Detection of lung neoplasia by analysis of methylated DNA

Abstract

Provided herein is technology for lung neoplasia screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of lung cancer.

What is claimed is: 
     
       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 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. 
 
     
     
       2. The method of  claim 1 , wherein said assaying comprises treating the eluted DNA with a methylation-sensitive restriction enzyme or with a reagent that selectively modifies unmethylated cytosine residues in the eluted DNA. 
     
     
       3. The method of  claim 1 , wherein said at least one methylation marker comprises a methylation marker selected from the group consisting of SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, EMX1, CYP26C1, SOBP, SUCLG2, SHOX2, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, and SKI. 
     
     
       4. The method of  claim 1 , wherein said at least one methylation marker comprises a group of methylation markers selected from:
 the group consisting of ZNF781, BARX1, and EMX1; 
 the group consisting of SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI; 
 the group consisting of SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, and EMX1; 
 the group consisting of SHOX2, SOBP, ZNF781, BTACT, CYP26C1, and DLX4; 
 the group consisting of SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI; and 
 the group consisting of ZNF781, BARX1, and EMX1, and further comprising SOBP and/or HOXA9. 
 
     
     
       5. The method of  claim 1 , wherein assaying the methylation state of a methylation marker in the sample comprises determining the extent of methylation at a plurality of bases. 
     
     
       6. The method of  claim 2 , wherein the reagent that selectively modifies unmethylated cytosine residues comprises bisulfite, and wherein said assaying comprises bisulfate converting methylation marker DNA and reference marker DNA. 
     
     
       7. The method of  claim 1 , wherein said eluted DNA is prepared from a plasma sample of at least one mL, and wherein the volume of said eluted DNA comprising a plurality of different DNA methylation marker target regions in the first reaction mixture is at least 20 to 50% of the total volume of the first reaction mixture. 
     
     
       8. 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, NKX62, 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; 
 wherein assaying said sample comprises analyzing multiple DNA methylation markers using a PCR pre-amplification and a PCR-flap assay by a process comprising: 
 I) combining DNA from the sample 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 the sample; 
 ii) thermostable DNA polymerase; 
 iii) dNTPs; and 
 iv) a buffer comprising Mg ++ ; 
 
 II) 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; 
 III) 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 I) 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 
 IV) detecting amplification of one or more different DNA methylation marker target regions from said DNA from said sample during PCR-flap assay reactions. 
 
     
     
       9. The method of  claim 1 , wherein said assaying comprises treating the DNA obtained from the sample with a methylation-sensitive restriction enzyme or with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA. 
     
     
       10. The method of  claim 9 , wherein the reagent that selectively modifies unmethylated cytosine residues comprises bisulfate. 
     
     
       11. The method of  claim 10 , wherein the assaying comprises bisulfite-converting methylation marker DNA and reference marker DNA. 
     
     
       12. The method of  claim 8 , wherein said at least one methylation marker comprises a methylation marker selected from the group consisting of SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, EMX1, CYP26C1, SOBP, SUCLG2, SHOX2, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, and SKI. 
     
     
       13. The method of  claim 8 , wherein said at least one methylation marker comprises a group of methylation markers selected from:
 the group consisting of ZNF781, BARX1, and EMX1; 
 the group consisting of SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI; 
 the group consisting of SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, and EMX1; 
 the group consisting of SHOX2, SOBP, ZNF781, BTACT, CYP26C1, and DLX4; 
 the group consisting of SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI; 
 
       and
 the group consisting of ZNF781, BARX1, and EMX1, and further comprising SOBP and/or HOXA9. 
 
     
     
       14. The method of  claim 8 , wherein assaying the methylation state of a methylation marker in the sample comprises determining the extent of methylation at a plurality of bases. 
     
     
       15. The method of  claim 8 , wherein said DNA from the sample is prepared from a plasma sample of at least one mL, and wherein the volume of said DNA from the sample comprising a plurality of different DNA methylation marker target regions in the first reaction mixture is at least 20 to 50% of the total volume of the first reaction mixture.