WO2017201331A2 - Séquences d'oligonucléotides pour la détection de séquences cibles à faible abondance et leurs kits - Google Patents

Séquences d'oligonucléotides pour la détection de séquences cibles à faible abondance et leurs kits Download PDF

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WO2017201331A2
WO2017201331A2 PCT/US2017/033401 US2017033401W WO2017201331A2 WO 2017201331 A2 WO2017201331 A2 WO 2017201331A2 US 2017033401 W US2017033401 W US 2017033401W WO 2017201331 A2 WO2017201331 A2 WO 2017201331A2
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seq
sequence
nucleic acid
mutation
cancer
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PCT/US2017/033401
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WO2017201331A3 (fr
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Vlada MELNIKOVA
Mark Erlander
Peter CROUCHER
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Trovagene, Inc.
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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
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention generally relates to compositions and methods for detecting specific nucleotide sequences in a sample. More specifically, provided are assay methods and oligonucleotide compositions for detecting a target nucleic acid sequence in a sample containing similar wild-type sequences.
  • PCR polymerase chain reaction
  • a major limitation of PCR-based methods is their low sensitivity and preferential amplification of normal (wild type) sequences due to the greater relative abundance of the wild type sequences over mutant sequences within a sample.
  • detection of a mutant allele is not possible until it represents greater than 1-10% of the total alleles present.
  • the ability to detect genetic mutations in a background of wild type DNA sequence where the variant sequence is present at a low percentage relative to non- variant (target) sequence is beneficial and highly desired.
  • Modified PCR methods allowing selective amplification of mutant genes without requiring post-amplification sequencing assays have been described. Such methods include restriction endonuclease-mediated selective PCR (see, e.g., Ward et al., 1998, Am J Pathol 153:373-379), locked nucleic acids (see, e.g., Sun et al., 2002, Nat Biotechnol 20:186-189) COLD-PCR (Li, J., et al., 2008, Nat Med 14:579-584; US Patent Publication 2013/0149695; US Patent 8,623,603; US Patent 8,455,190; PCT Patent Publication WO2003072809).
  • the COLD- PCR technique is relatively simple to perform, but has a low amplification factor (3-100x) and a low sensitivity towards minute temperature changes (Li, J., et al., 2008, Id.).
  • Quantitative threefold allele-specific PCR (QuanTAS-OCR), an assay for detection of minimal residual disease employing quantitative PCR has been recently described, but thus far only for the mutant allele JAK2 V617F associated with myeloproliferative neoplasms (Zapparoli et al. BMC Cancer 2013, 13:206; http://www.biomedcentral.com/1471-2407/13/206).
  • Nucleic acids in cancerous tissues, circulating cells, and cell-free (cf) nucleic acids present in bodily fluids can aid in identifying and selecting individuals with cancer or other diseases associated with such genetic alterations.
  • Mutations in BRAF, KRAS and EGFR are examples of genetic alterations that confer a survival and growth advantage to cancer cells and can be used for selection of targeted cancer therapies.
  • the alterations are frequently present with a large excess of non-altered, wild type sequences making detection difficult. See, e.g., Spindler et al., 2012, Clin. Cancer Res. 18:1177-1185; Benesova et al., 2013, Anal. Biochem.
  • the instant disclosure is based in part on the development of methods for amplification of short target sequences in cell-free nucleic acids in a biological sample, e.g., blood or urine, for example low-abundance nucleic acid sequences (e.g., a target sequence) such as altered, mutant, non-wild type nucleic acid sequences or other nucleic acids not normally present in biological samples having a background of native nucleic acid sequences.
  • a biological sample e.g., blood or urine
  • cell-free nucleic acids are generally in the range of 40-400 bp, so assays that amplify a short nucleic acid sequence, for example, 200 base pairs (bp) or less, 110 bp or less (e.g., 51-110 bp, as described in US Patent Publication 2016/0002740), or 50 bp or less (e.g., 20-50 bp, as described in US Patent Publication 2010/0068711) can advantageously be utilized to achieve greater sensitivity than assays that amplify a longer sequence.
  • the methods are also applicable for amplification of less fragmented sequences, such as, for example, more than 400 bp.
  • an oligonucleotide comprising a sequence of any one of SEQ ID NOs:l-34 is provided.
  • composition comprising a set of two primers for detecting a sequence encoding a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , or 32.
  • the method comprises
  • reaction mixture subjecting the reaction mixture to one or more cycles of an amplification reaction to create amplified sample nucleic acids
  • the specific mutant nucleic acid sequence encodes a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • the method comprises obtaining a sample of a bodily fluid from the subject; and testing for the presence of the second mutation in the cancer by the above method.
  • FIG. 1 illustrates aspects of various embodiments of invention methods.
  • FIG. 2 illustrates one embodiment of use of a composition provided in the present disclosure in a method for enrichment and detection of a low-abundance target sequence (EGFR Exon 19 deletion) within a high background of wild type sequence.
  • EGFR Exon 19 deletion a low-abundance target sequence
  • FIG. 3 illustrates one embodiment of use of a composition provided in the present disclosure in a method for enrichment and detection of a low-abundance target sequence (EGFR Exon 20 T790M) within a high background of wild type sequence.
  • EGFR Exon 20 T790M low-abundance target sequence
  • FIG. 4 illustrates one embodiment of use of a composition provided in the present disclosure in a method of a low abundance target enrichment assay for KRAS Exon 2 mutations.
  • FIG. 5 is a graph showing quantitation of KRAS G12/13 mutant fragments in clinical samples and analytical DNA blend samples.
  • FIG. 6 is graphs of urinary mutant KRAS and sum of longest tumor diameters (SLD) for 8 cancer patients at various dates.
  • PD progressive disease
  • SD stable disease
  • PR partial response.
  • FIG. 7A and 7B are an illustration and graphs outlining aspects of the Enrichment PCR- NGS assays used in Example 6.
  • FIG. 7A illustrates the assay design;
  • FIG. 7B shows the mutant allele fold-enrichment estimates.
  • FIG. 8 is graphs showing T790M clinical sensitivity and DNA yield from urine of different volumes.
  • FIG. 9 is graphs showing concordance between determinations of T790M in tissue vs. two different volumes of urine.
  • FIG. 10 is an illustration of a clinical study design using a mutation enrichment PCR - next generation sequencing assay for detecting KRAS G12/13 mutants in the urine of colorectal cancer patients.
  • FIG. 11 is a graph showing percentage change from baseline (before treatment) of KRAS G12/13 mutants in urine (left bars) and tumor size (right bars) of five patients with KRAS exon 2 mutation-positive colorectal cancer treated with FOLFOX.
  • FIG. 12 is a graph showing urinary KRAS G12/13 mutants at two- week intervals in four patients treated with FOLFOX.
  • FIG. 13 is a graph showing urinary KRAS G12/13 mutants in a patient superimposed with treatment data and results by imaging.
  • FIG. 14 is graphs showing quantification of EGFR mutant and Wild-Type DNA blends by mutation enrichment NGS.
  • the analysis is of a dilution series of indicated mutant EGFR variants spiked into 60 ng ( ⁇ 18, 180 GEq) of WT DNA.
  • An analysis algorithm was applied to transform the mutant EGFR sequencing reads into the absolute mutant copies detected.
  • the box-and- whisker plots show the median (center line), 25th and 75th percentiles (box) with the connecting "whiskers” extending from the first quartile minus 1.5 of the interquartile range (IQR, the third quartile less the first quartile) and the third quartile plus 1.5 of the IQR.
  • IQR interquartile range
  • FIG. 15 is a Venn diagram showing T790M-positive status of 60 cases with available matched tumor, plasma and urine specimens. Four cases not identified as T790M-positive by either tumor, plasma or urine are not depicted in the diagram.
  • FIG. 16A and 16B is graphs showing the dynamics of T790M cfDNA signal in urine of patients treated with rociletinib, a third generation anti-EGFR Tyrosine Kinase Inhibitor drug targeting T790M mutation positive tumors.
  • FIG 16A shows T790M presence in patients with partial response or stable disease.
  • FIG. 16B shows T790M presence in patients with progressive disease.
  • compositions and methods of the present invention provide the ability to rapidly determine response to therapy in cancer patients, transplant patients and patients infected with a chronic infectious disease in a non-invasive manner and with a high level of sensitivity, specificity and ease.
  • circulating tumor DNA having EGFR activating and resistance mutations was detectable in ctDNA from patient urine months before progression on anti-EGFR TKI.
  • Kinetic Monitoring of EGFR and KRAS mutations in Urinary Circulating Tumor DNA Predicts Radiographic Progression and Response in Patients with Metastatic Lung Adenocarcinoma Collaborating Institution: University of California, San Diego School of Medicine, Hatim Husain, M.D. Poster Presentation September 27, 2015 European Cancer Congress 2015).
  • compositions and methods for detecting sequences for example low abundance mutant sequences, in cell-free DNA using polymerase chain reaction (PCR) amplification of short (e.g., less than 50 nt) target sequences.
  • PCR polymerase chain reaction
  • nucleic acid sequence amplification protocols using the primers described herein to amplify short target sequences in nucleic acids in biological samples.
  • target sequence refers to a nucleic acid that is in low- abundance or is less prevalent in a nucleic acid sample than a corresponding wild type sequence. In one embodiment, the target sequence will make up less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the total amount of wild-type sequence plus target sequence in a sample.
  • the target sequence may be an abnormal or mutant allele. In those embodiments, the target sequence is a "specific mutant nucleic acid sequence".
  • the target sequence must have, for example, at least 50% (but may be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or greater) in homology to the corresponding wild-type sequence, but must differ by at least one nucleotide from the wild-type sequence. It is understood in the art that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions wherein specific binding is desired.
  • Target sequences are amplifiable via PCR with the same pair of primers as those used for the wild-type sequence, but need not be so restricted. Target sequences may also be amplifiable via PCR with primer pairs not used for the wild-type sequence so long as the primers are selected to amplify at the region of the sequence containing the target sequence.
  • wild-type sequence refers to a nucleic acid that is more prevalent in a nucleic acid sample than a corresponding target sequence.
  • the wild-type sequence makes up over 50% of the total wild-type plus target sequences in a sample.
  • the wild-type sequence is expressed at the DNA and/or RNA level at 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x 100x, 150x, 200x or more than the target sequence.
  • a wild-type strand refers to a single nucleic acid strand of a double- stranded wild-type sequence.
  • the wild-type sequence is the wild type version of the mutant sequence.
  • the target sequence is from another individual, e.g., when detecting an allogeneic transplant-specific sequence in the recipient, or when detecting a fetus-specific sequence in a maternal subject's urine or plasma, the "wild-type sequence" is the sequence from the subject/sample donor.
  • amplicon refers to a nucleic acid that is the product of amplification.
  • an amplicon may be homologous to a wild-type sequence, a target sequence, or any sequence of nucleic acid that has been subjected to amplification.
  • concentration of an amplicon sequence will be significantly greater than the concentration of original template nucleic acid sequence.
  • homology refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two polypeptides.
  • An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.
  • wild type refers to the most common or native polynucleotide sequence or allele for a certain gene in a population. Generally, the wild type allele is from normal cells.
  • mutant refers to a nucleotide change (i.e., a single or multiple nucleotide substitution, deletion, or insertion) in a nucleic acid sequence from a wild type sequence.
  • a nucleic acid that bears a mutation has a nucleic acid sequence (mutant allele) that is different in sequence from that of the corresponding wild type polynucleotide sequence.
  • oligonucleotides of these embodiments that comprise any one of SEQ ID NOs 1-34 are useful as primers in various embodiments of the assay methods described herein.
  • a “primer sequence” includes nucleic acid sequences of about 9 - 30 bp, or about 10-25 bp, or about 11-22 bp or about 13-16 bp in length.
  • a primer sequence is a synthetically engineered nucleic acid sequence that anneals to opposite strands of a target and wild-type sequence so as to form an amplification product during a PCR reaction.
  • the target and the wild- type sequence can be about 25 bases or more, about 30 bases or more, about 35 bases or more, about 40 bases or more, or greater than about 45 bases, in order to facilitate primer attachment.
  • a primer sequence may include a common sequence tag ("CS-tag”), also called a “common sequence” (“CS").
  • CS-tag common sequence tag
  • Common sequences are part of adapters and facilitate massively parallel PCR amplification and sequencing of multiple target amplicons and samples.
  • Adapters are platform-specific engineered nucleic acid sequences that facilitate downstream analysis and quantitation by next generation high-throughput sequencing (NGS).
  • NGS next generation high-throughput sequencing
  • Adapters can also include "barcodes” or "indices” to identify individual samples, as is known in the art.
  • Adapters may be about 6-80 nucleotides, 15-30 nt, about 20-25 nt, or about 18-23 nt in length.
  • Non-limiting examples of such CS-tags include CSl ACACTGACGACATGGTTCTACA (SEQ ID NO: 1) and CS2 TACGGTAGCAGAGACTTGGTCT (SEQ ID NO: 2) (Fluidigm, South San Francisco, CA US), or any equivalent region from any adaptor compatible with an NGS platform, including any adapter provided in Illumina Adapter Sequences/Oligonucleotide sequences ( ⁇ 2016 Mumina, Inc.), as used in the Examples with a MiSeq system (Illumina, Inc., San Diego, CA, US) with platform specific "P5" and "P7" sequences; any adapter useful for an Ion TorrentTM system (Thermo Fisher Scientific, Waltham, MA, US), e.g., Adapter PI, Adapter A, and/or Adapter A with a barcode, listed, e.g., at Application Note - Amplicon Sequencing, Ion Torrent, 2011, Life Technologies Corporation and BarcodeUpload.csv at
  • primer pair refers to two primer sequences designed so as to anneal to and extend from complementary nucleic acid strands and may be up to about 10 base pairs in length or more, about 15 base pairs in length or more, about 20 base pairs in length or more, about 35 base pairs in length or more, about 40 base pairs in length or more, about 45 base pairs in length or more, or between about 10 to about 60 base pairs in length.
  • a primer can include a CS- tag that is non-homologous to the target sequence.
  • the CS-tag (“synthetic tail") is incorporated into the resulting amplicon.
  • the CS-tag aids as a bridge sequence.
  • sequencing adapters may also be incorporated in additional amplification to aid in subsequent sequencing reactions.
  • CS-tag sequences may be, if desired, one or more common sequence allowing sequencing adapters to attach or bind.
  • Such adapters may be standard reagents and have indexes ("labels") while having partial complementarity to the initial CS-tag.
  • CS tags and adapters may be the same for each assay and may have fixed length, thereby allowing their use with a variety of target sequences.
  • the primer comprises a modified nucleotide moiety, a non- nucleotide moiety and/or additional nucleotides to form a sequence with the oligonucleotide that does not occur in nature (e.g., an adapter sequence).
  • the primer comprises a fluorescent molecule, a modified nucleotide (e.g., an XNA), or a phosphoramidite spacer.
  • the PCR amplifies a sequence of less than about 50 nucleotides (nt), e.g., 20-50 nt as described in US Patent Publication US/2010/0068711, or 50-110 nt, as described in US Patent Publication 2016/0002740.
  • the PCR is performed using a blocking oligonucleotide that suppresses amplification of a wild type version of the gene, e.g., as described in US Patent 8,623,603 or PCT Patent Publication WO 2015/073163.
  • one or more primers contains an exogenous or heterologous sequence (such as an adapter or "tag" sequence), as is known in the art, such that the resulting amplified molecule has a sequence that is not naturally occurring.
  • a disclosed primer pair are two oligonucleotide primers wherein each contains a sequence at its 3'-end that is complementary to one strand of a duplex target sequence. Additionally, one or both of the oligonucleotide primers contain a heterologous sequence at its 5'-end that is not found in the target sequence.
  • the heterologous sequence may be artificial, synthetic, manmade, or from a source that is exogenous to the target sequence. The use of such a primer results converts the target sequence into a chimeric molecule that is artificial and the result of performing the disclosed synthesis of nucleic acid molecules.
  • a primer may be up to 45 bp or about 9 - 30, about 10-25, about 11-22 or about 13-16 bp in length.
  • a primer may include an adapter sequence. An adapter sequence may be about 15 - 30 bp, about 20-25 bp or about 18-23bp in length.
  • the present invention is also directed to composition
  • composition comprising a set of two primers for detecting a sequence encoding a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • at least one of the two primers comprises a modified nucleotide moiety, or a non-nucleotide moiety.
  • At least one of the two primers comprises a fluorescent molecule, a modified nucleotide, or a phosphoramidite spacer. Additionally, in some embodiments, at least one of the two primers comprises an XNA, and/or an adapter sequence.
  • both primers comprise an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • the two primers comprise
  • the low abundance target sequence, amplified using the primers described herein, can be identified and, optionally, quantitated by any means known in the art.
  • the target sequence is detected after amplification using a nucleic acid probe, as is known in the art. See, e.g., Wetmur 1991, Crit Rev Biochem Mol Biol 26: 227- 259.
  • the probe can be immobilized, for example on a microarray, a gel, or another solid substrate, where the probe binds to PCR amplicons comprising the target sequence but not amplicons comprising the wild-type sequence.
  • the probe is in solution.
  • the probe binds to amplicons having the target sequence as well as to amplicons having the wild-type sequence.
  • the amplicons having the target sequence can then be interrogated using next- generation sequencing (NGS).
  • NGS next- generation sequencing
  • the probes bind to amplicons having the target sequence but not the wild-type sequence, and the probe-target sequence complex is separated from the wild-type sequence, and the separated probe-target sequence is identified and optionally quantified. See, e.g., U.S. Patent 8,529,744.
  • probes can be of any length useful to carry out the detection of the target sequence.
  • the probe is about the same length or longer than the amplicon having the target sequence, for example 90%, 100%, 200%, 500%, 1000%, 2000%, or any length in between or longer, than the amplicon.
  • Cell-free nucleic acids can also be amplified using the primers described herein with methods and reagents that enrich for amplicons having the target sequence over amplicons having the wild-type sequence.
  • a non-limiting example of such a method is described in PCT Patent Publication WO 2015/073163 and summarized in FIGS. 1 and 7 A.
  • a blocker sequence homologous to a wild-type sequence but not to a mutant sequence, preferentially binds to the wild-type sequence, sterically preventing the annealing or extension of primers in the wild-type sequence but not the mutant sequence.
  • the sample is enriched for the mutant sequence over the wild-type sequence.
  • the denaturation step of the PCR cycle is above the calculated Tm below which the blocker anneals to the wild-type sequence.
  • the blocker binds to the wild-type strand before the primers, since the primer Tm is designed to be below the blocker-wild-type Tm.
  • primers anneal and immediately extend on both mutant strands.
  • the blocker binding to the mutant strands (which occurs at a lower temperature) is further prevented by the annealed and extended primers. The blocker thus prevents extension of the wild- type sequence but not the mutant sequence.
  • oligonucleotides comprising a sequence of any one of SEQ ID NOs: 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, useful as primers or blockers for practicing the above assays.
  • the sequences are also listed in Example 3 below. They are useful in the above assays for detecting a sequence encoding a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • oligonucleotides of these embodiments that comprise any one of SEQ ID NOs: 5, 8, 17, 20 or 33 are useful as wild-type blocking sequences in some embodiments of the assays described herein. Additional blockers can be determined without undue experimentation from an evaluation of the wild-type sequence corresponding to the mutant sequence.
  • a blocker for an assay for detecting an EGFR exon 20 mutation can be devised as an oligonucleotide comprising a sequence complementary to a portion of SEQ ID NO:34.
  • a blocker for an assay for detecting an EGFR exon 19 mutation can be devised as an oligonucleotide comprising a sequence complementary to a portion of SEQ ID NO:35.
  • a wild-type blocking sequence (also referred to herein as “blocking sequence”, “blocker”, “wild-type blocking oligonucleotide”, “blocking oligonucleotide”, or “wild- type blocker”) is an engineered single stranded or double stranded nucleic acid sequence that, in various assays described herein, is fully complementary to a section of a wild-type sequence (the "wild-type sequence") but not the corresponding mutant sequence (the "target sequence”).
  • the wild-type blocking sequence is shorter than the amplicon sequence. Also, in most embodiments, the blocker does not exceed the length of a primer sequence.
  • a wild-type blocking sequence is designed to allow a differential between the melting temperature of blocking sequence-wild-type sequence and melting temperature of blocking sequence-target mutant sequence, with the melting temperature of a primer in the reaction mixture being higher than the melting temperature of the blocking sequence-target mutant sequence.
  • the length of a wild-type blocking sequence has no maximum or upper limit as the kinetics of the method are based in part on a relationship between a primer-blocker binding temperature and a native-denatured conformation of a target nucleic acid.
  • a blocker having a high melting temperature, and present in excess quantity in a reaction mixture allows achievement of its preferential binding or annealing to a wild-type sequence.
  • Both the high primer melting temperature and long primer length ensure efficient annealing and immediate extension on the target mutant template, prior to annealing of a blocker oligonucleotide to the target mutant template.
  • a target:blocker melting temperature is lower or substantially lower due to nucleic acid sequence mismatch, a less favorable kinetics or binding rate ensues, allowing the forward or reverse primer to preferentially anneal to the target nucleic acid sequence relative to (or as compared to) the kinetics or rate of blocker binding/annealing to a target sequence.
  • the kinetics of the enrichment assay are primer- and/or blocker-centric rather than based upon denaturation temperature of wild type sequence and mutant (target) sequences.
  • a “short blocker” or “short blocking sequence” is a wild-type blocking sequence having a shorter length than prior art blocking sequences.
  • a short blocking sequence if desired, may have a length of about 30 bp or less, about 25 bp or less, about 20 bp or less, about 15 bp or less, about 14 bp or less, about 13 bp or less, about 12 bp or less, about 11 bp or less, about 10 bp or less, between about 10 bp and 80 bp in length, or any length in between.
  • a short blocker sequence may include sequences having a melting temperature that is above the melting temperature of the wild-type sequence or a WT-WT duplex nucleotide strand.
  • a short blocking sequence when duplexed with a wild-type sequence, has a blocker:wild-type melting temperature that is greater than the melting temperature of at least one primer included in a reaction mixture.
  • Short blocking sequences may be complementary to either the forward or reverse wild-type strand.
  • the short blockers have a melting temperature that is about at, or above, the melting temperature of its corresponding wild-type oligonucleotide sequence.
  • the short sequence blocking oligonucleotide may contain one or more non-natural amino acid (XNA) as desired and more fully described below.
  • XNA non-natural amino acid
  • the wild-type blocking oligonucleotide sequences may include a 3 '-end that is blocked to inhibit extension.
  • the 5' end of the same oligonucleotide may also be blocked.
  • the blocking oligonucleotide strand(s) may include a 5 '-end comprising a nucleotide that prevents 5' to 3' exonucleolysis by Taq DNA polymerases.
  • the wild-type blocking sequence may be (a) a single stranded nucleic acid wild-type blocking sequence; (b) a double-stranded nucleic acid wild-type blocking sequence which denatures to form single strand wild-type blocking sequences when the reaction mixture is heated to the first denaturing temperature; (c) a single stranded DNA, RNA, peptide nucleic acid (PNA), bridged nucleic acid (BNA), altritol nucleic acid (ANA), 1,5- anhydrohexitol nucleic acid (HNA), cyclohexane nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), or locked nucleic acid (LNA), or any other xeno nucleic acid (XNA) now known or later discovered (see, e.g., Wang et al., 2013, Theranostics 3:395-408); or (d) a chimera
  • the wild-type blocking sequence or short blocking sequence may be fully complementary with one strand of the wild-type sequence (between primer binding sites or partially overlapping the primer binding sites).
  • the wild-type blocking sequence and short blocking sequence in one embodiment, are shorter than the amplicon sequence.
  • a wild-type blocking sequence or short blocking sequence may also be designed so as to allow amplification of mutant fragments (amplicons) of any size length. Such blocking sequences are preferably designed so as to have a length sufficient to amplify short fragmented mutant nucleic acids such as those fragmented DNA sequences present in a cell-free DNA sample.
  • a wild-type blocking sequence is preferably designed so as to allow a differential between the melting temperature of blocking sequence:wild-type sequence and melting temperature of blocking sequence:target mutant sequence and melting temperature of a primer in the reaction mixture.
  • a phosphoramidite spacer may be included at one or both ends of a blocker sequence ("C3 Spacer" or "SpC3").
  • spacers allow creation of an "arm" to which another molecule may be attached, or may prevent blocker sequence from being extended by polymerase enzyme.
  • the length of a wild-type blocking sequence has no maximum or upper limit.
  • the blocker has a length smaller than the amplified section of the target sequence. In some embodiments, the blocker overlaps with the portions of the wild-type sequence where the primers bind to the wild-type sequence. In some of these embodiments, the wild-type blocking sequence is only a few bases smaller than the amplified section of the wild-type sequence, so that the primers do not bind appreciably to the wild-type sequence due to blocking of the primer binding sites by the wild-type blocking sequence.
  • a XNA e.g., a BNA, an ANA, an HNA, a CeNA, a TNA, a GNA, a PNA, or an LNA
  • another modified nucleotide is used in the blocking sequence at a position that flanks and/or includes the nucleotide in the wild-type sequence that differs from that in the target sequence.
  • Such a construction will increase the difference in the melting temperature of the wild-type blocking sequence-wild-type sequence and the wild-type blocking sequence- target sequence heteroduplexes to further favor denaturation of wild-type blocking sequence- target sequence heteroduplexes at the denaturation temperature (T sd ) and enrichment of the target sequence.
  • XNA modifications may be added to other positions with the wild-type blocking sequence as to elevate and adjust the melting temperatures of the wild-type blocking sequence with the wild-type sequence and with the target-sequence.
  • the position of the modified nucleotide(s) or XNA may be selected to match at least one position where a mutation (i.e. a difference in sequence between the target and wild-type sequences) is suspected to be present.
  • a mutation i.e. a difference in sequence between the target and wild-type sequences
  • the difference between the temperature needed to denature duplexes of the wild-type blocking sequence and the complementary wild-type strand and that required to denature heteroduplexes of the wild-type blocking sequence and the partially complementary target sequence is maximized.
  • more than one nucleotide is modified to further affect melting temperatures and enhance amplification reaction sensitivity.
  • the blocker contains DNA residues with one or more LNA nucleotides having a ribose sugar moiety that is "locked" in the 3'-endo conformation.
  • LNA blocking oligonucleotide may be used to increase the melting temperature of the oligonucleotide for both a wild-type sequence and target sequence of the disclosure.
  • the position(s) of the XNA nucleotide on the chimeric blocking oligonucleotide is selected to match position(s) where mutations are suspected or known to be present, thereby increasing the difference between the temperature needed to denature heteroduplexes of the wild-type blocking sequence and target strands (wild-type:target) and the temperature needed to denature heteroduplexes of the wild-type blocking sequence and the complementary wild-type strand (wild-type: wild-type).
  • composition comprising one blocker oligonucleotide, and two primer oligonucleotides, each comprising one of the above sequences.
  • the three oligonucleotides are useful together in an assay for detecting a sequence encoding the mutation, e.g., as described herein.
  • oligonucleotides work particularly well together in the assay methods described herein. Included are the three oligonucleotides in each of the following (a), (b), (c), (d), (e), (f), or (g) (forward primer, reverse primer and blocker, respectively):
  • kits for detection of a low-abundance mutant or target nucleotide sequence in a sample comprises any of the above primer compositions.
  • the kit optionally also comprises a blocker, e.g., one of the blockers identified above.
  • the kit can also include e.g., control reagents (e.g., positive and/or negative control target nucleic acid and positive and/or negative control wild-type nucleic acid at a standard concentration) and/or instructions for using the kit to detect and optionally quantitate one or more low-abundance target nucleic acid.
  • the kit may also include various chemical reagents or appliances, as well as a unit for detection comprising a solution and/or a substance reactable with a dye, tag, fluorescent label or other such marker; the solution containing a dye which binds to a nucleic acid.
  • the sample comprises a nucleic acid comprising a specific wild- type nucleic acid sequence that differs from the specific mutant nucleic acid sequence by at least one nucleotide.
  • the method comprises
  • reaction mixture comprises
  • oligonucleotide having a sequence of any of SEQ ID NOs:3-33; and subjecting the reaction mixture to one or more cycle of an amplification reaction, wherein the specific mutant nucleic acid sequence encodes a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • the oligonucleotide is the target specific primer.
  • the amplification reaction is polymerase chain reaction.
  • the reaction mixture further comprises a second oligonucleotide having a sequence of any of SEQ ID NOs: 3-33.
  • sample refers to any composition that may contain a target sequence.
  • the sample in a biological sample such as a biological fluid or a biological tissue from a subject.
  • biological fluids include urine, blood, plasma, serum, saliva, pancreatic juice, semen, stool, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid or the like.
  • Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
  • a “sample” also includes a sample of in vitro cell culture, natural isolates (such as drinking water, seawater, solid materials), microbial specimens or specimens that have been "spiked” with nucleic acid tracer molecules.
  • the bodily fluid is urine and the specific mutant nucleic acid sequence is transrenal DNA.
  • a "patient” or “subject” is a mammal.
  • the mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. In many cases, the mammal is a human being.
  • the disclosed methods are used with human subjects, such as those undergoing therapy or treatment for a disease or disorder associated with a gene alteration as described herein, or subjects surveyed for residual disease or recurrence.
  • Subjects may be any individual of any age, sex or race.
  • Target nucleic acid sequences of the invention can be amplified from genomic DNA.
  • Genomic DNA can be isolated from tissues or cells according to the following method or an alternative method. Such methods are well known in the art.
  • nucleic acids sequences of the invention can be isolated from blood, urine or another fluid by methods known in the art.
  • the inventors have also discovered that, when detecting a mutant gene in transrenal DNA in urine, utilizing nucleic acids from a sample volume of 90 ml or more provides greater sensitivity for detecting the mutant gene than sample volumes of less than 90 ml. See Examples 5 and 6.
  • the sensitivity of assays described herein using a 90 ml urine sample is equivalent or better than the sensitivity of assays using tissue or plasma.
  • the urine can be processed by any means known in the art.
  • the urine is unfractionated (e.g., not centrifuged to remove cells) during processing.
  • the transrenal DNA is isolated from the urine using an anion exchange medium, e.g., a quaternary ammonium anion exchange medium, for example as described in US Patent 8,222,370.
  • the transrenal DNA may be eluted from the anion exchange medium substantially free of larger DNA, using a salt having a molarity of less than 2.0M.
  • the PCR preferentially amplifies the specific mutant nucleic acid sequence over the specific wild-type nucleic acid sequence, for example by any of the methods described in WO 2015/073163, i.e., using a blocker and a PCR reaction cycle using a melting temperature above the calculated Tm of the blocker to the wild-type sequence.
  • the instant disclosure is also based in part on the discovery that for short amplicons, a significant differential in melting temperature can be obtained between wild-type blocker- wild- type sequence melting temperature ("T m ”) and wild-type blocker-target sequence T m due to a mismatch at the position with variable sequence.
  • T m wild-type blocker- wild- type sequence melting temperature
  • the instant disclosure also provides a method for enriching and detecting low-abundance nucleic acid sequences (e.g., a target sequence) utilizing short blockers of about 80 bp or less, or about 60 bp or less, or about 40 bp or less, or between about 20-50 bp, or about 12 to about 20 bp in length.
  • the disclosure provides a quantitative method for substantially enriching and detecting low-abundance nucleic acid sequence(s) present in a sample having a greater abundance of non-target sequences such as a native, wild type sequence.
  • the instant disclosure also provides a method of detecting one or more target sequences (for example, multi-target) in a sample using oligonucleotide sequences designed with consideration of the binding kinetics of wild-type blocker and primer oligonucleotides.
  • a wild-type blocker may be a short oligonucleotide sequence, allowing for a amplification cycle(s), which reduces method reaction times while substantially enriching for and detecting low-abundance nucleic acid sequences (target sequences) contained in a high background of non-target nucleic acid sequences.
  • the reaction mixture further comprises a blocking sequence that is fully complementary with a region of the wild-type sequence, the region of the wild-type sequence being within or overlapping the specific mutant nucleic acid sequence, wherein the blocking sequence is in excess relative to the wild-type sequence.
  • the reaction mixture is subjected to two or more cycles of:
  • the term “enriching a target sequence” refers to increasing the amount of a target sequence and increasing the ratio of a target sequence relative to the corresponding wild- type sequence in a sample.
  • a “selective denaturation temperature” or “Tsd” or “T sd” is a temperature determined utilizing a preselected design including parameters and calculated Tm according to one aspect of an embodiment as disclosed herein.
  • a selective denaturation temperature will be a preselected temperature that is above the melting temperature of a blocker:wild-type sequence.
  • the Tm can be estimated by a number of methods, for example by a nearest-neighbor calculation as per Wetmur, 1991, Crit Rev Biochem Mol Biol 26: 227-259, or by commercial programs including OligoTM Primer Design. Alternatively, the Tm can be determined though actual experimentation. For example, double-stranded DNA binding or intercalating dyes, such as ethidium bromide or SYBR-green (MOLECULAR PROBES) can be used in a melting curve assay to determine the actual Tm of the nucleic acid. Additional methods for determining the Tm of a nucleic acid are well known in the art.
  • compositions provided herein may be used in detection of target sequence as part of a method described, for example, in PCT Patent Publication WO2015/073163 and WO/2003/072809, U.S. Patents No. 8,623,603 and 8,455,190, and US Patent Publication 2014/0106362, describing enrichment methods for determining the amount of a target sequence in a sample containing a wild-type sequence.
  • the method may comprise performance of a disclosed enrichment method followed by an additional analysis of the reaction mixture with enriched target sequence using one or more methods selected from MALDI-TOF, HR-melting, dideoxy-sequencing, single-molecule sequencing, pyrosequencing, NGS, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative- PCR.
  • methods selected from MALDI-TOF, HR-melting, dideoxy-sequencing, single-molecule sequencing, pyrosequencing, NGS, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative- PCR.
  • These analytical techniques may be used to detect specific target (mutant) sequences within synthesized nucleic acids as described herein.
  • the sample is urine
  • the target sequence is cfDNA and/or ctDNA.
  • the described method may also be performed as a quantitative assay allowing for quantification of the detected target (mutant) sequences.
  • the quantification provides a means for determining a calculated input percentage of the target sequence prior to enrichment based upon the output signal (optionally as a percentage) from the assessment. This may be performed by reference to a fitted curve.
  • the actual output from an assessment of a test sample is determined in combination with one or more control reactions containing a known quantity of target sequence DNA.
  • the outputs from the test sample and the control(s) are compared to a fitted curve to interpolate or extrapolate a calculated input for the test sample. This permits a quantitative determination of the amount of a target sequence in a sample pre- enrichment based upon a post-enrichment detection.
  • the detection limits for the presence of a gene alteration (mutation) in cf nucleic acids may be determined by assessing data from one or more negative controls (e.g. from healthy control subjects or verified cell lines) and a plurality of patient samples.
  • the limits may be determined based in part on minimizing the percentage of false negatives as being more important than minimizing false positives.
  • One set of non-limiting thresholds for BRAF V600E is defined as less than about 0.05% of the mutation in a sample of cf nucleic acids for a determination of no mutant present or wild-type only; the range of about 0.05% to about 0.107% as "borderline", and greater than about 0.107% as detected mutation.
  • a no- detection designation threshold for the mutation is set at less than about 0.001%, less than about 0.005%, less than about 0.01%, less than about 0.05%, less than about 0.1%, less than about 0.15%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% detection of the mutation relative to a corresponding wild type sequence.
  • these methods are capable of detecting one copy of the target sequence in a urine, plasma or tissue sample.
  • the sample is from a subject having a cancer associated with a mutant in BRAF, KRAS, EGFR, NRAS, PIK2CA or Alk.
  • the specific mutant nucleic acid sequence encodes a mutation at KRAS G12 or KRAS G13, and the subject has colorectal cancer or pancreatic cancer.
  • the specific mutant nucleic acid sequence encodes a mutation at EGFR L858, EGFR T790, EGFR19del, EGFR G719 or EGFR L861, and the subject has non-small cell lung cancer.
  • nucleic acids from a sample volume of 90 ml or more provides greater sensitivity for detecting the mutant gene than sample volumes of less than 90 ml.
  • a method of detecting a specific mutant nucleic acid sequence in a human urine sample comprises a specific wild-type nucleic acid sequence that differs from the specific mutant nucleic acid sequence by at least one nucleotide.
  • the method comprises
  • reaction mixture comprises the cell-free nucleic acids from the sample and primers that are suitable for amplifying the specific mutant nucleic acid sequence, to produce an amplification reaction product comprising an amplified specific mutant nucleic acid sequence if the specific mutant nucleic acid sequence was present in the sample;
  • the method is capable of detecting one copy of the mutant sequence in the sample.
  • the amplification reaction is a polymerase chain reaction (PCR) that preferentially amplifies the specific mutant nucleic acid sequence over the specific wild-type nucleic acid sequence.
  • the reaction mixture further comprises a blocking sequence that is fully complementary with a region of the wild-type sequence, the region of the wild-type sequence being within or overlapping the specific mutant nucleic acid sequence, wherein the blocking sequence is in excess relative to the wild-type sequence.
  • reaction mixture is subjected to two or more cycles of:
  • step (ii) the blocker anneals to the wild-type sequence at a higher temperature than the primers anneal to the mutant sequence.
  • the sequence of at least one of the primers at least partially overlaps with the blocking sequence.
  • the specific mutant nucleic acid sequence is associated with a cancer
  • the specific wild-type nucleic acid sequence is a wild-type version of the specific mutant nucleic acid sequence.
  • These methods can provide information to accurately predict patient prognosis and response to therapy of a variety of cancer types, including but not limited to, adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain or a nervous system cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal cancer, Hodgkin Disease, intestinal cancer, Kaposi Sarcoma, kidney cancer, large intestine cancer, laryngeal cancer, hypopharyngeal cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), non-HCL lymphoid
  • Non-limiting examples of non-HCL lymphoid malignancy include, but are not limited to, hairy cell variant (HCL-v), splenic marginal zone lymphoma (SMZL), splenic diffuse red pulp small B-cell lymphoma (SDRPSBCL), splenic leukemia/lymphoma unclassifiable (SLLU), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, low grade lymphoma, systemic mastocytosis, and splenic lymphoma/leukemia unclassifiable (SLLU).
  • HCL hairy cell variant
  • SDRPSBCL splenic diffuse red pulp small B-cell lymphoma
  • SLLU splenic leukemia/lymphoma unclassifiable
  • CLL chronic lymphocytic leukemia
  • prolymphocytic leukemia low grade lymphoma
  • systemic mastocytosis systemic mastocytosis
  • the specific mutant nucleic acid sequence encodes a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • the mutant nucleic acid sequence is a KRAS mutation, e.g., a KRAS mutation is at a codon encoding amino acid position 12 or 13.
  • the cancer is metastatic colorectal cancer or pancreatic cancer.
  • the mutant nucleic acid sequence is an EGFR mutation, e.g., a T790M, L858R, Exon 19 or Exon 20 mutation.
  • the cancer is non-small cell lung cancer.
  • the reaction mixture comprises at least one oligonucleotide comprising a sequence of any one of SEQ ID NOs: 3-33 and/or one or two oligonucleotide primers comprising any one of SEQ ID NOs: 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • the reaction mixture comprises three oligonucleotides from SEQ ID NO: 3-33, for example the three oligonucleotides in (a), (b), (c), (d), (e), (f), or (g): (a) for KRAS G12 or G13 mutations: SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5;
  • the quantity of a mutation before and after treatment has been correlated with response to treatment. See, e.g., Examples 5 and 9, in particular FIGS 6 and 16.
  • the quantity of the specific mutant nucleic acid in a post-treatment sample is less than 50%, 40%, 30%, 25%, 20%, 15% or 10%, depending on the disease and mutation, of the quantity of the specific mutant nucleic acid pre-treatment, then the patient has stable disease, partial response or complete response.
  • the quantity of the specific mutant nucleic acid in the post-treatment sample is greater than 50%, 40%, 30%, 25%, 20%, 15% or 10% of the quantity of the specific mutant nucleic acid in the pre-treatment sample, then the patient has progressive disease.
  • the threshold for discriminating response from non-response for any cancer can be determined without undue experimentation, e.g., by the criteria of Eisenhauer et al., 2009, Eur. J. Cancer 45:228-247.
  • the quantity of the mutant will generally decrease at the initiation of treatment whether the subject's cancer is responding or not. The decrease is often preceded by a significant increase in the mutant.
  • a method for determining, prior to imaging, whether a subject is responding to a cancer treatment.
  • the method comprises
  • the patient has stable disease, partial response or complete response, and
  • the patient has progressive disease.
  • the method further comprises continuing the cancer treatment if the subject is responding to the treatment, or discontinuing the cancer treatment and starting a different treatment if the subject is not responding to the treatment.
  • the method comprises
  • this method further comprises changing treatment if the cancer is progressing.
  • This method can also be used to detect secondary mutations that arise after treatment for a cancer associated with a first mutation. See Examples 6 and 9, where the secondary mutation, EGFR T790M, was detected in NSCLC patients after treatment for a cancer with a different primary EGFR mutation.
  • the cancer has a first mutation.
  • the method comprises
  • the cancer is non-small cell lung cancer.
  • the first mutation is of EGFR, e.g., EGFR L858 or EGFR 19del
  • the secondary mutation is EGFR T790M.
  • the subject is treated with a treatment specific for the secondary mutation. For example, where the mutation is EGFR T790M, the subject may be treated with osimertinib.
  • a method of treating a cancer patient that has a cancer comprises detecting and quantifying the specific mutant nucleic acid associated with the cancer in a first sample of bodily fluid from the patient by any of the above methods, and treating the cancer as appropriate based on the results of the detecting method.
  • the subject is undergoing a cancer treatment and the method further comprises
  • the first sample is taken from the patient before the treatment and the second sample is taken from the patient after the treatment has started.
  • both the first sample and the second sample are taken from the patient after the treatment has started.
  • these methods further comprise
  • the cancer is non-small cell lung cancer and the secondary mutation is EGFR T790M.
  • the sample comprises a nucleic acid comprising a specific wild-type nucleic acid sequence that differs from the specific mutant nucleic acid sequence by at least one nucleotide.
  • the method comprises
  • reaction mixture subjecting the reaction mixture to one or more cycle of amplification reaction to create amplified sample nucleic acids
  • the specific mutant nucleic acid sequence encodes a mutation at BRAF V600; EGFR Exon 19, Exon 20, T790, or L858; or KRAS G12 or G13.
  • the sample comprises cell-free DNA.
  • the specific mutant nucleic acid sequence in the sample is cell-free DNA.
  • the bodily fluid is blood, plasma, serum or urine.
  • the amplification reaction is polymerase chain reaction (PCR).
  • the specific mutant nucleic acid sequence in the sample is quantified.
  • the cancer is metastatic colorectal cancer, non-small cell lung cancer, or pancreatic cancer.
  • the mutant nucleic acid sequence is an EGFR mutation, e.g., a T790M, L858R, Exon 20 or Exon 19 mutation. In other aspects, the mutant nucleic acid sequence is a KRAS exon 2 mutation.
  • Also provided herewith is a method of determining, prior to imaging, whether a subject is responding to a cancer treatment.
  • the method comprises
  • detecting and quantifying a specific mutant nucleic acid associated with the cancer in the bodily fluid by the method described above that utilizes a set of two primers where at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • the patient has stable disease, partial response or complete response, and if the quantity of the specific mutant nucleic acid in the second sample is greater than 50%, 40%, 30%, 25%, 20%, 15% or 10% of the quantity of the specific mutant nucleic acid in the first sample, then the patient has progressive disease.
  • this method further comprises continuing the cancer treatment if the subject is responding to the treatment, or discontinuing the cancer treatment and starting a different treatment if the subject is not responding to the treatment.
  • the first sample and the second sample are blood, plasma, serum or urine samples.
  • the method comprises
  • detecting and quantifying a specific mutant nucleic acid associated with the cancer in the bodily fluid by the method described above that utilizes a set of two primers where at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • this method further comprises changing treatment if the cancer is progressing.
  • This method comprises obtaining a sample of a bodily fluid from the subject; and
  • oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • the bodily fluid is blood, plasma, serum or urine.
  • the cancer is non-small cell lung cancer.
  • the first mutation is of EGFR
  • the secondary mutation is EGFR T790M. In some of those embodiments, the first mutation is EGFR L858 or EGFR 19del. In various embodiments where the secondary mutation is EGFR T790M, the method further comprises treating the patient with osimertinib if EGFR T790M is present.
  • the method comprises detecting and quantifying a specific mutant nucleic acid associated with the cancer in a first sample of bodily fluid from the patient by the method described above that utilizes a set of two primers where at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • This method further comprises treating the cancer as appropriate based on the results of the detecting method.
  • the sample is blood, plasma, serum or urine.
  • the subject is undergoing a cancer treatment and the method further comprises
  • detecting and quantifying the specific mutant nucleic acid in the second sample by the method described above that utilizes a set of two primers where at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32; and
  • the first sample is taken from the patient before the treatment and the second sample is taken from the patient after the treatment has started. In other embodiments, both the first sample and the second sample are taken from the patient after the treatment has started.
  • this method further comprises testing for the presence of the secondary mutation in the cancer bythe method described above that utilizes a set of two primers where at least one of the two primers comprises an oligonucleotide comprising any one of SEQ ID NOs 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.
  • the cancer is non-small cell lung cancer.
  • the first mutation is of EGFR, e.g., EGFR L858 or EGFR 19del, and the secondary mutation is EGFR T790M.
  • the patient is treated with osimertinib if EGFR T790M is present.
  • transplant rejection or other diseases
  • diseases such as chronic viral (e.g., HIV, HCV, herpes), bacterial (e.g., tuberculosis) or other pathogen infections (e.g., parasitic infections such as by Enterobius vermicularis, Giardia lamblia, Ancylostoma duodenale, Necator americanus, and Entamoeba histolytica.
  • pathogen infections e.g., parasitic infections such as by Enterobius vermicularis, Giardia lamblia, Ancylostoma duodenale, Necator americanus, and Entamoeba histolytica.
  • pathogen infections e.g., parasitic infections such as by Enterobius vermicularis, Giardia lamblia, Ancylostoma duodenale, Necator americanus, and Entamoeba histolytica.
  • parasitic infections such as by Enterobius vermicularis, Giardia
  • tuberculosis or parasitic nucleic acids
  • transplant e.g., nucleic acids characteristic of the transplated tissue
  • FIG. 2 An example of a two-step PCR enrichment assay (EGFR Exon 19 deletions) is provided in FIG. 2.
  • a selective denaturation step precedes the annealing step.
  • the reaction ramps to the annealing temperature of 62.4°C, any complementary wild type strands generated in the previous PCR cycle bind blocker before the primers anneal.
  • blockentarget and wild-type (wt):target heteroduplex formation is likely to be extremely low (below detectable or calculable levels) because only a minor percentage will be complementary.
  • Table 1 Table 1.
  • Table 2 shows the fold enrichment of the mutation sequence when specific numbers of mutant copies are added with various amounts of background DNA. Under all conditions tested, the mutant sequence was enriched more than 1500 fold.
  • Table 3 shows the fold enrichment of the mutant sequence with greater amounts of mutant input than shown in Table 2.
  • Table 4 shows the fold enrichment of a pooled mutant sequences.
  • FIG. 3 A schematic example of an alternative enrichment assay is provided in FIG. 3.
  • a 98°C denaturation step ensures that all duplexes denature.
  • blocker-wild type duplexes form, but few blocker-mutant duplexes form (as 70°C is above the blocker-mutant Tm).
  • selective denaturation many of the blocker-wild type will denature (along with any blocker-mutant duplexes that may exist).
  • the reaction ramps back to the annealing temperature of 64.0°C
  • complementary wild type strands are bound by the excess blocker before the primers anneal.
  • the primers then anneal to the complementary mutant strand and bar the possibility of blocker binding to that mutant strand. Short amplicon length allows extension without the need for an additional elongation step.
  • the level of enrichment for EGFR T790M is provided in Table 5.
  • FIG. 4 provides an example of a low abundance target enrichment assay for KRAS Exon 2 single-base substitution.
  • This assay proceeds in a similar fashion as Example 2 except that the selective denaturation step is chosen below the wild type-blocker T m .
  • the temperature differential between the blocker-wild type T m , the primer-template T m and the blocker-mutant T m is much greater than in Example 2, leading to a more efficient enrichment.
  • Oligonucleotide sequences useful for detection of low abundance target nucleotide sequences are provided in Table 6.
  • Example 5 PCR mutation enrichment-next-generation sequencing method for absolute quantitation of circulating tumor DNA fragments in urine or plasma
  • KRAS G12/13 mutants were detected and quantified in clinical urine samples of cancer patients undergoing treatment.
  • the accuracy of the quantification procedure and the copy number determination of various clinical samples is shown in FIG. 5.
  • the sensitivity and specificity of the assay was determined (Table 7). The sensitivity determinations were divided into two groups, where the volume of urine analyzed was 40-110 ml in one group, and 90-110 ml in the second group. As shown in Table 7, the sensitivity was much higher in the 90-110 ml group than in the 40-110 ml group, when multiple cancers were evaluated together, as well as when metastatic colorectal cancer (mCRC) patients were evaluated separately.
  • mCRC metastatic colorectal cancer
  • Table 7 Summary of clinical sensitivity of KRAS G12/13 detection in urine and plasma.
  • FIG. 6 shows results of evaluations of disease course for eight patients, where urinary KRAS mutants are quantified and compared to the sum of longest diameters (SLD) of tumors present.
  • Example 6 Assessment of EGFR mutations in matched urine, plasma and tumor tissue in non- small cell lung cancer (NSCLC) patients treated with rociletinib (CO- 1686)
  • Rociletinib (CO- 1686) is a novel, oral, selective covalent inhibitor of EGFR mutations in NSCLC. Rociletinib inhibits key activating mutations along with the T790M mutation (Walter et al. Cancer Discov. 2013;3:1404-1415; Sequist et al. N Engl J Med. 2015;372:1700-1709).
  • Pretreatment urine or plasma was obtained from 68 patients with available tumor biopsy result in TIGER-X. Matched urine or plasma was available for 51 of these patients. Urine was available for 63 of these patients. Patients enrolled in TIGER-X were required to have documented evidence of an EGFR-activating mutation in their medical record.
  • Tissue Therascreen® EGFR RGQ polymerase chain reaction (PCR) test.
  • Urine and plasma Trovagene quantitative PCR next-generation sequencing (NGS) EGFR T790M assay, using MiSeq NGS sequencing.
  • NGS Trovagene quantitative PCR next-generation sequencing
  • FIG. 7B Gene-specific primers (GSP1, GSP2) with noncomplementary "common sequence tails"
  • CSl CS2
  • WT Wild-type
  • ASCC Allele-specific cycling conditions
  • WT amplification WT amplification.
  • Table 8 shows various characteristics of the EGFR assay with urine and plasma.
  • Urine testing for T790M has high sensitivity with prespecified urine volume acceptance criteria and identifies some patients missed by tissue testing Recommended urine volumes for testing are 90-1 OOmL (approximately half of normal void). Nineteen of 63 patients provided the recommended volume of 90-100 mL; 14 of these patients had T790M-positive tissue.
  • Table 9 shows the sensitivity of this assay analyzing the EGFR mutations Exon 19 del (Exl9del), L858R, and T790M.
  • Table 9 Analytical sensitivity of EGFR PCR-NGS mutation enrichment assays. Verification of a single copy detection sensitivity using Poisson distribution statistics.
  • Clinical sensitivity and DNA yields increase with urine volume (FIG. 8). Clinical sensitivity of the assays by mutations using two different volumes of urine and plasma are provided in Table 10. Clinical sensitivity was increased in the group with a urine sample size of 90-100 ml over the 40-100 ml urine sample size group. Table 10. Summary of clinical sensitivity of EGFR detection in urine and plasma.
  • Table 11 shows the urine/plasma/tissue concordance for the three EGFR mutations evaluated.
  • FIG. 9 provides a graphical illustration of the concordance between urine and tissue for T790M where the urine volume is 90-100 ml or 10-100 ml.
  • the urine volume is 90-100 ml or 10-100 ml.
  • 13/14 urine samples identified a corresponding positive tissue sample (93% positive percent agreement (PPA)).
  • the corresponding PPA for 10-100 ml urine volume was 72% (34/47).
  • FFPE formalin-fixed, paraffin-embedded
  • Table 12 shows T790M copies per sample and % T790M fragments in urine and plasma of patients with NSCLC. 100 ml of urine had more copies of T790M than 2 ml of plasma. Table 12.
  • Rates of T790M detection by M stage are similar in 90-100 ml urine samples as in plasma
  • the number of T790M fragments in urine was higher for Mlb patients than for M0/Mla patients.
  • Example 7 Use of urinary circulating tumor mutant KRAS DNA for monitoring treatment response in patients with metastatic colorectal cancer
  • Colorectal cancer is the third leading cause of cancer mortality in the United States. Despite advances in early detection, each year more than 50,000 patients are diagnosed with metastatic disease. Combination chemotherapy, targeted drugs, and surgical interventions have revolutionized the treatment landscape and improved survival of these patients. Clonal evolution is considered a major cause of drug resistance and non-invasive strategies to detect new and evolving mutations can impact the delivery of personalized treatment. Moreover, non-invasive techniques have the potential to transform the standard of response assessment in metastatic colorectal cancer (mCRC) and reduce the need for imaging in the management of CRC.
  • mCRC metastatic colorectal cancer
  • Urinary circulating tumor DNA (ctDNA) was extracted using quaternary ammonium anion exchange with elution of DNA fragments primarily ⁇ 400 bp using 1.8M NaCl, as described in US Patent 9,163,229.
  • the primers have 5' barcode adapters for compatibility with NGS (MiSeq).
  • the assay design is the same as in Example 6, using the above KRAS primers.
  • the clinical study design is illustrated in FIG. 10.
  • the study was an interim analysis of 13 metastatic CRC patients with known KRAS tissue status.
  • Urine was collected every two weeks on treatment and with each radiologic scan (at 6-8 weeks).
  • Table 14 summarizes the assay performance. Table 14.
  • the KRAS detection concordance between tissue and ctDNA was as follows:
  • Urine Concordant KRAS G12/13 mutation was detected in 5 of 7 patients. In one additional patient (patient with lung metastases), a different KRAS mutation was detected in urine after surgical resection of the primary tumor.
  • Plasma Concordant KRAS G12/13 mutation was detected in 6 of 7 patients. One of 7 tests failed.
  • the five patients treated with FOLFOX underwent CT scans and urine sampling at Cycle 4 Day 1. Partial response was observed in three of the patients and stable disease was observed in two patients (RECIST 1.1 criteria).
  • the KRAS mutation burden in urine decreased by more than 90% in response to FOLFOX treatment (FIG. 11).
  • the assay detected a decrease in urinary KRAS mutation burden at two weeks (the first time point sampled) after commencement of treatment (FIG. 12).
  • Clinical response could be predicted three months in advance of imaging, as shown in FIG. 13. With that patient, the response by imaging was preceded by a rapid decrease of urinary KRAS mutants of more than 99%. A relapse to progressive disease was also preceded by a large increase in urinary KRAS mutants, six months prior to imaging. That increase led to the decision to administer additional treatment more than a month prior to imaging.
  • the KRAS G12/13 assay described in Example 7 was used in a clinical study to examine KRAS G12/13 detection rate in plasma of patients with unresectable, locally advanced or metastatic pancreatic cancer (PC). Also evaluated was the association between baseline KRAS levels in plasma and patient outcomes (overall survival), and the correlation between changes in KRAS levels in plasma and changes in tumor size by radiographic assessment following treatment with chemotherapy.
  • the patients were 84 females (48.1%) and 92 males (51.9%); median age 67 years (range 45-89 years). Fifty four (20.4%) patients had locally advanced PC, 172 (79.6%) patients had metastatic PC. The patients were on palliative treatment with gemcitabine or FOLFIRINOX.
  • Example 6 Samples from 63 patients were studied.
  • DNA derived from NSCLC tumors can be detected with high sensitivity in urine, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive "liquid biopsy” samples.
  • ctDNA circulating tumor DNA
  • NSCLC non-small cell lung cancer
  • EGFR epidermal growth factor receptor
  • WT wild-type
  • NGS next-generation sequencing
  • CV% coefficient of variation percent
  • GEq genome equivalents
  • PCR polymerase chain reaction
  • EBV Epstein-Bar Virus
  • H&E Hematoxylin and Eosin
  • FFPE formalin-fixed paraffin- embedded
  • RECIST response evaluation criteria in solid tumors
  • PR partial response
  • SD stable disease
  • PD progressive disease.
  • NSCLC non-small cell lung cancer
  • NSCLC patients receiving first-line tyrosine kinase inhibitors (TKIs) targeting EGFR mutation-positive tumors i.e. erlotinib, gefitinib, afatinib
  • TKIs first-line tyrosine kinase inhibitors
  • EGFR mutation-positive tumors i.e. erlotinib, gefitinib, afatinib
  • T790M second mutation in EGFR
  • Rebiopsy of these patients is still an emerging standard of care, and up to 25% of cases may be medically ineligible due to comorbidities or the lack of an accessible lesion.
  • Those who do undergo rebiopsy may be at substantial risk of a false negative result due to the underlying intra- and inter-tumoral heterogeneity often associated with resistance mechanisms such as T790M.
  • ctDNA circulating tumor DNA
  • Example 6 Using the methods of Example 6, the clinical performance of this platform was evaluated in matched pretreatment urine and plasma was evaluated, and the feasibility of longitudinal monitoring of EGFR mutations from the urine of NSCLC patients was examined with data from TIGER-X, a phase 1/2 study of the third generation EGFR-TKI rociletinib (CO-1686).
  • TIGER-X A blinded, retrospective study was conducted on matched urine and plasma specimens collected from 63 Stage IIIB-iV patients enrolled in the TIGER-X trial (NCT01526928). Patients in TIGER-X were required to have histologically or cytologically confirmed NSCLC and documented evidence of >1 EGFR mutations. All patients signed an EC/IRB-approved consent prior to any procedures. Further details regarding TIGER-X study
  • Tissue biopsies were collected within 60 days of initiation of treatment with rociletinib.
  • tumor content was assessed by board-certified pathologists using Hematoxylin and Eosin (H&E) stained slides. Tumor specimens were considered evaluable if any tumor cells were identified present.
  • DNA was extracted from one 5 ⁇ section and central laboratory tissue testing was performed with the Cobas® EGFR Mutation Test (Roche Molecular Systems, CA).
  • DNA was extracted from two 5 ⁇ m sections and central laboratory tissue testing was performed with the Therascreen ® EGFR RGQ Polymerase Chain Reaction (PCR) Kit (Qiagen, CA).
  • PCR Therascreen ® EGFR RGQ Polymerase Chain Reaction
  • Blood and urine samples were obtained serially, prior to administration of the first dose and with every 21 -day cycle of treatment with rociletinib.
  • Blood samples were collected in K2 EDTA BD Vacutainer tubes, processed into plasma within 30 minutes (1800 xg for 10 min at 18- 23 °C), and stored at or below -70°C.
  • Plasma DNA analysis 1.5-4 mL of plasma was extracted using the QIAamp DNA Circulating Nucleic Acid Kit (Manchester, U.K.) according to manufacturer's instructions.
  • Urine samples between 10-100 mL were collected into 120-mL cups, supplemented with preservative, and stored at or below -70°C.
  • urine was concentrated to 4 mL using Vivacell 100 concentrators (Sartorius Corp, Bohemia, NY) and incubated with 700 ⁇ L of Q-sepharose Fast Flow quaternary ammonium resin (GE Healthcare, Pittsburg, PA). Tubes were spun to collect sepharose and bound DNA. The pellet was resuspended in a buffer containing guanidinium hydrochloride and isopropanol, and the eluted DNA was collected as a flow-through using polypropylene chromatography columns (BioRad Laboratories, Irvine, CA). The DNA was further purified using QiaQuick columns (Qiagen, Germany). Plasma and urine DNA was quantitated using a droplet digital PCR (ddPCR) assay that amplifies a single copy RNaseP reference gene (QX200 ddPCR system, Bio-Rad, CA) as described previously.
  • ddPCR droplet digital PCR
  • Urine and Plasma EGFR Mutation Analysis Quantitative analysis of the T790M resistance mutation and EGFR activating mutations (L858R and 69 deletion variants in exon 19) was performed using a mutation enrichment PCR coupled with next-generation sequencing detection (MiSeq, Mumina Inc., San Diego, CA). Selective amplification of mutant fragments was accomplished via short amplicon (42-44bp) kinetically driven PCR that amplifies the mutant fragments while suppressing the amplification of the wild-type (WT) sequence using a blocker oligonucleotide. PCR primers contained a 3' gene-specific sequence and a 5' common sequence that was used in the subsequent sample-barcoding step. The PCR enrichment cycling conditions utilized the initial 98°C denaturation step followed by the assay specific 5-15 cycles of pre- amplification PCR and 17-32 cycles of mutation enrichment PCR.
  • Custom DNA sequencing libraries were constructed and indexed using the Access Array
  • the analysis output files (FASTQ) from the runs were processed using custom sequencing reads counting and variant calling algorithms to tally the sums of total target gene reads, wild-type or mutant EGFR reads, which passed predetermined sequence quality criteria (qscore > 20).
  • a custom quantification algorithm was developed to accurately determine the absolute number of mutant DNA molecules in the source ctDNA sample.
  • each single multiplexed MiSeq NGS run contained a set of standard curve samples in addition to clinical samples and controls. For each run the standard sample set was assayed in parallel with patient samples starting with PCR enrichment of mutant EGFR DNA followed by NGS.
  • GEq WT DNA genome equivalents
  • ultra-short footprint PCR assays were developed to increase the likelihood of amplifying highly degraded ctDNA.
  • Three ultra-short footprint assays were developed to detect the most common EGFR mutations: (a) a 42bp EGFR exon 19 deletion assay recognizing 69 annotated deletions, (b) a 46bp EGFR exon 21 L858R assay, and (c) a 44bp EGFR exon 20 T790M assay.
  • mutant ctDNA fragments were enriched by a PCR-based method to maximize sensitivity for detecting ctDNA mutations having rare prevalence (i.e., ⁇ 0.01%).
  • Preferential PCR enrichment of mutant EGFR ctDNA was accomplished by using WT EGFR oligonucleotides that block the ability of PCR primers to anneal and amplify WT EGFR DNA thereby increasing the likelihood of amplifying mutant EGFR templates (Patients and Methods).
  • absolute ctDNA mutation copy numbers from a patient sample were quantitated by next generation sequencing (NGS) methodology. This was achieved with the aid of a standard sample set spiked with known copies of mutant EGFR molecules.
  • NGS next generation sequencing
  • Enrichment performance of EGFR mutant DNA was assessed by spiking 5 to 500 copies of mutant DNA into 18,181 GEq of WT DNA (0.028% to 2.7%). Fold-enrichment of EGFR mutant fragments increased as the proportion of mutant versus WT fragments decreased from 2.7% to 0.028% (Table 15 and FIG. 7B).
  • the resulting sequencing libraries were comprised of 24% to 99.9% mutant reads thus enabling sensitive mutation detection by NGS (Table 15).
  • 857 to 3,214 fold enrichment of EGFR mutation signal was obtained for an input of 5 copies of mutant EGFR DNA within 60 ng (18,181 GEq) of WT DNA (FIG. 14). Mutant reads in a test sample were converted to mutant copy number in the original sample by interpolation to the standard curve (Patients and Methods).
  • the lower limit of detection (LLoD) for the EGFR mutation assays was determined by using a statistical model based on the Poisson distribution of rare mutant DNA molecules within a series of highly diluted EGFR mutant DNA samples.
  • the frequency distribution of the number of DNA molecules that will be present for measurement in each PCR tube can be predicted by the Poisson distribution. If, for example, a PCR reaction is expected to contain a single molecule of target DNA, Poisson distribution predicts probabilities of 36.8, 36.8, 18.4, and 6.1% for 0, 1, 2, and 3 molecules, respectively, to be actually present in the PCR tube.
  • the LLoD was defined as the lowest number of copies for which the frequency distribution of the copy number events upon repeated measurements fell within the 95% confidence interval of expected frequency distribution determined by Poisson statistics.
  • LLoD finding and verification 80 repeated measurements were performed on a single multiplexed NGS run for each spike-in level of 1, 2 or 3 mutant EGFR copies within 18,181 genome equivalents (60 ng) of EGFR WT DNA, and the observed frequency distribution for mutant copy events was compared to the expected frequency.
  • stock DNA solutions of 100 mutant copies per ⁇ L were prepared using cell line DNA quantified using ddPCR (RainDance, Billerica, MA), and then diluted serially to a target copy level in 18,181 GEq of EGFR WT DNA.
  • the number of mutant EGFR copies in each measured dilution sample was determined by interpolating the number of NGS reads to a standard curve, with reference standards at 5, 10, 50, 100 and 250 copy level prepared from a different stock solution.
  • Lower Limit of Blank (LLoB) was calculated for each EGFR assay using samples containing EGFR WT DNA.
  • the analytical accuracy and reproducibility of the EGFR mutation assays were determined by a dilution series of six replicates of 0, 5, 10, 50, 100 and 250 copies in a background of 18,181 WT DNA copies, spanning the linear range of the assays.
  • the entire workflow was replicated six times with three dilution series replicates prepared by two different operators on three different days for a total of 18 measurements at each copy level; NGS analysis was on two different Illumina MiSeq instruments.
  • the Spearman correlation between spiked-in absolute copy numbers (quantified by ddPCR) versus detected copy numbers (quantified by mutation enrichment NGS) ranged from 0.967 to 0.981 for the EGFR mutation assays.
  • Table 19 shows the inter-run reproducibility of the EGFR exon 19 deletions, L858R and T790M mutation enrichment NGS assays for a 250 copies to 5 copies dilution series.
  • CV% denotes Coefficient of Variation Percent (CV%).
  • the mean coefficient of variation percentage was 34.5% across the reportable range of 5 to 250 for all three EGFR mutation assays yielding an adjusted quantifiable range of 27.5 to 1,375 copies per 100,000 GEq), with the highest CV% of 47.4-60.7% observed at the lowest input of 5 copies likely due to Poisson limitations (Table 19).
  • Assay fold-discrimination performance was examined by comparing the expected copy ratio between two consecutive dilutions to the observed copy ratio.
  • Table 20 shows the quantification of 2-fold differences between subsequent mutant copy input levels.
  • the expected fold ratio was calculated as the ratio of the two input mutant copy levels.
  • the observed fold ratio was calculated as the ratio of two means for two measured mutant copy levels with a 95% confidence interval.
  • Baseline tumor tissue biopsies and urine samples were obtained from 63 Stage IIIB/iV NSCLC patients enrolled in TIGER-X, a phase 1/2 trial of rociletinib in patients treated with at least one prior EGFR inhibitor and who have an EGFR activating mutation in their medical record. Tumor tissue was processed by a central laboratory for EGFR mutation testing. Of the 63 tumor tissue biopsies, 60 samples were adequate for analysis with 47 positive for T790M, 167 positive for L858R and 42 positive for exon 19 deletion mutations.
  • Urine volumes ranged from 10-100 mL with 19 of 63 samples meeting the pre-specified criteria for the recommended urine volume of 90-100 mL.
  • Table 21 shows contingency tables for the analysis of EGFR T790M mutation in matched tumor, urine and plasma samples from patients enrolled in TIGER-X clinical trial.
  • A Urine versus tumor analysis of T790M in 63 matched tumor and urine specimens.
  • B Plasma versus tumor analysis of T790M in 60 matched tumor and plasma specimens.
  • C Urine versus plasma analysis of T790M in 60 matched urine and plasma specimens. Table 21.
  • the sensitivity of the urine assays was 93% (13/14) for T790M, 80% (4/5) for L858R, and 83% (10/12) for exon 19 deletions for 90-100 mL sample volumes (Table 21).
  • sensitivity of EGFR mutation detection was 72% (34/47) for T790M, 7% (1/1) for L858R, and 67% (28/42) for exon 19 deletion mutations (Table 21).
  • the specificity of the EGFR urine assays was determined using urine samples obtained from healthy donors and patients with non-NSCLC metastatic cancers (Patients and Methods) and was 96% for T790M, 100% for L858R, and 94% for the exon 19 deletion mutations (Table 21).
  • Plasma was available for 60 of the 63 patients. Using tumor tissue testing results as a reference standard, the detection sensitivity of the assays in plasma was 93% (38/41; 3 of 44 available plasma samples failed NGS) for T790M, 100% (17/17) for L858R, and 87% (34/39) for exon 19 deletions (Table 22).
  • the specificity of the EGFR plasma tests was determined using plasma samples obtained from healthy donors and patients with non-NSCLC metastatic cancers and was 94% for the T790M, 100% for the L858R and 96% for the exon 19 deletion mutations (Table 21).
  • FIG. 15 shows overlapping positive T790 cases determined by urine, tissue and plasma samples. For all urine sample volumes, there were 11 cases that were urine T790M- positive but tumor tissue T790M-negative or tissue sample inadequate (Table 19). Of these 11 cases, 10 were also T790M positive in plasma (1 sample was T790M negative in plasma). Similarly, of the 11 discordant cases that were plasma T790M positive but tissue T790M negative or tissue sample inadequate, 10 were also positive by urine T790M testing (1 sample was T790M negative in urine). Together, urine and plasma T790M testing identified a higher proportion of positive cases (89%, 56/63) than tissue (75%, 47/63).
  • the systemically derived DNA fragments in urine can range from approximately 35 to 250 bp.
  • Assay sensitivity in the present study was calculated using tumor as the reference sample type. This method has limitations, particularly when applied to resistance mutations such as T790M which will have a significant false negative rate in biopsies due to tumor heterogeneity and low tumor cellularity. 9 ' 10 ' 13
  • the combination of urine and plasma testing identified 12 EGFR T790M positive cases that were undetectable by central lab testing of tumor tissue. While 10 of the 12 cases were positive by both ctDNA specimen types, one was unique to plasma and one was unique to urine. Urine may therefore provide unique and complementary information about a patient's mutational status that is not captured by plasma or tissue tests. These results indicate for the first time that either urine or plasma T790M testing may be considered as an alternative to tissue biopsy testing. Urine may be particularly attractive for patients with poor health status because it represents a truly non-invasive alternative that can be collected in a patient's own home.
  • Thress KS Brant R, Carr TH, et al. EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer 2015;90:509-515.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in exemplary embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and biologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne la détection de séquences d'acides nucléiques cibles présentes en faible abondance par rapport à une séquence d'acides nucléiques non cibles ou de type sauvage dans un échantillon. En particulier, des compositions comprenant des séquences d'oligonucléotides pouvant être utilisées dans des procédés d'enrichissement et d'amplification permettent un niveau sensiblement plus élevé de sensibilité de détection d'une séquence mutante ou cible à l'intérieur d'un arrière-plan élevé de séquence de type sauvage.
PCT/US2017/033401 2016-05-20 2017-05-18 Séquences d'oligonucléotides pour la détection de séquences cibles à faible abondance et leurs kits WO2017201331A2 (fr)

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