15 Exact Sciences Corporation Dosage multiplex pour la détection de mutations kras
US9127318B2 (en) 2011-10-18 2015-09-08 Exact Sciences Corporation Multiplexed KRAS mutation detection assay
US9783856B2 (en) 2011-10-18 2017-10-10 Exact Sciences Corporation Multiplexed KRAS mutation detection assay
US10626465B2 (en) 2011-10-18 2020-04-21 Exact Sciences Development Company, Llc Multiplexed KRAS mutation detection assay
US11352674B2 (en) 2011-10-18 2022-06-07 Exact Sciences Development Company, Llc Multiplexed KRAS mutation detection assay
US12060620B2 (en) 2011-10-18 2024-08-13 Exact Sciences Corporation Multiplexed KRAS mutation detection assay
EP2607495A1 (fr) * 2011-12-23 2013-06-26 Genomica S.A.U. Procédé de détection des mutations KRAS
WO2013092839A1 (fr) 2011-12-23 2013-06-27 Genomica S.A.U. Procédé de détection de mutations de kras
CN102747157B (zh) * 2012-07-16 2014-01-22 武汉海吉力生物科技有限公司 用于检测人类egfr基因突变的引物、探针、试剂盒及检测方法
CN102747157A (zh) * 2012-07-16 2012-10-24 武汉海吉力生物科技有限公司 用于检测人类egfr基因突变的引物、探针、试剂盒及检测方法
EP2898093B1 (fr) * 2012-09-21 2018-12-05 Genomica S.A.U. Procédé de détection des mutations de braf et pi3k

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