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Detection of lung neoplasia by analysis of methylated DNA

  • US 10,385,406 B2
  • Filed: 03/28/2017
  • Issued: 08/20/2019
  • Est. Priority Date: 05/05/2016
  • Status: Active Grant
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1. A method of processing a sample, the method comprising:

  • a) assaying a sample from a subject for an amount of at least one methylation marker selected from the group consisting of BARX1, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, ZNF671, ST8SIA1, NKX6_2, FAM59B, DIDO1, MAX_Chr1.110, AGRN, SOBP, MAX_chr10.226, ZMIZ1, MAX_chr8.145, MAX_chr10.225, PRDM14, ANGPT1, MAX.chr16.50, PTGDR_9, ANKRD13B, DOCK2, MAX_chr19.163, ZNF132, MAX chr19.372, HOXA9, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, ZNF781, PTGDR, GRIN2D, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, EMX1, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, B3GALT6, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329;

    b) assaying said sample for an amount of a reference marker;

    c) comparing the amount of said at least one methylation marker to the amount of reference marker in said sample to determine a methylation state for said at least one methylation marker in said sample; and

    optionallyd) generating a record reporting the methylation state for said at least one methylation marker in said sample;

    wherein said sample is a plasma sample obtained from a subject having or suspected of having a lung neoplasm, and wherein said method comprises;

    A) combining the plasma sample with;

    i) protease; and

    ii) a first lysis reagent, said first lysis reagent comprisingguanidine thiocyanate; and

    non-ionic detergent;

    to form a mixture wherein proteins are digested by said protease;

    B) to the mixture of step a) addingiii) silica particles, andiv) a second lysis reagent, said second lysis reagent comprising;

    guanidine thiocyanate;

    non-ionic detergent; and

    isopropyl alcohol;

    under conditions wherein DNA is bound to said silica particles;

    C) separating silica particles with bound DNA from the mixture of B);

    D) to the separated silica particles with bound DNA adding a first wash solution, said first wash solution comprising a) guanidine hydrochloride or guanidine thiocyanate, and b) ethyl alcohol;

    E) separating the silica particles with bound DNA from said first wash solution;

    F) to the separated silica particles with bound DNA adding a second wash solution, said second wash solution comprising a buffer and ethyl alcohol;

    G) separating washed silica particles with bound DNA from said second wash solution; and

    H) eluting DNA from the washed silica particles with bound DNA separated in step G);

    I) assaying eluted DNA for an amount of at least one methylated methylation marker and for an amount of reference marker in said eluted DNA; and

    J) comparing the amount of said at least one methylated methylation marker to the amount of reference marker in said DNA to determine a methylation state for said at least one methylation marker in said plasma sample;

    wherein assaying said eluted DNA comprises analyzing multiple DNA methylation markers using a PCR pre-amplification and a PCR-flap assay by a process comprising;

    K) combining eluted DNA comprising a plurality of different DNA methylation marker target regions in a first reaction mixture with PCR amplification reagents, wherein said PCR amplification reagents comprise;

    i) a plurality of different primer pairs for amplifying said plurality of different target regions, if present in said sample, from said eluted DNA;

    ii) thermostable DNA polymerase;

    iii) dNTPs; and

    iv) a buffer comprising Mg++L) exposing said first reaction mixture to thermal cycling conditions wherein a plurality of different DNA methylation marker target regions, if present in the sample, are amplified to produce a pre-amplified mixture, and wherein said thermal cycling conditions are limited to a number of thermal cycles that maintain amplification in an exponential range;

    M) partitioning said pre-amplified mixture into a plurality of PCR-flap assay reaction mixtures, wherein each PCR-flap assay reaction mixture comprises;

    i) an additional amount of a primer pair selected from said plurality of different primer pairs of step K) i);

    ii) thermostable DNA polymerase;

    iii) dNTPs;

    iv) said buffer comprising Mg++v) a flap endonuclease;

    vi) a flap oligonucleotide; and

    vi) a hairpin oligonucleotide comprising a region that is complementary to a portion of said flap oligonucleotide;

    andN) detecting amplification of one or more different DNA methylation marker target regions from said eluted DNA during PCR-flap assay reactions.

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