Detection of lung neoplasia by analysis of methylated DNA

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 optionally
• d) 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 comprising
• guanidine thiocyanate;
• and
• non-ionic detergent;
• to form a mixture wherein proteins are digested by said protease;
• B) to the mixture of step a) adding
• iii) silica particles, and
• iv) 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;
• and
• N) detecting amplification of one or more different DNA methylation marker target regions from said eluted DNA during PCR-flap assay reactions.