Characterization of methylated DNA, RNA, and proteins in subjects suspected of having lung neoplasms.

Abstract

This invention provides a method for measuring the amounts of multiple gene expression products in blood samples taken from a subject. [Solution] The method provides a method comprising: a) extracting from blood sampled from a subject i) at least one gene expression marker, which is the product of the expression of a marker gene selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1, and ii) at least one reference marker; b) measuring the amount of the at least one gene expression marker and the amount of the at least one reference marker extracted in a); and c) calculating a value for the amount of the at least one gene expression marker as a percentage of the amount of the at least one reference marker, wherein the value is calculated to represent the amount of the at least one gene expression marker in the blood sampled from the subject.

Description

[Technical Field]

[0001] This application claims priority under U.S. Provisional Patent Application No. 62 / 892,426, filed on 27 August 2019, which is incorporated herein by reference.

[0002] This specification provides technologies relating to the detection of neoplasms, and in particular, methods, compositions, and technologies relating to related uses for the detection of neoplasms such as lung cancer. Aspects of the present invention relate to systems and methods for detecting lung cancer by assaying extracts from a patient's blood. In particular, embodiments include systems and methods for determining the progression of lung cancer at different stages by detecting immune cell RNA expression or circulating cell-free RNA levels. [Background technology]

[0003] Lung cancer remains the leading cause of cancer deaths in the United States, and effective screening methods are urgently needed. Lung cancer alone accounts for 221,000 deaths annually. While treatments exist, they are often only administered to patients when the disease has progressed to a point where treatment effectiveness is compromised.

[0004] A major challenge in cancer treatment is identifying patients in the early stages of the disease. This is difficult with current methods because early cancerous or precancerous cell populations can be asymptomatic and may be located in areas that are difficult to access by biopsy. Therefore, a robust, minimally invasive assay that can be used to identify all stages of the disease, including the early stages which may be asymptomatic, would bring substantial benefits to cancer treatment. [Overview of the Initiative]

[0005] Each of the systems, devices, kits, compositions, and methods disclosed herein has several embodiments, and no single embodiment is the sole reason for their desirable attributes. Without limiting the scope of the claims, some notable features are briefly described here. Numerous other embodiments are also envisioned, including embodiments with fewer, additional, and / or different components, steps, features, purposes, benefits, and advantages. The components, embodiments, and steps may be arranged and ordered in different ways. Considering this description, and especially by reading the section titled “Modes for Carrying Out the Invention,” it will be understood how the features of the devices and methods disclosed herein offer advantages over other known devices and methods.

[0006] This technology provides a method for characterizing a sample or combination of samples derived from a subject, which includes analyzing the sample(s) for multiple different types of marker molecules. For example, in some embodiments, the technology provides a method which includes measuring the amount of at least one methylated marker gene in DNA in a sample obtained from a subject, further including measuring the amount of at least one RNA marker in a sample obtained from a subject, and assaying for the presence or absence of at least one protein marker in a sample obtained from a subject. In some embodiments, a single sample derived from a subject is analyzed for methylated marker DNA(s), marker RNA(s), and marker protein(s).

[0007] The analysis of DNA, RNA, and / or protein markers is not limited to the use of any particular technique. Methods for analyzing DNA and RNA include, but are not limited to, nucleic acid detection assays, including amplification and probe hybridization. Methods for analyzing proteins include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) detection, protein immunoprecipitation, Western blotting, and immunostaining.

[0008] One embodiment is a method for characterizing a sample derived from a subject, such as blood sampled from a subject, as a means for detecting lung cancer and / or determining the risk of lung cancer in a subject, such as a human. This method includes preparing a human-derived blood sample, detecting the target gene expression levels of the target genes S100 calcium-binding protein A9 (S100A9), selectin L (SELL), peptidylarginine deiminase 4 (PADI4), apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBE3CA), S100 calcium-binding protein A12 (S100A12), matrix metallopeptidase 9 (MMP9), formyl peptide receptor 1 (FPR1), thymidine phosphorylase (TYMP), and / or spermidine / spermine N1-acetyltransferase 1 (SAT1) in the blood sample, detecting the reference gene expression level of a reference gene in the blood sample, and comparing the detected target gene expression levels with the detected reference gene expression levels to determine the presence or absence of lung neoplasms or to determine the risk of a person having lung cancer.

[0009] In some embodiments, the technology is a method for measuring the amount of one or more gene expression products in blood sampled from a subject, a) From the blood sample taken from the subject, i) At least one gene expression marker, which is the product of the expression of a marker gene selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1, and ii) At least one reference marker Extracting and b) Measure the amount of at least one gene expression marker extracted in a) and the amount of at least one reference marker, c) Calculate a value for the amount of at least one gene expression marker as a percentage of the amount of at least one reference marker, wherein the value represents the amount of at least one gene expression marker in the blood sampled from the subject. This provides a method that includes this.

[0010] In some embodiments, extraction involves extracting a marker from a sample selected from whole blood, blood products containing leukocytes, and blood products containing plasma. In certain embodiments, at least one gene expression marker comprises a protein or RNA, and in certain preferred embodiments, the RNA extracted from blood sampled from a subject comprises circulating cell-free RNA. In some embodiments, the RNA extracted from blood sampled from a subject comprises RNA expressed by immune cells. In any of the embodiments described herein, the RNA extracted from blood sampled from a subject may comprise mRNA.

[0011] This technology is not limited to the measurement of a single gene expression marker, but encompasses the measurement of multiple gene expression markers so that the measurement data can be combined and analyzed, for example, as described in detail below. In some embodiments, this technology is applied to the measurement of a limited set of markers, for example, for convenience or efficiency in applying this technology. For example, in any of the embodiments described above, at least one gene expression marker may preferably consist of 2, 3, 4, 5, 6, 7, 8, or 9 gene expression markers.

[0012] In any of the embodiments described above, at least one reference marker may include RNA or protein expressed from a gene selected from PLGLB2, GABARAP, NACA, EIF1, UBB, UBC, CD81, TMBIM6, MYL12B, HSP90B1, CLDN18, RAMP2, MFAP4, FABP4, MARCO, RGL1, ZBTB16, C10orf116, GRK5, AGER, SCGB1A1, HBB, TCF21, GMFG, HYAL1, TEK, GNG11, ADH1A, TGFBR3, INPP1, ADH1B, STK4, ACTB, HNRNPA1, CASC3, and SKP1. In a particular preferred embodiment, at least one reference marker includes RNA. In a particular embodiment, the reference marker includes RNA selected from U1 snRNA and U6 snRNA.

[0013] When applied to any of the embodiments described above, the technology encompasses embodiments in which the amount of at least one gene expression marker is measured using one or more of the following methods: reverse transcription, polymerase chain reaction, nucleic acid sequencing, mass spectrometry, mass-based separation and target capture, quantitative pyrosequencing, flap endonuclease assay, PCR-flap assay, enzyme-linked immunosorbent assay (ELISA) detection, and protein immunoprecipitation. In certain embodiments, the measurement includes multiplex amplification.

[0014] In some embodiments, DNA is also analyzed. Provided herein is a set of methylation markers assayed in tissue or plasma that achieve highly accurate differentiation of all types of lung cancer while remaining negative in normal lung tissue and benign nodules. Markers selected from this set can be used individually or in a panel, for example, to characterize blood or body fluids, and are applied to lung cancer screening and the differentiation of malignant nodules from benign nodules. In some embodiments, markers in a panel are used to distinguish one form of lung cancer from another, for example, to distinguish the presence of adenocarcinoma or large cell carcinoma from the presence of small cell carcinoma, or to detect mixed pathological cancers. Provided herein is a technique for screening markers that, when detected in a sample taken from a subject, result in a high signal-to-noise ratio and low background levels.

[0015] EMX1, GRIND2, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX Panels of methylation markers and / or markers (e.g., chromosomal regions) with annotations selected from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329 have been identified in multiple studies by comparing the methylation status of methylation markers in lung cancer samples with the corresponding markers in normal (non-cancerous) samples.

[0016] As described herein, the technology provides several methylation markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more markers) that highly identify lung cancer and, in some embodiments, identify types of lung cancer.

[0017] Therefore, any of the techniques of the above embodiments for measuring the amount of one or more gene expression products in blood sampled from a subject, d) Extracting at least one methylated marker DNA and at least one reference marker DNA from blood sampled from the subject, e) measuring the amount of at least one methylated marker DNA, wherein the at least one methylated marker DNA comprises a nucleotide sequence related to at least one of EMX1, GRIND2, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329; f) measuring the amount of at least one reference marker DNA; g) calculating a value of the amount of at least one methylated marker DNA as a percentage relative to the amount of the reference marker DNA, the value indicating the amount of at least one methylated marker DNA in blood sampled from the subject; may further comprise.

[0018] The present technology is not limited to the measurement of a certain methylated marker DNA. The present technology includes, for example, the measurement of multiple methylated marker DNAs so that the measurement data of different methylated marker DNAs can be combined with each other and / or combined with the measurement data of RNA and / or protein gene expression markers, as will be described in detail below in this specification. In some embodiments, the present technology is applied to the measurement of a set of limited methylated marker DNAs, for example, for the convenience or efficiency in applying the present technology. For example, in any of the above-described embodiments, at least one methylated marker DNA may preferably consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 methylated marker DNAs. In a specific embodiment, at least one methylated marker DNA includes a nucleotide sequence related to at least one of BARX1, FLJ45983, HOPX, ZNF781, FAM59B, HOXA9, SOBP, and IFFO1. In any specific embodiment among the above, at least one gene expression marker includes the product of the expression of a marker gene selected from FPR1, PADI4, and SELL.

[0019] In a specific embodiment, the DNA extracted from the blood sampled from the subject includes cell-free circulating DNA. In other embodiments, the DNA includes cellular DNA. In any of the above-described embodiments, at least one reference marker DNA used to calculate the value of the amount of at least one methylated marker DNA may preferably be selected from B3GALT6 DNA and β-actin DNA.

[0020] Any of the above-described embodiments for measuring the methylated marker DNA includes an embodiment in which the methylated marker DNA is treated with a reagent that selectively modifies the DNA in a manner specific to the methylation status of the DNA. In some embodiments, the reagent includes a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme. In a specific preferred embodiment, the bisulfite reagent includes ammonium bisulfite.

[0021] While the Art is not limited to any particular method for measuring the amount of methylated marker DNA, in some embodiments, measuring the amount of at least one methylated marker DNA includes using one or more of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and target capture, and in certain preferred embodiments, the measurement includes multiplex amplification. In some embodiments, measuring the amount of at least one methylated marker DNA includes using one or more methods selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assays, PCR-flap assays, and bisulfite genome sequencing PCR.

[0022] An embodiment of this technology is a method for characterizing blood sampled from a subject, i) Processing blood sampled from the subject to generate extracted DNA and extracted RNA, ii) Measuring the amount of two or more marker RNAs in the extracted RNA, wherein the marker RNAs are selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1 RNA. iii) Measuring the amount of at least one reference RNA in the extracted RNA, wherein the reference RNA is selected from CASC3A, SKP1, and STK4. iv) Calculating the value of each of two or more marker RNAs as a percentage of the amount of at least one reference RNA, wherein the value of each marker RNA represents the amount of marker RNA in the blood sampled from the subject. v) Treat the extracted DNA with a bisulfite reagent to produce bisulfite-treated DNA, vi) Measuring the amount of two or more methylated marker DNAs in bisulfite-treated DNA, wherein the methylated marker DNAs are EMX1, GRIND2, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The following genes are selected for measurement: chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. vii) Measuring the amount of at least one reference DNA in bisulfite-treated DNA, wherein the at least one reference DNA is selected from B3GALT6 DNA and β-actin DNA. viii) Calculating the value of each of two or more methylated marker DNAs as a percentage of the amount of reference DNA measured in bisulfite-treated DNA, wherein the value of each methylated marker DNA represents the amount of methylated marker DNA in blood sampled from the subject. This provides a method that includes this.

[0023] The embodiments described herein that include DNA and RNA analysis encompass embodiments in which DNA and RNA are isolated from blood collected in a single blood collection device, including but not limited to a single blood collection tube or blood collection bag.

[0024] Any of the embodiments described herein include embodiments in which the subject has or is suspected of having a lung neoplasm, and / or embodiments in which the technology assesses the risk of lung cancer in the subject based on values ​​calculated using the measurement methods described above. For example, in some embodiments, the amount of at least one gene expression marker and / or the amount of at least one methylated marker DNA in the blood sampled from the subject indicates the lung cancer risk of the subject.

[0025] In some embodiments, the design for assaying the methylation status of a marker involves analyzing background methylation at individual CpG loci in the target region of the marker being examined by the assay technique. For example, in some embodiments, a large number of individual copies (e.g., more than 10,000, preferably more than 100,000 individual copies) of marker DNA in a sample isolated from a subject diagnosed with a disease, e.g., cancer, are examined to determine the frequency of methylation, and these data are compared to a similarly large number of individual copies of marker DNA in a sample isolated from a subject without the disease. The frequencies of disease-related methylation and background methylation at individual CpG loci in the marker DNA in the sample can be compared so that CpG loci with higher signal-to-noise ratios, e.g., high detectable methylation and / or reduced background methylation, can be selected for use in the assay design. See, for example, U.S. Patents 9,637,792 and 10,519,510, each incorporated herein by reference as a whole. In some embodiments, a group of CpG loci with high signal-to-noise ratios (e.g., 2, 3, 4, 5, or more individual CpG loci within a marker region) are simultaneously examined by the assay so that all CpG loci must have a pre-determined methylation status (e.g., all must be methylated, or none can be methylated).

[0026] In some embodiments, a kit is provided comprising reagents or materials for an assay, selected from measuring the amount or presence or absence of at least one gene expression marker and / or at least one methylation marker DNA. The at least one gene expression marker may be an RNA marker or a protein marker.

[0027] For example, a particular kit embodiment is: a) A reagent set for measuring the amount of at least one gene expression marker in blood sampled from a subject, wherein the at least one gene expression marker is generated from the expression of a marker gene selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1. b) A reagent set for measuring the amount of at least one reference marker in blood sampled from a subject. To provide.

[0028] In some embodiments, the kit further comprises a set of reagents for extracting at least one gene expression marker and at least one reference marker from blood. In some embodiments, the at least one gene expression marker comprises one or more RNAs and proteins, and the at least one reference marker comprises one or more RNAs, DNAs, and proteins. In certain embodiments, the kit includes, i) at least one first oligonucleotide, wherein at least a portion of the at least one first oligonucleotide specifically hybridizes to a nucleic acid chain containing a nucleotide sequence associated with a gene expression marker selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1. ii) At least one second oligonucleotide, wherein at least a portion of the at least one second oligonucleotide specifically hybridizes to a reference marker, and the reference marker is a reference nucleic acid. It is equipped with.

[0029] In the embodiments of the kit described above, the nucleic acid chain containing the nucleotide sequence associated with the gene expression marker is selected from RNA, cDNA, or amplified DNA. In certain embodiments, the reference nucleic acid includes RNA or DNA, and in some embodiments, the reference gene expression marker preferably includes RNA or protein expressed from a gene selected from PLGLB2, GABARAP, NACA, EIF1, UBB, UBC, CD81, TMBIM6, MYL12B, HSP90B1, CLDN18, RAMP2, MFAP4, FABP4, MARCO, RGL1, ZBTB16, C10orf116, GRK5, AGER, SCGB1A1, HBB, TCF21, GMFG, HYAL1, TEK, GNG11, ADH1A, TGFBR3, INPP1, ADH1B, STK4, ACTB, HNRNPA1, CASC3, and SKP1.

[0030] In any of the embodiments described above, the kit of this technology is c) A reagent set for measuring the amount of at least one methylated marker DNA in blood sampled from a subject, wherein the at least one methylated marker DNA is EMX1, GRIND2, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX A reagent set containing nucleotide sequences associated with at least one of the following: chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. It can also be equipped with additional features.

[0031] In some embodiments, a reagent set for measuring the amount of at least one methylated marker DNA is provided. i) at least one third oligonucleotide, at least a portion of the at least one third oligonucleotide being EMX1, GRIND2, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX At least one third oligonucleotide that specifically hybridizes to nucleic acid chains containing nucleotide sequences associated with the methylation maker genes chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. Includes.

[0032] The embodiments of the kit described above may further comprise at least one fourth oligonucleotide, at least a portion of which specifically hybridizes to a reference marker DNA, preferably a reference marker DNA selected from B3GALT6 DNA and β-actin DNA. In some embodiments, at least one of the nucleic acid strands containing a nucleotide sequence related to the methylation maker gene and the reference marker DNA comprises bisulfite-treated DNA.

[0033] In some embodiments, the kit described above further comprises reagents for selectively modifying DNA in a manner specific to the DNA methylation status. In certain embodiments, the reagents for selectively modifying DNA in a manner specific to the DNA methylation status include bisulfite reagents, methylation-sensitive restriction enzymes, or methylation-dependent restriction enzymes. In certain preferred embodiments, the bisulfite reagent includes ammonium bisulfite.

[0034] Embodiments of the kits provided above further encompass kits in which at least one or more of the first, second, third, and fourth oligonucleotides are selected from capture oligonucleotides, nucleic acid primer pairs, nucleic acid probes, and invasive oligonucleotides, and in certain embodiments the capture oligonucleotide is bound to a solid support by, for example, covalent or non-covalent bonds (e.g., biotin-streptavidin bonds or antigen-antibody bonds). In preferred embodiments, the solid support is a magnetic bead.

[0035] Any embodiment of the kit of the technology described herein is i) A first primer pair for generating a first amplified DNA from the gene expression marker product of a marker gene selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1, ii) A first probe containing a sequence complementary to the region of the first amplified DNA, iii) A second primer pair for generating a second amplified DNA, iv) A second probe containing a sequence complementary to the region of the second amplified DNA, v) Reverse transcriptase and, vi) Heat-resistant DNA polymerase and Includes a kit equipped with the following features.

[0036] In some embodiments, the second amplified DNA is generated from a methylated marker gene or a reference marker nucleic acid.

[0037] In certain embodiments, the first probe further comprises a flap portion having a first flap sequence that is substantially not complementary to the first amplified DNA, and in some embodiments, the second probe further comprises a flap portion having a second flap sequence that is substantially not complementary to the second amplified DNA. The kit of this technology is vii) A FRET cassette having an array complementary to the first flap array, viii) A FRET cassette containing an array complementary to the second flap array. It may further include one or more of the following.

[0038] Any of the kits described herein may further comprise a flap endonuclease. In certain preferred embodiments, the flap endonuclease is a FEN-1 endonuclease, for example, a heat-stable FEN-1 endonuclease derived from archaea.

[0039] The application of this technology further provides compositions. For example, in some embodiments, this technology provides, i) A first primer pair for generating a first amplified DNA from the gene expression marker product of a gene selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1, ii) A first probe containing a sequence complementary to the region of the first amplified DNA, iii) A second primer pair for generating a second amplified DNA, iv) A second probe containing a sequence complementary to the region of the second amplified DNA, v) Reverse transcriptase and, vi) Heat-resistant DNA polymerase and The present invention provides a composition containing the following:

[0040] In some embodiments, the composition further comprises nucleic acids extracted from blood sampled from a subject, preferably having or suspected of having a lung neoplasm or being at risk of having lung cancer. In some embodiments of the composition, the nucleic acids are - cellular RNA, - Circulating cell-free RNA, - cellular DNA, - Circulating cell-free DNA Includes one or more of the following.

[0041] In some embodiments, the second primer pair generates a second amplified DNA from a methylated marker gene or a reference marker nucleic acid. In certain preferred embodiments, the second primer pair - RNA expressed from genes selected from PLGLB2, GABARAP, NACA, EIF1, UBB, UBC, CD81, TMBIM6, MYL12B, HSP90B1, CLDN18, RAMP2, MFAP4, FABP4, MARCO, RGL1, ZBTB16, C10orf116, GRK5, AGER, SCGB1A1, HBB, TCF21, GMFG, HYAL1, TEK, GNG11, ADH1A, TGFBR3, INPP1, ADH1B, STK4, ACTB, HNRNPA1, CASC3, and SKP1, - RNA selected from U1 snRNA and U6 snRNA, - DNA selected from B3GALT6 DNA and β-actin DNA A second amplified DNA is generated from a reference nucleic acid selected from the above.

[0042] In certain embodiments, the second primer pair is EMX1, GRIND2, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX A methylation marker gene selected from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329 is selected to generate a second amplified DNA.

[0043] Those skilled in the art will recognize that the above composition is not limited to two primer pairs, but encompasses compositions comprising several different primer pairs for generating amplified DNA from multiple different gene expression markers, and / or several different primer pairs for generating amplified DNA from multiple different methylation marker genes. The composition may further comprise several different primer pairs for generating amplified DNA from multiple different reference marker nucleic acids.

[0044] In the compositions described above, the first probe and / or the second probe includes a detection portion containing a fluorophore. In certain embodiments, the probes of the art may be labeled with a fluorophore and a quenching portion such that when the probe is intact, for example, when it has not been cleaved by a 5' nuclease, the emission from the fluorophore is quenched.

[0045] In some embodiments, the first probe further comprises a flap portion having a first flap sequence that is substantially not complementary to the first amplified DNA, and / or the second probe further comprises a flap portion having a second flap sequence that is substantially not complementary to the second amplified DNA. In certain embodiments, the composition is vii) A FRET cassette containing an array complementary to the first flap array, viii) FRET cassette containing a complementary array to the second flap array This further includes one or more of the following.

[0046] Any of the above compositions may further contain a flap endonuclease, preferably a FEN-1 endonuclease, such as a heat-resistant FEN-1 derived from archaeal organisms.

[0047] In certain embodiments, the above-described composition is Mg ++ For example, it includes a buffer containing MgCl2. Preferably, the composition has a relatively high Mg compared to a standard PCR buffer. ++ and low KCl (for example, 6-10 mM, preferably 7.5 mM Mg) ++ It includes a PCR-flap assay buffer containing 0.0-0.8 mM KCl.

[0048] Embodiments of this technology further include a reaction mixture comprising any one of the compositions described herein.

[0049] In some embodiments, the kit comprises reagents or materials for at least two assays, the assays being selected from measuring the amount or presence or absence of 1) at least one methylated DNA marker; 2) at least one RNA marker; and / or 3) at least one protein marker. In a preferred embodiment, at least one methylated DNA marker is 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 The group is selected from 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, SLC12a, BHLHE23, CAPN2, FGF14, FLJ34208, B3GALT6, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, ZDHHC1, ZNF329, IFFO1, and HOPX. In certain preferred embodiments, at least the RNA expression marker is expressed from a gene selected from the group consisting of S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1. In some embodiments, at least one protein comprises an antigen, e.g., a cancer-related antigen, and in some embodiments, at least one protein comprises an antibody, e.g., an autoantibody against a cancer-related antigen.

[0050] In some embodiments, the oligonucleotide in the mixture comprises a reporter molecule, and in preferred embodiments, the reporter molecule comprises a fluorophore. In some embodiments, the oligonucleotide comprises a flap sequence. In some embodiments, the mixture further comprises one or more of a FRET cassette, FEN-1 endonuclease, and / or a heat-stable DNA polymerase, preferably a bacterial DNA polymerase.

[0051] [Definition] To facilitate understanding of the technology of this invention, several terms and phrases are defined below. Further definitions are provided throughout the detailed description.

[0052] Throughout this specification and the claims, the following terms have the meanings expressly associated with this specification unless otherwise specified in the context. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, but it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, but it may. Thus, as will be described later, various embodiments of the present invention can be readily combined without departing from the scope or spirit of the invention.

[0053] Furthermore, as used herein, the term "or" is an inclusive "or" operator unless otherwise specified in the context, and is equivalent to the terms "and / or". The term "based on" is not exclusive and allows to be based on additional unspecified elements unless otherwise specified in the context. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include multiple referents. The meaning of "in" includes "in" and "on."

[0054] The transitional phrase “essentially consisting of” as used in the claims of this application limits the scope of the claims to specific materials or steps of the claimed invention and “that which does not substantially affect the basic and novel properties (plural).” For example, a composition “essentially consisting of” the enumerated elements may contain impurities to such an extent that their presence does not alter the function of the enumerated composition compared to a pure composition, i.e., a composition “consisting of” the enumerated components.

[0055] Conditional statements such as “can,” “could,” “might,” or “may,” unless otherwise specified or understood in the context in which they are used, are generally intended to indicate that a particular embodiment includes certain features, elements, and / or steps, but other embodiments do not. Thus, such conditional statements are not generally intended to mean that features, elements, and / or steps are required in any way in one or more embodiments, or that one or more embodiments necessarily include logic for determining whether these features, elements, and / or steps are included in or performed in any particular embodiment, with or without user input or prompting.

[0056] Conjunctions such as "at least one of X, Y, and Z" are generally understood in their context to mean that an item, term, etc., may be X, Y, or Z, unless otherwise specified. Therefore, such conjunctions are not generally intended to mean that a particular embodiment requires the presence of at least one X, at least one Y, and at least one Z.

[0057] As used herein, terms such as “approximately,” “about,” “roughly,” and “substantially” refer to values, quantities, or characteristics that are close to the stated values, quantities, or characteristics and still perform the desired function or achieve the desired result.

[0058] As used herein, “methylation” refers to cytosine methylation of cytosine at the C5 or N4 position, adenine at the N6 position, or methylation of other types of nucleic acids. Typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplification template, so the DNA amplified in vitro is usually unmethylated. However, “unmethylated DNA” or “methylated DNA” may also refer to amplified DNA whose original template was unmethylated, or amplified DNA whose original template was methylated, respectively.

[0059] Therefore, as used herein, “methylated nucleotide” or “methylated nucleotide base” refers to a nucleotide base in which a methyl moiety is present, and this methyl moiety is not present in typical nucleotide bases where it is recognized. For example, cytosine does not contain a methyl moiety in its pyrimidine ring, but 5-methylcytosine does contain a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide, while 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, since thymine is a typical nucleotide base in DNA, for the purposes of this specification, thymine is not considered a methylated nucleotide when present in DNA.

[0060] As used herein, “methylated nucleic acid molecule” refers to a nucleic acid molecule containing one or more methylated nucleotides.

[0061] As used herein, “methylation state,” “methylation profile,” and “methylation status” of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated. In some embodiments, a nucleic acid may be characterized as “unmethylated” if it is not methylated at a particular locus (e.g., a locus for a particular single CpG dinucleotide) or a particular combination of loci, even if it is methylated at other loci in the same gene or molecule.

[0062] The methylation status of a particular nucleic acid sequence (e.g., a gene marker or DNA region as described herein) may indicate the methylation status of all bases in the sequence, or the methylation status of a subset of bases in the sequence (e.g., one or more cytosines), or may indicate information about the methylation density of a region in the sequence, whether or not it provides precise information about the location where methylation occurs within the sequence. As used herein, the terms “marker gene” and “marker” are used synonymously to refer to DNA, RNA, or protein (or other sample component) associated with a certain condition, e.g., cancer, whether or not the marker region is located within the coding region of DNA. Markers may include, for example, regulatory regions, flanking regions, intergenetic regions, etc. Similarly, the term “marker” as used in reference to any component of a sample, e.g., protein, RNA, carbohydrate, small molecule, etc., refers to a component that can be assayed (e.g., measured or otherwise characterized) in the sample and is associated with a condition of the sample or a sample derived from the sample. The term “methylation marker” refers to a gene or DNA whose methylation status is associated with a certain condition, e.g., cancer.

[0063] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus within the nucleic acid molecule. For example, the methylation state of cytosine at the seventh nucleotide in a nucleic acid molecule is methylated when the nucleotide present at the seventh nucleotide is 5-methylcytosine. Similarly, the methylation state of cytosine at the seventh nucleotide in a nucleic acid molecule is unmethylated when the nucleotide present at the seventh nucleotide is cytosine (and not 5-methylcytosine).

[0064] Methylation status may, in some cases, be expressed or indicated by a "methylation value" (e.g., representing methylation frequency, proportion, ratio, percentage, etc.). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with methylation-dependent restriction enzymes, by comparing amplification profiles after bisulfite reactions, or by comparing the sequences of bisulfite-treated nucleic acids with those of untreated nucleic acids. Therefore, a value, such as a methylation value, represents the methylation status and can thus be used as a quantitative indicator of methylation status across multiple copies of a gene locus. This is particularly useful when it is desirable to compare the methylation status of sequences in a sample to a threshold or reference value.

[0065] As used herein, “methylation frequency” or “methylation percentage (%)” refers to the number of molecules or loci that are methylated compared to the number of molecules or loci that are not methylated.

[0066] Therefore, methylation status describes the state of methylation of a nucleic acid (e.g., a genome sequence). Furthermore, methylation status refers to characteristics related to the methylation of a nucleic acid segment at a particular genomic locus. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated C in any particular region of the nucleic acid, and allele differences in methylation, for example, due to differences in allele origin. The terms “methylation status,” “methylation profile,” and “methylation status” also refer to the relative, absolute, or pattern of methylated or unmethylated C in any particular region of the nucleic acid in a biological sample. For example, if cytosine (C) residue(s) in a nucleic acid sequence are methylated, it may be described as “hypermethylated” or “increased methylation,” while if cytosine (C) residue(s) in a DNA sequence are not methylated, it may be described as “hypomethylated” or “decreased methylation.” Similarly, if cytosine (C) residues(s) in a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), that sequence is considered to be highly methylated or have increased methylation compared to the other nucleic acid sequence. Alternatively, if cytosine (C) residues(s) in a DNA sequence are not methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), that sequence is considered to be hypomethylated or have decreased methylation compared to the other nucleic acid sequence. Furthermore, as used herein, the term “methylation pattern” refers to the aggregate site of methylated and unmethylated nucleotides across a region of nucleic acid. Two nucleic acids may have the same or similar methylation frequency or methylation percentage, but different methylation patterns, if the number of methylated and unmethylated nucleotides is the same or similar across the region, but the positions of the methylated and unmethylated nucleotides are different.Sequences are said to have "variable methylation," "methylation differences," or "different methylation states" if they exhibit different degrees of methylation (e.g., one methylation is increased or decreased compared to another), different frequencies, or different patterns. The term "variable methylation" refers to differences in the level or pattern of nucleic acid methylation in cancer-positive samples compared to the level or pattern of nucleic acid methylation in cancer-negative samples. It may also refer to differences in level or pattern between patients whose cancer recurred after surgery and those whose cancer did not. Variable methylation, and specific levels or patterns of DNA methylation, can serve as prognostic and predictive biomarkers, for example, if precise cutoffs or predictive characteristics are defined.

[0067] The frequency of methylation states can be used to represent a population or a sample from a single individual. For example, a nucleotide locus with a 50% methylation frequency is methylated in 50% of cases and unmethylated in 50% of cases. Such frequencies can be used to represent, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a population or a collection of nucleic acids. Thus, if the methylation in a first population or pool of nucleic acid molecules differs from that in a second population or pool of nucleic acid molecules, the frequency of methylation states in the first population or pool will differ from that in the second population or pool. Such frequencies can also be used to represent, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such frequencies can be used to represent the degree to which a population of cells in a tissue sample is methylated or unmethylated at a nucleotide locus or nucleic acid region.

[0068] As used herein, "nucleotide locus" refers to the position of a nucleotide within a nucleic acid molecule. For methylated nucleotides, the nucleotide locus refers to the position of a methylated nucleotide within the nucleic acid molecule.

[0069] Typically, methylation of human DNA occurs in adjacent guanine and cytosine-containing dinucleotide sequences (also known as CpG dinucleotide sequences) where cytosine is located at the 5' position of guanine. In the human genome, most cytosines within CpG dinucleotides are methylated, but some remain unmethylated within certain CpG dinucleotide-rich genomic regions known as CpG islands (see, for example, Antequera, et al. (1990) Cell 62: 503-514).

[0070] As used herein, “CpG island” refers to a G:C-rich region of genomic DNA containing an increased number of CpG dinucleotides compared to the whole genomic DNA. A CpG island may be at least 100, 200, or more base pairs long, where the G:C content of the region is at least 50%, and the ratio of observed CpG frequency to expected frequency is 0.6; in some cases, a CpG island may be at least 500 base pairs long, where the G:C content of the region is at least 55%, and the ratio of observed CpG frequency to expected frequency is 0.65. The ratio of observed CpG frequency to expected frequency can be calculated according to the method presented in Gardiner-Garden et al (1987) J. Mol. Biol. 196: 261-281. For example, the observed CpG frequency relative to the expected frequency can be calculated according to the formula R = (A × B) / (C × D), where R is the ratio of the observed CpG frequency to the expected frequency, A is the number of CpG dinucleotides in the sequence being analyzed, B is the total number of nucleotides in the sequence being analyzed, C is the total number of C nucleotides in the sequence being analyzed, and D is the total number of G nucleotides in the sequence being analyzed. Methylation status is typically determined within CpG islands, for example, in promoter regions. However, it is understood that other sequences in the human genome are prone to DNA methylation such as CpA and CpT (Ramsahoye (2000) Proc. Natl.). Acad. Sci. USA 97: 5237-5242, Salmon and Kaye (1970) Biochim. Biophys. Acta. 204: 340-351, Grafstrom (1985) Nucleic Acids Res. 13: 2827-2842, Nyce (1986) Nucleic Acids Res. 14: 4353-4367, Woodcock (1987) See Biochem. Biophys. Res. Commun. 145: 888-894.

[0071] As used herein, “methylation-specific reagent” refers to a reagent that modifies the nucleotides of a nucleic acid molecule in accordance with the methylation state of the nucleic acid molecule; that is, a methylation-specific reagent refers to a compound, composition, or other agent that can alter the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. A method of treating a nucleic acid molecule with such a reagent may include contacting the nucleic acid molecule with the reagent and, together with additional steps as necessary, achieving the desired alteration of the nucleotide sequence. Such a method may be applied in such a manner that unmethylated nucleotides (e.g., each unmethylated cytosine) are modified into different nucleotides. For example, in some embodiments, such a reagent can deaminate unmethylated cytosine nucleotides to produce deoxyuracil residues. An exemplary reagent is a bisulfite reagent.

[0072] The term “bisulfite reagent” refers to reagents comprising bisulfites, disulfites, bisulfites, or combinations thereof that are useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences, as disclosed herein. Methods for such treatments are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each incorporated herein by reference as a whole). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent, such as but not limited to n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as a chroman derivative, e.g., 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid, or trihydroxybenzoic acid and its derivatives, e.g., gallic acid (see PCT / EP2004 / 011715 incorporated herein as a whole by reference). In certain preferred embodiments, the bisulfite reaction includes treatment with ammonium bisulfite, as described, for example, in WO2013 / 116375.

[0073] Modification of nucleic acid nucleotide sequences by methylation-specific reagents can sometimes result in nucleic acid molecules in which each methylated nucleotide is modified to a different nucleotide.

[0074] The term "methylation assay" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0075] As used herein, the “sensitivity” of a given marker (or a set of markers used together) refers to the percentage of samples that report DNA methylation values ​​above a threshold that distinguishes neoplastic samples from non-neoplastic samples. In some embodiments, a positive result is defined as a histologically confirmed neoplasia reporting a DNA methylation value above a threshold (e.g., a range associated with the disease), and a false negative result is defined as a histologically confirmed neoplasia reporting a DNA methylation value below a threshold (e.g., a range associated with no disease). Thus, the sensitivity value reflects the probability that a DNA methylation measurement for a given marker obtained from known disease samples falls within the range of disease-related measurements. As defined herein, the clinical relevance of the calculated sensitivity value represents an estimate of the probability of detecting the presence of a clinical condition when a given marker is applied to a subject with that condition.

[0076] As used herein, “specificity” of a given marker (or set of markers used together) refers to the percentage of non-neoplastic samples that report DNA methylation values ​​below a threshold that distinguishes neoplastic samples from non-neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample that reports DNA methylation values ​​below a threshold (e.g., a range associated with no disease), and a false negative is defined as a histologically confirmed non-neoplastic sample that reports DNA methylation values ​​above a threshold (e.g., a range associated with disease). Thus, the value of specificity reflects the probability that a DNA methylation measurement for a given marker obtained from a known non-neoplastic sample falls within the range of non-disease-related measurements. As defined herein, the clinical relevance of the calculated specificity value represents an estimate of the probability of detecting the absence of a clinical condition when a given marker is applied to a patient without that condition.

[0077] As used herein, “selected nucleotide” means one of the four nucleotides typically present in nucleic acid molecules (C, G, T, and A in DNA, and C, G, U, and A in RNA), and may include methylated derivatives of the typically present nucleotides (for example, if C is the selected nucleotide, the meaning of selected nucleotide includes both methylated and unmethylated forms of C), but “methylated selected nucleotide” specifically refers to a nucleotide that is typically methylated, and “unmethylated selected nucleotide” specifically refers to a nucleotide that typically exists in the unmethylated form.

[0078] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids depending on the methylation status of its recognition site. In the case of a restriction enzyme that specifically cleaves when the recognition site is unmethylated or hemimethylated (methylation-sensitive enzyme), if the recognition site is methylated on one or both strands, cleavage does not occur (or occurs with significantly reduced efficiency). In the case of a restriction enzyme that specifically cleaves only when the recognition site is methylated (methylation-dependent enzyme), if the recognition site is not methylated, cleavage does not occur (or occurs with significantly reduced efficiency). Preferably, the recognition sequence is a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). Further preferred in some embodiments is a restriction enzyme that does not cleave when the cytosine in this dinucleotide is methylated at carbon atom C5.

[0079] The term "primer" refers to an oligonucleotide that can act as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid template strand (e.g., in the presence of an inducer such as nucleotides and DNA polymerase, and at a suitable temperature and pH), whether it occurs spontaneously as a nucleic acid fragment from a restriction digest or is produced synthetically. Primers are preferably single-stranded for maximum amplification efficiency, but may alternatively be double-stranded. In the case of double-stranded primers, they are first treated to separate their strands before being used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. Generally, the primer is long enough to prime the synthesis of the extension product in the presence of an inducer. The exact length of the primer depends on many factors, including temperature, primer source, and the method used.

[0080] The term “probe” refers to an oligonucleotide (e.g., a sequence of nucleotides) that can hybridize to another oligonucleotide of interest, whether it occurs naturally, such as in purified restriction digests, or is produced synthetically, recombinantly, or by PCR amplification. Probes may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of specific gene sequences (e.g., “capture probes”). Any probe used in the present invention may, in some embodiments, be labeled with any “reporter molecule” so as to be detectable in any detection system, including but not limited to enzymes (e.g., ELISA, and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. The present invention is not intended to be limited to any particular detection system or labeling.

[0081] As used herein, the term “target” refers to a nucleic acid that is to be sorted from other nucleic acids, for example, by probe binding, amplification, isolation, or capture. For example, when used in relation to polymerase chain reactions, “target” refers to a region of nucleic acid to which a primer used in the polymerase chain reaction binds. On the other hand, when used in assays where the target DNA is not amplified, for example in some embodiments of invasive cleavage assays, the target includes a site to which a probe and an invasive oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure, thereby enabling the detection of the presence of the target nucleic acid. “Segment” is defined as a region of nucleic acid within a target sequence. When used in relation to double-stranded nucleic acids, the term “target” is not limited to a specific strand of the double-stranded target, for example, the coding strand, but can be used in relation to, for example, one or both strands of a double-stranded gene or reference DNA.

[0082] As used herein, the terms “cell-free” and “circulating cell-free” as used with respect to nucleic acids derived from blood are used synonymously and refer to nucleic acids found in blood but not in cells within blood, such as DNA and RNA species. As used herein with respect to nucleic acids extracted from blood, these terms refer to the properties and location of the nucleic acid before a sample is taken from the subject and before nucleic acids are extracted from the blood sample.

[0083] As used herein, the term "marker" refers to a substance (e.g., a nucleic acid, or a region of a nucleic acid, or a protein) that can be used to distinguish abnormal cells (e.g., cancer cells) from normal cells (non-cancerous cells) based on the presence, absence, or state (e.g., methylation status) of a marker substance. As used herein, "normal" methylation of a marker refers to the degree of methylation typically found in normal cells, e.g., non-cancerous cells.

[0084] As used herein, the term “neoplasm” refers to any new or abnormal growth of tissue, including but not limited to cancer. Therefore, a neoplasm may be a pre-malignant neoplasm or a malignant neoplasm.

[0085] The term “neoplasm-specific marker,” as used herein, refers to any biological material or element that may be used to indicate the presence of a neoplasm. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells. In some cases, the marker is a specific nucleic acid region (e.g., a gene, an intragenic region, a specific gene locus, etc.). A nucleic acid region that is a marker may be called, for example, a “marker gene,” “marker region,” “marker sequence,” or “marker locus.”

[0086] The term “sample” is used in its broadest sense. In one sense, it may refer to animal cells or tissues or bodily fluids. In another sense, it refers to specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and include, for example, liquids, solids, tissues, and gases. Environmental samples include environmental substances such as surface materials, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples to which the present invention is applicable. Where used herein with respect to a sample, the term “sample” taken from a source or subject, for example from a patient, is not limited to a single physical specimen but also includes samples taken in multiple parts. For example, a “sample” of blood may be taken in two, three, four, or more different blood collection tubes or other blood collection devices (e.g., bags), or combinations of different blood collection devices.

[0087] As used herein, the terms “patient” or “subject” refer to the organism that is the subject of the various tests provided by this technology. The term “subject” includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In a more preferred embodiment, the subject is a human. With respect to diagnostic methods, the preferred subject is a vertebrate subject. The preferred vertebrate is warm-blooded, and the preferred warm-blooded vertebrate is a mammal. The preferred mammal is most preferably a human. As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Accordingly, the technology of the present invention is provided for the diagnosis of mammals such as humans, as well as animals that are socially important to humans, such as mammals such as the Siberian tiger, which is important because it is endangered; economically important animals such as animals raised on farms for human consumption; and / or animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores such as cats and dogs, pigs including pigs, boars, and wild boars, ruminants and / or ungulates such as cattle, bulls, sheep, giraffes, deer, goats, bison, and camels, as well as pinnipeds and horses. Thus, the diagnosis and treatment of livestock, including but not limited to domesticated pigs, ruminants, ungulates, and horses (including racehorses), are also provided. The subject matter disclosed herein further includes a system for diagnosing lung cancer in a subject. This system may be provided, for example, as a commercially available kit that can be used for screening for lung cancer risk or for diagnosing lung cancer in a subject from which a biological sample is taken. An exemplary system provided in accordance with the art of the present invention includes evaluating the methylation status of markers described herein.

[0088] In the context of nucleic acids, the term “amplify” typically refers to the generation of multiple copies of a polynucleotide or a portion of a polynucleotide, starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule), where the amplification product or amplicon is generally detectable. Polynucleotide amplification encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or more copies of a target or template DNA molecule in polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, for example, U.S. Patent No. 5,494,810, incorporated herein by reference as a whole, is a form of amplification. Further types of amplification include allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611, incorporated herein by reference as a whole), assembly PCR (see, e.g., U.S. Patent No. 5,965,408, incorporated herein by reference as a whole), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594, incorporated herein by reference as a whole), hot-start PCR (see, e.g., U.S. Patents No. 5,773,258 and No. 5,338,671, each incorporated herein by reference as a whole), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, each incorporated herein by reference as a whole), and ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, 25:1854-1858, each incorporated herein by reference as a whole). (See U.S. Patent No. 5,508,169, 1997), methylation-specific PCR (see, for example, Herman, et al., (1996) PNAS 93(13) 9821-9826, which is incorporated herein by reference as a whole), miniprimer PCR, multiplex ligation-dependent probe amplification (see, for example, Schouten, et al., which is incorporated herein by reference as a whole).See (2002) Nucleic Acids Research 30(12): e57), multiplex PCR (see, for example, Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156, Ballabio, et al., (1990) Human Genetics 84(6) 571-573, Hayden, et al., (2008) BMC Genetics 9:80, each incorporated herein by reference as a whole), nested PCR, overlap extension PCR (see, for example, Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367, each incorporated herein by reference as a whole), real-time PCR (see, for example, Higuchi, et al., (1992) Biotechnology, each incorporated herein by reference as a whole 10:413-417, Higuchi, et al., (1993) Biotechnology. See 11:1026-1030), reverse transcription PCR (see, for example, Bustin, SA (2000) J. Molecular Endocrinology 25:169-193, which is incorporated herein by reference as a whole), solid-phase PCR, TAIL (thermal asymmetric interlaced) PCR, and touchdown PCR (see, for example, Don, et al., Nucleic Acids Research, each of which is incorporated herein by reference as a whole). See, but are not limited to, (1991) 19(14) 4008, Roux, K. (1994) Biotechniques 16(5) 812-814, Hecker, et al., (1996) Biotechniques 20(3) 478-485. Amplification of polynucleotides can also be achieved using digital PCR (see, for example, Kalinina, et al., Nucleic Acids Research. 25;1999-2004, (1997), Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96;9236-41, (1999), International Patent Publication WO05023091A2, and U.S. Patent Application Publication 20070202525; these are each incorporated herein by reference as a whole).

[0089] The term "polymerase chain reaction" ("PCR") refers to the methods described in KBMullis's U.S. Patents 4,683,195, 4,683,202, and 4,965,188, which describe a method for increasing the concentration of a segment of a target sequence in a genome or other mixture of DNA or RNA without cloning or purification. This process of amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by thermal cycling in a specific sequence in the presence of DNA polymerase. The two primers are complementary to each strand of the double-stranded target sequence. To bring about amplification, the mixture is denatured, and then the primers are annealed to the complementary sequence within the target molecule. After annealing, the primers are extended with polymerase to form a new complementary strand pair. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times to obtain an amplified segment of the desired target sequence at a high concentration (e.g., denaturation, annealing, and extension constitute one “cycle”; many “cycles” may be used). The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers to each other, and therefore this length is a controllable parameter. Due to the repeatable nature of the process, this method is called a “polymerase chain reaction” (“PCR”). Since the desired amplified segment of the target sequence becomes the dominant sequence (in terms of concentration) in the mixture, they are called “PCR amplified” and are “PCR products” or “amplicons”. Those skilled in the art will understand that the term “PCR” encompasses many variations of the initially described method, such as real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single-primer PCR, and AP-PCR (arbitrarily primed PCR).

[0090] As used herein, the term “nucleic acid detection assay” refers to any method for determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assays include DNA sequencing methods, probe hybridization methods, and structure-specific cleavage assays (e.g., INVADER assay (Hologic, Inc.)), each of which is incorporated herein by reference in whole for any purpose, for example, U.S. Patents 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816, Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and U.S. Patent No. 9,096,893), enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Patents No. 6,110,684, No. 5,958,692, and No. 5,851,770, which are incorporated herein by reference as a whole), polymerase chain reaction (PCR) as described above, branched hybridization methods (e.g., Chiron, U.S. Patents No. 5,849,481, No. 5,710,264, No. 5,124,246, and No. 5,624,802, which are incorporated herein by reference as a whole), rolling circle replication (e.g., U.S. Patents No. 6,210,884, No. 6, which are incorporated herein by reference as a whole), U.S. Patent Nos. 183,960 and 6,235,502), NASBA (e.g., U.S. Patent No. 5,409,818, incorporated herein by reference as a whole), Molecular Beacon Technology (e.g., U.S. Patent No. 6,150,097, incorporated herein by reference as a whole), E-sensor Technology (Motorola, U.S. Patents Nos. 6,248,229, 6,221,583, 6,013,170 and 6,063,573, incorporated herein by reference as a whole), Cycling Probe Technology (e.g., U.S. Patents Nos. 5,403,711, 5,011,769 and 5,660,988, incorporated herein by reference as a whole), Dade This includes, but is not limited to, Behring signal amplification methods (e.g., U.S. Patents 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated herein by reference as a whole), ligase chain reactions (e.g., Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)), and sandwich hybridization methods (e.g., U.S. Patent 5,288,609, which is incorporated herein by reference as a whole).

[0091] In some embodiments, a target nucleic acid is amplified (e.g., by PCR), and the amplified nucleic acid is simultaneously detected using an invasive cleavage assay. Assays configured to perform a detection assay (e.g., an invasive cleavage assay) in combination with an amplification assay are described in U.S. Patent No. 9,096,893, which is incorporated herein by reference in whole for all purposes. Further detection configurations combining amplification and invasive cleavage, known as the QuARTS method, are described, for example, in U.S. Patents No. 8,361,720, 8,715,937, 8,916,344, 9,212,392, and U.S. Patent Application No. 15 / 841,006, which are each incorporated herein by reference for all purposes. As used herein, the term “invasive cleavage structure” refers to a cleavage structure comprising i) a target nucleic acid, ii) an upstream nucleic acid (e.g., an invasive or “INVADER” oligonucleotide), and iii) a downstream nucleic acid (e.g., a probe), wherein the upstream and downstream nucleic acids anneal to a continuous region of the target nucleic acid, resulting in duplication between the 3' portion of the upstream nucleic acid and the resulting double helix between the downstream nucleic acid and the target nucleic acid. The duplication occurs where one or more bases in the upstream and downstream nucleic acids occupy the same position relative to the target nucleic acid bases, regardless of whether the duplicated base(s) of the upstream nucleic acid are complementary to the target nucleic acid, and regardless of whether these bases are native or non-native bases. In some embodiments, the 3' portion of the upstream nucleic acid that duplicates with the downstream double helix is ​​a non-basic chemical portion, such as an aromatic ring structure, as disclosed, for example, in U.S. Patent No. 6,090,543, which is incorporated herein by reference in its entirety. In some embodiments, one or more nucleic acids may be linked to each other via covalent bonds, such as nucleic acid stem-loops, or via non-nucleic acid chemical bonds (e.g., multi-carbon chains). As used herein, the term “flap endonuclease assay” includes the “INVADER” invasive dissection assay and the QuarTS assay, as described above.

[0092] The terms "probe oligonucleotide" or "flap oligonucleotide," when used in relation to flap assays, refer to oligonucleotides that interact with a target nucleic acid in the presence of an invasive oligonucleotide to form a cleavage structure.

[0093] The term "invasive oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a position adjacent to the hybridization region between the probe and the target nucleic acid, where the 3' end of the invasive oligonucleotide includes a portion (e.g., a chemical moiety or one or more nucleotides) that overlaps with the hybridization region between the probe and the target. The 3' terminal nucleotide of the invasive oligonucleotide may or may not form a base pair with the target nucleotide. In some embodiments, the invasive oligonucleotide includes a sequence at its 3' end that is substantially identical to the sequence located at the 5' end of a portion of the probe oligonucleotide that anneals to the target chain.

[0094] As used herein, the terms “flap endonuclease” or “FEN” refer to a class of nucleases, typically 5' nucleases, that act as structure-specific endonucleases on DNA structures having a double helix containing a single-stranded 5' overhang, or flap, on one strand, displacing this one strand with the other nucleic acid strand (for example, so that there are duplicate nucleotides at the junction between single-stranded and double-stranded DNA). FENs catalyze the hydrolytic cleavage of phosphodiester bonds at the junction between single-stranded and double-stranded DNA, releasing the overhang, or flap. Flap endonucleases are described by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al (Annu. This is outlined in Rev. Biochem. 2004 73: 589-615 (which is incorporated herein in whole by reference). FENs may exist as individual enzymes, multi-subunit enzymes, or as the activity of another enzyme or protein complex (e.g., DNA polymerase).

[0095] Flap endonucleases can be heat-resistant. For example, FEN-1 flap endonucleases from thermophilic organisms in the archives are typically heat-resistant. As used herein, the term "FEN-1" refers to non-polymerase flap endonucleases of eukaryotic or archaeal origin. See, for example, WO02 / 070755, U.S. Patent No. 7,122,364, and Kaiser MW, et al. (1999) J. Biol. Chem., 274:21387. All of these are incorporated herein by reference in whole for all purposes.

[0096] As used herein, the term “cleaved flap” refers to a single-stranded oligonucleotide that is a cleavage product of a flap assay.

[0097] When used in relation to a flap cleavage reaction, the term "cassette" refers to an oligonucleotide or combination of oligonucleotides configured to generate a detectable signal in response to the cleavage of a flap or probe oligonucleotide in a primary or first cleavage structure formed in a flap cleavage assay. In a preferred embodiment, the cassette hybridizes with a non-target cleavage product generated by the cleavage of the flap oligonucleotide to form a second overlapping cleavage structure, thereby allowing the cassette to be cleaved by the same enzyme, such as FEN-1 endonuclease.

[0098] In some embodiments, the cassette is a single oligonucleotide containing a hairpin portion (i.e., a region in which a portion of the cassette oligonucleotide hybridizes to a second portion of the same oligonucleotide under reaction conditions to form a double helix). In other embodiments, the cassette contains at least two oligonucleotides, each containing a complementary portion that can form a double helix under reaction conditions. In preferred embodiments, the cassette contains a label, such as a fluorophore. In particularly preferred embodiments, the cassette contains a labeled portion that produces the FRET effect.

[0099] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, i.e., the process by which multiple parts (e.g., fluorophores) transfer energy, for example, between them, or from one fluorophore to another (e.g., a quencher molecule). In some situations, FRET involves an excited donor fluorophore transferring energy to a lower-energy acceptor fluorophore via short-range (e.g., less than approximately 10 nm) dipole interactions. In other situations, FRET involves a loss of fluorescence energy from the donor and an increase in fluorescence from the acceptor fluorophore. In yet another form of FRET, energy may be exchanged from an excited donor fluorophore to a non-fluorescent molecule (e.g., a "dark" quench molecule, e.g., "BHQ" quencher, Biosearch Technologies). FRET is known and described to those skilled in the art (for example, each is incorporated herein by reference as a whole: Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50, Olivier, 2005 Mutant Res 573, See pages 103-110).

[0100] In an exemplary flap detection assay, an invasive oligonucleotide and a flap oligonucleotide are hybridized to a target nucleic acid to generate a first complex with the duplication described above. An unpaired "flap" is contained in the 5' end of the flap oligonucleotide. The first complex is a substrate for a flap endonuclease, such as FEN-1 endonuclease, which cleaves the flap oligonucleotide to release the 5' flap portion. In a secondary reaction, the released 5' flap product functions as an invasive oligonucleotide on the FRET cassette, creating a structure that is recognized by the flap endonuclease so that the FRET cassette is cleaved. When the fluorophores and quencher are separated by the cleavage of the FRET cassette, a detectable fluorescence signal is generated that exceeds the background fluorescence.

[0101] As used herein, the term "PCR-flap assay" refers to an assay configuration that combines targeted amplification by PCR with the detection of the amplified DNA by the formation of a second duplicate cleavage structure comprising a first duplicate cleavage structure containing the amplified target DNA, a 5' flap cleaved from the first duplicate cleavage structure, and a labeled reporter oligonucleotide, such as a "FRET cassette" or a 5' hairpin FRET reporter oligonucleotide. In the PCR-flap assay as used herein, the assay reagent comprises a mixture containing DNA polymerase, FEN-1 endonuclease, a primary probe containing a portion complementary to the target nucleic acid, and a FRET cassette or a 5' hairpin FRET reporter, wherein the target nucleic acid is amplified by PCR and the amplified nucleic acid is detected simultaneously (i.e., detection occurs during the process of targeted amplification). PCR-flap assays include the QuARTS assay described in U.S. Patent Nos. 8,361,720, 8,715,937, and 8,916,344, and the probe oligonucleotide with a longer target-specific region described in U.S. Patent No. 10,648,025 (Long probe). This includes flap assays using Quantitative Amplified Signal ("LQAS") and amplification assays such as those described in U.S. Patent No. 9,096,893 (for example, as illustrated in Figure 1 of the said patent), each of which is incorporated herein by reference in its entirety.

[0102] As used herein, the term "PCR-flap assay reagent" means one or more reagents for detecting a target sequence in a PCR-flap assay, the reagent comprising a mixture containing DNA polymerase, FEN-1 endonuclease, and a FRET cassette or 5' hairpin FRET reporter, and a nucleic acid molecule capable of participating in the amplification of a target nucleic acid and the formation of a flap cleavage structure in the presence of the target sequence.

[0103] As used herein with respect to the detection of nucleic acid amplification or signal amplification, the term “real-time” refers to the detection or measurement of product or signal accumulation during the course of a reaction, for example, in a reaction during incubation or thermal cycling. Such detection or measurement may be performed sequentially, at multiple distinct points in time during the amplification reaction, or in combination. For example, in a polymerase chain reaction, detection (e.g., detection of fluorescence) may be performed sequentially throughout or in part of the thermal cycling, or transiently at one or more points in one or more cycles. In some embodiments, real-time detection of a PCR or QuARTS reaction is achieved by determining the level of fluorescence at the same point (e.g., at a point in time during a cycle, or at a temperature step during a cycle) in each or all of multiple cycles. Real-time detection of amplification is sometimes referred to as detection “during” the amplification reaction.

[0104] As used herein, the term “quantitative amplification dataset” refers to data obtained during quantitative amplification of a target sample, such as target DNA. In the case of quantitative PCR or QuARTS assays, the quantitative amplification dataset is a collection of fluorescence values ​​obtained during amplification, for example, during multiple or all thermal cycles. The quantitative amplification data is not limited to data collected at any particular point in the reaction; fluorescence may be measured at individual points in each cycle or continuously throughout each cycle.

[0105] As used herein with respect to data collected during real-time PCR and PCR+INVADER assays, the abbreviations "Ct" and "Cp" refer to the cycle in which a signal (e.g., a fluorescence signal) exceeds a predetermined threshold indicating a positive signal. Various methods are used to calculate the threshold used as a determinant of signal versus concentration, and this value is generally expressed as either the "excess threshold" (Ct) or the "excess point" (Cp). In embodiments of the methods presented herein, either the Cp or Ct value can be used to analyze the real-time signal and determine the percentage of variant and / or non-variant components in the assay or sample.

[0106] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of a reaction assay, such a delivery system includes a system that enables the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc., in appropriate containers) and / or auxiliary materials (e.g., buffers, instructions for performing the assay, etc.). For example, a kit includes one or more storage means (e.g., a box) for containing the relevant reaction reagents and / or auxiliary materials. As used herein, the term “fragmentation kit” refers to a delivery system comprising two or more separate containers, each containing a small portion of the entire kit components. The containers may be delivered together or separately to the intended recipient. For example, the first container may contain an enzyme for use in an assay, and the second container may contain an oligonucleotide.

[0107] As used herein, the term “system” refers to a collection of articles for use for a particular purpose. In some embodiments, the articles include, for example, instructions for use as information provided on articles, paper, or a recordable medium (e.g., DVD, CD, flash drive, etc.). In some embodiments, the instructions direct the user to an online location, such as a website.

[0108] As used herein, the term “information” refers to any collection of facts or data. With respect to information stored or processed using computer systems, including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information relating to a subject” refers to facts or data relating to a subject (e.g., human, plant, or animal). The term “genomic information” refers to information relating to a genome, including but not limited to nucleic acid sequences, genes, methylation percentages, allele frequencies, RNA expression levels, protein expression, and phenotypes correlated with genotype. “Allele frequency information” refers to facts or data relating to allele frequencies, including but not limited to allele identity, statistical correlations between the presence of an allele and a trait of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, and the percentage of likelihood that an allele is present in an individual having one or more specific traits. [Brief explanation of the drawing]

[0109] [Figure 1-1] A table comparing the results of RRBS (Reduced Representation Bisulfite Sequencing) for selecting lung cancer-related markers described in Example 2 is presented, with each row showing the mean value of the indicated marker region (identified by chromosome and start and stop positions). The ratio of the mean methylation of each histological type (normal (Norm), adenocarcinoma (Ad), large cell carcinoma (LC), small cell carcinoma (SC), squamous cell carcinoma (SQ), and unidentified carcinoma (UND)) compared to the mean methylation of buffy coat samples (WBC or BC) derived from normal subjects is shown for each region, and the genes and transcripts identified in each region are indicated. A table comparing the results of RRBS for selecting lung adenocarcinoma-related markers is presented. [Figure 1-2] Refer to the column in Figure 1-1. [Figure 1-3] Refer to the column in Figure 1-1. [Figure 1-4] Refer to the column in Figure 1-1. [Figure 1-5] Refer to the column in Figure 1-1. [Figure 1-6] Refer to the column in Figure 1-1. [Figure 2-1] A table comparing the results of RRBS (Reduced Representation Bisulfite Sequencing) for selecting lung cancer-related markers described in Example 2 is presented, with each row showing the mean value of the indicated marker region (identified by chromosome and start and stop positions). The ratio of the mean methylation of each histological type (normal (Norm), adenocarcinoma (Ad), large cell carcinoma (LC), small cell carcinoma (SC), squamous cell carcinoma (SQ), and unidentified carcinoma (UND)) compared to the mean methylation of buffy coat samples (WBC or BC) derived from normal subjects is shown for each region, and the genes and transcripts identified in each region are shown. A table comparing the results of RRBS for selecting markers related to large cell lung cancer is presented. [Figure 2-2] Refer to the column in Figure 2-1. [Figure 2-3] Refer to the column in Figure 2-1. [Figure 2-4] Refer to the column in Figure 2-1. [Figure 2-5] Refer to the column in Figure 2-1. [Figure 2-6] Refer to the column in Figure 2-1. [Figure 2-7] Refer to the column in Figure 2-1. [Figure 2-8] Refer to the column in Figure 2-1. [Figure 2-9] Refer to the column in Figure 2-1. [Figure 2-10] Refer to the column in Figure 2-1. [Figure 2-11] Refer to the column in Figure 2-1. [Figure 2-12] Refer to the column in Figure 2-1. [Figure 2-13] Refer to the column in Figure 2-1. [Figure 2-14] Refer to the column in Figure 2-1. [Figure 2-15] Refer to the column in Figure 2-1. [Figure 2-16] Refer to the column in Figure 2-1. [Figure 3-1]A table comparing the results of RRBS (Reduced Representation Bisulfite Sequencing) for selecting lung cancer-related markers described in Example 2 is presented, with each row showing the mean value of the indicated marker region (identified by chromosome and start and stop positions). The ratio of the mean methylation of each histological type (normal (Norm), adenocarcinoma (Ad), large cell carcinoma (LC), small cell carcinoma (SC), squamous cell carcinoma (SQ), and unidentified carcinoma (UND)) compared to the mean methylation of buffy coat samples (WBC or BC) derived from normal subjects is shown for each region, and the genes and transcripts identified in each region are shown. A table comparing the results of RRBS for selecting markers related to small cell lung cancer is presented. [Figure 3-2] Refer to the column in Figure 3-1. [Figure 3-3] Refer to the column in Figure 3-1. [Figure 3-4] Refer to the column in Figure 3-1. [Figure 3-5] Refer to the column in Figure 3-1. [Figure 3-6] Refer to the column in Figure 3-1. [Figure 3-7] Refer to the column in Figure 3-1. [Figure 3-8] Refer to the column in Figure 3-1. [Figure 3-9] Refer to the column in Figure 3-1. [Figure 3-10] Refer to the column in Figure 3-1. [Figure 3-11] Refer to the column in Figure 3-1. [Figure 3-12] Refer to the column in Figure 3-1. [Figure 3-13] Refer to the column in Figure 3-1. [Figure 3-14] Refer to the column in Figure 3-1. [Figure 3-15] Refer to the column in Figure 3-1. [Figure 3-16] Refer to the column in Figure 3-1. [Figure 3-17] Refer to the column in Figure 3-1. [Figure 3-18] Refer to the column in Figure 3-1. [Figure 3-19] Refer to the column in Figure 3-1. [Figure 3-20] Refer to the column in Figure 3-1. [Figure 3-21] Refer to the column in Figure 3-1. [Figure 3-22] Refer to the column in Figure 3-1. [Figure 3-23] Refer to the column in Figure 3-1. [Figure 3-24] Refer to the column in Figure 3-1. [Figure 3-25] Refer to the column in Figure 3-1. [Figure 3-26] Refer to the column in Figure 3-1. [Figure 3-27] Refer to the column in Figure 3-1. [Figure 3-28] Refer to the column in Figure 3-1. [Figure 3-29] Refer to the column in Figure 3-1. [Figure 3-30] Refer to the column in Figure 3-1. [Figure 3-31] Refer to the column in Figure 3-1. [Figure 3-32] Refer to the column in Figure 3-1. [Figure 3-33] Refer to the column in Figure 3-1. [Figure 3-34] Refer to the column in Figure 3-1. [Figure 4-1] A table comparing the results of RRBS (Reduced Representation Bisulfite Sequencing) for selecting lung cancer-related markers described in Example 2 is presented, with each row showing the mean value of the indicated marker region (identified by chromosome and start and stop positions). The ratio of the mean methylation of each histological type (normal (Norm), adenocarcinoma (Ad), large cell carcinoma (LC), small cell carcinoma (SC), squamous cell carcinoma (SQ), and unidentified carcinoma (UND)) compared to the mean methylation of buffy coat samples (WBC or BC) derived from normal subjects is shown for each region, and the genes and transcripts identified in each region are shown. A table comparing the results of RRBS for selecting markers related to lung squamous cell carcinoma is presented. [Figure 4-2] Refer to the column in Figure 4-1. [Figure 4-3] Refer to the column in Figure 4-1. [Figure 4-4] Refer to the column in Figure 4-1. [Figure 4-5] Refer to the column in Figure 4-1. [Figure 4-6] Refer to the column in Figure 4-1. [Figure 4-7] Refer to the column in Figure 4-1. [Figure 4-8] Refer to the column in Figure 4-1. [Figure 5-1] A table is presented showing the nucleic acid sequences of the assay target region in both the unconverted and bisulfite-converted forms, as well as the detection oligonucleotides along with their corresponding sequence numbers. While the target nucleic acid, particularly the target DNA (including bisulfite-converted DNA), is shown as single-stranded for convenience, it is understood that embodiments of this technique may include complementary strands of the shown sequences. For example, primers and flap oligonucleotides may be selected to hybridize to the shown target strand or a strand complementary to the shown target strand. [Figure 5-2] Refer to the column in Figure 5-1. [Figure 5-3] Refer to the column in Figure 5-1. [Figure 5-4] Refer to the column in Figure 5-1. [Figure 5-5] Refer to the column in Figure 5-1. [Figure 5-6] Refer to the column in Figure 5-1. [Figure 5-7] Refer to the column in Figure 5-1. [Figure 5-8] Refer to the column in Figure 5-1. [Figure 5-9] Refer to the column in Figure 5-1. [Figure 5-10] Refer to the column in Figure 5-1. [Figure 5-11] Refer to the column in Figure 5-1. [Figure 5-12] Refer to the column in Figure 5-1. [Figure 5-13] Refer to the column in Figure 5-1. [Figure 5-14] Refer to the column in Figure 5-1. [Figure 5-15] Refer to the column in Figure 5-1. [Figure 5-16] Refer to the column in Figure 5-1. [Figure 5-17] Refer to the column in Figure 5-1. [Figure 6]This document illustrates an exemplary workflow for analyzing blood samples to determine a person's lung cancer risk. [Figure 7-1] This panel presents experimental data focusing on FPR1 gene expression detected by RNA. Panel A is a line chart of the training set of data showing the relationship between true positive and false positive cancer rates. Panel B is a line chart of the validation dataset showing the relationship between true positive and false positive cancer rates. Panel C is a dot plot showing FPR1 RNA expression levels in leukocytes taken from non-smokers, normal smokers, and lung cancer patients at different stages, demonstrating the slight sensitivity of normal smokers to tobacco. [Figure 7-2] Refer to the column in Figure 7-1. [Figure 8-1] This panel presents experimental data focusing on the S100A12 gene. Panel A is a line chart of the training set of data showing the relationship between true positive and false positive cancer rates. Panel B is a line chart of the validation dataset showing the relationship between true positive and false positive cancer rates. Panel C is a dot plot showing S100A12 RNA expression levels in leukocytes collected from non-smokers, normal smokers, and lung cancer patients at different stages. [Figure 8-2] Refer to the column in Figure 8-1. [Figure 9-1] This panel presents experimental data focusing on the MMP9 gene. Panel A is a line chart of the training set of data showing the relationship between true positive and false positive cancer rates. Panel B is a line chart of the validation dataset showing the relationship between true positive and false positive cancer rates, demonstrating improvement compared to FPR1. Panel C is a dot plot showing MMP9 RNA expression levels in leukocytes collected from non-smokers, normal smokers, and lung cancer patients at different stages. [Figure 9-2] Refer to the column in Figure 9-1. [Figure 10-1]This panel presents experimental data focusing on the SAT1 gene. Panel A is a line chart of the training set of data showing the relationship between true positive and false positive cancer rates. Panel B is a line chart of the validation dataset showing the relationship between true positive and false positive cancer rates. Panel C is a dot plot showing SAT1 RNA expression levels in leukocytes taken from non-smokers, normal smokers, and lung cancer patients at different stages. [Figure 10-2] Refer to the column in Figure 10-1. [Figure 11] The results of experiments using FPR1 as the target gene and STK4 as the reference gene are shown. Panel A is a dot plot showing the relationship between the FPR1 ratio and the normalization (FPKM) of FPR1 fragments per kilobase per million. Panel B is a line graph showing the ratio of true positive rate to false positive rate for FPR1 compared to STK4. [Figure 12] An exemplary embodiment of the method using S100A12 as the target gene and STK4 as the reference gene is shown. Panel A is a dot plot showing the relationship between the S100A12 ratio and S100A12 FPKM. Panel B is a line graph showing the ratio of true positive rate to false positive rate for S100A12 compared to STK4. [Figure 13] An exemplary embodiment of the method using MMP9 as the target gene and STK4 as the reference gene is shown. Panel A is a dot plot showing the relationship between the MMP9 ratio and MMP9 FPKM. Panel B is a line graph showing the ratio of true positive rate to false positive rate for MMP9 compared to STK4. [Figure 14] This scatter plot shows data comparing RNA expression levels of both S100A12 and MMP9 as target genes in different stages of lung cancer. FPKM normalization was used, and the data includes all samples from both the training and validation sets. [Figure 15] This scatter plot shows data comparing RNA expression levels of both S100A12 and SAT1 as target genes in cancer patients, benign patients, and healthy patients. FPKM normalization was used. The dashed separator lines are for visualization purposes only. [Figure 16] This scatter plot shows data comparing RNA expression levels of both S100A12 and TYMP as target genes in cancer patients, benign patients, and healthy patients. STK4 normalization was used. The dashed separator lines are for visualization purposes only. [Modes for carrying out the invention]

[0110] Provided herein are techniques relating to methods for characterizing a sample or combination of samples derived from a subject, including the selection of marker analytes and the analysis of a sample(s) for multiple different types of marker analytes, e.g., DNA, RNA, and proteins. For example, in some embodiments, the techniques provide a method comprising measuring the amount of at least one methylated marker gene in DNA having a specific methylation status (e.g., methylated or unmethylated) in a sample obtained from a subject, further comprising measuring the amount of at least one RNA marker in a sample obtained from a subject, and assaying for the presence or absence or amount of at least one protein marker in a sample obtained from a subject. In some embodiments, a single sample derived from a subject is analyzed for methylated marker DNA(s), marker RNA(s), and marker protein(s).

[0111] In this detailed description of various embodiments, numerous specific details are described for illustrative purposes so that the disclosed embodiments may be fully understood. However, those skilled in the art will understand that these various embodiments may be practiced with or without these specific details. In other examples, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily understand that the particular order in which the methods are presented and practiced is illustrative, and that any change in this order may still be included within the spirit and scope of the various embodiments disclosed herein.

[0112] All patents, applications, published applications, and other publications referenced herein are incorporated herein by reference as a whole by reference. If any term or phrase is used herein in a manner that contradicts or otherwise distorts the definition given in the patents, applications, published patents, and other publications incorporated herein by reference, the use herein shall prevail over the definition incorporated herein by reference. The following explanation is divided into the following sections: I. RNA marker analysis (including quantitative RNA analysis and quantitative protein analysis), and II. Analysis of Methylation Markers I. RNA Marker Analysis A. Quantitative RNA analysis Embodiments relate to a system and method for determining whether a patient at risk of cancer may have the disease by analyzing nucleic acid expression in the blood, particularly circulating cell-free nucleic acid or nucleic acid expression in immune cells. Determination of a patient who may have cancer can be performed using blood-derived samples for assaying RNA accumulation or expression levels, such analysis can be performed by expression microarrays, nucleic acid sequencing, nCounter, or real-time PCR. In some embodiments, the expression levels of a subset of reference nucleic acids are compared to the expression levels of a subset of target nucleic acids known to be elevated in patients with cancer. A subset of reference nucleic acids may be found by analyzing blood from many disease-free patients and selecting genes that are expressed at stable levels within these patients. A subset of reference nucleic acids can also be found by analyzing solid tissue samples taken from multiple tissue types (e.g., colon, lung, kidney, liver, etc.) and selecting genes that are expressed at stable levels in the patient's blood.

[0113] One embodiment is shown in the flowchart of Figure 6. As shown, process 100 begins with initiation step 105 and then moves to step 110, in which a blood sample is obtained from a human. The blood sample may be taken from a human patient suspected of having lung cancer, but if the patient is known to have lung cancer, a more thorough analysis of the type or stage of cancer may be desired. Process 100 then moves to step 115, where the blood sample to be analyzed is transported to the laboratory in a blood collection tube at room temperature or on ice to minimize sample degradation as much as possible. Once the blood sample is received in the laboratory, process 100 moves to step 120, in which RNA is extracted from the blood, as will be described in more detail below. After the RNA has been extracted, process 100 moves to step 125, where the gene expression levels of one or more target genes and, optionally, one or more reference genes are detected by measuring the level of specific RNA in the sample. The method for detecting gene expression and selecting target and reference genes will be described in more detail below. Once the gene expression level of a specific target gene is determined, process 100 moves to step 130, in which an analysis is performed to determine the risk of patients having or developing lung cancer based on the measurement of the target gene expression level in the patient. Process 100 then terminates in termination step 135.

[0114] In some embodiments, a subset of target genes may be selected by analyzing genes that show increased transcript accumulation or elevated expression levels in blood or solid tumor samples taken from individuals with cancer.

[0115] In some embodiments, the target gene subset includes genes whose transcripts accumulate or whose expression levels decrease in blood or solid tumor samples taken from individuals with cancer.

[0116] In some embodiments, the subset of reference genes includes genes whose transcript accumulation or expression levels are not altered in normal individuals compared to cancer patients. In these embodiments, a subset of target genes whose accumulation or expression levels are elevated in blood or solid tumor specimens is selected in combination with one or more reference genes.

[0117] In some embodiments, aspects of the technology of this disclosure relate to the finding that the RNA expression levels of the formyl peptide receptor gene (FPR1), S100A12, MMP9, SAT1, and TYMP are altered in patients with cancer. For example, the RNA levels of FPR1, S100A12, MMP9, SAT1, and TYMP were found to be elevated in patients with lung cancer, as described below. Furthermore, the RNA level of FPR1 was shown to be elevated compared to the RNA levels of other reference genes such as STK4, ACTB, and HNRNPA1.

[0118] In some embodiments, once the target gene is known, the reference gene can be selected by analyzing a large number of candidates from multiple samples and selecting the one that shows the greatest difference between the target gene and the reference gene in gene expression from cancer patients. In some embodiments, the reference gene may be selected by examining the accumulation or expression levels of transcripts of many genes and finding the one with the lowest variability. In some embodiments, the reference gene is selected not based on its individual accumulation or expression levels, but on the absence of a relative change in its accumulation or expression level in cancer.

[0119] Once the target gene (and, in some embodiments, the reference gene) for a given cancer type is known, its expression profile can be measured in blood samples taken from cancer patients and patients whose cancer is being assayed. Since plasma or leukocytes can be collected and prepared in many primary care clinics without presenting any risks beyond standard blood collection, the relative RNA accumulation or expression levels between the target gene and the reference gene in some embodiments can be valuable cancer biomarkers. Furthermore, if the target gene and reference gene in some embodiments can be assayed with high reliability, they may offer several advantages over current cancer assays. For example, in some embodiments, this method can detect cancers in the early stages of development, cancers with few symptoms, cancers that are difficult to distinguish from benign conditions, or cancers that may be occurring in areas of the body that are not accessible by conventional biopsy assays.

[0120] Increased RNase activity is commonly observed in tumors. This RNase activity may inhibit tumor growth and may be part of the immune system's response to cancer. Cytotoxic T cells may induce apoptosis in cancer cells via IFN-γ, and this apoptosis can lead to the activation of RNases such as RNase L. Cell death via necrosis, which can be caused by hypoxia due to tumor growth, may also contribute to RNase release. Plasma from lung cancer patients is known to have increased RNase activity (Marabella). et al., (1976) “Serum ribonuclease in patients with lung carcinoma,” Journal of Surgical Oncology, 8(6):501-505; Reddi et al. (1976) “Elevated serum ribonuclease in patients with pancreatic cancer,” Proc. Nat'l.Acad. Sci. USA 73(7):2308-2310). It is also known that lung cells contain RNases similar to those found in plasma (Neuwelt et al., (1978) “Possible Sites of Origin of Human Plasma Ribonucleases as Evidenced by Isolation and Partial Characterization of Ribonucleases from Several Human Tissues,” Cancer Research 38:88-93).

[0121] The higher the level of RNase present in plasma, the more rapidly free RNA is degraded. Therefore, the release of RNase may result in a decrease in detectable RNA in plasma RNA preparations. While all RNA can exist at reduced levels, this difference can only be detected with high accuracy if the normal variability of the gene is low. For example, if the normal range of gene expression is 10-100 units, accurately detecting a decrease of 1 unit may be difficult. However, if gene expression is typically between 10-11 units, a decrease of 1 unit is easily detectable (e.g., any value less than 10 units indicates a decrease).

[0122] In some embodiments, the target gene is FPR1. FPR1 plays multiple roles in the lung and cancer. FPR1 is expressed in lung fibroblasts (VanCompernolle et al. (2003) J Immunol. 171(4):2050-6) and is required for wound repair in the lung (Shao (2011) Am J Respir Cell Mol Biol 44:264-269). Fibroblasts are known to be important in both attracting immune cells to fight tumors (Gemperle (2012) PLOSOne 7(11):1-7, e50195) and generating tumor-protective stroma (Wang (2009) Clin Cancer Res 15(21) 6630-6638). FPR1 may also exacerbate the activity of other oncogenes within tumors (Huang (2007) Cancer Res 67(12):5906-5913). Although there is no evidence of overexpression in lung cancer, FPR1 is known to be regulated by RNA stabilization (Mandal (2007) J Immunol 178:2542-2548, Mandal (2005) J Immunol 175:6085-6091). Given these roles, FPR1 RNA may be intentionally secreted by tumor cells to increase tumor growth (e.g., by activating the wound repair system for proliferation or by promoting the growth of protective stroma) or by immune cells to increase the immune response (e.g., by attracting more immune cells).

[0123] In some embodiments, the target gene is S100 calcium-binding protein A12 (S100A12), also known as cargranulin C and EN-RAGE (a newly identified extracellular RAGE-binding protein), particularly associated with innate immune function. S100A12 is expressed by phagocytic cells and released at tissue inflammation sites. It is an endogenous DAMP that becomes pro-inflammatory after being released into the extracellular space following brain injury. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily and is a specific cell surface reaction site for advanced glycation end products (AGEs), which increase with age. The interaction between AGEs and RAGE is associated with chronic inflammation. When RAGE interactions occur in inflammatory and vascular cells, MMP expression increases. The human s100A12 mRNA sequence is publicly available under GenBank acceptance number NM005621. The human S100A12 amino acid sequence is publicly available under GenPept acceptance number NP05612.

[0124] In some embodiments, target genes include myeloid-associated proteins (MRPs) involved in the process of neutrophil migration to inflammatory sites. MRP proteins are a subfamily of S100 proteins, and three members of the MRP family have been further characterized: S100A8, S100A9, and S100A12, with molecular weights of 10.6, 13.5, and 10.4 kDa, respectively, which are abundantly expressed in the neutrophil cytosol and expressed at lower levels in monocytes. S100A8 and S100A9 are also expressed by activated endothelial cells, certain epithelial cells, keratinocytes, and differentiated HL-60 and THP-1 in neutrophils and monocytes. Because MRPs lack a signal peptide sequence, they reside in the cytosol rather than as granules and account for up to 40% of cytosolic proteins. The three MRPs exist as non-covalent homodimers. Furthermore, in the presence of calcium, S100A8 and S100A9 associate to form a non-covalent heterodimer called S100A8 / A9; these are also known as the MRP-8 / 14 complex, calprotectin, p23, and cystic fibrosis antigen. S100A8 is also called MRP-8, L1 antigen light chain, and cargranulin A, and S100A9 is called MRP-14, L1 antigen heavy chain, cystic fibrosis antigen, cargranulin B, and BEE22. Other names for S100A12 include p6, CAAF1, CGRP, MRP-6, EN-RAGE, and cargranulin C.

[0125] The S100 protein family includes 19 members of small (10-14 kDa) acidic calcium-binding proteins. These are characterized by the presence of two EF-hand type calcium-binding motifs, one having two more amino acids than the other. These intracellular proteins contribute to protein phosphorylation, enzymatic activity, and Ca 2+They are involved in regulating homeostasis and the polymerization of intermediate filaments. S100 proteins generally exist as homodimers, but some can form heterodimers. More than half of S100 proteins are also found in the extracellular space, where they exert cytokine-like activity via specific receptors; one was recently characterized as the receptor for advanced glycation end products (RAGE). S100A8 and S100A9 belong to a subset of the S100 protein family called myeloid-related proteins (MRPs) because their expression is almost completely restricted to neutrophils and monocytes, which are products of myeloid precursors.

[0126] High concentrations of MRP in serum can occur in conditions associated with increased circulating neutrophil counts or their activity. Elevated levels of S100A8 / A9 (greater than 1 μg / ml) are observed in the serum of patients with various infectious and inflammatory conditions, including cystic fibrosis, tuberculosis, and juvenile rheumatoid arthritis. These are also expressed at very high levels in the synovial fluid and plasma of patients with rheumatoid arthritis and gout. High levels of MRP (up to 13 μg / ml) are also known to be present in the plasma of patients with chronic myeloid leukemia and chronic lymphocytic leukemia. The presence of these proteins further preceded the appearance of leukemic cells in the blood of relapsed patients. The extracellular presence of S100A8 / A9 suggests that MRP may be released actively or during cell necrosis.

[0127] MRPs are expressed in the cytosol, and it is suggested that they are secreted via alternative pathways. When released into the extracellular environment, MRPs exert pro-inflammatory functions. These activities are common to several other S100 proteins. For example, S100 stimulates the release of the pro-inflammatory cytokine IL-6 from neurons and promotes neurite extension. S100L (S100A2) is chemotactic to eosinophils, and soriacin (S100A7) is chemotactic to neutrophils and T lymphocytes, but not to monocytes. S100A8, S100A9, and S100A8 / A9 are chemotactic to neutrophils, and 10 -9 ~10 -10It shows maximum activity in M. The mouse S100A8, also called CP-10, is 10 -12 M is known to be a good and potent chemotactic factor for mouse myeloid cells due to its activity.

[0128] Furthermore, S100A12 exhibits chemotaxis towards monocytes and neutrophils, and induces the expression of TNF-α and IL-1β in mouse macrophage cell lines. MRP also stimulates leukocyte adhesion to endothelium. S100A9 stimulates neutrophil adhesion to fibrinogen by activating β2 integrin Mac-1.

[0129] Recently, S100A8, S100A12, and S100A8 / A9 have also been demonstrated to stimulate neutrophil adhesion to fibrinogen. Endothelial cells incubated with S100A12 showed increased surface expression of ICAM-1 and VCAM-1, resulting in lymphocyte adhesion to endothelial cells. This induction occurs after NF-κB activation. MRP inhibits oxidative bursts, either directly or by reacting with oxygen metabolites. S100A9 reduces the level of H2O2 released by peritoneal BCG-stimulated macrophages. This effect can be observed using human and mouse S100A9 but not with S100A8. Unlike S100A9, S100A8 stimulates OCl - It can be efficiently oxidized by anions, leading to the formation of a covalently bonded S100A8 homodimer and the loss of its chemotactic activity (as demonstrated in mouse S100A8).

[0130] Alternatively, because MRPs are cytosolic proteins, they can protect neutrophils from the harmful effects of their own oxidative bursts. S100A9 is also known to be involved in the control of inflammatory pain through its nociceptive activity. The function of MRPs has also been investigated in vivo. When injected intraperitoneally into mice, mouse S100A8 stimulated the accumulation of neutrophils and macrophages within 4 hours. Inhibition of S100A12 reduced acute inflammation in a mouse model of delayed-type hypersensitivity and chronic inflammation in colitis. All MRPs induce an inflammatory response when injected into a mouse air sac model.

[0131] In some embodiments, the target gene encodes a protein of the matrix metalloproteinase (MMP) family, which is involved in the degradation of the extracellular matrix in normal physiological processes such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes such as arthritis and metastasis. Most MMPs are secreted as inactive proproteins that are activated when cleaved by extracellular proteinases. The enzyme encoded by this gene degrades type IV and type V collagen. Studies in rhesus monkeys suggest that this enzyme is involved in IL-8-induced recruitment of hematopoietic progenitor cells from bone marrow, and studies in mice suggest a role in tumor-associated tissue remodeling.

[0132] MMPs, particularly MMP9, MMP2, and MMP3, have been linked to cancer for over 40 years. Growing evidence suggests their roles in angiogenesis, lymphangiogenesis, and vascularization—crucial for cancer cell invasion and metastasis—in addition to their role in ECM degradation. For example, MMP9 increases the bioavailability of isolated VEGF bound to its receptor in several cancers, including colorectal and pancreatic cancer. MMP9 also mediates the proteolytic activation of TGF-β, a key growth factor for HCC. Matrix metalloproteinases (MMPs) are proteases that promote the proliferation, migration, invasion, and metastasis of cancer cells (Egeblad and Werb, 2002). Overexpression of MAN1A1 increased MMP9 mRNA expression levels, while overexpression of MAN1C1 decreased MMP9 mRNA expression levels. Because MMPs can degrade all types of extracellular matrix proteins, decreased MMP9 expression implies impaired cell migration and invasion capabilities. Genes known to be involved in metastasis include MMP9 and CTTN. MMP9 is a member of a group of secreted zinc metalloproteinases that degrade collagen in the extracellular matrix in mammals. Elevated expression of MMP9 has been associated with metastasis in many different cancer types (Turner et al. 2000, Osman et al. 2002). CTTN has been shown to be an oncogene located in the 11q13 region that is frequently amplified in squamous cell carcinoma of head and neck cancer and breast cancer (Schuuring et al. 1992, Schuuring et al. 1998).

[0133] In some embodiments, target genes may include tumorigenetic genes, including BMP2 and EGFR. BMP2 is a member of the transforming growth factor beta superfamily, which regulates proliferation, differentiation, and other functions in many cell types. EGFR is one of the most frequently amplified and mutated genes in many different types of cancer, including head and neck SCC (Santani et al. 1991, Dassonville et al. 1993, Grandis and Tweardy 1993). Other identified candidate genes whose role in the metastatic process is not clearly defined include GTSE1 and EEF1A1. GTSE1 is a microtubule-localized protein. Its expression is regulated by the cell cycle, and overexpression can induce accumulation in the G2 / M phase (Monte et al. 2000). GTSE1 has been shown to downregulate the levels and activity of the p53 tumor suppressor protein and suppress its ability to induce apoptosis after DNA damage (Monte et al. 2004). The EEF1A1 gene encodes the alpha subunit of elongation factor 1, which is involved in the binding of aminoacyl-tRNA to the 80S ribosome. The involvement of this gene in tumorigenesis is unclear.

[0134] In some embodiments, the target gene is SAT1. The protein encoded by the SAT1 gene belongs to the acetyltransferase family and is the rate-limiting enzyme in the catabolic pathway of polyamine metabolism. It catalyzes the acetylation of spermidine and spermine and is involved in regulating the intracellular concentration and extracellular transport of polyamines. Deficiency of this gene is associated with bald, spinous follicular keratosis (KFSD). Alternative splice transcripts have been found for this gene.

[0135] In some embodiments, the target gene is TYMP. The TYMP gene (formerly known as ECGF1) provides instructions for producing an enzyme called thymidine phosphorylase. Thymidine is a molecule known as a nucleoside, which is used as a building block of DNA (after chemical modification). Thymidine phosphorylase converts thymidine into two small molecules, 2-deoxyribose 1-phosphate and thymine. This chemical reaction is a crucial step in the degradation of thymidine, which helps regulate the levels of nucleosides within cells. Thymidine phosphorylase plays a vital role in maintaining adequate amounts of thymidine in cellular structures called mitochondria. Mitochondria convert energy from food into a form that cells can use. While most DNA is packaged in chromosomes within the nucleus, mitochondria also have small amounts of their own DNA (called mitochondrial DNA or mtDNA). Mitochondria use nucleosides, including thymidine, to construct new molecules of mtDNA as needed. Approximately 50 mutations in the TYMP gene have been identified in humans with mitochondrial neurogastroenteritis (MNGIE). TYMP mutations significantly reduce or eliminate thymidine phosphorylase activity. A deficiency in this enzyme allows thymidine to accumulate in the body at extremely high levels. Excess thymidine is thought to damage mtDNA, interfering with its normal maintenance and repair. As a result, mutations can accumulate in mtDNA, leading to mtDNA instability. Mitochondria may also have less mtDNA than normal (mtDNA depletion). These genetic changes impair the normal function of mitochondria. While mtDNA abnormalities underlie the gastrointestinal and neurological problems characteristic of MNGIE disorders, it remains unclear how mitochondrial deficiencies cause the specific features of the disorder.

[0136] In some embodiments, the reference gene is STK4. The protein encoded by the STK4 gene is a cytoplasmic kinase structurally similar to yeast Ste20p kinase, acting upstream of the stress-induced mitogen-activated protein kinase cascade. The encoded protein can phosphorylate myelin basic proteins and undergoes autophosphorylation. Caspase cleavage fragments of the encoded protein have been shown to phosphorylate histone H2B. Specific phosphorylation catalyzed by this protein correlates with apoptosis, suggesting that this protein may induce the chromatin condensation observed in this process.

[0137] In some embodiments, the assay uses the following reference genes: PLGLB2, GABARAP, NACA, EIF1, UBB, UBC, CD81, TMBIM6, MYL12B, HSP90B1, CLDN18, RAMP2, MFAP4, FABP4, MARCO, RGL1, ZBTB16, C10orf116, GRK5, AGER, SCGB1A1, HBB, TCF21, GMFG, HYAL1, TEK, GNG 11, one or more of ADH1A, TGFBR3, INPP1, ADH1B, STK4, ACTB, CASC3, SKP1, and HNRNPA1; and one or more of the following target genes: CTSS, FPR1, FPR2, FPRL1, FPRL2, CXCR2, NCF2, S100A12, MMP9, SAT1, TYMP, APOBEC3A, SELL, S100A9, and PADI4.

[0138] Regression can be used to fit data points generated from patient samples to a standard so that the results are expressed in units of the standard. In some embodiments, the standard consists of RNA derived from one or more cell lines. In some embodiments, the standard may consist of synthetic RNA. The number of fragments of each RNA in the standard may be known, and the standardized unit may be the number of RNA molecules present in each target.

[0139] The assay may include components with different detectable labels targeting components of different sequences or similar regions, components targeting different regions of the same gene, or components targeting regions of genes other than those listed in the R1a assay above.

[0140] The results can be evaluated using Viomics' cancer testing decision rules, such as Viomics' NSCLC test. A plot can be created where one axis represents the ratio of a specific target gene to a first reference gene, and the other axis represents the ratio of the target gene to a second reference gene.

[0141] When using cell line controls, the results for NSCLC and normal samples differ significantly from each other. Despite some overlap, NSCLC samples consistently show significantly higher target gene expression-to-reference gene expression ratios than non-cancer samples when compared to cell line controls.

[0142] Similar results can be obtained when using synthetic RNA standards instead of cell line controls. The reduction in duplication may be due to a decrease in the variability of the standards resulting from a reduction in the number of serial dilutions (from 6 to 3). Each step in serial dilution may introduce error.

[0143] The results can also be interpreted as a single ratio between the linear combination of the first target gene expression and the linear combination of the second target gene expression. The decision rule may specify that a score above a given threshold indicates cancer, and a score below the threshold indicates the absence of cancer. A synthetic standard can be designed such that the coefficient of each marker is 1, and each score is calculated as score = target gene / (reference gene 1 + reference gene 2).

[0144] For example, the gene expression levels of genes selected from the list above can be determined from the sample and compared to levels determined from a set of synthetic standards (e.g., a serial dilution series) spanning a range of typically obtained values. For each gene, the gene expression level determined from the patient sample is compared to the gene expression level determined by performing regression analysis on a synthetic standard template and fitting the accumulated level value to each gene. The regression and fitted values ​​are obtained individually for each gene. Once the fitted values ​​are obtained, additional analysis (e.g., calculation of ratios) can be performed.

[0145] These scores can be compared to a threshold, such that a score above the threshold indicates a higher lung cancer risk for the patient sample.

[0146] The correct concentrations, coefficients, and thresholds for each standard can be determined by collecting data from small sample sets from both cancer and non-cancer patients, and then separating them using a linear model. The linear model may be generated via statistical methods such as logistic regression or support vector machines using linear kernel functions, or the linear model may be generated by testing.

[0147] Exclusion criteria may be implemented to ensure that samples meeting the exclusion criteria do not report results. These exclusion criteria may include other tests performed before or after one of the embodiments described. Exclusion criteria may also be based on the results of the tests themselves. For example, in some embodiments, very small amounts of a marker may indicate sample degradation, and an unexpectedly large ratio between the expression levels of two reference genes may indicate contamination. In some embodiments, a sample is excluded if the ratio of two reference genes differs by more than 10, 5, 4, 3, or 2 times compared to the median ratio of the gene accumulation levels.

[0148] In some embodiments, this method may include statistical distance determination. In some embodiments, this method determines assay results (e.g., positive or negative results) based on statistical distance, in contrast to a fixed cutoff determined solely by the ROC curve.

[0149] Based on specificity, the results can be divided into groups (e.g., high confidence, low confidence). This number may be transformed using some simple formula to obtain a numerical confidence score.

[0150] In some embodiments, this method may include models and derivations for predicting the type of cancer present in a patient based on the results of RNA expression combined with demographic or lifestyle attributes.

[0151] RNA extraction method General methods for RNA extraction are disclosed in standard molecular biology textbooks, including Ausubel et al. (1997) Current Protocols of Molecular Biology, John Wiley and Sons. In particular, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, following the manufacturer's instructions (QIAGEN Inc., Valencia, Calif.). For example, total RNA can be isolated from cultured cells using a Qiagen RNeasy mini-column. Numerous RNA isolation kits are commercially available and can be used in the methods of the techniques described herein.

[0152] In some embodiments, RNA in whole blood samples can be extracted using the QIAamp® RNA Blood Mini Kit (Qiagen, Germantown, MD). To purify total RNA from biological materials such as whole blood, the biological material is brought into contact with an RNA lysis / binding solution, and then into contact with a solid support. The biological material is lysed using the RNA lysis / binding solution to release the RNA, which is then added to the solid support. Furthermore, the RNA lysis / binding solution prevents the adverse effects of harmful enzymes such as RNases. The RNA lysis / binding solution can be successfully used to lyse cultured cells or pelletized leukocytes, or to lyse cells adhered to or collected on culture plates such as standard 96-well plates. If the biological material consists of tissue clumps or small particles, the RNA lysis / binding solution can be effectively used to crush such tissue clumps into a slurry due to its effective lysis capacity. The volume of RNA lysis / binding solution may be scaled up or down depending on the cell number or tissue size. Once the biological material is dissolved, the lysate may be added directly to the solid support, or it may be passed through a pre-clear membrane to remove larger particles from the lysate. An example of a suitable product is Gentra Solid Phase RNA Pre-Clear Column (Gentra Systems, Inc., Minneapolis, Minn.).

[0153] Alternatively, the RNA lysis / binding solution may be added directly to the solid support, thereby omitting a step and further simplifying the method. In this latter method, the RNA lysis / binding solution may be applied to the solid support, then dried on the solid support, and then the treated solid support may be brought into contact with the biological material. For example, in one embodiment, a suitable volume of RNA lysis / binding solution is added directly to a solid support placed in a Spin-X® basket (Costar, Corning NY), and this is then placed in a 2 ml spin tube. The solid support is heated at a temperature of 40-80°C for at least 12 hours until dry, then any excess unbound RNA lysis / binding solution is removed, and then stored under dry conditions. The biological material may be added directly to the solid support pretreated with the RNA lysis / binding solution and incubated for at least 1 minute, for example, at least 5 minutes, until it is suitably lysed and the nucleic acid is released and bound to the solid support.

[0154] When biological materials contain cellular or viral material, direct contact with an RNA lysis / binding solution, or contact with a solid support pretreated with an RNA lysis / binding solution, solubilizes and / or ruptures the cell and nuclear membrane or viral coat, thereby releasing nucleic acids and other contaminants such as proteins and phospholipids. The released nucleic acids selectively bind to the solid support in the presence of an RNA-complexed lithium salt. The use of an optional reducing agent can help reduce RNase activity, which may be necessary in tissues rich in RNases.

[0155] After this incubation period, any remaining biological material is optionally removed by suitable means such as centrifugation, pipetting, pressure, or vacuum, or by using these means in combination with an RNA washing solution so that the nucleic acids remain bound to the solid support. Any remaining biological material other than nucleic acids, including proteins and phospholipids, can be removed first by centrifugation. This separates any unbound contaminants in the lysate from the solid support. Multiple washing steps remove substantially all contaminants from the solid support, leaving only the RNA that preferentially bound to the solid support.

[0156] Subsequently, the bound RNA can be eluted using an appropriate amount of RNA eluent known to those skilled in the art. The RNA can then be released from the solid support by centrifugation or by applying pressure or vacuum, and then collected in a suitable container.

[0157] In some embodiments, this method may begin by extracting cfRNA from a patient's sample and assaying the extracted cfRNA. For example, see O'Driscoll, L. et al. (2008) “Feasibility and relevance of global expression profiling of gene transcripts,” which is incorporated herein by reference in its entirety. See "in serum from breast cancer patients using whole genome microarrays and quantitative RT-PCR." Cancer Genomics Proteomics 5:94-104. In some embodiments, consistent and repeatable methods are used to isolate cfRNA from plasma or other RNA sources to ensure data reliability. The protocols listed below may be used to obtain cfRNA from blood, but other methods are also conceivable.

[0158] cfRNA molecules can be purified from plasma or other samples, for example, using Qiagen's QIAamp® Circulating Nucleic Acid Kit. The protocol for this kit is described in the document “QIAamp Circulating Nucleic Acid Handbook,” 2nd edition, January 2011, which is incorporated herein by reference in its entirety. This protocol provides one embodiment of a method for purifying circulating total nucleic acids from 1 mL of plasma. Briefly, a lysis reagent and protease are added together with inactive carrier RNA. Total nucleic acids (DNA and RNA) are bound to a column, the column is washed multiple times, and then eluted from the column.

[0159] For example, the protocol may be performed by following these steps: Pipette 100 μl, 200 μl, or 300 μl of QIAGEN® proteinase K into a 50 ml centrifuge tube. Add 1 ml, 2 ml, or 3 ml of serum or plasma to the 50 ml tube. Add 0.8 ml, 1.6 ml, or 2.4 ml of buffer ACL (containing 1.0 μg of carrier RNA). Close the cap and pulse vortex for 30 seconds to mix, ensuring that a visible vortex forms in the tube. Mix the sample and buffer ACL thoroughly to ensure efficient lysis and obtain a homogeneous solution. Do not interrupt the procedure at this point.

[0160] To begin the lysis incubation, incubate at 60°C for 30 minutes. Return the tube to the bench and add 1.8 ml, 3.6 ml, or 5.4 ml of buffer ACB to the lysate in the tube. Close the cap and pulse vortex for 15-30 seconds to mix thoroughly. Incubate the lysate-buffer ACB mixture in the tube on ice for 5 minutes. Insert the QIAamp® Mini column into the VacConnector of the QIAvac® 24 Plus. Insert the 20 ml tube extender into the open QIAamp® Mini column. Ensure the tube extender is securely inserted into the QIAamp® Mini column to prevent sample leakage.

[0161] Set aside the collection tube for the dry spin described below. Apply the lysate-buffer ACB mixture to the tube extender of the QIAamp® Mini column. Switch on the vacuum pump. Once all the lysate has been completely aspirated into the column, switch off the vacuum pump and release the pressure to 0 mbar. Carefully remove and discard the tube extender. Note that a large amount of sample lysate (approximately 11 ml if starting with a 3 ml sample) may take up to 10 minutes to pass through the QIAamp® Mini membrane under vacuum pressure. To quickly and easily release the vacuum pressure, a vacuum regulator (part of the QIAvac® Connecting System) should be used. To avoid cross-contamination, be careful not to move the tube extender above a nearby QIAamp® Mini column.

[0162] Apply 600 μl of Buffer ACW1 to the QIAamp(registered trademark) Mini column. Leave the column lid open and switch on the vacuum pump. After all of Buffer ACW1 has been drawn into the QIAamp(registered trademark) Mini column, switch off the vacuum pump and release the pressure to 0 mbar. Apply 750 μl of Buffer ACW2 to the QIAamp(registered trademark) Mini column. Leave the column lid open and switch on the vacuum pump. After all of Buffer ACW2 has been drawn into the QIAamp(registered trademark) Mini column, switch off the vacuum pump and release the pressure to 0 mbar. Apply 750 μl of ethanol (96-100%) to the QIAamp(registered trademark) Mini column. Leave the column lid open and switch on the vacuum pump. Once all of the ethanol has been drawn into the spin column, switch off the vacuum pump and release the pressure to 0 mbar. Close the lid of the QIAamp(registered trademark) Mini column. Remove from the vacuum manifold and discard the VacConnector. Place the QIAamp(registered trademark) Mini column into a clean 2 ml collection tube and centrifuge at full speed (20,000 × g; 14,000 rpm) for 3 minutes.

[0163] Place the QIAamp® Mini column into a new 2 ml collection tube. Open the lid and incubate the assembly at 56°C for 10 minutes to allow the membrane to dry completely. Place the QIAamp® Mini column into a clean 1.5 ml elution tube (provided) and discard the 2 ml collection tube from step 14. Carefully apply 20-150 μl of elution buffer AVE to the center of the QIAamp® Mini membrane. Close the lid and incubate at room temperature for 3 minutes. Ensure that the elution buffer AVE is equilibrated to room temperature (15-25°C). When eluting with a small amount (<50 μl), the elution buffer must be dispensed to the center of the membrane to ensure complete elution of bound DNA. The elution volume is flexible and can be adapted according to the requirements of the downstream application. The recovered elution volume will be up to 5 μl less than the elution volume applied to the QIAamp® Mini column. Elute the nucleic acids by centrifugation at full speed (20,000 × g; 14,000 rpm) for 1 minute in a microcentrifuge. The above example, QIAamp® Circulating Nucleic Acid Handbook 1 / 2011, represents the knowledge of those skilled in the art and is illustrative, not limiting. This specification envisions alternative embodiments, including variations of the above method or different methods of cfRNA purification, and the methods and compositions disclosed herein are not limited to any particular cfRNA purification method. An exemplary RNA method is further described in Example 1 below.

[0164] i. Sequencing-based methods for detecting gene expression levels In some embodiments, RNA levels may be assayed using sequencing techniques. Examples of sequencing techniques include pyrosequencing, e.g., the "454" method (Margulies et al., (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376-380, Ronaghi, et al. (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242:84-89), and "Solexa" or Illumina-type sequencing (Fedurco et al., (2006) BTA, a novel reagent for DNA attachment of glass and efficient generation of solid-phase amplified DNA colonies. Nucleic Acid Research 34, e22, Turcatti et al. (2008), A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic Acid Research 36, e25), SOLiD sequencing technology (Shendure, J. et al. (2005) Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728-1732, McKernan, K. et al, (2006) Reagents, methods, and libraries for bead-based sequencing, U.S. Patent Application No. 20080003571), Heliscope technology (Harris, TD et al. (2008) Single-molecule DNA sequencing of a viral genome. Science 320, 106-109), Ion Torrent technology (Rothberg et al., This includes, but is not limited to, one or more technologies such as (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348-352), SMRT sequencing technology (Pacific Biosciences), or GridION nanopore-based sequencing (Oxford Nanopore Technologies, http: / / www.nanoporetech.com / technology / the-gridion-system / the-gridion-system).In some embodiments, any number of so-called “next-generation” DNA sequencing methods may be used, such as those described in Shendure and Ji, “Next-generation DNA sequencing”, Nature Biotechnology 26(10):1135-1145 (2008), or in other techniques available to those skilled in the art. Other methods for determining DNA sequences are also applicable, and the embodiments disclosed herein are not limited to any particular method for determining base identity at a specific locus, but are not limited to any other method.

[0165] In some embodiments, next-generation sequencing (NGS) techniques are used, enabling massively parallel sequencing of cloned amplified molecules and single nucleic acid molecules. Non-limiting examples of NGS include sequencing by synthesis using reversible dye terminators and sequencing by ligation.

[0166] In some embodiments, a ligation reaction composition is formed comprising at least one RNA molecule to be detected, at least one first adapter, at least one second adapter, and a double-stranded RNA ligase. The first adapter comprises a first oligonucleotide containing at least two ribonucleosides at its 3' end and a second oligonucleotide containing a single-stranded portion when the first and second oligonucleotides hybridize together. The second adapter comprises a third oligonucleotide containing a 5' phosphate group and a fourth oligonucleotide containing a single-stranded portion when the third and fourth oligonucleotides hybridize together. The first and second adapters are ligated to the RNA molecule in the ligation reaction composition by the double-stranded RNA ligase to form a ligation product. The first and second adapters anneal to the RNA molecule in a directional manner due to their structure, and each adapter is ligated to the RNA molecule it anneals to simultaneously or nearly simultaneously, rather than sequentially (for example, the second adapter and the RNA molecule are combined with a ligase, and the second adapter is ligated to the 3' end of the RNA molecule, then the first adapter is combined with the ligated RNA molecule and the second adapter, and then the first adapter is ligated to the RNA molecule and the 5' end of the second adapter, with an intervening purification step between the ligation of the second adapter to the RNA molecule and the ligation of the first adapter to the RNA molecule. See, e.g., Elbashir et al, Genes and Development 15: 188-200, 2001, Berezikov et al.) See al., Nat. Genet. Supp. 38: S2-S7, 2006. It should be understood that the order in which the components are added to the ligation reaction composition is not limited, and the components can be added in any order. It should also be understood that during the process of adding the components, the adapter may be ligated with the corresponding RNA molecule in the presence of a ligase before adding all the components of the reaction composition, for example, a second adapter may be ligated with the corresponding RNA molecule in the presence of a ligase before adding the first adapter, and that such a reaction is within the scope intended by this instruction as long as there is no purification step between the ligation of one adapter to the RNA molecule and the ligation of the other adapter to the RNA molecule. RNA-directed DNA polymerase (sometimes called RNA-dependent DNA polymerase) is combined with the ligation product to form a reaction mixture, which is incubated under conditions suitable for the reverse transcript. When a reverse transcript is combined with a ribonuclease, typically ribonuclease H (RNase H), at least a portion of the ribonucleoside is digested from the reverse transcript, forming an amplification template.

[0167] Next, the amplification template is combined with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase (sometimes called a DNA-dependent DNA polymerase) to form an amplification reaction composition. The amplification reaction composition is thermocycled under conditions suitable for enabling the generation of amplification products. In some embodiments, at least one species of amplification product is detected. In some embodiments, a reporter probe and / or nucleic acid dye is used to indirectly detect the presence of at least one RNA species in the sample. In certain embodiments, the amplification reaction composition further includes a reporter probe, such as but not limited to TaqMan® probes, molecular beacons, or Scorpion® primers, or a nucleic acid dye, such as SYBR® Green or other nucleic acid-binding dyes or nucleic acid insertion dyes. In certain embodiments of this teaching, detection includes, but is not limited to, quantitative PCR, real-time or endpoint detection techniques. In some embodiments, the sequences of at least a portion of the amplification product are determined, thereby allowing identification of the corresponding RNA molecule. In some embodiments, a library of amplification products containing library-specific nucleotide sequences is generated from RNA molecules in the starting material, where at least some of the amplification product species share a library-specific identifier, which includes, but is not limited to, a barcode sequence or hybridization tag, or a general marker or affinity tag, such as a library-specific nucleotide sequence. In some embodiments, two or more libraries are combined and analyzed, and the results are then deconvolved based on the library-specific identifier.

[0168] In some embodiments, only one polymerase, i.e., a DNA polymerase possessing both DNA-directed and RNA-directed DNA polymerase activity, is used in the reverse transcription reaction composition, and no additional polymerase is used. In embodiments of other methods, both RNA-directed DNA polymerase and DNA-directed DNA polymerase are added to the reverse transcription reaction composition, and no additional polymerase is added to the amplification reaction composition.

[0169] In some embodiments, a method for detecting RNA molecules in a sample comprises combining a sample with a polypeptide comprising at least one first adapter, at least one second adapter, and double-strand specific RNA ligase activity to form a ligation reaction composition, wherein at least one first adapter and at least one second adapter are ligated to the RNA molecules of the sample to form a ligation product in the same ligation reaction composition, and detecting the RNA molecules of the ligation product or a substitute thereof. In some embodiments, at least one first adapter comprises a first oligonucleotide having a length of 10 to 60 nucleotides and containing at least two ribonucleosides at its 3' end, and a second oligonucleotide having a nucleotide sequence substantially complementary to the first oligonucleotide, further comprising a single-stranded 5' portion of 1 to 8 nucleotides when the first and second oligonucleotides are double-stranded. In some embodiments, at least one second adapter comprises a third oligonucleotide having a length of 10 to 60 nucleotides and containing a 5' phosphate group, and a fourth oligonucleotide having a nucleotide sequence substantially complementary to the third oligonucleotide, and further comprising a single-stranded 3' portion of 1 to 8 nucleotides when the third and fourth oligonucleotides form a double helix. In some embodiments, the single-stranded portion independently has a degenerate nucleotide sequence or a sequence complementary to a portion of the RNA molecule. In some embodiments, the first and third oligonucleotides have different nucleotide sequences. In the ligation reaction composition, the RNA molecule to be detected hybridizes with a single-stranded portion of at least one first adapter and a single-stranded portion of at least one second adapter.

[0170] In some embodiments, detecting an RNA molecule or a substitute comprises: combining a ligation product with i) an RNA-directed DNA polymerase, ii) a DNA polymerase having DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase activity, or iii) an RNA-directed DNA polymerase and a DNA-directed DNA polymerase; reverse transcribing the ligation product to form a reverse transcript; digesting at least some of the ribonucleosides from the reverse transcript using ribonuclease H to form an amplification template; combining the amplification template with at least one forward primer, at least one reverse primer, and, if the ligation product is combined as in i), a DNA-directed DNA polymerase to form an amplification reaction composition; and cycling the amplification reaction composition to form at least one amplification product, determining the sequence of at least a portion of the amplification product, thereby detecting an RNA molecule.

[0171] In some embodiments, a method for generating an RNA library includes combining several different RNA molecules with several first adapter species, several second adapter species, and a double-strand specific RNA ligase to form a ligation reaction composition, wherein at least one first adapter comprises a first oligonucleotide having at least two ribonucleosides at its 3' end and a second oligonucleotide having a single-stranded portion when the first and second oligonucleotides hybridize together, and at least one second adapter comprises a third oligonucleotide having a 5' phosphate group and a fourth oligonucleotide having a single-stranded portion when the third and fourth oligonucleotides hybridize together, and ligating at least one first adapter and at least one second adapter to RNA molecules to form several different ligation product species, wherein the first and second adapters are ligated to RNA molecules in the same ligation reaction composition. The method further comprises combining multiple ligation product species with RNA-directed DNA polymerase, reverse transcribing at least some of the multiple ligation product species to form multiple reverse transcriptate species, digesting at least some ribonucleosides from at least some of the multiple reverse transcriptates using ribonuclease H (RNase H) to form multiple amplification template species, combining the multiple amplification template species with at least one forward primer, at least one reverse primer, and DNA-directed DNA polymerase to form an amplification reaction composition, and cycling the amplification reaction composition to form a library containing multiple amplification product species, wherein at least some of the amplification product species contain identification sequences common to at least some of the other amplification product species in the library.

[0172] In some embodiments, the sequence of at least a portion of the amplified product is determined, thereby detecting the RNA molecule of interest. The term “sequencing” is used herein in a broad sense and refers to any technique known in the Art that enables the identification of the sequence of at least some consecutive nucleotides in at least a portion of RNA, including but not limited to at least a portion of the elongation product or vector insert. Some non-limiting examples of sequencing techniques include Sanger’s dideoxy-terminator method and Maxam and Gilbert’s chemical cleavage method, and variations of these methods, namely, sequencing by hybridization, including but not limited to hybridization of amplified products to microarrays or beads, such as bead arrays, pyrosequencing (see, e.g., Ronaghi et al., Science 281:363-65, 1998), and restriction mapping. Some sequencing methods include electrophoresis, including but not limited to capillary electrophoresis and gel electrophoresis, mass spectrometry, and single-molecule detection. In some embodiments, sequencing includes direct sequencing, duplex sequencing, cycle sequencing, single-base extension sequencing (SBE), solid-phase sequencing, or a combination thereof. In some embodiments, sequencing is performed using, for example, ABI PRISM® 377 DNA Sequencer, ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730xl. The process includes detecting sequencing products using instruments such as a Genetic Analyzer, ABI PRISM® 3700 DNA Analyzer, or Applied Biosystems SOLiD® System (all manufactured by Applied Biosystems), Genome Sequencer 20 System (Roche Applied Science), or a mass spectrometer, but not limited to these. In certain embodiments, sequencing includes emulsion PCR (see, e.g., Williams et al., Nature Methods 3(7):545-50, 2006). In certain embodiments, sequencing includes high-throughput sequencing techniques, such as massively parallel signature sequencing (MPSS), but not limited to these. Descriptions of MPSS can be found, in particular, in Zhou et al., Methods of Molecular Biology 331:285-311, Humana Press Inc., Reinartz et al., Briefings in Functional Genomics and Proteomics, 1:95-104, 2002, and Jongeneel et al., Genome Research 15:1007-14, 2005. In some embodiments, sequencing involves incorporating dNTPs, including but not limited to dATP, dCTP, dGTP, dTTP, dUTP, dITP, or combinations thereof, including the dideoxyribonucleotide version of dNTPs, into the amplification product.

[0173] Further exemplary techniques useful for determining the sequence of at least a portion of a nucleic acid molecule include, but are not limited to, emulsion-based PCR followed by any suitable massively parallel sequencing or other high-throughput techniques. In some embodiments, determining the sequence of at least a portion of the amplified product to detect the corresponding RNA molecule includes quantifying the amplified product. In some embodiments, sequencing is performed using the SOLiD® System (Applied Biosystems) as described, for example, in PCT Patent Application Publication WO06 / 084132 entitled "Reagents, Methods, and Libraries For Bead-Based Sequencing" and WO07 / 121489 entitled "Reagents, Methods, and Libraries for Gel-Free Bead-Based Sequencing". In some embodiments, quantification of the amplified product includes real-time or endpoint quantitative PCR, or both. In some embodiments, quantification of the amplified product includes generating an expression profile of the detected RNA molecule, such as an mRNA expression profile or a miRNA expression profile. In certain embodiments, quantification of amplification products may include, for example, one or more 5'-nuclease assays, including but not limited to TaqMan® Gene Expression Assays and TaqMan® miRNA Assays, which may include, but not limited to, microfluidics devices, including low-density arrays. Any suitable expression profiling technique known in the art can be used in various embodiments of the methods of this disclosure.

[0174] Those skilled in the art will understand that the sequencing methods used are typically not limiting to the methods of the present invention. Rather, any sequencing technique that provides a sequence of at least several consecutive nucleotides of at least a portion of the corresponding amplification product or RNA to be detected, or at least a portion of the vector insert derived from the amplification product, can typically be used in the methods of the present invention. Descriptions of sequencing techniques can be found, among others, in McPherson, particularly Chapter 5; Sambrook and Russell, Ausubel et al.; Siuzdak, The Expanding Role of Mass Spectrometry in Biotechnology, MCC Press, 2003, particularly Chapter 7; and Rapley. In some embodiments, unincorporated primers and / or dNTPs are removed before the sequencing step by enzymatic digestion, including but not limited to digestion with exonuclease I and shrimp alkaline phosphatase, for example, ExoSAP-IT® reagent (USB Corporation). In some embodiments, unincorporated primers, dNTPs, and / or ddNTPs are removed as needed by gel or column purification, sedimentation, filtration, beading, magnetic separation, or hybridization-based pull-out (e.g., ABI PRISM® Duplex® 384 Well F / R Sequence Capture Kit, Applied Biosystems P / N (See 4308082).

[0175] Those skilled in the art will understand that, in certain embodiments, the read length of the sequencing / re-sequencing technique used may be a factor in the size of the RNA molecule that can be effectively detected (see, for example, Kling, Nat. Biotech. 21(12):1425-27). In some embodiments, amplification products generated from RNA molecules in a first sample are labeled with a first identification sequence (sometimes referred to herein as a “barcode”) or other marker, and amplification products generated from RNA molecules in a second sample are labeled with a second identification sequence or a second marker. Amplification products containing the first identification sequence and those containing the second identification sequence are pooled before determining the sequence of the corresponding RNA molecule in the corresponding sample. In certain embodiments, three or more different RNA libraries are combined, each containing an identifier sequence specific to the library in question. In some embodiments, the first adapter, the second adapter, forward primers, reverse primers, or a combination thereof, include an identification sequence or a complement of an identification sequence.

[0176] In some embodiments, sequencing includes the use of commercially available technologies, such as the hybridization sequencing platform from Affymetrix Inc. (Sunnyvale, Calif.), the synthesis sequencing platforms from 454 Life Sciences (Bradford, Conn.), Illumina / Solexa (Hayward, Calif.), and Helicos Biosciences (Cambridge, Mass.), and the ligation sequencing platform from Applied Biosystems (Foster City, Calif.), as described below. In addition to single-molecule sequencing performed using synthesis sequencing from Helicos Biosciences, other single-molecule sequencing technologies include, but are not limited to, Pacific Biosciences' SMRT® technology, ION TORRENT® technology, and nanopore sequencing developed, for example, by Oxford Nanopore Technologies.

[0177] In some embodiments, this method involves (a) hybridizing the multiple first primers to the RNA sample under conditions that a complex is formed between the 3' regions of two or more first primers in a plurality of first primers and two or more RNA molecules in the RNA sample, and the 3' regions of the first primers contain a random nucleotide sequence and a first nucleotide sequence tag; (b) extending the multiple first primers of the complex by reverse transcription, thereby generating complementary DNA (cDNA) molecules for two or more RNA molecules; (c) converting multiple double-stranded polynucleotide molecules containing a second nucleotide sequence tag to two or more cDNA molecules, (i) forming a complex between the 3' overhang of a double-stranded polynucleotide molecule in a plurality of double-stranded polynucleotide molecules and the 3' region of a cDNA molecule, and the 3' overhang contains a second random nucleotide sequence The method includes (ii) hybridizing multiple double-stranded polynucleotide molecules under the condition that (ii) the 5' end of the complementary second strand of a double-stranded polynucleotide molecule in a plurality of double-stranded polynucleotide molecules is adjacent to the 3' end of a cDNA molecule; (d) ligating the 5' end of the complementary second strand of a double-stranded polynucleotide molecule to the 3' ends of two or more cDNA molecules, thereby generating an unbound strand of a double-stranded polynucleotide molecule; (e) removing the unbound strand of a double-stranded polynucleotide molecule, thereby forming a plurality of single-stranded cDNA molecules containing first and second nucleotide sequence tags; and (f) converting the plurality of single-stranded cDNA molecules into double-stranded cDNA molecules, thereby creating a cDNA library representing a specific strand of an RNA molecule in an RNA sample, thereby creating a complementary DNA (cDNA) library representing a specific strand of an RNA molecule in an RNA sample.

[0178] In other embodiments, this method involves (a) hybridizing the multiple first primers to the RNA sample under conditions where a complex is formed between the 3' regions of two or more first primers in a plurality of first primers and two or more RNA molecules in the RNA sample, and the 3' regions of the single-stranded primers contain a random nucleotide sequence and a first nucleotide sequence tag; (b) extending the first primers of the complex by reverse transcription, thereby generating complementary DNA (cDNA) molecules of two or more RNA molecules; and (c) binding double-stranded polynucleotide molecules to cDNA molecules under certain conditions. The method includes (c) binding a double-stranded polynucleotide molecule to a cDNA molecule under conditions where the 5' end of the double-stranded polynucleotide molecule is bound to a cDNA molecule, the RNA molecule is not bound to the 3' end of the double-stranded polynucleotide molecule, and the double-stranded DNA molecule contains a second nucleotide sequence tag; (d) removing the RNA molecule; and (e) synthesizing a complementary second strand DNA molecule from the cDNA molecule, thereby forming a cDNA library that represents a specific strand of the RNA molecule in the RNA sample.

[0179] In some embodiments, the primer may hybridize to the polynucleotide using a non-random sequence, such as a poly-T or poly-A sequence, and the non-random sequence may, in some forms of this embodiment, terminate in a random or non-random non-poly-T or non-poly-T sequence that hybridizes with the target. As another example, the primer may include a sequence that corresponds to either substantially complementary or substantially identical to the exon sequence. When multiple polynucleotides are targeted simultaneously, the primer may be the same as or different from the one used to target multiple polynucleotides.

[0180] In some embodiments, ultra-parallel sequencing utilizes sequencing chemistry based on Illumina's synthesis and reversible terminators (e.g., as described in Bentley et al., Nature 6:53-59

[2009] ). In some embodiments, Illumina's sequencing technique relies on the binding of complementary DNA (cDNA) of an RNA transcript to a planar, optically transparent surface to which an oligonucleotide anchor is bound. The template cDNA is repaired at the ends to produce a 5'-phosphorylated blunt end, and a single A base is added to the 3' end of the blunt phosphorylated DNA fragment using the polymerase activity of a Klenow fragment. This addition prepares a DNA fragment for ligation to an oligonucleotide adapter, having a single T base overhang at the 3' end to increase ligation efficiency. The adapter oligonucleotide is complementary to the flow cell anchor. Under limiting dilution conditions, the adapter-modified single-stranded template DNA is added to the flow cell and immobilized by hybridization to the anchor. By extending the bound DNA fragments and performing bridge amplification, an ultra-high-density sequencing flow cell is created with hundreds of millions of clusters, each containing approximately 1,000 copies of the same template. In one embodiment, complementary DNA (cDNA) is amplified using PCR before being subjected to cluster amplification.

[0181] In some embodiments, templates are sequenced using a robust four-color DNA sequencing technique by synthesis with a reversible terminator equipped with a removable fluorescent dye. High-sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. Short sequence reads of approximately 20–40 bp, e.g., 36 bp, are aligned to a repeat-masked reference genome, and the unique mapping of the short sequence reads to the reference genome is identified using specially developed data analysis pipeline software. An unrepeat-masked reference genome can also be used. Regardless of whether a repeat-masked or unrepeat-masked reference genome is used, only reads that uniquely map to the reference genome are counted. After the completion of the first read, the template can be reconstructed in situ to enable a second read from the opposite end of the fragment. Thus, either single-end sequencing or paired-end sequencing of the DNA fragment may be used. Partial sequencing of DNA fragments present in the sample is performed, and sequence tags containing reads of a predetermined length, e.g., 36 bp, are mapped to a known reference genome and counted. In one embodiment, one end of a cloned amplified copy of a cDNA molecule is sequenced and processed by bioinformatics alignment analysis on an Illumina Genome Analyzer using Efficient Large-Scale Alignment of Nucleotide Databases (ELAND) software.

[0182] ii. PCR-based methods for detecting RNA expression levels Samples generated by RNA extraction methods are highly pure and may be free of PCR inhibitors, making them suitable for qPCR, which is used in some embodiments to assay RNA relative expression as an assay for various types of cancer.

[0183] In some embodiments, these methods involve performing PCR or qPCR to generate amplicons. PCR and qPCR protocols are illustrated herein and can be directly applied to or adapted for use using the compositions described herein for the detection and / or identification of target genes and reference genes.

[0184] Some embodiments provide methods involving quantitative PCR (qPCR) (also known as real-time PCR). qPCR can also offer the advantages of quantitative measurement and reduced time and noise. As used herein, “quantitative PCR” (“qPCR” or more specifically “real-time qPCR”) refers to the direct monitoring of the progress of PCR amplification in progress without the need for repeated sampling of reaction products. In qPCR, reaction products can be monitored via signaling mechanisms (e.g., fluorescence) as they are produced, and tracked after the signal rises above background levels, but before the reaction reaches a plateau. The number of cycles required to achieve a detectable or “threshold” level of fluorescence (referred herein to as the cycle threshold or “CT”) is directly proportional to the concentration of the amplifiable target at the start of the PCR process, enabling the measurement of signal intensity, which provides a real-time measure of the amount of target nucleic acid in the sample.

[0185] To prepare PCR and qPCR reactions, the reaction mixture contains a minimum amount of template nucleic acid (e.g., present in the test sample, except for the negative control described later) and oligonucleotide primers and / or probes, combined with a suitable buffer, salt, etc., and an appropriate concentration of nucleic acid polymerase. As used herein, “nucleic acid polymerase” refers to an enzyme that catalyzes the polymerization of nucleoside triphosphates. Generally, this enzyme initiates synthesis at the 3' end of a primer annealed to the target sequence and proceeds along the template in the 5'-3' direction until synthesis is completed. An appropriate concentration includes those that catalyze this reaction in the methods described herein. Known DNA polymerases useful in the methods disclosed herein include, for example, E. coli DNA polymerase I, T7 DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase, Thermococcus litoralis DNA polymerase, Thermococcus aquaticus (Taq) DNA polymerase, and Pyrococcus furiosus (Pfu) DNA polymerase, FASTSTART (trademark) Taq DNA polymerase, APTATAQ (trademark) DNA polymerase (Roche), KLENTAQ 1 (trademark) DNA polymerase (AB peptides Inc.), HOTGOLDSTAR (trademark) DNA polymerase (Eurogentec), KAPATAQ (trademark) HotStart DNA polymerase, and KAPA2G (trademark) Fast Includes HotStart DNA polymerase (Kapa Biosystems), PHUSION® HotStart DNA polymerase (Finnzymes), etc.

[0186] In addition to the above components, the reaction mixture of the method of the present invention comprises a primer, a probe, and deoxyribonucleoside triphosphate (dNTP).

[0187] Typically, the reaction mixture further comprises four different types of dNTPs corresponding to four naturally occurring nucleoside bases, such as dATP, dTTP, dCTP, and dGTP. In some embodiments, each dNTP is typically present in amounts ranging from about 10 to 5000 μM, usually about 20 to 1000 μM, about 100 to 800 μM, or about 300 to 600 μM.

[0188] The reaction mixture may further include an aqueous buffer medium containing a monovalent ion source, a divalent cation source, and a buffering agent. Any convenient monovalent ion source such as potassium chloride, potassium acetate, ammonium acetate, potassium glutamate, ammonium chloride, or ammonium sulfate can be used. The divalent cation may be magnesium, manganese, or zinc, where the cation is typically magnesium. Any convenient magnesium cation source, such as magnesium chloride or magnesium acetate, can be used. The amount of magnesium present in the buffer may be in the range of 0.5 to 10 mM, and can be in the range of about 1 to about 6 mM, or about 3 to about 5 mM. Typical buffers or salts that may be present in the buffer include Tris, Trisine, HEPES, MOPS, etc., and the amount of buffer is typically in the range of about 5 to 150 mM, usually about 10 to 100 mM, and more commonly about 20 to 50 mM. In certain preferred embodiments, the buffer is present in an amount sufficient to yield a pH in the range of about 6.0 to 9.5, for example, about pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5. Other agents that may be present in the buffer medium include chelating agents such as EDTA and EGTA. In some embodiments, the reaction mixture may include BSA, etc. Furthermore, in some embodiments, the reactants may include antifreeze agents such as trehalose, especially when the reagents are provided as a master mix that can be stored for extended periods.

[0189] When preparing a reaction reaction, various components can be combined in any convenient order. For example, the buffer may be combined with the primer, polymerase, and then the template nucleic acid, or all of the various components may be combined simultaneously to generate the reaction reaction.

[0190] Alternatively, for example, Quantifast PCR Mix (Qiagen), TAQMAN® Universal PCR Master Mix (Applied Biosystems), OMNIMIX® or SMARTMIX® (Cepheid), IQ™ Supermix (Bio-Rad Commercially available pre-mixed reagents, including those from Laboratories, LIGHTCYCLER® FastStart (Roche Applied Science, Indianapolis, IN), or BRILLIANT® QPCR Master Mix (Stratagene, La Jolla, CA), may be used in the methods disclosed herein, either in accordance with the manufacturer's instructions or modified to improve reaction conditions (e.g., by modifying buffer concentration, cation concentration, or dNTP concentration as needed).

[0191] The reaction mixture can be subjected to primer extension reaction conditions ("conditions sufficient to yield a polymerase-based nucleic acid amplification product"), for example, conditions that allow polymerase-mediated primer extension by adding nucleotides to the ends of a primer molecule using a template strand as a template. In many embodiments, the primer extension reaction conditions are amplification conditions, which comprise multiple reaction cycles, each cycle comprising (1) a denaturation step, (2) an annealing step, and (3) a polymerization step. As will be discussed later, in some embodiments, the amplification protocol does not include a specific time dedicated to annealing, but instead includes only specific times dedicated to denaturation and extension. The number of reaction cycles varies depending on the application being performed, but is typically at least 15, more commonly at least 20, and may be up to 60 or more, with the number of different cycles typically ranging from about 20 to 40. In methods where more than about 25, usually more than about 30 cycles are performed, it may be convenient or desirable to introduce additional polymerase into the reaction mixture to maintain conditions suitable for enzymatic primer extension.

[0192] The denaturation step involves heating the reaction mixture to a high temperature and maintaining the mixture at a high temperature for a sufficient amount of time for any double-stranded or hybridized nucleic acids present in the reaction mixture to dissociate. For denaturation, the temperature of the reaction mixture is typically raised to a range of about 85–100°C, usually about 90–98°C, and more commonly about 93–96°C, and maintained at this temperature for a range of about 3–120 seconds, usually starting from about 3 seconds.

[0193] Following denaturation, the reaction mixture can be subjected to conditions sufficient for primer annealing to the template nucleic acid present (if any) in the mixture, and polymerization of nucleotides to the primer ends, such as conditions sufficient for enzymatic production of the primer extension product, in which the primer is extended in the 5' to 3' direction using the nucleic acid to be hybridized as a template. In some embodiments, the annealing and extension processes occur in the same step. The temperature of the reaction mixture, which is lowered to achieve these conditions, is usually selected to yield optimal efficiency and specificity, and is generally in the range of about 50–85°C, typically about 55–70°C, and more commonly about 60–68°C. In some embodiments, the annealing conditions can be maintained for a period of time ranging from about 15 seconds to 30 minutes, typically about 20 seconds to 5 minutes, or about 30 seconds to 1 minute, or for about 30 seconds.

[0194] This step may optionally include one annealing step and one extension step, with the temperature and duration of each step being modified and optimized. In the case of a two-step annealing and extension, the annealing step may proceed as described above. Following the annealing of the primers to the template nucleic acid, the reaction mixture is further subjected to conditions sufficient to polymerize nucleotides at the primer ends as described above. To achieve polymerization conditions, the temperature of the reaction mixture is raised or maintained at a temperature typically in the range of about 65–75°C, usually about 67–73°C, for a period of about 15 seconds–20 minutes, usually about 30 seconds–5 minutes. In some embodiments, the methods disclosed herein do not include separate annealing and extension steps. Rather, these methods include denaturation and extension steps and do not include a step specifically dedicated to annealing.

[0195] The above cycles of denaturation, annealing, and extension can be performed using an automated device, typically known as a thermal cycler. Thermal cyclers that can be used are described elsewhere in this specification, as well as in U.S. Patent Nos. 5,612,473, 5,602,756, 5,538,871, and 5,475,610, the disclosures of which are incorporated herein by reference.

[0196] The methods described herein can also be used to detect a target nucleic acid sequence in non-PCR-based applications, where the target can be immobilized on a solid support. Methods for immobilizing nucleic acid sequences on solid supports are described in Ausubel et al, eds. (1995) Current Protocols in Molecular Biology (Greene Publishing and Wiley-Interscience, NY), and also in protocols provided by manufacturers, e.g., for membranes by Pall Corporation, Schleicher & Schuell, for magnetic beads by Dynal, for culture plates by Costar, Nalgenunc, for bead array platforms by Luminex and Becton Dickinson, and for other supports useful according to the embodiments provided herein by CPG, Inc.

[0197] Changes in the exact amounts of various reagents and conditions (e.g., buffer conditions, cycling times, etc.) for PCR or other suitable amplification procedures that result in similar amplification or detection / quantification results are considered equivalents. In one embodiment, the qPCR detection of a target has a sensitivity to detect less than 50 copies (preferably less than 25 copies, more preferably less than 15 copies, even more preferably less than 10 copies, e.g., 5, 4, 3, 2, or 1 copy) of the target nucleic acid in a sample.

[0198] In some embodiments, this method may include PCR amplification of a template RNA. DNase treatment may be performed to remove DNA contaminants from the RNA sample. The target RNA may be converted to cDNA using reverse transcriptase, and this step may use one or more of the same primers used in the PCR reaction. The target cDNA may be amplified, for example, by a consistent and repeatable method for amplifying plasma-derived cDNA or other cDNA. In some embodiments, one or more targets in the cDNA may be amplified and quantified via Taqman® chemistry. This protocol may not be the only suitable protocol for detecting RNA quantity. However, since changes in the protocol can significantly affect the final result, it may be important to use a consistent protocol for cDNA synthesis and amplification.

[0199] In some embodiments, the Qiagen assay #QF00119602 may be used for qPCR with primers / probes provided according to the manufacturer's protocol. Agilent's Universal RNA may be used as a standard for qPCR.

[0200] RNA standards can be used to normalize results across multiple runs. This standard can be run at different dilutions. In some embodiments, synthetic standards can be used. For example, the normal range and cut-off of one or more markers may be examined, a synthetic standard may be obtained and used directly, or it may be diluted or combined to achieve a level similar to the predicted level, such as the predicted level of a marker. In some embodiments, the synthetic standard is present at a level that is the predicted level in the patient sample, or within one order of magnitude (e.g., 10-fold higher or 10-fold lower) of this level. In some embodiments, the synthetic standard is present at a level that is within a 5-fold difference (5-fold higher or 5-fold lower) of the predicted level for the patient sample. In some embodiments, the synthetic standard is present at a level that is within a 2-fold difference (2-fold higher or 2-fold lower) of the predicted level for the patient sample.

[0201] Many methods can be used to determine the appropriate level of each synthetic RNA in the synthetic standard. In one embodiment, several representative samples may be run and the results (e.g., Ct values or fit values relative to the standard) may be recorded. Then, each synthetic RNA may be run in the same assay and the results may be measured on the same scale as the samples (e.g., Ct score or fit value relative to the standard). Inspection can determine which standard should be used. For example, 50 samples may be run and Ct scores in the range of 33 - 38 may be obtained for a given gene. Standards of 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 copies may result in Ct scores of 24, 28, 32, 36, 40, or 44. Thus, during assay preparation, the 10 5 standard is used, and 10 4 and 10 3Dilution can be decided upon. Using this strategy, only the original standard and two dilutions are needed to cover future samples. A similar method can be used to select appropriate concentrations of other standards in the same multiplex. Using this method, a single standard can be used even if there are large discrepancies between different genes in the multiplex, as different concentrations can be used for each transcript to be assayed. By using the method disclosed herein, a wide range of accumulation levels of transcripts can be assayed by reducing the number of amplification reactions with standard templates.

[0202] For example, if gene A is expected to be in the range of 100–10,000 copies / μl and gene B is expected to be in the range of 1,000,000–100,000,000 copies, a synthetic standard can be created by mixing 10,000 copies of gene A and 100,000,000 copies of gene B, thereby requiring only three standards in a 10-fold dilution series to cover the entire expected range for the sample. Using such a synthetic standard dramatically reduces, in some embodiments, the number of standards or control samples that need to be run in the qPCR reaction plate to generate a standard curve that covers the expected ranges for both gene a and gene B. This method also minimizes the risk of small errors introduced by pipetting due to formulation during serial dilutions.

[0203] In some embodiments, reverse transcriptase PCR (RT-PCR) can be used to determine the RNA levels of biomarkers, such as mRNA or miRNA levels. Using RT-PCR, it is possible to compare such RNA levels of biomarkers in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize gene expression patterns, to distinguish closely related RNAs, and to analyze RNA structure.

[0204] Typically, the first step is to isolate RNA, such as mRNA, from the sample. The starting material may be total RNA isolated from a human sample, such as a human tumor or tumor cell line, and the corresponding normal tissue or cell line, respectively. Therefore, RNA may be isolated from a sample, such as tumor cells or a tumor cell line, and compared to pooled DNA derived from a healthy donor. If the mRNA source is a primary tumor, mRNA can be extracted.

[0205] Gene expression profiling by RT-PCR may involve reverse transcribing an RNA template to cDNA, which is then amplified in a PCR reaction. Commonly used reverse transcriptases include, but are not limited to, avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney's mouse leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, stem-loop primers, or oligo-dT primers, depending on the context and the purpose of expression profiling. For example, extracted RNA can be reverse transcribed using the GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA) according to the manufacturer's instructions. The resulting cDNA can then be used as a template in a subsequent PCR reaction.

[0206] In some embodiments, the PCR step uses a Taq DNA polymerase that has 5'-3' nuclease activity but lacks 3'-5' proofreading end nuclease activity. TaqMan PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equal 5' nuclease activity may be used. Two oligonucleotide primers are used to generate an amplicon typical of the PCR reaction. A third oligonucleotide or probe is designed to detect a nucleotide sequence located between the two PCR primers. This probe is not extendable by the Taq DNA polymerase enzyme and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Laser-induced luminescence from the reporter dye is quenched by the quench dye if the two dyes are on the probe and located close to each other. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resulting probe fragments dissociate in solution, and the signal from the released reporter dye does not exhibit the quenching effect of the second fluorophore. One molecule of the reporter dye is released for each new molecule synthesized, and the detection of the unquenched reporter dye provides a basis for quantitative interpretation of the data.

[0207] In some embodiments, TaqMan® RT-PCR is used, for example, ABI The procedure can be performed using commercially available instruments such as the PRISM 7700® Sequence Detection System® (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA) or a Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In one embodiment, the 5' nuclease procedure is performed using a real-time quantitative PCR device such as the ABI PRISM 7700® Sequence Detection System®. This system consists of a thermocycler, laser, charge-coupled device (CCD), camera, and computer. The system amplifies a 96-well format sample in the thermocycler. During amplification, a laser-induced fluorescence signal is collected in real time through fiber optic cables in all 96 wells and detected by the CCD. The system includes software to operate the instrument and analyze the data. TaqMan data is first expressed as Ct, or threshold cycle. Fluorescence values ​​are recorded for every cycle and represent the amount of product amplified up to that point in the amplification reaction. The first point at which the fluorescence signal is recorded as statistically significant is the threshold cycle (Ct).

[0208] In some embodiments, RT-PCR is performed using an internal standard to minimize the effects of error and inter-sample variability. An ideal internal standard is expressed at a consistent level across different tissues and is unaffected by experimental processing. RNAs frequently used to normalize gene expression patterns are the mRNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin.

[0209] In some embodiments, real-time quantitative PCR can measure the accumulation of PCR products using a double-labeled FRET fluorescence-generating probe (e.g., TaqMan® probe). Real-time PCR is suitable for both quantitative competitive PCR, where internal competitors for each target sequence are used for normalization, and quantitative comparative PCR, which uses normalization genes or housekeeping genes for RT-PCR present in the sample. See, for example, Held et al. (1996) Genome Research 6:986-994.

[0210] In some embodiments, PCR flap assays can be used to measure RNA in a sample. As described in detail in Example 1, the QuARTS and LQAS / TELQAS flap assay technologies combine a polymerase-based targeted DNA amplification process with an invasive cleavage-based signal amplification process. What is described herein is an assay that combines reverse transcription with these flap assay technologies for the quantification of RNA from a sample.

[0211] iii. Alternative methods for detecting gene expression levels In some embodiments, RNA levels may be assayed via hybridization to microarrays, nCounter, or similar methods. For example, one class of arrays commonly used in differential expression studies includes microarrays or oligonucleotide arrays. These arrays utilize numerous probes that are synthesized directly on substrates and used to examine complex RNA or message populations based on the principle of complementary hybridization. Typically, these microarrays provide 16 to 20 sets of oligonucleotide probe pairs of relatively short length (20-25 nucleotides) across selected regions of the gene or nucleotide sequence of interest. Probe pairs used in oligonucleotide arrays also include perfect-match probes and mismatch probes designed to hybridize to the same RNA or message strand. Perfect-match probes contain known sequences that are perfectly complementary to the message of interest, while mismatch probes are similar to perfect-match probes in terms of their sequence, except that they contain at least one mismatch nucleotide that differs from the perfect-match probe. During expression analysis, the hybridization efficiency of messages from a sample nucleotide population is evaluated against perfect match and mismatch probes to simultaneously verify and quantify the expression levels of multiple messages. In some embodiments, the entire gene array is printed on a microarray. In some embodiments, a subset of genes, including at least one of the target genes and at least one of the reference genes, is included in the microarray.

[0212] In some embodiments, the analysis can be facilitated using a device such as the nCounter, provided by Nanostring Technologies, for example. The nCounter analysis system is an integrated system comprising a fully automated preparation station, a digital analyzer, a CodeSet (molecular barcode), and all the reagents and consumables necessary to perform the analysis. Analysis in the nCounter system consists of a single, simple workflow: hybridization in solution, post-hybridization processing, acquisition of digital data, and normalization. In some embodiments, the process is automated. In some embodiments, a custom or pre-designed barcode probe set may be pre-mixed with a comprehensive system control set as part of the analysis.

[0213] In some embodiments, gene expression levels are detected using an in situ hybridization assay. In an in situ hybridization assay, cells are immobilized on a solid support, typically a glass slide. In some embodiments, the cells may be denatured with heat or alkali. The cells are then brought into contact with a hybridization solution at a moderate temperature to allow annealing of a specific labeled probe. The probe is preferably labeled with a radioisotope or a fluorescent reporter.

[0214] In some embodiments, FISH (fluorescence in situ hybridization) uses a fluorescent probe that binds only to portions of sequences that exhibit a high degree of sequence similarity. FISH is a cytogenetic technique used in some embodiments to detect and localize specific polynucleotide sequences within cells. For example, FISH can be used to detect DNA sequences on chromosomes. FISH can also be used to detect and localize specific RNAs, such as mRNA, within tissue samples. FISH shows a fluorescent probe that binds to a specific nucleotide sequence that exhibits a high degree of sequence similarity. A fluorescence microscope can be used to examine whether and where the fluorescent probe is bound. In addition to detecting specific nucleotide sequences, such as translocations, fusions, cleavage, duplication, and other chromosomal abnormalities, FISH helps define spatiotemporal patterns of specific gene copy numbers and / or gene expression within cells and tissues.

[0215] In some embodiments, comparative genome hybridization (CGH) is performed in The dynamics of situ hybridization are used to compare the copy numbers of different DNA or RNA sequences from a sample, or to compare the copy numbers of different DNA or RNA sequences in one sample with the copy numbers of substantially identical sequences in another sample. In many useful applications of CGH, the DNA or RNA is isolated from the cells or cell population of interest. The comparison may be qualitative or quantitative. Copy number information arises from a comparison of hybridization signal intensities between different locations on a reference genome. Methods, techniques, and uses of CGH are described in U.S. Patent No. 6,335,167 and U.S. Patent Application No. 60 / 804,818, the relevant portions of which are incorporated herein by reference.

[0216] B. Quantitative Protein Analysis In some embodiments, the level of gene expression is determined by detecting the level of protein expression. Protein-based detection techniques include immunoaffinity assays. Antibodies can be used to immunoprecipitate a specific protein from a solution sample or to immunoblot a protein separated, for example, by a polyacrylamide gel. Immunocytochemical methods can also be used when detecting specific protein polymorphisms in tissues or cells.

[0217] In other embodiments, alternative antibody-based techniques can be used, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA), as well as sandwich assays using monoclonal or polyclonal antibodies. See, for example, U.S. Patent Nos. 4,376,110 and 4,486,530, which are hereby incorporated by reference in their entireties.

[0218] In some embodiments, immunohistochemistry is used to detect protein levels. Immunohistochemistry (IHC) is a process by which a localized antigen (e.g., a protein) in the cells of a tissue specifically binds an antibody to the antigen within the tissue. The antigen-binding antibody can be conjugated or fused to a tag that enables detection, for example, via visualization. In some embodiments, the tag is an enzyme that can catalyze a colorimetric reaction, such as alkaline phosphatase or horseradish peroxidase. The enzyme can be fused to the antibody or non-covalently attached, for example, using a biotin-avidin system. Alternatively, the antibody may be tagged with a fluorophore such as fluorescein, rhodamine, DyLight Fluor, or Alexa Fluor. The antigen-binding antibody may be directly tagged or may recognize itself to a detection antibody having a tag. Using IHC, one or more proteins can be detected. The expression of a gene product can be related to its staining intensity compared to a control level.

[0219] In some embodiments, protein levels can be detected using liquid chromatography or mass spectrometry. In the HPLC-microscopy tandem mass spectrometry technique, proteolytic digestion is performed on the protein, and the resulting peptide mixture is separated by reverse-phase chromatography. Tandem mass spectrometry is then performed, and the data collected therefrom is analyzed. See Gatlin et al., Anal. Chem., 72:757-763 (2000).

[0220] Several methods and devices for obtaining gene expression level data necessary for carrying out the methods disclosed herein and for use in compositions and kits, as well as single data storage methods or devices, should not be considered as limitations.

[0221] II. Analysis of Methylation Markers In some embodiments, the marker is a region of 100 bases or less, a region of 500 bases or less, a region of 1000 bases or less, a region of 5000 bases or less, or in some embodiments, the marker is 1 base. In some embodiments, the marker is located in a promoter with a high CpG density.

[0222] This technique is not limited by the type of sample. For example, in some embodiments, the sample may be a stool sample, tissue sample, sputum, blood sample (e.g., plasma, serum, whole blood), excrement, or urine sample.

[0223] Furthermore, this technique is not limited to the methods used to determine the methylation status. In some embodiments, the assay includes using methylation-specific polymerase chain reactions, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or targeted capture. In some embodiments, the assay includes the use of methylation-specific oligonucleotides. In some embodiments, this technique uses massively parallel sequencing (e.g., next-generation sequencing), such as synthesis sequencing, real-time (e.g., single-molecule) sequencing, bead emulsion sequencing, or nanopore sequencing, to determine the methylation status.

[0224] This technology provides reagents for detecting variable methylation regions (DMRs).In some embodiments, oligonucleotides are provided, including EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX Chromosomal regions having annotations selected from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329; or the group consisting of ZNF781, BARX1, and EMX1; or SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI The group consists of; SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, and EMX1; SHOX2, SOBP, ZNF781, BTACT, CYP26C1, and DLX4; SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI; ZNF781, BARX1, and EMX1, and SOBP and / or HOXA9; BARX1, FLJ45983, SOBP, HOPX, IFFO1, and ZNF781; and a sequence complementary to a marker selected from any subset of markers defining the group consisting of BARX1, FAM59B, HOXA9, SOBP, and IFFO1.

[0225] One embodiment of the kit is, for example, a bisulfite reagent and EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX A kit is provided comprising a control nucleic acid having a methylation state associated with a subject that does not have cancer (e.g., lung cancer), comprising a chromosomal region having annotations preferably selected from any subset of the markers listed above, from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. In some embodiments, the kit comprises a bisulfite reagent and an oligonucleotide, as described herein. In some embodiments, the kit comprises a bisulfite reagent and a control nucleic acid comprising a sequence of such chromosomal region and having a methylation state associated with a subject having lung cancer.

[0226] This technology relates to embodiments of compositions (e.g., reaction mixtures). In some embodiments, EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX A composition is provided comprising a nucleic acid having an annotation preferably selected from one of the subsets of markers listed above, from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329, and a bisulfite reagent.Some embodiments include EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The present invention provides a composition comprising a nucleic acid having an annotation selected from any subset of markers listed above, preferably from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329, and an oligonucleotide described herein.Some embodiments include EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The present invention provides a composition comprising a nucleic acid having an annotation preferably selected from any subset of the markers listed above, including chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329, and a methylation-specific restriction enzyme.Some embodiments include EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The present invention provides a composition comprising a nucleic acid having an annotation preferably selected from any subset of the markers listed above, including chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329, and a polymerase.

[0227] Further relevant method embodiments for screening neoplasms (e.g., lung cancer) in samples obtained from subjects, e.g., EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12. 526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, T A method is provided which includes: determining the methylation status of a marker in a sample containing a base in a chromosomal region having annotations preferably selected from one of the subsets of markers listed above from BX15 and ZNF329; comparing the methylation status of the marker in a control sample with the methylation status of the marker in a normal control sample derived from a subject without lung cancer; and determining the confidence interval and / or p-value of the difference in methylation status between the control sample and the normal control sample. In some embodiments, the confidence intervals are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.Some embodiments of the method include EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX From chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329, preferably the markers listed above. The present invention provides a method for generating nucleic acid after a bisulfite reaction by reacting a nucleic acid containing a chromosomal region having annotations selected from one of a subsets with a bisulfite reagent; sequencing the nucleic acid after the bisulfite reaction to obtain the nucleotide sequence of the nucleic acid after the bisulfite reaction; comparing the nucleotide sequence of the nucleic acid after the bisulfite reaction with the nucleotide sequence of a nucleic acid containing a chromosomal region derived from a subject without lung cancer to identify the difference between the two sequences; and, if a difference exists, identifying the subject as having a neoplasm.

[0228] This technology provides a system for screening lung cancer in samples obtained from subjects. An exemplary embodiment of the system includes, for example, a system for screening lung cancer in samples obtained from subjects, comprising: an analytical component configured to determine the methylation status of the sample; a software component configured to compare the methylation status of the sample with the methylation status of a control or reference sample recorded in a database; and an alert component configured to warn the user of cancer-related methylation status. In some embodiments, the alert is triggered by multiple assays (e.g., multiple markers, e.g., EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The software component receives the results of determining the methylation status of chromosomal regions having annotations preferably selected from one of the subsets of markers listed above, such as chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329, and calculates a value or result to report based on multiple results.Some embodiments of the EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX are provided herein for use in calculating values ​​or results and / or alerts to be reported to a user (e.g., a physician, nurse, clinician, etc.). A database is provided of weighted parameters associated with each chromosomal region having annotations preferably selected from any subset of the markers listed above, including chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. In some embodiments, all results from multiple assays are reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a composite of one or more results from multiple assays indicating the lung cancer risk of the subject.

[0229] In some embodiments of the system, the samples are EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The system comprises nucleic acids having annotations preferably selected from any subset of markers listed above, including chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. In some embodiments, the system further comprises components for isolating nucleic acids, components for collecting samples, for example, components for collecting stool samples.In some embodiments, the system includes EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX The database includes nucleic acid sequences comprising chromosomal regions having annotations preferably selected from any subset of markers listed above, such as chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. In some embodiments, the database includes nucleic acid sequences derived from subjects who do not have lung cancer.Nucleic acids, for example, each nucleic acid is EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX A set of nucleic acids is also provided that includes sequences containing chromosomal regions having annotations preferably selected from any subset of the markers listed above, such as chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329.

[0230] Embodiments of the related system include a set of nucleic acids as described, and a database of nucleic acid sequences associated with the set of nucleic acids. Some embodiments further include a bisulfite reagent. And some embodiments further include a nucleic acid sequencer.

[0231] In a particular embodiment, a method for characterizing a sample obtained from a human subject, comprising: a) obtaining a sample from a human subject; and b) assaying the methylation status of one or more markers in the sample, wherein the markers are from the following group of markers: EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK 2, TSC22D4, MAX.chr8.124, RASSF1, ST8SIA1, NKX6_2, FAM59B, DIDO1, MAX_Chr1.110, AGRN, SOBP, MAX_chr10.226, Z MIZ1, MAX_chr8.145, MAX_chr10.225, PRDM14, ANGPT1, MAX.chr16.50, PTGDR_9, DOCK2, MAX_chr19.163, ZNF132, MAX A method is provided which includes: a) assaying a base in a chromosomal region having an annotation preferably selected from any subset of markers listed above, from chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329; and c) comparing the methylation status of the assayed marker with the methylation status of the marker assayed in a subject without neoplasms.

[0232] In some embodiments, this technique relates to evaluating the presence and methylation status of one or more of the markers identified herein in a biological sample. These markers include one or more variable methylation regions (DMRs), as described herein. In embodiments of this technique, the methylation status is evaluated. Therefore, the techniques provided herein are not limited to methods for measuring the methylation status of genes. For example, in some embodiments, the methylation status is measured by genome scanning. For example, one method includes restriction landmark genome scanning (Kawai et al. (1994) Mol. Cell. Biol. 14: 7421-7427), and another example includes MSAP-PCR (methylation-specific arbitrarily primed PCR) (Gonzalgo et al. (1997) Cancer Res. 57: 594-599). In some embodiments, changes in the methylation pattern at a specific CpG site are monitored by performing Southern blot analysis of the region of interest after digestion of genomic DNA with methylation-specific restriction enzymes, particularly methylation-sensitive enzymes (digested Southern blot analysis). In some embodiments, the analysis of changes in methylation patterns involves a process that includes digestion of genomic DNA with one or more methylation-specific restriction enzymes and analysis of cleaved or uncleaved regions indicating the methylation status of the region to be analyzed. In some embodiments, the analysis of the processed DNA includes PCR amplification, the amplification result indicating whether or not the DNA was cleaved by the restriction enzyme. In some embodiments, the methylation status of the DNA of interest is analyzed by evaluating one or more of the presence, absence, quantity, size, and sequence of the generated amplification product. For example, Melnikov, et al., (2005) Nucl. Acids Res, 33(10):e93, Hua, et al., (2011) Exp. Mol. Pathol. 91(1):455-60, and Singer-Sam See et al. (1990) Nucl. Acids Res. 18: 687. Furthermore, other techniques utilizing bisulfite treatment of DNA as a starting point for methylation analysis have been reported. These include methylation-specific PCR (MSP) of PCR products amplified from bisulfite-converted DNA (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93: 9821-9826) and restriction enzyme digestion (Sadri and Hornsby (1996) Nucl. Acids Res. 24: 5058-5059, and Xiong and This includes Laird (1997) Nucl. Acids Res. 25: 2532-2534), detection of gene mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1143-1147), and quantification of allele-specific expression (Szabo and Mann (1995)). Genes Dev. 9: 3097-3108, and Singer-Sam et al. PCR techniques have been developed for this purpose (al. (1992) PCR Methods Appl. 1: 160-163). Such techniques use internal primers that anneal to a PCR-generated template and terminate immediately 5' to the single nucleotide being assayed. Methods using the "Quantitative Ms-SNuPE Assay" described in U.S. Patent No. 7,037,650 are used in some embodiments.

[0233] In some embodiments, the design for assaying the methylation status of a marker involves analyzing background methylation at individual CpG loci in the target region of the marker being examined by the assay technique. For example, in some embodiments, a large number of individual copies (e.g., more than 10,000, preferably more than 100,000 individual copies) of marker DNA in a sample isolated from a subject diagnosed with a disease, e.g., cancer, are examined to determine the frequency of methylation, and these data are compared to a similarly large number of individual copies of marker DNA in a sample isolated from a subject without the disease. The frequencies of disease-related methylation and background methylation at individual CpG loci in the marker DNA in the sample can be compared so that CpG loci with higher signal-to-noise ratios, e.g., high detectable methylation and / or reduced background methylation, can be selected for use in the assay design. See, for example, U.S. Patents 9,637,792 and 10,519,510, each incorporated herein by reference as a whole. In some embodiments, a group of CpG loci with high signal-to-noise ratios (e.g., 2, 3, 4, 5, or more individual CpG loci within a marker region) are simultaneously examined by the assay so that all CpG loci must have a pre-determined methylation status (e.g., all must be methylated, or none can be methylated).

[0234] When evaluating methylation status, it is often expressed as the proportion or percentage of individual strands of DNA that are methylated at a specific site (e.g., at a single nucleotide, at a specific region or locus, at a longer sequence of interest, e.g., at a subsequence of approximately 100 bp, 200 bp, 500 bp, 1000 bp or longer) relative to the total population of DNA in the sample containing that particular site. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using a calibrator. A known amount of DNA is then treated with bisulfite, and the resulting methylation-specific sequences are determined using either real-time PCR or other exponential amplification methods, such as the QuARTS assay (e.g., provided by U.S. Patents No. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, and U.S. Patent Application No. 15 / 841,006).

[0235] For example, in some embodiments, the method includes generating a standard curve for an unmethylated target by using an external standard. This standard curve consists of at least two points and relates the real-time Ct value for unmethylated DNA to a known quantitative standard. A second standard curve for methylated targets is then constructed from at least two points and an external standard. This second standard curve relates the Ct of methylated DNA to a known quantitative standard. Next, the Ct values ​​of the test sample are determined for methylated and unmethylated populations, and the genomic equivalent of the DNA is calculated from the standard curves generated by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, for example, (number of methylated DNAs) / (number of methylated DNAs + number of unmethylated DNAs) × 100.

[0236] Furthermore, compositions and kits for carrying out these methods are provided herein. For example, in some embodiments, reagents specific to one or more markers (e.g., primers, probes) are provided individually or in sets (e.g., sets of primer pairs for amplifying multiple markers). Additional reagents for performing detection assays (e.g., QuARTS, PCR, sequencing, bisulfite, or enzymes, buffers, positive and negative controls for performing other assays) may also be provided. In some embodiments, kits are provided containing one or more reagents that are sufficient or useful to carry out the method. Reaction mixtures containing reagents are also provided. In addition, master mix reagent sets are provided, containing multiple reagents that can be added to each other and / or to the test sample to complete the reaction mixture.

[0237] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments include the isolation of nucleic acids described in U.S. Patent Application No. 13 / 470,251, "Isolation of Nucleic Acids," which is incorporated herein by reference in its entirety.

[0238] Genomic DNA can be isolated by any means, including the use of commercially available kits. Briefly, if the DNA of interest is encapsulated in a cell membrane, the biological sample is generally disrupted and lysed by enzymatic, chemical, or mechanical means. Proteins and other contaminants can then be removed from the DNA solution, for example, by digestion with proteinase K. The genomic DNA is then recovered from the solution. This can be carried out by a variety of methods, including salting out, organic extraction, or binding of the DNA to a solid support. The choice of method is influenced by several factors, including time, cost, and the amount of DNA required. All clinical sample types containing neobiotic or preneobiotic material, such as cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, stool, colonic effluent, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof, are suitable for use in the methods of the present invention.

[0239] This technique is not limited to the methods used to prepare samples and prepare nucleic acids for testing. For example, in some embodiments, DNA is isolated from a stool sample, or from a blood or plasma sample, using direct gene capture, such as detailed in U.S. Patent Application No. 61 / 485386, or by related methods.

[0240] This technique relates to the analysis of any sample that may be associated with lung cancer or that may be examined to determine the absence of lung cancer. For example, in some embodiments, the sample includes tissue and / or biological fluids obtained from a patient. In some embodiments, the sample includes secretions. In some embodiments, the sample includes sputum, blood, serum, plasma, gastric secretions, lung tissue samples, lung cells, or lung DNA recovered from feces. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art, as will be obvious to those skilled in the art.

[0241] A. Methylation assay for detecting lung cancer Candidate methylated DNA markers were identified by unbiased whole-methylome sequencing of selected lung cancer cases and lung control tissues. Top candidate markers were further evaluated in 255 independent patients, 119 of whom were controls, 37 of whom were from benign nodules, and 136 cases included all lung cancer subtypes. DNA extracted from patient tissue samples was treated with bisulfite, and then candidate markers and β-actin (ACTB) as a normalization gene were amplified using QuARTS (Quantitative Amplified). The assay was performed using Allele-Specific Real-time Target and Signal amplification. QuARTS assay chemistry provides advanced identification for methylation marker selection and screening.

[0242] Receiver operating characteristic analysis of individual marker candidates showed area under the curve (AUC) in the range of 0.512–0.941. At 100% specificity, a composite panel of eight methylation markers (SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.12.526, HOXB2, and EMX1) yielded 98.5% sensitivity for all subtypes of lung cancer. Furthermore, the use of the eight-marker panel did not result in false positives from benign lung nodules.

[0243] B. Methylation detection assays and kits The markers described herein are used in a variety of methylation detection assays. The most frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is the bisulfite method for the detection of 5-methylcytosine in DNA, described by Frommer et al. (expressly incorporated herein in whole by reference for all purposes, Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-31) or a variation thereof. The bisulfite method for mapping 5-methylcytosine is based on the observation that cytosine reacts with bisulfite ions (also known as bisulfites), but 5-methylcytosine does not. This reaction typically proceeds in the following steps: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, spontaneous deamination of the sulfonation reaction intermediate results in sulfonated uracil. Finally, sulfonated uracil is desulfonated under alkaline conditions to form uracil. Uracil forms a base pair with adenine (and therefore behaves like thymine), while 5-methylcytosine forms a base pair with guanine (and therefore behaves like cytosine), making detection possible. This allows detection by, for example, bisulfite genome sequencing (Grigg G, & Clark S, Bioessays (1994) 16: 431-36, Grigg G, DNA Seq. (1996) 6: 189-98), methylation-specific PCR (MSP) as disclosed in, for example, U.S. Patent No. 5,786,146, or assays including sequence-specific probe cleavage, such as the QuARTS flap endonuclease assay (e.g., Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific Using the technology (see Clin Chem 56: A199, and U.S. Patents No. 8,361,720, 8,715,937, 8,916,344, and 9,212,392), it becomes possible to distinguish methylated cytosine from unmethylated cytosine.

[0244] Some conventional techniques involve encapsulating the DNA to be analyzed in an agarose matrix, thereby preventing DNA diffusion and unraveling (bisulfites only react with single-stranded DNA), and replacing the precipitation and purification steps with rapid dialysis (Olek A, et al. (1996) “A modified and improved method for bisulfite based cytosine methylation analysis” Nucleic Acids Res. 24: 5064-6). Thus, it is possible to analyze the methylation status of individual cells, illustrating the usefulness and sensitivity of this method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26: 2255.

[0245] Bisulfite techniques typically involve amplifying specific short fragments of known nucleic acids following bisulfite treatment, and then assaying the product by either sequencing (Olek & Walter (1997) Nat. Genet. 17: 275-6) or primer extension reaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25: 2529-31, WO95 / 00669, U.S. Patent No. 6,251,594) to analyze individual cytosine positions. Some methods utilize enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25: 2532-4). Detection by hybridization has also been described in the art (Olek et al., WO99 / 28498). Furthermore, the use of bisulfite techniques for detecting methylation in individual genes is described (Grigg & Clark (1994) Bioessays 16: 431-6, Zeschnigk et al. (1997) Hum Mol Genet. 6: 387-95, Feil et al. (1994) Nucleic Acids Res. 22: 695, Martin et al. (1995) Gene 157: 261-4, WO9746705, WO9515373).

[0246] Various methylation assay procedures can be used in conjunction with the bisulfite treatment according to the present invention. These assays enable the determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays include, among many techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific restriction enzymes, such as methylation-sensitive or methylation-dependent enzymes.

[0247] For example, bisulfite treatment has simplified genome sequencing for the analysis of methylation patterns and 5-methylcytosine distribution (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-1831). Furthermore, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used to assess methylation status, as described, for example, by Sadri & Hornsby (1997) Nucl. Acids Res. 24: 5058-5059, or as embodied by the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25: 2532-2534).

[0248] COBRA® analysis is a useful quantitative methylation assay for determining DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, it uses restriction enzyme digestion to reveal methylation-dependent sequence differences in the PCR product of sodium bisulfite-treated DNA. The methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment, following the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). Then, the bisulfite-converted DNA is amplified by PCR using primers specific to the target CpG island, followed by restriction endonuclease digestion, gel electrophoresis, and detection using a specificly labeled hybridization probe. The methylation levels in the original DNA sample are expressed in a linearly quantitative manner across a wide range of DNA methylation levels, as measured by the relative amounts of digested and undigested PCR products. Furthermore, this technique can be applied with high reliability to DNA obtained from micro-dissected paraffin-embedded tissue samples.

[0249] Typical reagents for COBRA® analysis (e.g., those found in a typical COBRA®-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. Furthermore, bisulfite conversion reagents may include DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0250] Assays such as "MethyLight®" (fluorescently labeled real-time PCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE® (Methylation-sensitive Single Nucleotide Primer Extension) reaction (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP", Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, U.S. Patent No. 5,786,146), and methylated CpG island amplification ("MCA", Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.

[0251] The "HeavyMethyl®" assay technique is a quantitative method for evaluating methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") that cover or are covered by the amplification primers at CpG positions between amplification primers enable methylation-specific selective amplification of nucleic acid samples.

[0252] The term "HeavyMethyl®MethyLight® assay" refers to the HeavyMethyl®MethyLight® assay, a variation of the MethyLight® assay, which combines the MethyLight® assay with a methylation-specific blocking probe that covers CpG positions between amplification primers. The HeavyMethyl® assay may also be used in combination with methylation-specific amplification primers.

[0253] Typical reagents for HeavyMethyl® analysis (e.g., those found in a typical MethyLight®-based kit) may include, but are not limited to, PCR primers for a specific locus (e.g., a specific gene, marker, gene region, marker region, bisulfite-treated DNA sequence, CpG island, or bisulfite-treated DNA sequence or CpG island); blocking oligonucleotides; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0254] Methylation-Specific PCR (MSP) allows for the evaluation of the methylation status of virtually all CpG sites within a CpG island, regardless of the use of methylation-specific restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, U.S. Patent No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated cytosine to uracil but not methylated cytosine. This product is then amplified with primers specific to methylated DNA and primers specific to unmethylated DNA. MSP requires only a small amount of DNA, shows sensitivity to 0.1% of methylated alleles in a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents for MSP analysis (e.g., those found in a typical MSP-based kit) may include, but are not limited to, methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxyribonucleotides; and specific probes.

[0255] The MethyLight® assay is a high-throughput quantitative methylation assay utilizing fluorescence-based real-time PCR (e.g., TaqMan®) that requires no further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight® process begins with a mixed sample of genomic DNA, which is converted into a mixed pool of methylation-dependent sequence differences in a sodium bisulfite reaction following a standard procedure (the bisulfite process converts unmethylated cytosine residues to uracil). Subsequently, fluorescence-based PCR is performed in a “biased” reaction, using PCR primers that overlap with known CpG dinucleotides, for example. Sequence identification is performed at both the amplification and fluorescence detection levels.

[0256] The MethyLight® assay is used as a quantitative test for methylation patterns in nucleic acids, such as genomic DNA samples, where sequence identification is performed at the level of probe hybridization. In the quantitative version, a PCR reaction results in methylation-specific amplification in the presence of a fluorescent probe that overlaps with a specific putative methylation site. A control is provided that is unbiased with respect to the amount of input DNA, as neither the primer nor the probe overlaps with any CpG dinucleotides. Alternatively, qualitative testing of genomic methylation can be achieved by probed a biased PCR pool using either a control oligonucleotide that does not cover known methylation sites (e.g., HeavyMethyl® and a fluorescence-based version of the MSP technique) or an oligonucleotide that covers potential methylation sites.

[0257] The MethyLight® process is used with any suitable probe (e.g., TaqMan® probes, Lightcycler® probes, etc.). For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan® probes, e.g., MSP primers and / or HeavyMethylblocker oligonucleotides and TaqMan® probes. TaqMan® probes are double-labeled with fluorescent "reporter" and "quencher" molecules and are designed to be specific to regions with relatively high GC content, so they melt at a temperature approximately 10°C higher than forward or reverse primers during the PCR cycle. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed TaqMan® probe. Subsequently, the 5'-to-3' endonuclease activity of Taq polymerase digests the TaqMan® probe, causing the probe to displace and release a fluorescent reporter molecule. This signal, no longer quenched, is then quantitatively detected using a real-time fluorescence detection system.

[0258] Typical reagents for MethyLight® analysis (e.g., those found in a typical MethyLight®-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxyribonucleotides; and Taq polymerase.

[0259] The QM® (Quantitative Methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, where sequence identification is performed at the level of probe hybridization. In this quantitative version, the PCR reaction provides unbiased amplification in the presence of a fluorescent probe that overlaps with specific putative methylation sites. The reaction, in which neither the primers nor the probe overlap with any CpG dinucleotides, provides an unbiased control relative to the amount of input DNA. Alternatively, qualitative testing of genomic methylation is achieved by probe a biased PCR pool using either a control oligonucleotide that does not cover known methylation sites (HeavyMethyl® and a fluorescence-based version of the MSP technique) or an oligonucleotide that covers potential methylation sites.

[0260] The QM® process can be used in the amplification process with any suitable probe, such as the "TaqMan®" probe or the Lightcycler® probe. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and the TaqMan® probe. The TaqMan® probe is double-labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific to regions with relatively high GC content, so that it melts at a temperature approximately 10°C higher than the forward or reverse primer during the PCR cycle. This makes it possible to keep the TaqMan® probe completely hybridized during the PCR annealing / extension step. As the Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed TaqMan® probe. Subsequently, the 5'-to-3' endonuclease activity of Taq polymerase digests the TaqMan® probe, displacing the probe and releasing a fluorescent reporter molecule, the signal of which is no longer quenched, which is quantitatively detected using a real-time fluorescence detection system. Typical reagents for QM® analysis (e.g., those that may be found in a typical QM®-based kit) may include, but are not limited to, PCR primers for a specific locus (e.g., a specific gene, marker, gene region, marker region, bisulfite-treated DNA sequence, CpG island, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffer and deoxyribonucleotides; and Taq polymerase.

[0261] The Ms-SNuPE™ technique is a quantitative method for evaluating differences in methylation at specific CpG sites based on bisulfite treatment of DNA followed by single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil, while 5-methylcytosine remains unchanged. Then, PCR primers specific to the bisulfite-converted DNA are used to amplify the desired target sequence, and the resulting product is isolated and used as a template for analysis of methylation at the target CpG site. Small amounts of DNA (e.g., micro-dissected lesion sections) can be analyzed, thus avoiding the use of restriction enzymes to determine the methylation status of CpG sites.

[0262] Typical reagents for Ms-SNuPE® analysis (e.g., those found in a typical Ms-SNuPE®-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffer and deoxyribonucleotides; gel extraction kits; positive control primers; Ms-SNuPE® primers for specific loci; reaction buffer (for Ms-SNuPE reactions); and labeled nucleotides. Furthermore, bisulfite conversion reagents may include DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffer; and DNA recovery components.

[0263] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion (e.g., by an enzyme that recognizes sites containing CG sequences, such as MspI), coupling with an adapter ligand, and then complete sequencing of the fragments. Restriction enzyme selection enriches the fragments for CpG-dense regions, reducing the number of extra sequences that could be mapped to multiple gene locations during analysis. Therefore, RRBS reduces the complexity of nucleic acid samples by selecting a subset of restriction fragments for sequencing (e.g., by size selection using preparative gel electrophoresis). In contrast to whole-genome bisulfite sequencing, all fragments produced by restriction enzyme digestion contain DNA methylation information for at least one CpG dinucleotide. Thus, RRBS enriches the sample for promoters, CpG islands, and other genomic features containing high-frequency restriction enzyme cleavage sites within these regions, thus providing an assay for evaluating the methylation status of one or more genomic loci.

[0264] A typical RRBS protocol includes the steps of digesting nucleic acid samples with restriction enzymes such as MspI, overhang filling and A-tailing, adapter ligation, bisulfite conversion, and PCR. For example, et al. (2005) “Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution” Nat Methods 7: 133-6, Meissner et al. (2005) “Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis” Nucleic Acids Res. 33: See 5868-77.

[0265] In some embodiments, the methylation status is evaluated using the QuARTS (quantitative allele-specific real-time target and signal amplification) assay. Three reactions occur sequentially in each QuARTS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in the first reaction, and FRET cleavage and fluorescence signal generation (reaction 3) in the second reaction. When the target nucleic acid is amplified with a specific primer, a specific detection probe containing a flap sequence loosely binds to the amplicon. If an invasive oligonucleotide specific to the target binding site is present, a 5' nuclease, such as FEN-1 endonuclease, releases the flap sequence by cleaving between the detection probe and the flap sequence. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence acts as an invasive oligonucleotide on the FRET cassette, resulting in cleavage between the fluorophores and quenchers of the FRET cassette, which generates a fluorescence signal. This cleavage reaction can cleave multiple probes per target and thus release multiple fluorophores per flap, resulting in exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes containing different dyes. See, for example, Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56: A199), each of which is incorporated herein by reference for any purpose, as well as U.S. Patents 8,361,720, 8,715,937, 8,916,344, and 9,212,392.

[0266] In some embodiments, bisulfite-treated DNA is purified before quantification. This can be done by any means known in the Art, such as but not limited to ultrafiltration, for example, by means of a Microcon® column (manufactured by Millipore®). Purification is carried out according to a modified manufacturer's protocol (see, for example, PCT / EP2004 / 011715, which is incorporated herein by reference in whole). In some embodiments, bisulfite-treated DNA is bound to a solid support, such as magnetic beads, and desulfonation and washing are performed while the DNA is bound to the support. Examples of such embodiments are provided, for example, in WO2013 / 116375, U.S. Patent No. 9,315,853, and U.S. Patent Application No. 63 / 058,179, each of which is incorporated herein by reference in whole. In certain preferred embodiments, the DNA bound to the support is ready for a methylation assay immediately after desulfonation and washing on the support. In some embodiments, the desulfonated DNA is eluted from the support before the assay.

[0267] In some embodiments, the treated DNA fragments are amplified using a set of primer oligonucleotides according to the present invention (see, for example, Figure 5) and an amplification enzyme. Amplification of several DNA segments can be carried out simultaneously in a single, identical reaction vessel. Typically, amplification is carried out using polymerase chain reaction (PCR).

[0268] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, several embodiments involve the isolation of nucleic acids, as described in U.S. Patents 9,000,146, 9,163,278, and 10,704,081, each of which is incorporated herein by reference as a whole.

[0269] In some embodiments, the markers described herein are used in QUARTS assays performed on stool samples. In some embodiments, methods are provided for generating DNA samples, particularly those containing small amounts (e.g., less than 100, less than 60 microliters) of highly purified and low-abundance nucleic acids, and substantially and / or virtually free of substances that inhibit assays used to examine the DNA sample (e.g., PCR, INVADER, QUARTS assays, etc.). Such DNA samples are used in diagnostic assays to qualitatively detect the presence of genes, gene variants (e.g., alleles), or gene modifications (e.g., methylation) present in a sample taken from a patient, or to quantitatively measure their activity, expression, or quantity. For example, some cancers correlate with the presence of specific variant alleles or specific methylation states, and therefore, the detection and / or quantification of such variant alleles or methylation states has predictive value in the diagnosis and treatment of cancer.

[0270] Many valuable genetic markers are present in very small amounts in samples, and the events that generate such markers are rare. Therefore, even highly sensitive detection methods such as PCR require a large amount of DNA to provide low-abundance targets in sufficient quantities to meet or replace the detection threshold of the assay. Furthermore, even the presence of small amounts of inhibitors impairs the accuracy and precision of these assays, which are intended to detect such low-abundance targets. Accordingly, this specification provides a method for controlling the volume and concentration requirements for generating such DNA samples.

[0271] In some embodiments, the sample includes blood, serum, plasma, or saliva. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art, as will be obvious to those skilled in the art. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to a variety of techniques known to those skilled in the art, including but not limited to centrifugation and filtration. While it is generally preferred not to use invasive techniques to obtain a sample, it may still be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. This technique is not limited to the methods used to prepare the sample and prepare nucleic acids for testing. For example, in some embodiments, DNA is isolated from a stool sample, or from a blood or plasma sample, using direct gene capture, as detailed in, for example, U.S. Patent Nos. 8,808,990 and 9,169,511, and WO2012 / 155072, or by related methods.

[0272] The analysis of markers can be performed separately or simultaneously with further markers within a single test sample. For example, combining several markers in a single test can efficiently process multiple samples and thereby improve the accuracy of diagnosis and / or prognosis. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at consecutive points in time). Testing such consecutive samples can enable the identification of changes in the methylation status of markers over time. Not only changes in methylation status, but also the absence of changes in methylation status can provide useful information about the disease state, including, but not limited to, the approximate time since the onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapy, the identification of the effectiveness of various therapies, and the identification of the subject's outcome, including the risk of future events.

[0273] Biomarker analysis can be performed in a variety of physical formats. For example, the use of microtiter plates or automation can facilitate the processing of a large number of test samples. Alternatively, a single sample format can be developed to facilitate timely emergency treatment and diagnosis, for example, in emergency transport or emergency room situations.

[0274] Embodiments of this technology are intended to be provided in the form of a kit. The kit comprises embodiments such as compositions, devices, and apparatus described herein, and instructions for use of the kit. Such instructions describe appropriate methods for preparing analytes from a sample, for example, for taking a sample and preparing nucleic acids from a sample. The individual components of the kit are packaged in appropriate containers and packages (e.g., vials, boxes, blister packs, ampoules, bottles, tubing, etc.), and the components are packaged together in an appropriate container (e.g., one or more boxes) for convenience of storage, transport, and / or use of the kit by the user. Liquid components (e.g., buffers) may be provided in a lyophilized form for reconstitution by the user. The kit may include controls or references for evaluating, verifying, and / or guaranteeing the performance of the kit. For example, a kit for assaying the amount of nucleic acid present in a sample may include a control containing the same nucleic acid or another nucleic acid at a known concentration for comparison, and in some embodiments, may include a detection reagent (e.g., primer) specific to the control nucleic acid. The kit is suitable for clinical use and, in some embodiments, for use at the user's home. The components of the kit, in some embodiments, provide the functionality of a system for preparing nucleic acid solutions from samples. In some embodiments, specific components of the system are provided by the user.

[0275] III. Application In some embodiments, the diagnostic assay identifies the presence of a disease or condition in an individual. In some embodiments, the disease is cancer (e.g., lung cancer).

[0276] In some embodiments, abnormal methylation is associated with lung cancer in markers (for example, one or more markers selected from the markers listed in Table 1, or preferably EMX1, GRIN2D, ANKRD13B, ZNF781, ZNF671, IFFO1, HOPX, BARX1, HOXA9, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, 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, DOCK2, MAX_chr19.163, ZNF132, MAX One or more of the following are used: chr19.372, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, PTGDR, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, and ZNF329. In some embodiments, the assay further includes the detection of a reference gene (e.g., β-actin, ZDHHC1, B3GALT6; see, for example, U.S. Patent No. 10,465,248 and WO2018 / 017740, each incorporated herein by reference for any purpose).

[0277] In some embodiments, markers whose abnormal expression is associated with lung cancer (preferably one or more markers listed in Table 3: S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1) are used and detected by measuring one or more RNA (e.g., mRNA) or proteins in the sample. In some embodiments, the assay further includes the detection of a reference gene (e.g., as shown in Table 3).

[0278] In some embodiments, the technique is applied to the treatment of a patient (e.g., a lung cancer patient, an early-stage lung cancer patient, or a patient who may develop lung cancer), the method comprising determining the methylation status of one or more markers provided herein and administering a treatment to the patient based on the results of determining the methylation status. The treatment may be the administration of a pharmaceutical compound, a vaccine, the performance of surgery, imaging of the patient, or the performance of another test. Preferably, the use is in methods of clinical screening, methods of prognosis assessment, methods of monitoring the outcomes of therapy, methods of identifying patients most likely to respond to a particular therapeutic procedure, methods of imaging patients or subjects, and methods of drug screening and development.

[0279] In some embodiments, this technique is applied to a method for diagnosing lung cancer in a subject. As used herein, the terms “diagnose” and “diagnose” refer to a method that enables a person skilled in the art to estimate, and even determine, whether a subject has a given disease or condition, or is likely to develop a given disease or condition in the future. A person skilled in the art often makes a diagnosis based on one or more diagnostic indicators, such as biomarkers, whose methylation status indicates the presence, severity, or absence of a condition.

[0280] In addition to diagnosis, clinical cancer prognosis involves determining the malignancy of the cancer and the likelihood of tumor recurrence in order to plan the most effective therapy. When the prognosis can be determined more accurately, or even when the potential risk of developing cancer can be assessed, appropriate therapy can be selected, and in some cases, a less burdensome therapy can be chosen for the patient. Evaluation of cancer biomarkers (e.g., determination of methylation status) is useful in differentiating between patients with a good prognosis and / or low risk of developing cancer who do not require treatment or require only limited treatment, and patients who are more likely to develop or recur cancer and may benefit from more intensive treatment.

[0281] Therefore, as used herein, “to make a diagnosis” or “to diagnose” further includes determining the risk of developing cancer or determining the prognosis, which may be based on the measurement of diagnostic biomarkers disclosed herein, to predict clinical outcomes (with or without medical intervention), select appropriate treatments (or determine whether treatments are effective), or monitor current treatments and potential changes to treatments.

[0282] Furthermore, in some embodiments of this technology, multiple determinations of biomarkers over time can be made to facilitate diagnosis and / or prognosis. Changes in biomarkers over time may be used to predict clinical outcomes, monitor the progression of lung cancer, and / or monitor the effectiveness of appropriate therapies for cancer. For example, in such embodiments, it may be expected that changes in the methylation status of one or more biomarkers (and, if monitored, one or more additional biomarkers) disclosed herein in a biological sample will be observed over time during the course of effective therapy.

[0283] This technology can be further applied to a method for determining whether to initiate or continue prevention or treatment of a target cancer. In some embodiments, the method includes: preparing a series of biological samples from a subject over a period of time; analyzing the series of biological samples to determine the methylation status of at least one biomarker disclosed herein in each biological sample; and comparing measurable changes in the methylation status of one or more of the biomarkers in each biological sample. Changes in the methylation status of biomarkers over a period of time can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to initiate or continue prevention or treatment of cancer, and determine whether the current treatment is effectively treating the cancer. For example, a first time point can be selected before the initiation of treatment, and a second time point can be selected at some point after the initiation of treatment. Methylation status can be measured in each of the samples taken at different time points, and qualitative and / or quantitative differences can be recorded. Changes in the methylation status of biomarker levels in different samples may correlate with the risk of developing lung cancer, prognosis, determination of treatment effectiveness in the subject, and / or cancer progression.

[0284] In preferred embodiments, the methods and compositions of the present invention are for treating or diagnosing diseases in their early stages, for example, before the symptoms of the disease appear. In some embodiments, the methods and compositions of the present invention are for treating or diagnosing diseases in their clinical stages.

[0285] As described above, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and the temporal changes in the markers can be used to determine the diagnosis or prognosis. For example, a diagnostic marker may be determined initially and again a second time. In such embodiments, an increase in the marker from the initial to the second determination may diagnose a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in the marker from the initial to the second determination may indicate a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change in one or more markers may be related to the severity of cancer and future adverse events. In certain embodiments, comparative measurements of the same biomarker can be made at multiple time points, or a given biomarker may be measured at one time point and a second biomarker at a second time point, and it will be understood by those skilled in the art that comparisons of these markers can provide diagnostic information.

[0286] As used herein, the phrase “determine prognosis” means a method that enables a person skilled in the art to predict the course or outcome of a condition in an object. The term “prognosis” does not mean that the course or outcome of a condition can be predicted with 100% accuracy, nor does it mean that the likelihood of a given course or outcome occurring can be predicted based on the methylation status of a biomarker. Rather, as will be understood by a person skilled in the art, the term “prognosis” means an increase in the probability of a particular course or outcome occurring, i.e., that the likelihood of a course or outcome occurring in an object exhibiting a given condition is higher than in an individual that does not exhibit that condition. For example, the probability of a given outcome (e.g., developing lung cancer) occurring may be very low in an individual that does not exhibit that condition.

[0287] In some embodiments, statistical analysis is used to associate prognostic indicators with predisposition to adverse outcomes. For example, in some embodiments, a methylation status different from that of a normal control sample obtained from patients without cancer may indicate that a subject is more likely to develop cancer than a subject with a more similar level of methylation status to that of the control sample, as determined by the level of statistical significance. Furthermore, changes in methylation status from baseline (e.g., "normal") levels may reflect the prognosis of a subject, and the degree of change in methylation status may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining confidence intervals and / or p-values. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is incorporated herein in whole by reference. The exemplary confidence intervals for the subject matter of the present invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, and the exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0288] In other embodiments, threshold values ​​of change in the methylation status of prognostic or diagnostic biomarkers disclosed herein can be established, and the degree of change in the methylation status of a biomarker in a biological sample is directly compared to the threshold value of change in the methylation status. Preferred threshold values ​​of change in methylation status for the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet another embodiment, a “nomogram” can be established, thereby directly relating the methylation status of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) to a relevant predisposition for a given outcome. Those skilled in the art are familiar with using such nomograms to relate two numerical values, with the understanding that the uncertainty in this measurement is the same as the uncertainty in marker concentration, since individual sample measurements rather than population mean are referenced.

[0289] In some embodiments, control samples are analyzed simultaneously with biological samples so that results obtained from biological samples can be compared with results obtained from control samples. Furthermore, standard curves may be provided so that assay results for biological samples can be compared. Such standard curves present the methylation status of biomarkers as a function of assay units, for example, as a function of fluorescence signal intensity if fluorescent labeling is used. When samples are taken from multiple donors, standard curves may be provided for the control methylation status of one or more biomarkers in normal tissue, and also for the "risk" level of one or more biomarkers in tissue taken from donors with lung cancer.

[0290] The analysis of markers can be performed separately or simultaneously with further markers within a single test sample. For example, combining several markers in a single test can efficiently process multiple samples and thereby improve the accuracy of diagnosis and / or prognosis. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at consecutive points in time). Testing such consecutive samples can enable the identification of changes in the methylation status of markers over time. Not only changes in methylation status, but also the absence of changes in methylation status can provide useful information about the disease state, including, but not limited to, the approximate time since the onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapy, the identification of the effectiveness of various therapies, and the identification of the subject's outcome, including the risk of future events.

[0291] Biomarker analysis can be performed in a variety of physical formats. For example, the use of microtiter plates or automation can facilitate the processing of a large number of test samples. Alternatively, a single sample format can be developed to facilitate timely emergency treatment and diagnosis, for example, in emergency transport or emergency room situations.

[0292] In some embodiments, if there is a measurable difference in the methylation status of at least one biomarker in the sample compared to a control methylation status, the subject is diagnosed with lung cancer. Conversely, if no change in methylation status is identified in the biological sample, the subject may be identified as not having lung cancer, having no risk of cancer, or having a low risk of cancer. In this regard, subjects with lung cancer or at risk can be distinguished from subjects with cancer or a low or substantially no risk. Subjects at risk of developing lung cancer can be placed under a more intensive and / or periodic screening schedule. On the other hand, subjects at low or substantially no risk can avoid screening procedures until the emergence of lung cancer risk in these subjects is indicated by future screening, for example, screening performed according to the techniques of the present invention.

[0293] As described above, depending on the embodiment of the method of the present invention, the detection of a change in the methylation state of one or more biomarkers may be a qualitative or quantitative determination. Therefore, the step of diagnosing a subject as having lung cancer or being at risk of developing lung cancer involves measuring a specific threshold, for example, indicating that the methylation state of one or more biomarkers in a biological sample differs from a predetermined control methylation state. In some embodiments of this method, the control methylation state is a detectable methylation state of any of the biomarkers. In other embodiments of the method, where a control sample is tested simultaneously with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of this method, the predetermined methylation state is identified based on and / or by a standard curve. In other embodiments of this method, the predetermined methylation state is a specific state or a range of states. Therefore, the predetermined methylation state may be selected, within acceptable limits that would be apparent to those skilled in the art, partly based on the embodiment of the method in practice and the desired specificity, etc.

[0294] In some embodiments, samples from subjects with or suspected of having lung cancer are screened using one or more methylation markers and a preferred assay method that provides data to distinguish between different types of lung cancer, such as non-small cell carcinoma (adenocarcinoma, large cell carcinoma, squamous cell carcinoma) and small cell carcinoma. For example, see marker reference number AC27 (Figure 2; PLEC), which is highly methylated in adenocarcinoma and small cell carcinoma (shown as mean methylation compared to mean methylation at the locus in the normal buffy coat), but not highly methylated in large cell or squamous cell carcinoma; marker reference number AC23 (Figure 1; ITPRIPL1), which is more highly methylated in adenocarcinoma than in all other sample types; marker reference number LC2 (Figure 2; DOCK2), which is more highly methylated in large cell carcinoma than in all other sample types; marker reference number SC221 (Figure 3; ST8SIA4), which is more highly methylated in small cell carcinoma than in all other sample types; and marker reference number SQ36 (Figure 4, DOK1), which is more highly methylated in squamous cell carcinoma than in all other sample types.

[0295] The methylation markers selected as described herein may be used alone or in combination (e.g., in a panel) to provide sufficient information for analysis of a sample derived from a subject to reveal the presence of lung neoplasms and to distinguish between types of lung cancer, such as small cell carcinoma and non-small cell carcinoma. In preferred embodiments, the markers or combinations of markers provide sufficient data to distinguish between adenocarcinoma, large cell carcinoma, and squamous cell carcinoma, and / or to characterize undetermined or mixed pathological carcinomas. In other embodiments, the methylation markers or combinations thereof are selected to provide a positive result (e.g., a result indicating the presence of lung neoplasms) without distinguishing between the types of lung cancer present.

[0296] In recent years, it has become clear that circulating epithelial cells representing metastatic tumor cells can be detected in the blood of many cancer patients. Molecular profiling of rare cells is important in biological and clinical research. Its applications are diverse, including the characterization of circulating epithelial cells (CEpCs) in the peripheral blood of cancer patients for disease prognosis and personalized treatment (e.g., Cristofanilli M, et al. (2004) N Engl J Med 351:781-791, Hayes DF, et al. (2006) Clin Cancer Res 12:4218-4224, Budd GT, et al., (2006) Clin Cancer Res 12:6403-6409, Moreno JG, et al. (2005) Urology 65:713-718, Pantel et al., (2008) Nat Rev 8:329-340, and Cohen SJ, et al. (2008) J Clin Oncol (See 26:3213-3221). Accordingly, embodiments of the present disclosure provide compositions and methods for detecting the presence of metastatic cancer in a subject by identifying the presence of methylation markers in plasma or whole blood.

[0297] Furthermore, this specification describes assays that include an LQAS PCR-flap assay following multiplex reverse transcription and pre-amplification (the LQAS assay combined with reverse transcription and pre-amplification is called the RT-TELQAS assay ("Reverse Transcription-Target Enrichment Long probe Quantitative Amplified Signal")). In the RT-TELQAS assay, target RNA, for example, total RNA from a sample, is treated with an RT-pre-amplification reaction mixture containing, for example, 20 U of MMLV reverse transcriptase, 1.5 U of GoTaq® DNA polymerase, 10 mM MOPS buffer, pH 7.5, 7.5 mM MgCl2, 250 μM of each dNTP, and oligonucleotide primers (e.g., 12 primer pairs / 24 primers, in equimolar amounts (e.g., 200 nM each primer) or amounts modified to adjust the amplification efficiency of different target RNAs for 12 targets). The mixture is incubated at a temperature suitable for reverse transcription (e.g., 42°C), and then pre-amplification of the target sequences corresponding to the included primer pairs is performed by a limited number of thermal cycles (e.g., 10 cycles at 95°C, 63°C, and 70°C). After thermal cycling, an aliquot (e.g., 10 μL) of the RT-pre-amplification reaction mixture is used in the LQAS PCR-flap assay as described later. The RNAs suitable for detection in RT-TELQAS and RT-LQAS assays are not limited to any particular type of RNA target. For example, RNA of any form derived from tissues or cells, or circulating cell-free RNA from blood, such as protein-coding messenger RNA (mRNA), microRNA (miRNA), piRNA, tRNA, and other non-coding RNA molecules (ncRNA) (see, for example, SU Umu, et al. “A comprehensive profile of circulating RNAs in human serum,” RNA Biology 15(2):242-250 (2018), which is incorporated herein by reference as a whole), can be assayed using the RT-TELQAS and RT-LQAS methods described herein.

[0298] In preferred embodiments, these methods use a relatively high Mg compared to standard PCR buffers. ++ and low KCl (for example, 6-10 mM, preferably 7.5 mM Mg) ++ The reaction is carried out in a reaction mixture containing a PCR-flap assay buffer having MgCl2, 20mM Tris-HCl, pH 8, and 50mM KCl. Typical PCR buffers include 1.5mM MgCl2, 20mM Tris-HCl, pH 8, and 50mM KCl, while PCR-flap assay buffers include 7.5mM MgCl2, 10mM MOPS, 0.3mM Tris-HCl, pH 8.0, 0.8mM KCl, 0.1μg / μL BSA, 0.0001% Tween-20, and 0.0001% IGEPAL CA-630. Surprisingly, in the RT-LQAS and RT-TELQAS methods described herein, all amplification steps, including reverse transcription of the RT-LQAS flap assay and RT-pre-amplification of the TELQAS method, are carried out in the same PCR-flap assay buffer. When using multiplex pre-amplification, the same primer pair can be used for pre-amplification target enrichment and quantitative PCR-flap assays; that is, the primers do not need to be nested primers. See, for example, U.S. Patent No. 10,704,081, incorporated herein by reference.

[0299] [Examples] The following embodiments are provided to illustrate the present invention, but are not limiting. Specific embodiments are provided to facilitate understanding and to assist in the interpretation of the technical proposal. That is, these embodiments are for illustrative purposes only and are by no means intended to limit the scope of the present invention. Unless otherwise specified, the embodiments do not represent specific conditions and are subject to conventional conditions or manufacturer's recommended conditions.

[0300] Example 1 Methods for isolating RNA, DNA, and proteins.

[0301] The following are exemplary methods for RNA isolation, DNA isolation, and protein sample preparation prior to analysis.

[0302] RNA isolation from blood Blood samples are collected in blood collection tubes suitable for subsequent RNA detection (e.g., PAXgene Blood RNA Tube, Qiagen, Inc.). Samples may be assayed immediately or frozen for future analysis. RNA is extracted from the sample using standard methods, e.g., the Qiasymphony PAXgene blood RNA kit (product number: 762635), according to the manufacturer's instructions. RNA samples may be diluted before testing with RT-LQAS (e.g., 1:50 in 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).

[0303] DNA isolation from cells and plasma For cell lines, for example, genomic DNA can be isolated from cell-conditioned medium using the "Maxwell® RSC ccfDNA Plasma Kit" (Promega Corp., Madison, WI). Following the kit protocol, use 1 mL of cell-conditioned medium (CCM) instead of plasma and process according to the kit instructions. The elution volume is 100 μL, of which 70 μL is typically used for bisulfite conversion.

[0304] An exemplary procedure for isolating DNA from a 4 mL plasma sample is as follows: ● Add 300 μL of proteinase K (20 mg / mL) to 4 mL of plasma sample and mix. ● Add 3 μL of 1 μg / μL fish DNA to the plasma-proteinase K mixture. ● Add 2 mL of plasma lysis buffer to the plasma.

[0305] The plasma lysis buffer is as follows: - 4.3M guanidine thiocyanate - 10% IGEPAL CA-630 (Octylphenoxypoly(ethyleneoxy)ethanol, branched) (A mixture of 5.3g of IGEPAL CA-630 and 45mL of 4.8M guanidine thiocyanate) ● Incubate the mixture at 55°C for 1 hour while shaking at 500 rpm. ● Add the following and mix: ○ 3 mL of plasma lysis buffer ○ 2 mL of 100% isopropanol ○ 200 μL of magnetic silica-bonded beads (16 μg / μL of beads) (In some cases, the dissolution buffer and isopropanol may be mixed after each addition, and / or pre-mixed before adding to the mixture.) ● Incubate at 30°C for 30 minutes while shaking at 500 rpm. ● Attach the tube(s) to a magnet and collect the beads. Aspirate the supernatant and discard it. ● Add 750 μL of GuHCl-EtOH to the container containing the bound beads and mix.

[0306] The GuHCl-EtOH washing buffer is as follows: - 3M GuHCl (Guanidine Hydrochloride) - 57% EtOH (ethyl alcohol) ● Shake at 400 rpm for 1 minute. ● Transfer the sample to a deep well plate or a 2 mL microcentrifuge tube. ● Attach the tube to a magnet and allow the beads to be collected for 10 minutes. Aspirate the supernatant and discard it. ● Add 1000 μL of washing buffer (10 mM Tris-HCl, 80% EtOH) to the beads and incubate at 30°C for 3 minutes while shaking. ● Attach the tube to a magnet and collect the beads. Aspirate the supernatant and discard it. ● Add 500 μL of washing buffer to the beads and incubate at 30°C for 3 minutes while shaking. ● Attach the tube to a magnet and collect the beads. Aspirate the supernatant and discard it. ● Add 250 μL of washing buffer and incubate at 30°C for 3 minutes while shaking. ● Attach the tube to a magnet and collect the beads. Aspirate and discard the remaining buffer. ● Add 250 μL of washing buffer and incubate at 30°C for 3 minutes while shaking. ● Attach the tube to a magnet and collect the beads. Aspirate and discard the remaining buffer. ● Dry the beads at 70°C for 15 minutes while shaking. ● Add 125 μL of elution buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA) to the beads and incubate at 65°C for 25 minutes while shaking. ● Attach the tube to a magnet and let it collect beads for 10 minutes. ● Aspirate the supernatant containing the DNA and transfer it to a new container or tube.

[0307] Bisulfite conversion I. Sulfonation of DNA using ammonium bisulfite 1. In each tube, combine 64 μL of DNA, 7 μL of 1N NaOH, and 9 μL of carrier solution containing 0.2 mg / mL of BSA and 0.25 mg / mL of fish DNA.

[0308] 2. Incubate at 42°C for 20 minutes.

[0309] 3. Add 120 μL of 45% ammonium bisulfite and incubate at 66°C for 75 minutes.

[0310] 4. Incubate at 4°C for 10 minutes.

[0311] II. Desulfonation using magnetic beads material ● Magnetic beads (Promega MagneSil Paramagnetic Particles, Promega catalog number AS1050, 16 μg / μL). ● Binding buffer: 6.5-7M guanidine hydrochloride. ● Post-conversion washing buffer: 80% ethanol containing 10 mM TrisHCl (pH 8.0). ● Desulfonation buffer: 70% isopropyl alcohol and 0.1N NaOH were selected as the desulfonation buffer.

[0312] The samples are mixed using any suitable device or technique for mixing or incubating the samples at the temperatures and mixing rates described below. For example, a Thermomixer (Eppendorf) can be used for mixing or incubating the samples. An example of desulfonation is as follows: 1. Thoroughly mix the bead stock by vortexing the bottle for 1 minute.

[0313] 2. Ali-coat 50 μL of beads into a 2.0 mL tube (e.g., USA Scientific).

[0314] 3. Add 750 μL of binding buffer to the beads.

[0315] 4. Add 150 μL of the sulfonated DNA from Step I.

[0316] 5. Mix (for example, 1000 RPM, 30°C for 30 minutes).

[0317] 6. Place the tube on the magnetic stand and leave it for 5 minutes. While the tube is still on the stand, remove the supernatant and discard it.

[0318] 7. Add 1,000 μL of washing buffer. Mix (e.g., 1000 RPM, 30°C for 3 minutes).

[0319] 8. Place the tube on the magnetic stand and leave it for 5 minutes. With the tube still on the stand, remove the supernatant and discard it.

[0320] 9. Add 250 μL of washing buffer. Mix (e.g., 1000 RPM, 30°C for 3 minutes).

[0321] 10. Place the tube in the magnetic rack; remove the supernatant after 1 minute and discard.

[0322] 11. Add 200 μL of desulfonation buffer. Mix (e.g., 1000 RPM, 30°C for 5 minutes).

[0323] 12. Place the tube in the magnetic rack; remove the supernatant after 1 minute and discard it.

[0324] 13. Add 250 μL of washing buffer. Mix (e.g., 1000 RPM, 30°C for 3 minutes).

[0325] 14. Place the tube in the magnetic rack; remove the supernatant after 1 minute and discard it.

[0326] 15. Add 250 μL of washing buffer to the tube. Mix (e.g., 1000 RPM, 30°C for 3 minutes).

[0327] 16. Place the tube in the magnetic rack; remove the supernatant after 1 minute and discard it.

[0328] 17. Incubate all tubes at 30°C for 15 minutes with the lids open.

[0329] 18. Remove the tube from the magnetic rack and add 70 μL of elution buffer directly to the beads.

[0330] 19. Incubate the beads with the elution buffer (e.g., 1000 RPM, 40°C for 45 minutes).

[0331] 20. Place the tube in the magnetic rack for about 1 minute; remove and set aside the supernatant.

[0332] The converted DNA is then used in a detection assay, such as a pre-amplification and / or flap endonuclease assay, as described later.

[0333] For further embodiments of bisulfite treatment of nucleic acids, see U.S. Patent No. 10,704,081 and U.S. Patent Application No. 63 / 058,179, filed July 29, 2020. These are incorporated herein by reference in whole for any purpose and may be applied in the art described herein.

[0334] In some embodiments, RNA and DNA are isolated from different blood samples of the subject. For example, blood may be collected into a first collection tube configured for optimal preservation and / or isolation of RNA and a second collection tube configured for optimal preservation and isolation of DNA, and RNA and DNA may be extracted from a portion of the blood collected in this manner. In other embodiments, both RNA and DNA are extracted from a single blood sample using, for example, collection tubes configured for optimal preservation and isolation of both DNA and RNA (e.g., NORGEN Biotek Corp.'s cf-DNA / cf-RNA Preservative Tubes (catalog no. 63950) for the preservation and isolation of both cell-free DNA and cell-free RNA).

[0335] In some embodiments, RNA and DNA are assayed together, for example, in an RT-LQAS / RT-TELQAS reaction. In some embodiments, RNA and DNA are isolated separately and / or treated separately, for example, with bisulfite, as described above, while in some embodiments, RNA and DNA are treated together, for example, both are present during bisulfite treatment and subsequent purification, and then added together to the assay reaction product.

[0336] Flap endonuclease assay QuARTS and LQAS / TELQAS flap assay technologies combine a polymerase-based targeted DNA amplification process with an invasive cleavage-based signal amplification process. QuARTS technology is described, for example, in U.S. Patents 8,361,720, 8,715,937, 8,916,344, and 9,212,392, while flap assays using probe oligonucleotides with longer target-specific regions (Long probe Quantitative Amplified Signal, "LQAS") are described in U.S. Patent 10,648,025, each incorporated herein by reference in whole for any purpose. In the QuARTS assays described herein, the flap oligonucleotide has a 12-base target-specific region, while the LQAS assay uses a flap oligonucleotide with at least a 13-base target-specific region, employing different thermal cycling procedures for amplification. The fluorescent signals generated by the QuARTS and LQAS reactions can be monitored in a manner similar to real-time PCR, allowing for the quantification of the amount of target nucleic acid in the sample.

[0337] An exemplary QuARTS reaction typically involves approximately 200–600 nmol / L (e.g., 500 nmol / L) of each primer and detection probe, approximately 100 nmol / L of invasive oligonucleotides, approximately 600–700 nmol / L of each FRET cassette (FAM, e.g., commercially available from Hologic, Inc.; HEX, e.g., commercially available from BioSearch Technologies; and Quasar 670, e.g., commercially available from BioSearch Technologies, containing a "black hole" quencher, e.g., BioSearch Technologies' BHQ-1, BHQ-2, or BHQ-3), 6.675 ng / μL of FEN-1 endonuclease (e.g., Cleavase® 2.0, Hologic, Inc.), and 1 unit of Taq DNA polymerase per 30 μL reaction volume (e.g., GoTaq® DNA polymerase, Promega (Corporate Corp., Madison, WI), contains 10 mmol / L of 3-(n-morpholino)propanesulfonic acid (MOPS), 7.5 mmol / L of MgCl2, and 250 μmol / L of each of dNTPs. Exemplary Quarts cycling conditions are shown in the table below. In some applications, the quantification cycle (C q Analysis of this provides a measure of the initial number (e.g., copy number) of the target DNA strand in the sample.

[0338] [Table 1]

[0339] An exemplary LQAS reactant typically comprises approximately 200–600 nmol / L of each primer, approximately 100 nmol / L of an invasive oligonucleotide, approximately 500 nmol / L of each flap oligonucleotide probe, and a FRET cassette. The LQAS reactant can be subjected to the following thermocycling conditions, for example:

[0340] [Table 2]

[0341] Multiplex Pre-amplification for Quarts and LQAS Assays Multiplex pre-amplification of bisulfite-converted DNA A large amount of bisulfite-treated DNA can be used in a single high-volume multiplex amplification reaction to pre-amplify most or all of the bisulfite-treated DNA from the input sample. For example, the DNA is extracted from cell lines (e.g., DFCI032 cell line (adenocarcinoma); H1755 cell line (neuroendocrine)) using, for example, the Maxwell Promega blood kit #AS1400, as described above. This DNA is then bisulfite-converted, for example, as described above.

[0342] Pre-amplification is performed in a reaction mixture containing, for example, 7.5 mM MgCl2, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, 0.0001% IGEPAL CA-630, 250 μM each of dNTPs, oligonucleotide primers (e.g., 12 primer pairs / 24 primers, equimolar amounts for 12 targets (e.g., including but not limited to 200-500 nM for each primer), or individual primer concentrations adjusted to balance the amplification efficiency of different target regions), 0.025 units / μL HotStart GoTaq concentrate, and 20-50 volume% bisulfite-treated target DNA (e.g., 10 μL of target DNA in 50 μL of reaction mixture, or 50 μL of target DNA in 125 μL of reaction mixture). The thermal cycling time and temperature should be selected to be appropriate for the reactants and the capacity of the amplification vessel. For example, the reactants may be subjected to the following cycle:

[0343] [Table 3]

[0344] After thermal cycling, an aliquot (e.g., 10 μL) of the pre-amplified reaction is diluted to 500 μL with 10 mM Tris and 0.1 mM EDTA, with or without fish DNA. The diluted aliquot (e.g., 10 μL) of the pre-amplified DNA is used in a QuARTS PCR-flap assay, for example, as described above. See also U.S. Patent Application No. 62 / 249,097 filed October 30, 2015, Application No. 15 / 335,096 filed October 26, 2016, and PCT / US16 / 58875 filed October 26, 2016. These are each incorporated herein by reference in whole for all purposes.

[0345] The assay combining pre-amplification and LQAS is the TELQAS assay ("Target It is called "Enrichment Long probe Quantitative Amplified Signal" (an abbreviation).

[0346] Using the pre-amplified sample, prepare the QuarTS and TELQAS reactions as follows:

[0347] [Table 4]

[0348] As described above, the flap oligonucleotides used in the QuARTS assay have a 12-base target-specific region, while the LQAS assay uses flap oligonucleotides with at least 13 bases in the target-specific region, and are subjected to different thermal cycling conditions.

[0349] The Quarts reactant is subjected to the following thermocycling conditions:

[0350] [Table 5]

[0351] The TELQAS reaction product is subjected to the following thermocycling conditions:

[0352] [Table 6]

[0353] LQAS / TELQAS for RNA detection ("RT-LQAS" or "RT-TELQAS") An exemplary RT-LQAS reaction product includes 20 U of MMLV reverse transcriptase (MMLV-RT), 219 ng of Cleavase® 2.0, 1.5 U of GoTaq® DNA polymerase, 200 nM of each primer, 500 nM of each probe and FRET oligonucleotide, 10 mM MOPS buffer, pH 7.5, 7.5 mM MgCl2, and 250 μM of each nNTP. An exemplary protocol is as follows: 1. Remove the required oligonucleotide mix from the -20°C freezer and thaw it.

[0354] 2. Thaw the control sample briefly from -80°C to room temperature, then place it on ice.

[0355] 3. Thaw the sample plate briefly from -80°C to room temperature, then place it on ice.

[0356] 4. Prepare the master mix of oligosaccharides in a tube of the appropriate size.

[0357] 5. Dilute MMLV-RT with H2O in a 1:20 ratio.

[0358] [Table 7]

[0359] 6. Using a matrix pipette or 8-channel P20 pipette, pipette 20 μL of the master mix into a 96-well RT-LQAS plate according to the plate layout.

[0360] 7. Load 10 μL of sample, control, and calibrator (following the plate layout).

[0361] 8. Seal the plate and briefly centrifuge it.

[0362] 9. Run the plate under the following reaction conditions. The reaction is typically run in a thermal cycler configured to collect fluorescence data in real time (e.g., continuously, or at the same time in some or all cycles). For example, a Roche LightCycler 480 instrument or an Applied Biosystem QuantStudioDX Real-Time PCR instrument may be used under the following conditions:

[0363] [Table 8]

[0364] In some embodiments, the RT-LQAS assay may include, as described above, a multiplex reverse transcription step and a pre-amplification step, e.g., a step of pre-amplifying 2, 5, 10, 12, or more targets (i.e., any number of targets more than one) in the sample, and may be referred to as "RT-TELQAS". In a preferred embodiment, the RT-pre-amplification is carried out in a reaction mixture containing, for example, 20 U of MMLV reverse transcriptase, 1.5 U of GoTaq® DNA polymerase, 10 mM MOPS buffer, pH 7.5, 7.5 mM MgCl2, 250 μM of each dNTP, and oligonucleotide primers (e.g., 12 primer pairs / 24 primers, equimolar amounts (e.g., 200 nM each primer) for 12 targets, or individual primer concentrations adjusted to balance the amplification efficiency of different targets). The thermal cycling time and temperature are selected to be appropriate for the reactants and the volume of the amplification vessel. For example, the reactants may be subjected to the following cycle:

[0365] [Table 9]

[0366] After thermal cycling, an aliquot (e.g., 10 μL) of the RT-pre-amplification reaction is diluted to 500 μL with 10 mM Tris and 0.1 mM EDTA, with or without fish DNA. An aliquot (e.g., 10 μL) of the diluted pre-amplification DNA is used in the LQAS / TELQAS PCR-flap assay as described above. In some embodiments, the LQAS / TELQAS PCR-flap assay is performed using an additional amount of the same primer pair.

[0367] Example 2 Selection and testing of methylation markers Marker selection process: Reduced Representation Bisulfite Sequencing (RRBS) data were obtained from 16 adenocarcinoma lung cancer tissues, 11 large cell lung cancer tissues, 14 small cell lung cancer tissues, 24 squamous cell lung cancer tissues, and 18 non-cancerous lung tissues. RRBS results were also obtained from buffy coat samples from 26 healthy patients.

[0368] After alignment with bisulfite-converted human genome sequences, mean methylation in each CpG island was calculated for each sample type (i.e., tissue or buffy coat), and marker regions were selected based on the following criteria: ● The region was selected to have at least 50 base pairs. ● In the design of the QuARTS flap assay, regions were selected such that there is at least one methylated CpG under each of the following regions: a) the probe region, b) the forward primer binding region, and c) the reverse primer binding region. For the forward and reverse primers, it is preferable that the methylated CpG is close to the 3' end of the primer but not at the 3' terminal nucleotide. An exemplary flap endonuclease assay oligonucleotide is shown in Figure 5. ● It is preferable that the methylation of the buffy coat in any CpG within the target region is >0.5% or less. ● Methylation of cancer tissue within the target region is preferably >10%. ● In assays designed for tissue analysis, methylation of normal tissue within the region of interest is preferably <0.5%.

[0369] RRBS data for different lung cancer histological types are shown in Figures 2-5. Based on the above criteria, the markers shown in the table below were selected, and the QuARTS flap assay was designed for them as shown in Figure 5.

[0370] [Table 10-1]

[0371] [Table 10-2]

[0372] [Table 10-3]

[0373] Analysis of cross-reactivity of selected markers with buffy coat.

[0374] 1) Screening of buffy coat The markers listed above were screened using DNA extracted from a buffy coat obtained from 10 mL of blood from healthy patients. The DNA was extracted using the Promega Maxwell RSC system (Promega Corp., Fitchburg, WI) and converted using the Zymo EZ DNA Methylation® Kit (Zymo Research, Irvine, CA). Cross-reactivity was examined using a biplex reaction with bisulfite-converted β-actin DNA ("BTACT"), with approximately 40,000 strands of target genomic DNA being used. The samples were then tested using the QuARTS flap endonuclease assay described above. The assays for three markers showed significant cross-reactivity:

[0375] [Table 11]

[0376] 2) Organizational screening 264 tissue samples were obtained from various commercial and non-commercial sources (Asuragen, BioServe, ConversantBio, Cureline, Mayo Clinic, MD Anderson, and PrecisionMed), as shown in Table 2 below.

[0377] [Table 12]

[0378] A pathologist examined the tissue sections, circled histologically distinct lesions, and instructed for microscopic dissection. Total nucleic acid extraction was performed using the Promega Maxwell RSC system. Formalin-fixed paraffin-embedded (FFPE) slides were rubbed, and DNA was extracted using the Maxwell® RSC DNA FFPE Kit (#AS1450) following the manufacturer's procedure, but omitting the RNase treatment step. The same procedure was used for FFPE curls. For frozen punch biopsy samples, a modified procedure was used with the RSC DNA FFPE Kit lysis buffer and the Maxwell® RSC Blood DNA Kit (#AS1400), omitting the RNase step. Samples were eluted with 10 mM Tris, 0.1 mM EDTA, pH 8.5, and 10 μL was used to prepare six multiplex PCR reactions.

[0379] The following multiplex PCR primer mix was prepared at a 10-fold concentration (10-fold = 2 μM each primer): ● Multiplex PCR reaction product 1 consisted of the following markers: BARX1, LOC100129726, SPOCK2, TSC22D4, PARP15, MAX.chr8.145105646-145105653, ST8SIA1_22, ZDHHC1, BIN2_Z, SKI, DNMT3A, BCL2L11, RASSF1, FERMT3, and BTACT. ● Multiplex PCR reaction product 2 consisted of the following markers: ZNF671, ST8SIA1, NKX6-2, SLC12A8, FAM59B, DIDO1, MAX_Chr1.110, AGRN, PRKCB_28, SOBP, and BTACT. ● Multiplex PCR reaction product 3 consisted of the following markers: MAX.chr10.22624430-22624544, ZMIZ1, MAX.chr8.145105646-145105653, MAX.chr10.22541891-22541946, PRDM14, ANGPT1, MAX.chr16.50875223-50875241, PTGDR_9, ANKRD13B, DOCK2, and BTACT. ● Multiplex PCR reaction product 4 consisted of the following markers: MAX.chr19.16394489-16394575, HOXB2, ZNF132, MAX.chr19.37288426-37288480, MAX.chr12.52652268-52652362, FLJ45983, HOXA9, TRH, SP9, DMRTA2, and BTACT. ● Multiplex PCR reaction product 5 consisted of the following markers: EMX1, ARHGEF4, OPLAH, CYP26C1, ZNF781, DLX4, PTGDR, KLHDC7B, GRIN2D, chr17_737, and BTACT. ● Multiplex PCR reaction product 6 consisted of the following markers: TBX15, MATK, SHOX2, BCAT1, SUCLG2, BIN2, PRKAR1B, SHROOM1, S1PR4, NFIX, and BTACT.

[0380] Each multiplex PCR reaction was prepared with 0.2 μM reaction buffer, 0.2 μM of each primer, and 0.05 μM Hotstart Go Taq (5 U / μL) to a final concentration. 40 μL of the master mix was then combined with 10 μL of DNA template to create a final reaction volume of 50 μL.

[0381] The thermal profile of the multiplex PCR involved a 5-minute pre-incubation stage at 95°C, 10 cycles of amplification at 95°C for 30 seconds, 64°C for 30 seconds, and 72°C for 30 seconds, and a cooling stage at 4°C for holding until further processing. After the multiplex PCR was complete, the PCR product was diluted 1:10 with a diluent of 20 ng / μL of fish DNA (e.g., in water or buffer; see U.S. Patent No. 9,212,392 incorporated herein by reference), and 10 μL of the diluted amplified sample was used for each Quarts assay reaction.

[0382] Each QuARTS assay was constructed in a triplex format, consisting of two methylation markers and BTACT as the reference gene. ● The following seven triplex Quarts assays were run from multiplex PCR product 1: (1) BARX1, LOC100129726, BTACT; (2) SPOCK2, TSC22D4, BTACT; (3) PARP15, MAXchr8145105646-145105653, BTACT; (4) ST8SIA1_22, ZDHHC1, BTACT; (5) BIN2_Z, SKI, BTACT; (6) DNMT3A, BCL2L11, BTACT; (7) RASSF1, FERMT3, and BTACT. ● The following five triplex Quarts assays were run from multiplex PCR product 2: (1) ZNF671, ST8SIA1, BTACT; (2) NKX6-2, SLC12A8, BTACT; (3) FAM59B, DIDO1, BTACT; (4) MAX_Chr1110, AGRN, BTACT; (5) PRKCB_28, SOBP, and BTACT. ● The following five triplex Quarts assays were run from multiplex PCR product 3: (1) MAXchr1022624430-22624544, ZMIZ1, BTACT; (2) MAXchr8145105646-145105653, MAXchr1022541891-22541946, BTACT; (3) PRDM14, ANGPT1, BTACT; (4) MAXchr1650875223-50875241, PTGDR_9, BTACT; (5) ANKRD13B, DOCK2, and BTACT. ● The following five triplex Quarts assays were run from multiplex PCR product 4: (1) MAXchr1916394489-16394575, HOXB2, BTACT; (2) ZNF132, MAXchr1937288426-37288480, BTACT; (3) MAXchr1252652268-52652362, FLJ45983, BTACT; (4) HOXA9, TRH, BTACT; (5) SP9, DMRTA2, and BTACT. ● The following five triplex Quarts assays were run from multiplex PCR product 5: (1) EMX1, ARHGEF4, BTACT; (2) OPLAH, CYP26C1, BTACT; (3) ZNF781, DLX4, BTACT; (4) PTGDR, KLHDC7B, BTACT; (5) GRIN2D, chr17_737, and BTACT. ● The following five triplex Quarts assays were run from multiplex PCR product 6: (1) TBX15, MATK, BTACT; (2) SHOX2, BCAT1, BTACT; (3) SUCLG2, BIN2, BTACT; (4) PRKAR1B, SHROOM1, BTACT; (5) S1PR4, NFIX, and BTACT.

[0383] 3) Data analysis: The analysis of tissue data included markers selected based on the RRBS criteria of <0.5% methylation in normal tissue and >10% methylation in cancerous tissue. This yielded 51 markers for further analysis.

[0384] To determine the sensitivity of the marker, the following was done: 1. The methylation percentage for each marker was calculated by dividing the value of the chain obtained for that specific marker by the value of the ACTB (β-actin) chain.

[0385] 2. The maximum methylation percentage for each marker was measured in normal tissue. This is defined as 100% specificity.

[0386] 3. Positivity for each marker in cancer tissue was determined by the number of cancer tissues with a higher percentage of methylation of that marker than the maximum methylation percentage in normal tissue.

[0387] The sensitivity of the 51 markers is shown below.

[0388] [Table 13-1]

[0389] [Table 13-2]

[0390] Specificity and sensitivity can be increased by using combinations of markers. For example, a combination of eight markers—SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, and EMX1—resulted in 100% specificity and 98.5% sensitivity (134 / 136 cancers) in all cancerous tissues examined.

[0391] In some embodiments, markers are selected for highly sensitive and specific detection of specific types of lung cancer tissue, such as adenocarcinoma, large cell carcinoma, squamous cell carcinoma, or small cell carcinoma, by using markers that exhibit sensitivity and specificity to specific cancer types or combinations of types.

[0392] This panel of methylated DNA markers assayed in tissue achieves highly accurate differentiation of all types of lung cancer while remaining negative in normal lung tissue and benign nodules. The assay of this marker panel can also be applied to blood or fluid-based tests, for example, in lung cancer screening and differentiation between malignant and benign nodules.

[0393] Example 3 Testing of a 30-marker set in plasma samples From the list of markers in Example 2, 30 markers were selected for DNA analysis in plasma samples from 295 subjects (64 with lung cancer and 231 normal controls). DNA was extracted from 2 mL of plasma from each subject and treated with bisulfite as described in Example 1. Aliquots of the bisulfite-converted DNA were used in two multiplex QuARTS assays as described in Example 1. The markers selected for analysis are as follows: 1. BARX1 2. BCL2L11 3. BIN2_Z 4. CYP26C1 5. DLX4 6. DMRTA2 7. DNMT3A 8. EMX1 9. FERMT3 10. FLJ45983 11. HOXA9 12. KLHDC7B 13.MAX.chr10.22624430-22624544 14.MAX.chr12.52652268-52652362 15. MAX.chr8.124173236-124173370 16. MAX.chr8.145105646-145105653 17. NFIX 18. OPLAH 19. PARP15 20. PRKCB_28 21. S1PR4 22. SHOX2 23. SKI 24. SLC12A8 25. SOBP 26. SP9 27. SUCLG2 28. TBX15 29. ZDHHC1 30. ZNF781 The target sequences, bisulfite-converted target sequences, and assay oligonucleotides for these markers are shown in Figure 5. The primers and flap oligonucleotides (probes) used for each conversion target were as follows:

[0394] [Table 14-1]

[0395] [Table 14-2]

[0396] [Table 14-3]

[0397] [Table 14-4]

[0398] [Table 14-5]

[0399] [Table 14-6]

[0400] [Table 14-7]

[0401] [Table 14-8]

[0402] [Table 14-9]

[0403] [Table 14-10]

[0404] * The B3GALT6 marker is used as both a cancer methylation marker and a reference target. See U.S. Patent Application No. 62 / 364,082, filed July 19, 2016, which is incorporated herein by reference in its entirety.

[0405] †For the zebrafish reference DNA, see U.S. Patent Application No. 62 / 364,049, filed July 19, 2016, which is incorporated herein by reference in its entirety.

[0406] As described above, DNA prepared from plasma was amplified using two multiplex pre-amplification reactions as described in Example 1. The multiplex pre-amplification reactions included reagents to amplify the following combination of markers.

[0407] [Table 15]

[0408] After pre-amplification, aliquots of the pre-amplification mixture were diluted 1:10 with 10 mM Tris-HCl and 0.1 mM EDTA, and then the triplex Quartz was prepared as described in Example 1. The assay was performed using a PCR-flap assay. For the first group of triplex reactions, pre-amplification material from multiplex mix 1 was used, and for the second group of reactions, pre-amplification material from multiplex mix 2 was used. The triplex combinations were as follows: Group 1: ZF_RASSF1-B3GALT6-BTACT (ZBA Triplex) BARX1-SLC12A8-BTACT (BSA2 Triplex) PARP15-MAX.chr8.124-BTACT (PMA Triplex) SHOX2-ZDHHC1-BTACT (SZA2 Triplex) BIN2_Z-SKI-BTACT (BSA Triplex) DNMT3A-BCL2L11-BTACT (DBA Triplex) TBX15-FERMT3-BTACT (TFA Triplex) PRKCB_28-SOBP-BTACT (PSA2 Tripleplex) Group 2: ZF_RASSF1-B3GALT6-BTACT (ZBA Triplex) MAX.chr8.145-MAX_chr10.226-BTACT(MMA2 Triplex) MAX.chr12.526-FLJ45983-BTACT (MFA Triplex) HOXA9-EMX1-BTACT (HEA Triplex) SP9-DMRTA2-BTACT (SDA Triplex) OPLAH-CYP26C1-BTACT (OCA Triplex) ZNF781-DLX4-BTACT (ZDA Triplex) SUCLG2-KLHDC7B-BTACT (SKA Triplex) S1PR4-NFIX-BTACT (SNA Triplex) The acronym for each triplex uses the first letter of each gene name (for example, the HOXA9-EMX1-BTACT combination = "HEA"). If an acronym is repeated for a different combination of markers or from another experiment, the second group having that acronym will include the number 2. The dye reporters used in the FRET cassette for each member of the above-mentioned triplexes are FAM-HEX-Quasar670, respectively.

[0409] The quantitative reaction was calibrated using plasmids containing the target DNA sequence. For each calibrator plasmid, 10-10 ng / mL of fish DNA per μl was used in fish DNA diluent (10 mM Tris-HCl, 0.1 mM EDTA). 6 A series of 10-fold calibrator-diluted stocks containing copies of the target strand were prepared. For the triplex reaction, a mixed stock containing plasmids with each of the triplex targets was used. 1 × 10⁶ per μL 5 A mixture containing each plasmid was prepared by copying, and this was used to create a 1:10 dilution series. The strands in unknown samples were inversely calculated using a standard curve generated by plotting Cp vs. Log(plasmid strand).

[0410] Receiver Operating Characteristic (ROC) curve analysis was used to calculate the area under the curve (AUC) for each marker. These results were sorted by the upper limit of the 95% coverage interval and are shown in the table below.

[0411] [Table 16]

[0412] These markers performed very well in distinguishing cancer patient samples from normal control samples (see ROC table above). Combining markers improved sensitivity. For example, using logistic fitting of the data, as well as a 6-marker fit using SHOX2, SOBP, ZNF781, BTACT, CYP26C1, and DLX4, ROC curve analysis yielded an area under the curve (AUC) of 0.973. This 6-marker fit yielded a sensitivity of 92.2% with a specificity of 93%. Using SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI, an ROC curve with an AUC of 0.97982 was obtained.

[0413] Example 4 Archived plasma from the second independent s...

Claims

[Claim 1] A method for measuring the amount of one or more gene expression products in blood sampled from a subject, a) From the blood sampled from the subject, i) at least one gene expression marker which is the product of the expression of a marker gene selected from S100A9, SELL, PADI4, APOBE3CA, S100A12, MMP9, FPR1, TYMP, and SAT1, and ii) At least one reference marker Extracting and b) Measuring the amount of at least one gene expression marker and at least one reference marker extracted in a), c) Calculating the amount of the at least one gene expression marker as a percentage of the amount of the at least one reference marker, wherein the value represents the amount of the at least one gene expression marker in the blood sampled from the subject. The method comprising the above.

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