Detecting breast cancer

JP2025106467A5Pending Publication Date: 2026-01-23MAYO FOUNDATION FOR MEDICAL EDUCATION & RESEARCH +1
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Patent Information

Application Number
JP2025064241
Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
Priority Date
2017-11-30
Filing Date
2025-04-09
Publication Date
2026-01-23

AI Technical Summary

Technical Problem

Current methods for breast cancer screening are inadequate in accurately identifying women at high risk, as they do not effectively utilize DNA methylation markers and are limited by biased approaches like microarray-based interrogation and enzyme recognition, which fail to provide single-nucleotide resolution.

Method used

Identification of 375 novel DNA methylation markers and panels that distinguish breast cancer tissue from benign tissue, utilizing techniques such as bisulfite treatment and methylation-specific PCR to assess methylation patterns in CpG sites, enabling high specificity and sensitivity in breast cancer detection.

Benefits of technology

The novel DNA methylation markers provide a high signal-to-noise ratio, allowing for accurate differentiation between breast cancer and benign tissues, and can be applied in various breast cancer types, including triple-negative, HER2+, luminal A, luminal B, BRCA1, and BRCA2, enhancing breast cancer screening efficacy.

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Abstract

To provide methods for detecting the presence of breast cancer.SOLUTION: Provided is a method for measuring the methylation level of CpG sites for one or more genes selected from a novel DNA methylation marker and / or panels of markers capable of distinguishing a breast cancer tissue from a benign breast tissue in a biological sample from a human individual.SELECTED DRAWING: None
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Description

Technical Field

[0001] Cross - reference to related applications This application claims priority and the benefit of U.S. Provisional Patent Application No. 62 / 592,828, filed on November 30, 2017, the entire content of which is incorporated herein by reference.

[0002] Provided herein are techniques related to breast cancer screening, and in particular, but not limited to, methods, compositions, and related uses for detecting the presence of breast cancer.

Background Art

[0003] Breast cancer affects approximately 230,000 U.S. women annually and kills about 40,000 lives each year. Although carriers of germline mutations in the BRCA1 and BRCA2 genes are known to have a high risk of breast cancer, the majority of women who develop breast cancer do not have a mutation in either of these genes, and there are limitations in the ability to accurately identify women at high risk of breast cancer. Although effective preventive therapies exist, current risk prediction models do not accurately identify the majority of women at high risk of breast cancer (see, for example, Pankratz VS, et al., J Clin Oncol 2008 Nov 20;26(33):5374 - 9).

[0004] There is a need for improved methods for detecting breast cancer.

[0005] The present invention addresses these needs.

Summary of the Invention

[0006] Methylated DNA has been studied as a promising class of biomarkers in most tumor types of tissues. In many instances, DNA methyltransferases add methyl groups to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically interesting mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence expression and thus contribute to carcinogenesis. DNA methylation may be a more chemically and biologically stable diagnostic tool than RNA or protein expression (Laird (2010) Nat Rev Genet 11:191-203). Furthermore, in other cancers such as sporadic colorectal cancer, methylation markers provide excellent specificity, have more extensive information than individual DNA mutations, and are highly sensitive (Zou et al (2007) Cancer Epidemiol Biomarkers Prev 16:2686-96).

[0007] Analysis of CpG islands has provided important insights when used in animal models and human cell lines. For example, Zhang and colleagues found that amplicons from different parts of the same CpG island can have different levels of methylation (Zhang et al. (2009) PLoS Genet 5:e1000438). Furthermore, methylation levels were bimodally distributed between highly methylated and unmethylated sequences, further supporting a binary switch-like pattern of DNA methyltransferase activity (Zhang et al. (2009) PLoS Genet 5:e1000438). Analysis of in vivo mouse tissues and in vitro cell lines demonstrated that only about 0.3% of promoters with high CpG density (HCP, defined as having >7% CpG sequences within a 300 base pair region) were methylated, whereas regions with low CpG density (LCP, defined as having <5% CpG sequences within a 300 base pair region) tended to be methylated at high frequencies in dynamic tissue-specific patterns (Meissner et al. (2008) Nature 454:766-70). HCP includes promoters for ubiquitous housekeeping genes and highly regulated developmental genes. Among the HCP sites that were methylated at >50%, there were several established markers, such as Wnt2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature 454:766-70).

[0008] Epigenetic methylation of DNA at cytosine-phosphate-guanine (CpG) island sites by DNA methyltransferases has been studied as a promising class of biomarkers in most tumor types of tissues. In a biologically interesting mechanism, epigenetic methylation events in the promoter regions of tumor suppressor genes are thought to silence expression and contribute to carcinogenesis. DNA methylation may be a more chemical and biologically stable diagnostic tool than RNA or protein expression. Furthermore, in other cancers such as sporadic colorectal cancer, abnormal methylation markers have broader information, higher sensitivity, and provide excellent specificity compared to individual DNA mutations.

[0009] Several methods are available for exploring novel methylation markers. Microarray-based interrogation of CpG methylation is a rational and high-throughput approach, but this strategy is biased towards known regions of interest, mainly established tumor suppressor promoters. Alternative methods for genome-wide analysis of DNA methylation have been developed over the past decade. There are three basic approaches. First, digestion of DNA with restriction enzymes that recognize specific methylation sites is used, followed by several possible analytical techniques to provide methylation data limited to enzyme recognition sites or primers, and these are used to amplify DNA in a quantification step (methylation-specific PCR, MSP, etc.). The second approach uses antibodies targeting methyl-cytosine or other methylation-specific binding domains to enrich the methylated fraction of genomic DNA, followed by microarray analysis or sequencing to map the fragments to a reference genome. This approach does not provide single-nucleotide resolution of all methylation sites within the fragment. The third approach begins with bisulfite treatment of DNA to convert all unmethylated thymines to uracil, followed by restriction enzyme digestion and complete sequencing of all fragments after ligation to adapter ligands. By the choice of restriction enzyme, fragments of CpG-dense regions can be enriched and the number of redundant sequences that may map to multiple gene locations during analysis can be reduced.

[0010] RRBS provides single nucleotide resolution CpG methylation state data for all CpG islands and 80 - 90% of most tumor suppressor promoters with medium to high read coverage. In cancer case - control studies, analysis of these reads results in the identification of differentially methylated regions (DMRs). In previous RRBS analyses of pancreatic cancer specimens, hundreds of DMRs have been discovered, many of which are never associated with carcinogenesis and many of which have not been annotated. Further validation tests on independent tissue sample sets confirmed marker CpGs that were 100% sensitive and specific in terms of performance.

[0011] Provided herein are technologies related to breast cancer screening, and in particular, but not limited to, methods, compositions, and related uses for detecting the presence of breast cancer.

[0012] Indeed, as described in Examples I, II, and III, experiments conducted during the process of identifying embodiments of the present invention identified a new set of differentially methylated regions (DMRs) to distinguish cancer in non - tumorigenic control DNA and breast - derived DNA.

[0013] Such experiments have enumerated and described 375 novel DNA methylation markers that distinguish breast cancer tissue from benign breast tissue (see Tables 2 and 18, Examples I, II, and III).

[0014] From these 375 novel DNA methylation markers, further experiments have identified the following markers and / or panels of markers that can distinguish breast cancer tissue from benign breast tissue: · ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1 (see Table 16E, Example II), and ·ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B (see Table 22, Example III).

[0015] From these 375 novel DNA methylation markers, the following markers and / or panels of markers for detecting breast cancer in a blood sample (e.g., a plasma sample, a whole blood sample, a serum sample) were identified by further experiments: ·CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B (see Table 27, Example III).

[0016] From these 375 novel DNA methylation markers, the following markers and / or panels of markers for distinguishing triple-negative breast cancer tissue from benign breast tissue were identified by further experiments: ·ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr11.14926602-14927148, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, MAX.chr4.8859253-8859329, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr6.157557371-157557657, MPZ, NKX2-6, PDX1, PLXNC1_A, PPARG, PRKCB, PTPRN2, RBFOX_A, SCRT2_A, SLC7A4, STAC2_B, STX16_A, STX16_B, TBX1, TRH_A, VSTM2B_A, ZBTB16, ZNF132, and ZSCAN23 (see Table 3, Example I), ·CALN1_A, LOC100132891, NACAD, TRIM67, ATP6V1B1, DLX4, GP5, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ST8SIA4, STX16_B ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, KCNK9, SCRT2_B, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, and DSCR6 (see Table 11, Example I), · ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4273906-4274012, MAX.chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4 (see Table 16A, Example II).

[0017] From these 375 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish breast cancer tissue from benign breast tissue: + HER2 ·ABLIM1, AFAP1L1, AKR1B1, ALOX5, AMN, ARL5C, BANK1, BCAT1, BEGAIN, BEST4, BHLHE23_B, BHLHE23_C, C17orf64, C1QL2, C7orf52, CALN1_B, CAV2, CD8A, CDH4_A, CDH4_B, CDH4_C, CDH4_D, CDH4_E, CDH4_F, CHST2_B, CLIP4, CR1, DLK1, DNAJC6, DNM3_A, EMX1_A, ESPN, FABP5, FAM150A, FLJ42875, GLP1R, GNG4, GYPC_A, HAND2, HES5, HNF1B_A, HNF1B_B, HOXA1_A, HOXA1_B, HOXA7_A, HOXA7_B, HOXA7_C, HOXD9, IGF2BP3_A, IGF2BP3_B, IGSF9B_A, IL15RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, LOC283999, LY6H, MAST1, MAX.chr1.158083198-158083476, MAX.chr1.228074764-228074977, MAX.chr1.46913931-46913950, MAX.chr10.130085265-130085312, MAX.chr11.68622869-68622968, MAX.chr14.101176106-101176260, MAX.chr15.96889069-96889128, MAX.chr17.8230197-8230314, MAX.chr19.46379903-46380197, MAX.chr2.97193163-97193287, MAX.chr2.97193478-97193562, MAX.chr20.1784209-1784461, MAX.chr21.44782441-44782498, MAX.chr22.23908718-23908782, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.180101084-180101094, MAX.chr5.42952185-42952280, MAX.chr5.42994866-42994936, MAX.chr6.27064703-27064783, MAX.chr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID2_B, NID2_C, ODC1, OSR2_B, PAQR6, PCDH8, PIF1, PPARA, PPP2R5C, PRDM13_A, PRHOXNB, PRKCB, RBFOX3_A, RBFOX3_B, RFX8, SNCA, STAC2_A, STAC2_B, STX16_B SYT5, TIMP2, TMEFF2, TNFRSF10D, TRH_B, TRIM67, TRIM71_C, USP44_A, USP44_B, UTF1, UTS2R, VSTM2B_A, VSTM2B_B, ZFP64, and ZNF132 (see Table 4, Example I). ·BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, NACAD, TRIM67, ATP6V1B1, C17orf64, CHST2_B, DLX4, DNM3_A, EMX1_A, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, COL23A1, KCNK9, LAYN, PLXNC1_A, RIC3, SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, and MAX.chr11.68622869-68622968 (see Table 11, Example I), ·ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, C10orf125 (see Table 16B, Example II).

[0018] From these 375 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish luminal A breast cancer tissue from benign breast tissue: ·ARL5C, BHLHE23_C, BMP6, C10orf125, C17orf64, C19orf66, CAMKV, CD1D, CDH4_E, CDH4_F, CHST2_A, CRHBP, DLX6, DNM3_A, DNM3_B, DNM3_C, ESYT3, ETS1_A, ETS1_B, FAM126A, FAM189A1, FAM20A, FAM59B, FBN1, FLRT2, FMN2, FOXP4, GAS7, GYPC_A, GYPC_B, HAND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931-46913950, MAX.chr11.68622869-68622968, MAX.chr12.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.73073682-73073814, MAX.chr20.1783841-1784054, MAX.chr21.47063802-47063851, MAX.chr4.8860002-8860038, MAX.chr5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MAX.chr8.688863-688924, MAX.chr9.114010-114207, MPZ, NID2_A, NKX2-6, ODC1, OSR2_A, POU4F1, PRDM13_B, PRKCB, RASGRF2, RIPPLY2, SLC30A10, ST8SIA4, SYN2, TRIM71_A, TRIM71_B, TRIM71_C, UBTF, ULBP1, USP44_B, and VSTM2B_A (see Table 5, Example I), ·BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf125, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, ST8SIA4, STX16_B UBTF, LOC100132891, ITPRIPL1, MAX.chr12.4273906-4274012, MAX.chr12.59990671-59990859, BHLHE23_D, COL23A1, KCNK9, OTX1, PLXNC1_A, HNF1B_B, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, DSCR6, MAX.chr11.68622869-68622968 (see Table 11, Example I), ·ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125 (see Table 16C, Example II).

[0019] From these 375 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish luminal B breast cancer tissue from benign breast tissue: · ACCN1, AJAP1_A, AJAP1_B, BEST4, CALN1_B, CBLN1_B, CDH4_E, DLX4, FOXP4, IGSF9B_B, ITPRIPL1, KCNA1, KLF16, LMX1B_A, MAST1, MAX.chr11.14926602-14927148, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B SYNJ2, TMEM176A, TSHZ3, and VIPR2 (see Table 6, Example I). · CALN1_A, LOC100132891, MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, DLX4, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, BHLHE23_D, HNF1B_B, TRH_A, ASCL2, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, AJAP1_B, and DSCR6 (see Table 11, Example I). ·ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D (see Table 16D, Example II).

[0020] From these 375 novel DNA methylation markers, the following markers and / or panels of markers that can distinguish BRCA1 breast cancer tissue from benign breast tissue were identified by further experiments: · C10orf93, C20orf195_A, C20orf195_B, CALN1_B, CBLN1_A, CBLN1_B, CCDC61, CCND2_A, CCND2_B, CCND2_C, EMX1_B, FAM150B, GRASP, HBM, ITPRIPL1, KCNK17_A, KIAA1949, LOC100131176, MAST1, MAX.chr1.8277285-8277316, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.30719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr6.157556793-157556856, MAX.chr8.124173030-124173395, MN1, MPZ, NR2F6, PDXK_A, PDXK_B, PTPRM, RYR2_B, SERPINB9_A, SERPINB9_B, SLC8A3, STX16_B TEPP, TOX, VIPR2, VSTM2B_A, ZNF486, ZNF626, and ZNF671 (see Table 7, Example I), ·BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6 (see Table 11, Example I).

[0021] From these 375 novel DNA methylation markers, the following markers and / or panels of markers that can distinguish BRCA2 breast cancer tissue from benign breast tissue were identified by further experiments: ·ANTXR2, B3GNT5, BHLHE23_C, BMP4, CHRNA7, EPHA4, FAM171A1, FAM20A, FMNL2, FSCN1, GSTP1, HBM, IGFBP5, IL17REL, ITGA9, ITPRIPL1, KIRREL2, LRRC34, MAX.chr1.239549742-239549886, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-151145743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64 (see Table 8, Example I). ·MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968 (see Table 11, Example I).

[0022] From these 375 novel DNA methylation markers, the following markers and / or panels of markers that can distinguish invasive breast cancer tissue from benign breast tissue were identified by further experiments: · CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MPZ, NKX2-6, PRKCB, RBFOX3_B, SALL3, and VSTM2B_A (see Table 2, Example I).

[0023] From these 375 novel DNA methylation markers, further experiments identified the following markers and / or marker panels that can distinguish between high-grade (DCIS-HG) breast cancer tissues and low-grade (DCIS-LG) breast tissues of ductal carcinoma in situ: · SCRT2_B, MPZ, MAX.chr8.124173030-124173395, ITPRIPL1, ITPRIPL1, DLX4, CALN1_A, and IGF2BP3_B (see Table 15, Example I), · SCRT2_B, ITPRIPL1, and MAX.chr8.124173030-12417339 (100% sensitivity with 91% specificity) (see Table 15, Example I), · DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, and ITPRIPL1 (see Table 17, Example II).

[0024] As described herein, the technology provides a number of methylated DNA markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, or 8 markers) that highly distinguish overall breast cancer and various breast cancer types (e.g., triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer). Experiments applied a selection filter to candidate markers to identify markers that provide a high signal-to-noise ratio and a low background level, providing high specificity for breast cancer screening or diagnosis.

[0025] In some embodiments, the technology relates to assessing the presence and methylation status of one or more of the markers identified herein in a biological sample (e.g., breast tissue, plasma sample). These markers include one or more differentially methylated regions (DMRs) as described herein, such as those provided in Tables 2 and 18. The methylation status is evaluated in embodiments of the technology. As such, the technology provided herein is not limited to methods by which the methylation status of a gene is measured. For example, in some embodiments, the methylation status is measured by a genome scanning method. For example, one method includes restriction landmark genome scanning (Kawai et al. (1994) Mol. Cell. Biol. 14:7421-7427), and another example includes methylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997) Cancer Res. 57:594-599). In some embodiments, changes in the methylation pattern at specific CpG sites are monitored by Southern analysis (digestion-Southern method) of the target region following digestion of genomic DNA with a methylation-sensitive restriction enzyme. In some embodiments, analyzing changes in the methylation pattern includes a PCR-based process that includes digestion of genomic DNA with a methylation-sensitive or methylation-dependent restriction enzyme prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18:687). Additionally, other techniques that utilize bisulfite treatment of DNA as a starting point for methylation analysis have been reported. These include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93:9821-9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl. Acids Res. 24:5058-5059, and Xiong and Laird (1997) Nucl. Acids Res. 25:2532-2534).The PCR technique has been developed for the detection of genetic mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) as well as the quantification of allele-specific expression (Szabo and Mann (1995) Genes Dev. 9:3097-3108, and Singer-Sam et al. (1992) PCR Methods Appl. 1:160-163). Such techniques use internal primers that anneal to the PCR-generated template and terminate immediately 5' of the single nucleotide being assayed. Methods using the "quantitative Ms-SNuPE assay" as described in U.S. Patent No. 7,037,650 are used in some embodiments.

[0026] When assessing methylation status, the methylation status is often expressed as the proportion or percentage of individual DNA strands that are methylated at a particular site (e.g., at a single nucleotide, at a particular region or locus, in a longer sequence of interest, e.g., in a DNA sequence of up to about 100 bp, 200 bp, 500 bp, 1000 bp or more) compared to the DNA population in the sample containing that site. Conventionally, the amount of unmethylated nucleic acid is determined by PCR using a calibrator substance. The known amount of DNA is then bisulfite-treated, and the resulting methylation-specific sequences are determined using either real-time PCR or other exponential amplification, e.g., the QuARTS assay (e.g., as provided by U.S. Patent No. 8,361,720, which is incorporated herein by reference, as well as U.S. Patent Application Publication Nos. 2012 / 0122088 and 2012 / 0122106).

[0027] For example, in some embodiments, the method includes generating a calibration curve for unmethylated targets by using an external standard. The calibration curve is composed of at least two points and relates to the real-time Ct values for unmethylated DNA relative to a known quantitative standard. A second calibration curve for methylated targets is then composed of at least two points and the external standard. This second calibration curve relates to the Ct values for methylated DNA relative to a known quantitative standard. Next, the test sample Ct values are determined for the methylated and unmethylated populations, and the genomic equivalent of the DNA is calculated from the calibration curves generated by the first two steps. The percentage of methylation at the target site is calculated from the amount of methylated DNA compared to the total amount of DNA in the population, e.g., (number of methylated DNA) / (number of methylated DNA + number of unmethylated DNA)×100.

[0028] Also provided herein are compositions and kits for practicing the methods. For example, in some embodiments, reagents specific for one or more markers (e.g., primers, probes) are provided alone or in sets (e.g., sets of primer pairs for amplifying multiple markers). Additional reagents for performing the detection assay (e.g., QuARTS, PCR, sequencing, bisulfite, or enzymes, buffers, positive and negative controls for performing other assays) may also be provided. In some embodiments, the kit contains reagents capable of modifying DNA in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents). In some embodiments, kits are provided that contain one or more reagents necessary, sufficient, or useful for practicing the methods. Also provided is a reaction mixture containing the reagents. Also provided is a master mix reagent set containing a plurality of reagents that may be added to each other and / or to a test sample to complete the reaction mixture.

[0029] In some embodiments, the techniques described herein are related to a programmable machine designed to perform a series of arithmetic or logical operations as provided by the methods described herein. For example, some embodiments of the techniques are related to (e.g., implemented by) computer software and / or computer hardware. In one aspect, the techniques relate to a computer including, in the form of memory, elements for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., methods as provided herein) for reading, manipulating, and storing data. In some embodiments, the microprocessor is part of a system for determining the methylation state (e.g., of one or more DMRs, e.g., DMRs 1-375 as provided in Tables 2 and 18), comparing the methylation state (e.g., of one or more DMRs, e.g., DMRs 1-375 as provided in Tables 2 and 18), generating a calibration curve, determining Ct values, calculating the ratio, frequency, or percentage of methylation (e.g., of one or more DMRs, e.g., DMRs 1-375 as provided in Tables 2 and 18), identifying CpG islands, determining the specificity and / or sensitivity of an assay or marker, calculating an ROC curve and related AUC, performing sequence analysis, all as described herein or as known in the art.

[0030] In some embodiments, the microprocessor or computer uses methylation state data in an algorithm for predicting the site of cancer.

[0031] In some embodiments, software or hardware components receive multiple assay results, determine a single-valued result, and report to a user indicating a cancer risk based on the results of multiple assays (e.g., determining the methylation status of multiple DMRs as provided in Tables 2 and 18). Related embodiments calculate a risk factor based on a mathematical combination (e.g., weighted combination, linear combination) of multiple assay results that determine the methylation status of multiple markers (e.g., multiple DMRs as provided in Tables 2 and 18). In some embodiments, the methylation status of a DMR defines a dimension and may have a value in a multi-dimensional space, and the coordinates defined by the methylation status of multiple DMRs are, for example, results related to cancer risk for reporting to a user.

[0032] Some embodiments include a storage medium and a memory component. The memory component (e.g., volatile and / or non-volatile memory) finds use in storing instructions (e.g., process embodiments as provided herein) and / or data (e.g., workpieces such as methylation measurements, arrays, and statistical descriptions related thereto). Some embodiments also relate to a system including one or more of a CPU, a graphics card, and a user interface (including an output device such as a display and an input device such as a keyboard).

[0033] Programmable machines related to the technology include conventional existing technologies and technologies under development or not yet developed (e.g., quantum computers, chemical computers, DNA computers, optical computers, computers based on spintronics, etc.).

[0034] In some embodiments, the technology includes a wired (e.g., metal cable, optical fiber) or wireless transmission medium for transmitting data. For example, some embodiments relate to data transmission via a network (e.g., local area network (LAN), wide area network (WAN), ad hoc network, Internet, etc.). In some embodiments, the programmable machine exists on a peer-like network, and in some embodiments, the programmable machine has a client / server relationship.

[0035] In some embodiments, the data is stored on a computer-readable storage medium, such as a hard disk, flash memory, optical medium, floppy disk, etc.

[0036] In some embodiments, the technology provided herein is associated with a plurality of programmable devices that operate in cooperation to implement the methods as described herein. For example, in some embodiments, multiple computers (e.g., connected by a network) may operate in parallel and collect and process data in an implementation of cluster computing or grid computing or some other distributed computer architecture that depends on full computers (equipped with an on-board CPU, storage device, power supply, network interface, etc.) connected to a network (private, public, or Internet) by a conventional network interface such as Ethernet, optical fiber, or by wireless network technology.

[0037] For example, some embodiments provide a computer that includes a computer-readable medium. The embodiments include a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Such a processor may include a microprocessor, an ASIC, a state machine, or other processors, and can be any of a number of computer processors, such as those made by Intel Corporation of Santa Clara, California and Motorola Corporation of Schaumburg, Illinois. Such a processor may include or communicate with a medium, such as a computer-readable medium, that stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

[0038] Embodiments of computer-readable media include, but are not limited to, electronic, optical, magnetic, or other storage or transmission devices that can provide a processor with computer-readable instructions. Other examples of suitable media include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, ASICs, configuration processors, all optical media, all magnetic tapes or other magnetic media, or any other media. Also, various other forms of computer-readable media may transmit or carry instructions to a computer and include both wired and wireless routers, private or public networks, or other transmission devices or channels. The instructions may include code from any suitable computer programming language, such as, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0039] The computer is connected to a network in some embodiments. The computer may also include a number of external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smart phones, pocket bells, digital tablets, laptop computers, Internet appliances, and other processor-based devices. Generally, the computers related to the technical aspects provided herein can support any operating system that can support one or more programs including the technologies provided herein, such as Microsoft Windows, Linux, UNIX, Mac It may be a platform based on any type of processor operating on OS X, etc. Some embodiments include personal computers that execute other application programs (e.g., applications). The applications can be stored in memory, and these can include, for example, word processing applications, spreadsheet applications, email applications, instant messenger applications, presentation applications, Internet browser applications, calendar / organizer applications, and any other applications executable by the client device.

[0040] All such components, computers, and systems described herein in connection with the technology may be logical or virtual.

[0041] Accordingly, provided herein is breast cancer and / or various breast cancer types (e.g., triple-negative breast cancer, HER2 +A technique related to a method for screening breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), the method assays the methylation status of markers in a sample (e.g., breast tissue) (e.g., plasma sample) obtained from a subject, and identifies the subject as having breast cancer and / or a specific breast cancer type when the methylation status of the marker is different from the methylation status of the marker assayed in a subject without breast cancer, and the marker includes bases in a DMR selected from the group consisting of variable methylation regions (DMRs) 1 to 375 as provided in Table 2 and Table 18.

[0042] In some embodiments, when the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in a subject without breast cancer, it indicates that the subject has breast cancer: ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1 (see Table 16E, Example II).

[0043] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer: ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B (see Table 22, Example III).

[0044] In some embodiments where the sample obtained from the subject is a blood sample (e.g., plasma, serum, whole blood) and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer: CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B (see Table 27, Example III).

[0045] In some embodiments where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, the subject is indicated as having triple-negative breast cancer: ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr11.14926602-14927148, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, MAX.chr4.8859253-8859329, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr6.157557371-157557657, MPZ, NKX2-6, PDX1, PLXNC1_A, PPARG, PRKCB, PTPRN2, RBFOX_A, SCRT2_A, SLC7A4, STAC2_B, STX16_A, STX16_B, TBX1, TRH_A, VSTM2B_A, ZBTB16, ZNF132, and ZSCAN23 (see Table 3, Example I).

[0046] In some embodiments, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, the subject is indicated as having triple-negative breast cancer: CALN1_A, LOC100132891, NACAD, TRIM67, ATP6V1B1, DLX4, GP5, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ST8SIA4, STX16_B ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, KCNK9, SCRT2_B, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, and DSCR6 (see Table 11, Example I).

[0047] In some embodiments, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, the subject is indicated as having triple-negative breast cancer: ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4273906-4274012, MAX.chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4 (see Table 16A, Example II).

[0048] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is HER2 +Indicating the presence of breast cancer: ABLIM1, AFAP1L1, AKR1B1, ALOX5, AMN, ARL5C, BANK1, BCAT1, BEGAIN, BEST4, BHLHE23_B, BHLHE23_C, C17orf64, C1QL2, C7orf52, CALN1_B, CAV2, CD8A, CDH4_A, CDH4_B, CDH4_C, CDH4_D, CDH4_E, CDH4_F, CHST2_B, CLIP4, CR1, DLK1, DNAJC6, DNM3_A, EMX1_A, ESPN, FABP5, FAM150A, FLJ42875, GLP1R, GNG4, GYPC_A, HAND2, HES5, HNF1B_A, HNF1B_B, HOXA1_A, HOXA1_B, HOXA7_A, HOXA7_B, HOXA7_C, HOXD9, IGF2BP3_A, IGF2BP3_B, IGSF9B_A, IL15RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, LOC283999, LY6H, MAST1, MAX.chr1.158083198-158083476, MAX.chr1.228074764-228074977, MAX.chr1.46913931-46913950, MAX.chr10.130085265-130085312, MAX.chr11.68622869-68622968, MAX.chr14.101176106-101176260, MAX.chr15.96889069-96889128, MAX.chr17.8230197-8230314, MAX.chr19.46379903-46380197, MAX.chr2.97193163-97193287, MAX.chr2.97193478-97193562, MAX.chr20.1784209-1784461, MAX.chr21.44782441-44782498, MAX.chr22.23908718-23908782, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.180101084-180101094, MAX.chr5.42952185-42952280, MAX.chr5.42994866-42994936, MAX.chr6.27064703-27064783, MAX.chr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID2_B, NID2_C, ODC1, OSR2_B, PAQR6, PCDH8, PIF1, PPARA, PPP2R5C, PRDM13_A, PRHOXNB, PRKCB, RBFOX3_A, RBFOX3_B, RFX8, SNCA, STAC2_A, STAC2_B, STX16_B SYT5, TIMP2, TMEFF2, TNFRSF10D, TRH_B, TRIM67, TRIM71_C, USP44_A, USP44_B, UTF1, UTS2R, VSTM2B_A, VSTM2B_B, ZFP64, and ZNF132 (see Table 4, Example I).

[0049] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more of the markers assayed in subjects without breast cancer, the subject is HER2 +Indicating having breast cancer: BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, NACAD, TRIM67, ATP6V1B1, C17orf64, CHST2_B, DLX4, DNM3_A, EMX1_A, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, COL23A1, KCNK9, LAYN, PLXNC1_A, RIC3, SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, and MAX.chr11.68622869-68622968 (see Table 11, Example I).

[0050] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more of the markers assayed in subjects without breast cancer, the subject is HER2 +Indicating the presence of breast cancer: ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, C10orf125 (see Table 16B, Example II).

[0051] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having luminal A breast cancer: ARL5C, BHLHE23_C, BMP6, C10orf125, C17orf64, C19orf66, CAMKV, CD1D, CDH4_E, CDH4_F, CHST2_A, CRHBP, DLX6, DNM3_A, DNM3_B, DNM3_C, ESYT3, ETS1_A, ETS1_B, FAM126A, FAM189A1, FAM20A, FAM59B, FBN1, FLRT2, FMN2, FOXP4, GAS7, GYPC_A, GYPC_B, HAND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931-46913950, MAX.chr11.68622869-68622968, MAX.chr12.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.73073682-73073814, MAX.chr20.1783841-1784054, MAX.chr21.47063802-47063851, MAX.chr4.8860002-8860038, MAX.chr5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MAX.chr8.688863-688924, MAX.chr9.114010-114207, MPZ, NID2_A, NKX2-6, ODC1, OSR2_A, POU4F1, PRDM13_B, PRKCB, RASGRF2, RIPPLY2, SLC30A10, ST8SIA4, SYN2, TRIM71_A, TRIM71_B, TRIM71_C, UBTF, ULBP1, USP44_B, and VSTM2B_A (see Table 5, Example I).

[0052] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is shown to have luminal A breast cancer: BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf125, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, ST8SIA4, STX16_B UBTF, LOC100132891, ITPRIPL1, MAX.chr12.4273906-4274012, MAX.chr12.59990671-59990859, BHLHE23_D, COL23A1, KCNK9, OTX1, PLXNC1_A, HNF1B_B, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, DSCR6, MAX.chr11.68622869-68622968 (see Table 11, Example I).

[0053] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having luminal A breast cancer: ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125 (see Table 16C, Example II).

[0054] In some embodiments where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, the subject is indicated as having luminal B breast cancer: ACCN1, AJAP1_A, AJAP1_B, BEST4, CALN1_B, CBLN1_B, CDH4_E, DLX4, FOXP4, IGSF9B_B, ITPRIPL1, KCNA1, KLF16, LMX1B_A, MAST1, MAX.chr11.14926602-14927148, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B SYNJ2, TMEM176A, TSHZ3, and VIPR2 (see Table 6, Example I).

[0055] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated to have luminal B breast cancer: CALN1_A, LOC100132891, MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, DLX4, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, BHLHE23_D, HNF1B_B, TRH_A, ASCL2, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, AJAP1_B, and DSCR6 (see Table 11, Example I).

[0056] In some embodiments where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, the subject is indicated as having luminal B breast cancer: ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D (see Table 16D, Example II).

[0057] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having BRCA1 breast cancer: C10orf93, C20orf195_A, C20orf195_B, CALN1_B, CBLN1_A, CBLN1_B, CCDC61, CCND2_A, CCND2_B, CCND2_C, EMX1_B, FAM150B, GRASP, HBM, ITPRIPL1, KCNK17_A, KIAA1949, LOC100131176, MAST1, MAX.chr1.8277285-8277316, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.30719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr6.157556793-157556856, MAX.chr8.124173030-124173395, MN1, MPZ, NR2F6, PDXK_A, PDXK_B, PTPRM, RYR2_B, SERPINB9_A, SERPINB9_B, SLC8A3, STX16_B TEPP, TOX, VIPR2, VSTM2B_A, ZNF486, ZNF626, and ZNF671 (see Table 7, Example I).

[0058] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having BRCA1 breast cancer: BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6 (see Table 11, Example I).

[0059] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having BRCA2 breast cancer: ANTXR2, B3GNT5, BHLHE23_C, BMP4, CHRNA7, EPHA4, FAM171A1, FAM20A, FMNL2, FSCN1, GSTP1, HBM, IGFBP5, IL17REL, ITGA9, ITPRIPL1, KIRREL2, LRRC34, MAX.chr1.239549742-239549886, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-151145743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64 (see Table 8, Example I).

[0060] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having BRCA2 breast cancer: MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968 (see Table 11, Example I).

[0061] In some embodiments where the sample obtained from the subject is breast tissue and the methylation status of one or more of the following markers is different from the methylation status of one or more markers assayed in subjects without breast cancer, the subject is indicated as having invasive breast cancer: CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MPZ, NKX2-6, PRKCB, RBFOX3_B, SALL3, and VSTM2B_A (see Table 9, Example I).

[0062] In some embodiments, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, distinguish between high-grade (DCIS-HG) breast cancer tissue and low-grade (DCIS-LG) breast tissue of ductal carcinoma in situ:SCRT2_B, MPZ, MAX.chr8.124173030-124173395, ITPRIPL1, ITPRIPL1, DLX4, CALN1_A, and IGF2BP3_B (see Table 15, Example I).

[0063] In some embodiments, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, distinguish between high-grade (DCIS-HG) breast cancer tissue and low-grade (DCIS-LG) breast tissue of ductal carcinoma in situ:SCRT2_B, ITPRIPL1, and MAX.chr8.124173030-12417339 (100% sensitivity with 91% specificity) (see Table 15, Example I).

[0064] In some embodiments, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers is different from the methylation state of one or more markers assayed in subjects without breast cancer, distinguish between high-grade (DCIS-HG) breast cancer tissue and low-grade (DCIS-LG) breast tissue of ductal carcinoma in situ:DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, and ITPRIPL1 (see Table 17, Example II).

[0065] The technology is for breast cancer and / or various breast cancer types (e.g., triple-negative breast cancer, HER2 +Relates to identifying and distinguishing breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer). Some embodiments provide methods that include assaying a plurality of markers, for example, methods that include assaying 2 to 11 to 100 or 120 or 375 markers.

[0066] The technology is not limited to the methylation state being evaluated. In some embodiments, evaluating the methylation state of a marker in a sample includes determining the methylation state of a single base. In some embodiments, assaying the methylation state of a marker in a sample includes determining the degree of methylation with multiple bases. Further, in some embodiments, the methylation state of a marker includes an increase in methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker includes a decrease in methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker includes a different pattern of methylation of the marker compared to the normal methylation state of the marker.

[0067] Further, in some embodiments, the marker is a region of 100 bases or less, the marker is a region of 500 bases or less, the marker is a region of 1000 bases or less, the marker is a region of 5000 bases or less, or in some embodiments, the marker is a single base. In some embodiments, the marker is present in a high CpG density promoter.

[0068] The technology is not limited by the type of sample. For example, in some embodiments, the sample is a fecal sample, a tissue sample (e.g., a breast tissue sample), a blood sample (e.g., plasma, serum, whole blood), excreta, or a urine sample.

[0069] Furthermore, the technology is not limited to the methods used to determine the methylation state. In some embodiments, assaying includes using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, assaying includes using methylation-specific oligonucleotides. In some embodiments, the technology uses next-generation sequencing (e.g., sequencing-by-synthesis, real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.) to determine the methylation state.

[0070] The technology provides reagents for detecting DMRs. For example, in some embodiments, a set of oligonucleotides comprising the sequences provided by SEQ ID NOs: 1-422 (see Tables 10, 19, and 20) is provided. In some embodiments, oligonucleotides are provided that include sequences complementary to chromosomal regions having bases in the DMR, e.g., oligonucleotides that are sensitive to the methylation state of the DMR.

[0071] The technology provides a panel of various markers used to identify breast cancer. For example, in some embodiments, the markers are ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1 (see Table 16E, Example II). The chromosome regions include annotations

[0072] The technology provides a panel of various markers used to identify breast cancer. For example, in some embodiments, the markers include chromosomal regions having the annotation of ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B (see Table 22, Example III).

[0073] The technology provides a panel of various markers used to identify breast cancer. For example, in some embodiments, the markers include chromosomal regions having the annotation of CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B (see Table 27, Example III).

[0074] The technology provides a panel of various markers used to identify triple-negative breast cancer. For example, in some embodiments, the markers are ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr11.14926602-14927148, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, MAX.chr4.8859253-8859329, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr6.157557371-157557657, MPZ, NKX2-6, PDX1, PLXNC1_A, PPARG, PRKCB, PTPRN2, RBFOX_A, SCRT2_A, SLC7A4, STAC2_B, STX16_A, STX16_B, TBX1, TRH_A, VSTM2B_A, ZBTB16, ZNF132, and ZSCAN23 (see Table 3, Example I) and includes chromosomal regions with the annotation.

[0075] The technology provides panels of various markers for use in identifying triple negative breast cancer. For example, in some embodiments, the markers include chromosomal regions having the annotation of CALN1_A, LOC100132891, NACAD, TRIM67, ATP6V1B1, DLX4, GP5, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ST8SIA4, STX16_B ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, KCNK9, SCRT2_B, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, and DSCR6 (see Table 11, Example I).

[0076] The technology provides panels of various markers for use in identifying triple negative breast cancer. For example, in some embodiments, the markers include chromosomal regions having the annotation of ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4273906-4274012, MAX.chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4 (see Table 16A, Example II).

[0077] The technology is HER2 +Provided is a panel of various markers used to identify breast cancer. For example, in some embodiments, the markers are ABLIM1, AFAP1L1, AKR1B1, ALOX5, AMN, ARL5C, BANK1, BCAT1, BEGAIN, BEST4, BHLHE23_B, BHLHE23_C, C17orf64, C1QL2, C7orf52, CALN1_B, CAV2, CD8A, CDH4_A, CDH4_B, CDH4_C, CDH4_D, CDH4_E, CDH4_F, CHST2_B, CLIP4, CR1, DLK1, DNAJC6, DNM3_A, EMX1_A, ESPN, FABP5, FAM150A, FLJ42875, GLP1R, GNG4, GYPC_A, HAND2, HES5, HNF1B_A, HNF1B_B, HOXA1_A, HOXA1_B, HOXA7_A, HOXA7_B, HOXA7_C, HOXD9, IGF2BP3_A, IGF2BP3_B, IGSF9B_A, IL15RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, LOC283999, LY6H, MAST1, MAX.chr1.158083198-158083476, MAX.chr1.228074764-228074977, MAX.chr1.46913931-46913950, MAX.chr10.130085265-130085312, MAX.chr11.68622869-68622968, MAX.chr14.101176106-101176260, MAX.chr15.96889069-96889128, MAX.chr17.8230197-8230314, MAX.chr19.46379903-46380197, MAX.chr2.97193163-97193287, MAX.chr2.97193478-97193562, MAX.chr20.1784209-1784461, MAX.chr21.44782441-44782498, MAX.chr22.23908718-23908782, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.180101084-180101094, MAX.chr5.Chromosomal regions having annotations of 42952185 - 42952280, MAX.chr5.42994866 - 42994936, MAX.chr6.27064703 - 27064783, MAX.chr7.152622607 - 152622638, MAX.chr8.145104132 - 145104218, MAX.chr9.136474504 - 136474527, MCF2L2, MSX2P1, NACAD, NID2_B, NID2_C, ODC1, OSR2_B, PAQR6, PCDH8, PIF1, PPARA, PPP2R5C, PRDM13_A, PRHOXNB, PRKCB, RBFOX3_A, RBFOX3_B, RFX8, SNCA, STAC2_A, STAC2_B, STX16_B SYT5, TIMP2, TMEFF2, TNFRSF10D, TRH_B, TRIM67, TRIM71_C, USP44_A, USP44_B, UTF1, UTS2R, VSTM2B_A, VSTM2B_B, ZFP64, and ZNF132 (see Table 4, Example I).

[0078] The technology is HER2 +Provided is a panel of various markers used to identify breast cancer. For example, in some embodiments, the markers are BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, NACAD, TRIM67, ATP6V1B1, C17orf64, CHST2_B, DLX4, DNM3_A, EMX1_A, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, COL23A1, KCNK9, LAYN, PLXNC1_A, RIC3, SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, and chromosomal regions having the annotation of MAX.chr11.68622869-68622968 (see Table 11, Example I).

[0079] The technology is HER2 +Provided is a panel of various markers used to identify breast cancer. For example, in some embodiments, the markers are ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, C10orf125 (see Table 16B, Example II), and include chromosomal regions with the annotation.

[0080] The technology provides a panel of various markers used to identify luminal A breast cancer. For example, in some embodiments, the markers are ARL5C, BHLHE23_C, BMP6, C10orf125, C17orf64, C19orf66, CAMKV, CD1D, CDH4_E, CDH4_F, CHST2_A, CRHBP, DLX6, DNM3_A, DNM3_B, DNM3_C, ESYT3, ETS1_A, ETS1_B, FAM126A, FAM189A1, FAM20A, FAM59B, FBN1, FLRT2, FMN2, FOXP4, GAS7, GYPC_A, GYPC_B, HAND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931 - 46913950, MAX.chr11.68622869 - 68622968, MAX.chr12.4273906 - 4274012, MAX.chr12.59990591 - 59990895, MAX.chr17.73073682 - 73073814, MAX.chr20.1783841 - 1784054, MAX.chr21.47063802 - 47063851, MAX.chr4.8860002 - 8860038, MAX.chr5.172234248 - 172234494, MAX.chr5.178957564 - 178957598, MAX.chr6.130686865 - 130686985, MAX.chr8.687688 - 687736, MAX.chr8.688863 - 688924, MAX.chr9.114010 - 114207, MPZ, NID2_A, NKX2 - 6, ODC1, OSR2_A, POU4F1, PRDM13_B, PRKCB, RASGRF2, RIPPLY2, SLC30A10, ST8SIA4, SYN2, TRIM71_A, TRIM71_B, TRIM71_C, UBTF, ULBP1, USP44_B, and VSTM2B_A (see Table 5, Example I), and includes chromosomal regions with the annotation.

[0081] The technology provides a panel of various markers used to identify luminal A breast cancer. For example, in some embodiments, the markers are BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf125, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, ST8SIA4, STX16_B UBTF, LOC100132891, ITPRIPL1, MAX.chr12.4273906-4274012, MAX.chr12.59990671-59990859, BHLHE23_D, COL23A1, KCNK9, OTX1, PLXNC1_A, HNF1B_B, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, DSCR6, MAX.chr11.68622869-68622968 (see Table 11, Example I), and include chromosomal regions with such annotations.

[0082] The technology provides a panel of various markers used to identify luminal A breast cancer. For example, in some embodiments, the markers are ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125 (see Table 16C, Example II).

[0083] The technology provides a panel of various markers used to identify luminal B breast cancer. For example, in some embodiments, the markers are ACCN1, AJAP1_A, AJAP1_B, BEST4, CALN1_B, CBLN1_B, CDH4_E, DLX4, FOXP4, IGSF9B_B, ITPRIPL1, KCNA1, KLF16, LMX1B_A, MAST1, MAX.chr11.14926602-14927148, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B SYNJ2, TMEM176A, TSHZ3, and VIPR2 (see Table 6, Example I).

[0084] The technology provides a panel of various markers used to identify luminal B breast cancer. For example, in some embodiments, the markers are CALN1_A, LOC100132891, MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, DLX4, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, BHLHE23_D, HNF1B_B, TRH_A, ASCL2, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, AJAP1_B, and DSCR6 (see Table 11, Example I), and includes chromosomal regions with the annotation.

[0085] The technology provides a panel of various markers used to identify luminal B breast cancer. For example, in some embodiments, the markers are ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D (see Table 16D, Example II), and includes chromosomal regions having the annotation.

[0086] The technology provides a panel of various markers used to identify BRCA1 breast cancer. For example, in some embodiments, the markers are C10orf93, C20orf195_A, C20orf195_B, CALN1_B, CBLN1_A, CBLN1_B, CCDC61, CCND2_A, CCND2_B, CCND2_C, EMX1_B, FAM150B, GRASP, HBM, ITPRIPL1, KCNK17_A, KIAA1949, LOC100131176, MAST1, MAX.chr1.8277285-8277316, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.30719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr6.157556793-157556856, MAX.chr8.124173030-124173395, MN1, MPZ, NR2F6, PDXK_A, PDXK_B, PTPRM, RYR2_B, SERPINB9_A, SERPINB9_B, SLC8A3, STX16_B TEPP, TOX, VIPR2, VSTM2B_A, ZNF486, ZNF626, and ZNF671 (see Table 7, Example I) and includes chromosomal regions with the annotation.

[0087] The technology provides a panel of various markers used to identify BRCA1 breast cancer. For example, in some embodiments, the markers are BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6 (see Table 11, Example I), and include chromosomal regions with the annotation.

[0088] The technology provides a panel of various markers used to identify BRCA2 breast cancer. For example, in some embodiments, the markers are ANTXR2, B3GNT5, BHLHE23_C, BMP4, CHRNA7, EPHA4, FAM171A1, FAM20A, FMNL2, FSCN1, GSTP1, HBM, IGFBP5, IL17REL, ITGA9, ITPRIPL1, KIRREL2, LRRC34, MAX.chr1.239549742-239549886, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-151145743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64 (see Table 8, Example I), and include chromosomal regions with the annotation.

[0089] The technology provides a panel of various markers used to identify BRCA2 breast cancer. For example, in some embodiments, the markers include chromosomal regions annotated as MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968 (see Table 11, Example I).

[0090] The technology provides a panel of various markers used to identify invasive breast cancer. For example, in some embodiments, the markers include chromosomal regions annotated as CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MPZ, NKX2-6, PRKCB, RBFOX3_B, SALL3, and VSTM2B_A (see Table 9, Example I).

[0091] The technology provides a panel of various markers used to distinguish between high-grade non-invasive ductal carcinoma in situ (DCIS-HG) breast cancer tissue and low-grade non-invasive ductal carcinoma in situ (DCIS-LG) breast tissue. For example, in some embodiments, the markers include chromosomal regions annotated as SCRT2_B, MPZ, MAX.chr8.124173030-124173395, ITPRIPL1, ITPRIPL1, DLX4, CALN1_A, and IGF2BP3_B (see Table 15, Example I).

[0092] The technology provides a panel of various markers used to distinguish between high-grade non-invasive ductal carcinoma in situ (DCIS-HG) breast cancer tissue and low-grade non-invasive ductal carcinoma in situ (DCIS-LG) breast tissue. For example, in some embodiments, the markers include chromosomal regions annotated as SCRT2_B, ITPRIPL1, and MAX.chr8.124173030-12417339 (100% sensitivity with 91% specificity) (see Table 15, Example I).

[0093] The technology provides a panel of various markers used to distinguish between high-grade non-invasive ductal carcinoma in situ (DCIS-HG) breast cancer tissue and low-grade non-invasive ductal carcinoma in situ (DCIS-LG) breast tissue. For example, in some embodiments, the markers include chromosomal regions annotated as DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, and ITPRIPL1 (see Table 17, Example II).

[0094] Embodiments of a kit are provided. For example, the kit can include reagents that can modify DNA by methylation-specific techniques (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents), and a control nucleic acid that includes the sequence of a DMR selected from the group consisting of DMR1-375 (from Tables 2 and 18) and has a methylation state associated with a subject without breast cancer. In some embodiments, the kit includes a bisulfite reagent and oligonucleotides as described herein. In some embodiments, the kit can include reagents that can modify DNA by methylation-specific techniques (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents), and a control nucleic acid that includes the sequence of a DMR selected from the group consisting of DMR1-375 (from Tables 2 and 18) and has a methylation state associated with a subject having breast cancer. Some kit embodiments include a sample collector for obtaining a sample (e.g., a fecal sample, a breast tissue sample, a plasma sample, a serum sample, a whole blood sample) from a subject, reagents that can modify DNA by methylation-specific techniques (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents), and oligonucleotides as described herein.

[0095] The technology relates to embodiments of a composition (e.g., a reaction mixture). In some embodiments, a composition is provided that includes a nucleic acid containing a DMR and reagents that can modify DNA by methylation-specific techniques (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents). Some embodiments provide a composition that includes a nucleic acid containing a DMR and oligonucleotides as described herein. Some embodiments provide a composition that includes a nucleic acid containing a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide a composition that includes a nucleic acid containing a DMR and a polymerase.

[0096] Additional related method embodiments include breast cancer and / or various breast cancer types (e.g., triple-negative breast cancer, HER2) in a sample obtained from a subject (e.g., a breast tissue sample, a plasma sample, a fecal sample) +Provided for screening for breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), for example, the method determines the methylation status of a marker in a sample containing a base in one or more DMRs of DMR1-375 (from Tables 2 and 18), the methylation status of the marker of the subject sample is compared with the methylation status of the marker of a normal control sample from a subject without breast cancer (e.g., breast cancer and / or breast cancer type: triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), and determining the confidence interval and / or p-value of the difference in the methylation status between the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of the method include reacting a nucleic acid containing a DMR with a reagent (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) that can modify the nucleic acid by a methylation-specific technique to generate, for example, a nucleic acid modified by a methylation-specific technique, sequencing the nucleic acid modified by a methylation-specific technique, providing the nucleotide sequence of the nucleic acid modified by a methylation-specific technique, comparing the nucleotide sequence of the nucleic acid modified by a methylation-specific technique with the nucleotide sequence of a nucleic acid containing a DMR from a subject without breast cancer and / or breast cancer type to identify the difference between the two sequences, and, if a difference exists, identifying that the subject has breast cancer (e.g., breast cancer and / or breast cancer type: triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer).

[0097] A system for screening for breast cancer in a sample obtained from a subject is provided by technology. Exemplary embodiments of the system include, for example, a system for screening for breast cancer and / or breast cancer type (e.g., triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer) in a sample obtained from a subject (e.g., a breast tissue sample, a plasma sample, a fecal sample), the system comprising an analysis component configured to determine the methylation state of the sample, a software component configured to compare the methylation state of the sample with the methylation state of a control sample or a reference sample recorded in a database, and a warning component configured to warn the user of a breast cancer-related methylation state. The warning is determined, in some embodiments, by a software component that receives results from a plurality of assays (e.g., a plurality of markers, e.g., determining the methylation state of a DMR, as provided in Tables 2 and 18), calculates a value or result, and reports based on the plurality of results. Some embodiments provide a database of weighting parameters associated with each DMR provided herein for use in calculating values or results and / or warnings to be reported to a user (e.g., a physician, a nurse, a clinician, etc.). In some embodiments, all results from a plurality of assays are reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a combination of one or more results from a plurality of assays indicative of the cancer risk in the subject.

[0098] In some embodiments of the system, the sample comprises a nucleic acid comprising a DMR. In some embodiments, the system further comprises a component for isolating the nucleic acid, a component for collecting the sample, e.g., a component for collecting a fecal sample, etc. In some embodiments, the system comprises a nucleic acid sequence comprising a DMR. In some embodiments, the database comprises breast cancer and / or a specific breast cancer type (e.g., triple-negative breast cancer, HER2 +It includes nucleic acid sequences from subjects without (breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer). Further provided is a nucleic acid, for example, a set of nucleic acids, each nucleic acid having a sequence containing a DMR. In some embodiments, the set of nucleic acids has sequences from subjects without breast cancer and / or a specific breast cancer type for each nucleic acid. Embodiments of related systems include a set of nucleic acids as described and a database of nucleic acid sequences related to the set of nucleic acids. Some embodiments further include reagents (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) that can modify DNA by methylation-specific techniques. Some embodiments also further include a nucleic acid sequencer.

[0099] In certain embodiments, provided is a method of characterizing a sample (e.g., a breast tissue sample, a plasma sample, a whole blood sample, a serum sample, a fecal sample) from a human patient. For example, in some embodiments, such embodiments include obtaining DNA from a sample of a human patient, assaying the methylation status of a DNA methylation marker containing a base in a DMR selected from the group consisting of variable methylation regions (DMRs) 1 - 375 from Tables 2 and 18, and + comparing the assayed methylation status of one or more DNA methylation markers to the methylation level criteria of one or more DNA methylation markers for human patients without (breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer).

[0100] Such methods are not limited to a particular type of sample derived from a human patient. In some embodiments, the sample is a breast tissue sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a fecal sample, a tissue sample, a breast tissue sample, a blood sample (e.g., a plasma sample, a whole blood sample, a serum sample), or a urine sample.

[0101] In some embodiments, such methods include assaying a plurality of DNA methylation markers. In some embodiments, such methods include assaying from 2 to 11 DNA methylation markers. In some embodiments, such methods include assaying from 12 to 120 DNA methylation markers. In some embodiments, such methods include assaying from 2 to 375 DNA methylation markers. In some embodiments, such methods include assaying the methylation state of one or more DNA methylation markers in a sample, including determining the methylation state of a single base. In some embodiments, such methods include assaying the methylation state of one or more DNA methylation markers in a sample, including determining the degree of methylation over a plurality of bases. In some embodiments, such methods include assaying the methylation state of the forward strand or assaying the methylation state of the reverse strand.

[0102] In some embodiments, the DNA methylation marker is a region of 100 bases or less. In some embodiments, the DNA methylation marker is a region of 500 bases or less. In some embodiments, the DNA methylation marker is a region of 1000 bases or less. In some embodiments, the DNA methylation marker is a region of 5000 bases or less. In some embodiments, the DNA methylation marker is a single base. In some embodiments, the DNA methylation marker is present in a high CpG density promoter.

[0103] In some embodiments, assaying includes using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or target capture.

[0104] In some embodiments, assaying comprises the use of methylation-specific oligonucleotides. In some embodiments, the methylation-specific oligonucleotides are selected from the group consisting of SEQ ID NOs: 1-422 (Tables 10, 19, and 20).

[0105] In some embodiments, chromosomal regions having an annotation selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1 (see Table 16E, Example II) contain DNA methylation markers.

[0106] In some embodiments, chromosomal regions having annotations selected from the group consisting of ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B (see Table 22, Example III) contain DNA methylation markers.

[0107] In some embodiments, chromosomal regions having annotations selected from the group consisting of CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B (see Table 27, Example III) contain DNA methylation markers.

[0108] In some embodiments, chromosomal regions having an annotation selected from the group consisting of ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr11.14926602-14927148, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, MAX.chr4.8859253-8859329, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr6.157557371-157557657, MPZ, NKX2-6, PDX1, PLXNC1_A, PPARG, PRKCB, PTPRN2, RBFOX_A, SCRT2_A, SLC7A4, STAC2_B, STX16_A, STX16_B, TBX1, TRH_A, VSTM2B_A, ZBTB16, ZNF132, and ZSCAN23 (see Table 3, Example I) contain DNA methylation markers.

[0109] In some embodiments, a chromosomal region having an annotation selected from the group consisting of CALN1_A, LOC100132891, NACAD, TRIM67, ATP6V1B1, DLX4, GP5, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ST8SIA4, STX16_B ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, KCNK9, SCRT2_B, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, and DSCR6 (see Table 11, Example I) contains a DNA methylation marker.

[0110] In some embodiments, a chromosomal region having an annotation selected from the group consisting of ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4273906-4274012, MAX.chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4 (see Table 16A, Example II) contains a DNA methylation marker.

[0111] In some embodiments, ABLIM1, AFAP1L1, AKR1B1, ALOX5, AMN, ARL5C, BANK1, BCAT1, BEGAIN, BEST4, BHLHE23_B, BHLHE23_C, C17orf64, C1QL2, C7orf52, CALN1_B, CAV2, CD8A, CDH4_A, CDH4_B, CDH4_C, CDH4_D, CDH4_E, CDH4_F, CHST2_B, CLIP4, CR1, DLK1, DNAJC6, DNM3_A, EMX1_A, ESPN, FABP5, FAM150A, FLJ42875, GLP1R, GNG4, GYPC_A, HAND2, HES5, HNF1B_A, HNF1B_B, HOXA1_A, HOXA1_B, HOXA7_A, HOXA7_B, HOXA7_C, HOXD9, IGF2BP3_A, IGF2BP3_B, IGSF9B_A, IL15RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, LOC283999, LY6H, MAST1, MAX.chr1.158083198-158083476, MAX.chr1.228074764-228074977, MAX.chr1.46913931-46913950, MAX.chr10.130085265-130085312, MAX.chr11.68622869-68622968, MAX.chr14.101176106-101176260, MAX.chr15.96889069-96889128, MAX.chr17.8230197-8230314, MAX.chr19.46379903-46380197, MAX.chr2.97193163-97193287, MAX.chr2.97193478-97193562, MAX.chr20.1784209-1784461, MAX.chr21.44782441-44782498, MAX.chr22.23908718-23908782, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.180101084-180101094, MAX.chr5.42952185-42952280, MAX.chr5.42994866-42994936, MAX.chr6.Chromosomal regions having an annotation selected from the group consisting of 27064703-27064783, MAX.chr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID2_B, NID2_C, ODC1, OSR2_B, PAQR6, PCDH8, PIF1, PPARA, PPP2R5C, PRDM13_A, PRHOXNB, PRKCB, RBFOX3_A, RBFOX3_B, RFX8, SNCA, STAC2_A, STAC2_B, STX16_B SYT5, TIMP2, TMEFF2, TNFRSF10D, TRH_B, TRIM67, TRIM71_C, USP44_A, USP44_B, UTF1, UTS2R, VSTM2B_A, VSTM2B_B, ZFP64, and ZNF132 (see Table 4, Example I) contain DNA methylation markers.

[0112] In some embodiments, chromosomal regions having annotations selected from the group consisting of BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, NACAD, TRIM67, ATP6V1B1, C17orf64, CHST2_B, DLX4, DNM3_A, EMX1_A, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, COL23A1, KCNK9, LAYN, PLXNC1_A, RIC3, SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, and MAX.chr11.68622869-68622968 (see Table 11, Example I) contain DNA methylation markers.

[0113] In some embodiments, chromosomal regions having an annotation selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, C10orf125 (see Table 16B, Example II) contain DNA methylation markers.

[0114] In some embodiments, chromosomal regions having annotations selected from the group consisting of ARL5C, BHLHE23_C, BMP6, C10orf125, C17orf64, C19orf66, CAMKV, CD1D, CDH4_E, CDH4_F, CHST2_A, CRHBP, DLX6, DNM3_A, DNM3_B, DNM3_C, ESYT3, ETS1_A, ETS1_B, FAM126A, FAM189A1, FAM20A, FAM59B, FBN1, FLRT2, FMN2, FOXP4, GAS7, GYPC_A, GYPC_B, HAND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931-46913950, MAX.chr11.68622869-68622968, MAX.chr12.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.73073682-73073814, MAX.chr20.1783841-1784054, MAX.chr21.47063802-47063851, MAX.chr4.8860002-8860038, MAX.chr5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MAX.chr8.688863-688924, MAX.chr9.114010-114207, MPZ, NID2_A, NKX2-6, ODC1, OSR2_A, POU4F1, PRDM13_B, PRKCB, RASGRF2, RIPPLY2, SLC30A10, ST8SIA4, SYN2, TRIM71_A, TRIM71_B, TRIM71_C, UBTF, ULBP1, USP44_B, and VSTM2B_A (see Table 5, Example I) contain DNA methylation markers.

[0115] In some embodiments, chromosomal regions having annotations selected from the group consisting of BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf125, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, ST8SIA4, STX16_B UBTF, LOC100132891, ITPRIPL1, MAX.chr12.4273906-4274012, MAX.chr12.59990671-59990859, BHLHE23_D, COL23A1, KCNK9, OTX1, PLXNC1_A, HNF1B_B, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, DSCR6, MAX.chr11.68622869-68622968 (see Table 11, Example I) contain DNA methylation markers.

[0116] In some embodiments, chromosomal regions having an annotation selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125 (see Table 16C, Example II) contain DNA methylation markers.

[0117] In some embodiments, chromosomal regions having annotations selected from the group consisting of ACCN1, AJAP1_A, AJAP1_B, BEST4, CALN1_B, CBLN1_B, CDH4_E, DLX4, FOXP4, IGSF9B_B, ITPRIPL1, KCNA1, KLF16, LMX1B_A, MAST1, MAX.chr11.14926602-14927148, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B SYNJ2, TMEM176A, TSHZ3, and VIPR2 (see Table 6, Example I) contain DNA methylation markers.

[0118] In some embodiments, a chromosomal region having an annotation selected from the group consisting of CALN1_A, LOC100132891, MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, DLX4, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, BHLHE23_D, HNF1B_B, TRH_A, ASCL2, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, AJAP1_B, and DSCR6 (see Table 11, Example I) contains a DNA methylation marker.

[0119] In some embodiments, chromosomal regions having an annotation selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D (see Table 16D, Example II) contain DNA methylation markers.

[0120] In some embodiments, chromosomal regions having annotations selected from the group consisting of C10orf93, C20orf195_A, C20orf195_B, CALN1_B, CBLN1_A, CBLN1_B, CCDC61, CCND2_A, CCND2_B, CCND2_C, EMX1_B, FAM150B, GRASP, HBM, ITPRIPL1, KCNK17_A, KIAA1949, LOC100131176, MAST1, MAX.chr1.8277285-8277316, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.30719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr6.157556793-157556856, MAX.chr8.124173030-124173395, MN1, MPZ, NR2F6, PDXK_A, PDXK_B, PTPRM, RYR2_B, SERPINB9_A, SERPINB9_B, SLC8A3, STX16_B TEPP, TOX, VIPR2, VSTM2B_A, ZNF486, ZNF626, and ZNF671 (see Table 7, Example I) contain DNA methylation markers.

[0121] In some embodiments, BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B A chromosomal region having an annotation selected from the group consisting of UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6 (see Table 11, Example I) contains a DNA methylation marker.

[0122] In some embodiments, chromosomal regions having annotations selected from the group consisting of ANTXR2, B3GNT5, BHLHE23_C, BMP4, CHRNA7, EPHA4, FAM171A1, FAM20A, FMNL2, FSCN1, GSTP1, HBM, IGFBP5, IL17REL, ITGA9, ITPRIPL1, KIRREL2, LRRC34, MAX.chr1.239549742-239549886, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-151145743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64 (see Table 8, Example I) contain DNA methylation markers.

[0123] In some embodiments, chromosomal regions having annotations selected from the group consisting of MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968 (see Table 11, Example I) contain DNA methylation markers.

[0124] In some embodiments, chromosomal regions having an annotation selected from the group consisting of CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MPZ, NKX2-6, PRKCB, RBFOX3_B, SALL3, and VSTM2B_A (see Table 9, Example I) contain DNA methylation markers.

[0125] In some embodiments, chromosomal regions having an annotation selected from the group consisting of SCRT2_B, MPZ, MAX.chr8.124173030-124173395, ITPRIPL1, ITPRIPL1, DLX4, CALN1_A, and IGF2BP3_B (see Table 15, Example I) contain DNA methylation markers.

[0126] In some embodiments, chromosomal regions having an annotation selected from the group consisting of DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, and ITPRIPL1 (see Table 17, Example II) contain DNA methylation markers.

[0127] In some embodiments, such methods include determining the methylation status of two DNA methylation markers. In some embodiments, such methods include determining the methylation status of pairs of DNA methylation markers provided in the rows of Table 2 and / or Table 18.

[0128] In certain embodiments, the technology provides a method for characterizing a sample obtained from a human patient (e.g., a breast tissue sample, a plasma sample, a whole blood sample, a serum sample, a fecal sample). In some embodiments, such methods include determining the methylation status of DNA methylation markers in a sample containing bases in a DMR selected from the group consisting of DMR1-375 from Tables 2 and 18, comparing the methylation status of the DNA methylation markers in the patient sample with the methylation status of the DNA methylation markers in a normal control sample from a human subject without breast cancer and / or a specific breast cancer type (e.g., triple negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), and determining the confidence interval and / or p-value of the difference in the methylation status between the human patient and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0129] In certain embodiments, the technology provides a method for characterizing a sample obtained from a human subject (e.g., a breast tissue sample, a plasma sample, a whole blood sample, a serum sample, a fecal sample), the method comprising reacting a nucleic acid comprising a DMR with a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) to generate a nucleic acid modified in a methylation-specific manner, sequencing the nucleic acid modified in a methylation-specific manner, providing a nucleotide sequence of the nucleic acid modified in a methylation-specific manner, and comparing the nucleotide sequence of the nucleic acid modified in a methylation-specific manner with the nucleotide sequence of a nucleic acid comprising a DMR from a subject without breast cancer to identify a difference between the two sequences.

[0130] In certain embodiments, the technology provides a system for characterizing a sample obtained from a human subject (e.g., a breast tissue sample, a plasma sample, a fecal sample), the system comprising an analysis component configured to determine the methylation state of the sample, a software component configured to compare the methylation state of the sample with the methylation state of a control sample or a reference sample recorded in a database, and a warning component configured to determine a single value based on the combination of methylation states and alert the user of a breast cancer-related methylation state. In some embodiments, the sample comprises a nucleic acid comprising a DMR.

[0131] In some embodiments, such a system further comprises a component for isolating nucleic acids. In some embodiments, such a system further comprises a component for collecting the sample.

[0132] In some embodiments, the sample is a fecal sample, a tissue sample, a breast tissue sample, a blood sample (e.g., a plasma sample, a whole blood sample, a serum sample), or a urine sample.

[0133] In some embodiments, the database comprises nucleic acid sequences comprising DMRs. In some embodiments, the database comprises nucleic acid sequences from subjects without breast cancer.

[0134] Further embodiments will be apparent to those skilled in the relevant arts based on the teachings contained herein.

[0135] Definitions To facilitate understanding of this technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0136] Throughout this specification and the claims, the following terms have the meanings explicitly associated herein unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, although it may, and further, the phrase "in another embodiment" as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, the various embodiments of the present invention may be readily combined without departing from the scope or spirit of the invention.

[0137] In addition, as used herein, the term "or" is an inclusive disjunctive operator and is synonymous with the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on additional factors not recited unless the context clearly dictates otherwise. Further, throughout this specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in" includes "in" and "on".

[0138] The transitional phrase "consisting essentially of", when used in the claims of this application, limits the claim to a particular substance or step and "those that do not materially affect the basic and novel characteristic(s) of the claimed invention", as stated in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition "consisting essentially of" the recited elements may contain impurities that do not change the function of the recited composition at a level that would change the function of the recited composition as compared to a pure composition, i.e., a composition "consisting of" the recited components.

[0139] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. Examples of "nucleic acids" include, but are not limited to, single-stranded and double-stranded nucleic acids. As used herein, the term "nucleic acid" also includes DNA as described above that contains one or more modified bases. Thus, DNA having a modified backbone for reasons of stability or otherwise is a "nucleic acid". The term "nucleic acid", as used herein, encompasses such chemical, enzymatic, or metabolic modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, such as simple and complex cells.

[0140] The terms "oligonucleotide", "polynucleotide", "nucleotide", or "nucleic acid" refer to molecules having two or more, preferably more than three, and usually more than ten deoxyribonucleotides or ribonucleotides. The exact size will depend on many factors and thus on the ultimate function or use of the oligonucleotide. Oligonucleotides may be produced by any method including chemical synthesis, DNA replication, reverse transcription, or combinations thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0141] As used herein, the terms "locus" or "region" of a nucleic acid refer to a small region of the nucleic acid, such as a gene, single nucleotide, CpG island, etc. on a chromosome.

[0142] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) that are associated by base pairing rules. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'. Complementarity can be "partial", in which case only some of the nucleic acid bases match according to the base pairing rules. Alternatively, "complete" or "total" complementarity may exist between nucleic acids. The degree of complementarity between nucleic acid strands gives rise to the efficiency and strength of hybridization between the nucleic acid strands. This is particularly important in amplification reactions and in detection methods that rely on binding between nucleic acids.

[0143] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes the coding sequence of RNA or is required for the production of a polypeptide or its precursor. A functional polypeptide can be encoded by the full-length coding sequence or by any part of the coding sequence as long as the desired activity or functional property of the polypeptide (e.g., enzyme activity, ligand binding, signal transduction, etc.) is retained. The term "part", when used with respect to a gene, refers to a fragment of that gene. The fragment can range in size from several nucleotides to one nucleotide less than the entire sequence of the gene. Thus, a "nucleotide comprising at least a part of a gene" can include a fragment of the gene or the entire gene.

[0144] The term "gene" also encompasses the coding region of a structural gene and includes sequences (e.g., including coding sequences, regulatory sequences, structural sequences, and other sequences) that flank the coding region at both the 5' and 3' ends, for example, located at a distance of about 1 kb on either end, so that the gene corresponds to the length of the full-length mRNA. Sequences located on the 5' side of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences located on the 3' side or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or 3' untranslated sequences. The term "gene" encompasses both cDNA and the genomic form of the gene. In some organisms (e.g., eukaryotes), the genomic form or clone of a gene contains coding regions interrupted by non-coding sequences referred to as "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA), and introns can contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nucleus or the primary transcript, and thus, introns are not present in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in the nascent polypeptide.

[0145] In addition to the intron content, the genomic form of a gene may also include sequences located at both the 5' and 3' ends of the sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located on the 5' side or 3' side of the untranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or affect the transcription of the gene. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation.

[0146] The term "wild-type" when referring to a gene refers to a gene having the characteristics of the gene isolated from a naturally occurring source. The term "wild-type" when referring to a gene product refers to a gene product having the characteristics of the gene product isolated from a naturally occurring source. The term "naturally occurring" when used with respect to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by human hand in the laboratory is naturally occurring. A wild-type gene is often the gene or allele that is observed at the highest frequency in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified" or "variant" when referring to a gene or gene product refer to a gene or gene product that exhibits a modification (e.g., a changed characteristic) in its sequence and / or functional properties when compared to the wild-type gene or gene product, respectively. Note that naturally occurring variants can be isolated and are identified by the fact that they have characteristics that are changed when compared to the wild-type gene or gene product.

[0147] The term "allele" refers to genetic diversity, which includes, but is not limited to, variants and mutants, polymorphic loci, as well as single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles may occur naturally in a population or may arise during the lifetime of any particular individual in the population.

[0148] Accordingly, the terms "variant" and "mutant", when used with respect to a nucleotide sequence, refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related nucleotide acid sequence. "Diversity" is the difference between two different nucleotide sequences, usually with one sequence being a reference sequence.

[0149] "Amplification" is a special case of nucleic acid replication that involves template specificity. It is in contrast to non-specific template replication (e.g., replication that is template-dependent but not dependent on a particular template). Template specificity is distinguished herein from replication fidelity (e.g., synthesis of the appropriate polynucleotide sequence) and nucleotide (ribonucleotide or deoxyribonucleotide) specificity. Template specificity is often described in terms of "target" specificity. Target sequences are "targets" in the sense that they are required to be selected from other nucleic acids. Amplification techniques are primarily designed for this selection.

[0150] The term "amplifying" or "amplification" in the context of nucleic acids typically refers to the production 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), and the amplification product or amplicon is generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The production of multiple DNA copies from one or a few copies of a target or template DNA molecule during polymerase chain reaction (PCR) or ligase chain reaction (LCR, see, e.g., U.S. Patent No. 5,494,810, which is hereby incorporated by reference in its entirety) is a form of amplification. Additional types of amplification include allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611, which is hereby incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Patent No. 5,965,408, which is hereby incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594, which is hereby incorporated by reference in its entirety), hot start PCR (see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671, each of which is hereby incorporated by reference in its entirety), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, which is hereby incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, See U.S. Patent No. 5,508,169 (25:1854 - 1858 (1997), which is hereby incorporated by reference in its entirety), methylation - specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821 - 9826, which is hereby incorporated by reference in its entirety), miniprimer PCR, multiplex ligation - dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57, which is hereby incorporated by reference in its entirety), multiplex PCR (see, e.g., 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, which are hereby incorporated by reference in their entireties), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15)7351 - 7367, which is hereby incorporated by reference in its entirety), real - time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413 - 417, Higuchi, et al., (1993) Biotechnology 11:1026 - 1030, which are hereby incorporated by reference in their entireties), reverse transcription PCR (see, e.g., Bustin, S.A. (2000) J. Molecular Endocrinology 25:169 - 193, which is hereby incorporated by reference in its entirety), solid - phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don, et al., Nucleic See, for example, Acids Research (1991) 19(14) 4008, Roux, K. (1994) Biotechniques 16(5) 812-814, Hecker, et al., (1996) Biotechniques 20(3) 478-485, each of which is incorporated herein by reference in its entirety, but not limited thereto. Polynucleotide amplification 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 No. WO05023091A2, US Patent Application Publication No. 20070202525, each of which is incorporated herein by reference in its entirety).

[0151] The term "polymerase chain reaction" ("PCR") refers to the methods of U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188 to K.B. Mullis that describe a method for increasing the concentration of a segment of a target sequence in a genomic or other DNA or RNA mixture without cloning or purification. This process for 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 performing thermal cycles in the correct order in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double-stranded target sequence. To effect amplification, the mixture is denatured and then the primers are annealed to their complementary sequences within the target molecules. After annealing, the primers are extended with a polymerase to form pairs of new complementary strands. The steps of denaturation, primer annealing, and polymerase extension are repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle" and there can be a number of "cycles"), and a high concentration of amplified segments of the desired target sequence can be obtained. The length of the amplified segment with respect to the desired target sequence is determined by the relative positions of the primers with respect to each other, and thus this length is a controllable parameter. Because of the iterative nature of the process, this method is referred to as the "polymerase chain reaction" ("PCR"). Since the desired amplified segments of the target sequence become the dominant sequences (with respect to concentration) in the mixture, they are said to be "amplified by PCR" and they are "PCR products" or "amplicons". One of ordinary skill in the art will understand that the term "PCR" encompasses many other forms of the described method that use, for example, real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer and arbitrary primer PCR, and the like.

[0152] Template specificity is achieved by enzyme selection in most amplification techniques. Amplification enzymes are enzymes that, under the conditions under which they are used, will process only the specific sequences of nucleic acids in a heterogeneous mixture of nucleic acids. For example, in the case of Q-beta replicase, MDV-1 RNA is the specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038

[1972] ). Other nucleic acids will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has stringent specificity for its own promoter (Chamberlin et al, Nature, 228:227

[1970] ). In the case of T4 DNA ligase, the enzyme will not ligate two oligonucleotides or polynucleotides if there is a mismatch between the substrate and the template of the oligonucleotide or polynucleotide in the ligation junction (Wu and Wallace (1989) Genomics 4:560). Finally, thermostable template-dependent DNA polymerases (e.g., Taq and Pfu DNA polymerases) have been found to exhibit high specificity for the sequences defined by primer binding by virtue of their ability to function at high temperatures, and thus high temperature provides thermodynamic conditions that are favorable for primer hybridization to the target sequence but not for hybridization to non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0153] 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, structure-specific cleavage assays (e.g., INVADER assays (Hologic, Inc.), e.g., U.S. Patent Nos. 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, each of which is incorporated herein by reference in its entirety for all purposes), the enzyme mismatch cleavage method (e.g., Variagenics, U.S. Patent Nos. 6,110,684, 5,958,692, 5,851,770, each of which is incorporated herein by reference in its entirety), the polymerase chain reaction (PCR) described above, the branched hybridization method (e.g., Chiron, U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, each of which is incorporated herein by reference in its entirety), rolling circle replication (e.g., U.S. Patent Nos. 6,210,884, 6,183,960, and 6,235,502, each of which is incorporated herein by reference in its entirety), NASBA (e.g., U.S. Patent No. 5,409,818, which is incorporated herein by reference in its entirety), molecular beacon technology (e.g., U.S. Patent No. 6,150,097, which is incorporated herein by reference in its entirety), E sensor technology (Motorola, U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, each of which is incorporated herein by reference in its entirety), cycling probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988, each of which is incorporated herein by reference in its entirety), Dade Behring signal amplification method (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, each of which is incorporated herein by reference in its entirety), ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88,189-93(1991)), and the sandwich hybridization method (e.g., U.S. Patent No. 5,288,609, which is incorporated herein by reference in its entirety), but are not limited thereto.

[0154] The term "amplifiable nucleic acid" refers to a nucleic acid that can potentially be amplified by any amplification method. It is contemplated that "amplifiable nucleic acids" will usually include "sample templates".

[0155] The term "sample template" refers to a nucleic acid derived from a sample that is analyzed for the presence of a "target" (defined below). In contrast, "background template" is used with respect to nucleic acids other than sample templates that may or may not be present in the sample. Background templates are most often incidental. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants that need to be purified away from the sample. For example, nucleic acids from organisms other than those being detected may be present as background in the test sample.

[0156] The term "primer" refers to any oligonucleotide, whether occurring naturally as, for example, a nucleic acid fragment from a restriction digest or produced synthetically, which is capable of functioning as an initiation point for synthesis of a primer extension product complementary to a nucleic acid template strand when placed under conditions (e.g., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at a suitable temperature and pH) in which synthesis of the extension product is induced. A primer is preferably single-stranded for maximum amplification efficiency but may alternatively be double-stranded. In the case of double-stranded primers, the primer is first treated to separate the strands and then used for preparation of the extension product. Preferably, the primer is an oligodeoxyribonucleotide. A primer must be of sufficient length to initiate synthesis of the extension product in the presence of the inducing agent. The exact length of the primer will depend on a number of factors including temperature, primer source, and method of use.

[0157] The term "probe" refers to any oligonucleotide (e.g., a nucleotide sequence) that occurs naturally, is synthesized, recombinantly produced, or generated by PCR amplification, similar to a purified restriction digest, which can hybridize to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). In some embodiments, any probe used in the present invention may be labeled with any "reporter molecule" such that it is detectable in any detection system, including, but not limited to, enzyme (e.g., enzyme-based histochemical assays in addition to ELISA), fluorescence, radiation, and luminescence systems. The present invention is not intended to be limited to any particular detection system or label.

[0158] The term "target", as used herein, refers to a nucleic acid that is required to be selected from other nucleic acids, e.g., by probe binding, amplification, isolation, capture, etc. For example, when used in connection with the polymerase chain reaction, "target" refers to the region of the nucleic acid that is bound by the primers used in the polymerase chain reaction, while when used in an assay where the target DNA is not amplified, e.g., in some embodiments of the invasive cleavage assay, the target includes the site where the probe and the invasive oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure, and thus the presence of the target nucleic acid can be detected. "Segment" is defined as a region of nucleic acid within the target sequence.

[0159] As used herein, "methylation" refers to cytosine methylation at the C5 or N4 position of cytosine, N6 of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is not normally methylated because typical in vitro DNA amplification methods do not retain the methylation pattern of the amplification template. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA where the original template was not methylated or was methylated, respectively.

[0160] Thus, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, which methyl moiety is not present in the typical nucleotide bases that are recognized. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, while 5-methylcytosine contains a methyl moiety at the 5-position of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, while 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5-position of its pyrimidine ring, but for the purposes herein, since thymine is a typical nucleotide base of DNA, thymine is not considered a methylated nucleotide when present in DNA.

[0161] As used herein, "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0162] As used herein, the "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 to be 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 to be unmethylated.

[0163] The methylation state of a particular nucleic acid sequence (e.g., a gene marker or DNA region as described herein) can indicate the methylation state of all bases in the sequence, or the methylation state of a subset of bases within the sequence (e.g., one or more cytosines), or can indicate information regarding the local methylation density within the sequence, with or without providing the exact position information where methylation occurs.

[0164] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of methylated nucleotides at a specific locus in the nucleic acid molecule. For example, the methylation state of cytosine at the 7th nucleotide in a nucleic acid molecule is methylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine. Similarly, the methylation state of cytosine at the 7th nucleotide in a nucleic acid molecule is not methylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (and not 5-methylcytosine).

[0165] The methylation state can optionally be represented or indicated by a "methylation value" (e.g., representing methylation frequency, ratio, proportion, percentage, etc.). The methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion using a methylation-dependent restriction enzyme, or by comparing amplification profiles after bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Thus, a value, e.g., a methylation value, represents the methylation state and can therefore be used as a quantitative measure of the methylation state across multiple copies of the locus. This is for particular applications where it is desirable to compare the methylation state of sequences in a sample to a threshold or reference value.

[0166] As used herein, "methylation frequency" or "methylation percentage (%)" refers to the number of instances where a molecule or locus is methylated compared to the number of instances where the molecule or locus is not methylated.

[0167] As such, the methylation state describes the methylation state of nucleic acids (e.g., genomic sequences). Further, the methylation state refers to the characteristics of nucleic acid segments at specific genomic loci related to methylation. Such characteristics include whether any of the cytosine (C) residues within this DNA sequence are methylated, the position(s) of the methylated C residue(s), the frequency or percentage of methylated C across any specific region of the nucleic acid, and allelic differences in methylation due to, for example, differences in allelic origin, but are not limited thereto. The terms “methylation state”, “methylation profile”, and “methylation status” also refer to the relative concentration, absolute concentration, or pattern of methylated C or unmethylated C across any specific region of a nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) within a nucleic acid sequence is methylated, it may be referred to as having “hypermethylation” or “an increase in methylation”, whereas if a cytosine (C) residue(s) within a DNA sequence is not methylated, it may be referred to as having “hypomethylation” or “a decrease in methylation”. Similarly, if a cytosine (C) residue(s) within a nucleic acid sequence is methylated when compared to another nucleic acid sequence (e.g., from a different region, or a different individual, etc.), that sequence is considered to have hypermethylation or an increase in methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) within a DNA sequence is not methylated when compared to another nucleic acid sequence (e.g., from a different region, or a different individual, etc.), that sequence is considered to have hypomethylation or a decrease in methylation compared to the other nucleic acid sequence. Further, the term “methylation pattern” as used herein refers to the collective sites of methylated and unmethylated nucleotides across a region of a nucleic acid. Two nucleic acids may have the same or similar methylation frequency or percentage of methylation, but may have 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.Arrays are said to have "variable methylation", or "differences in methylation", or "different methylation states" if they differ in the degree of methylation (e.g., one has an increase or decrease in methylation compared to the other), frequency, or pattern. The term "variable methylation" refers to the difference in the level or pattern of nucleic acid methylation in cancer-positive samples compared to that in cancer-negative samples. It may also refer to the difference in level or pattern between patients in whom cancer has recurred after surgery and those in whom it has not. Variable methylation and specific levels or patterns of DNA methylation can be prognostic and predictive biomarkers, for example, when an exact cut-off or predictive characteristic is defined.

[0168] Methylation state frequency can be used to describe samples from a population of individuals or a single individual. For example, a nucleotide locus with a 50% methylation state frequency is methylated in 50% of cases and not methylated in 50% of cases. Such frequencies can be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or nucleic acid aggregate. Thus, if the methylation in a first population or pool of nucleic acid molecules is different from that in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from that of the second population or pool. Such frequencies can also be used, for example, to describe 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 describe the degree to which a cell population from a tissue sample is methylated or not methylated at a nucleotide locus or nucleic acid region.

[0169] As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. A nucleotide locus of a methylated nucleotide refers to the position of the methylated nucleotide in a nucleic acid molecule.

[0170] Typically, methylation of human DNA occurs on dinucleotide sequences containing adjacent guanine and cytosine where the cytosine is located on the 5’ side of the guanine (also referred to as CpG dinucleotide sequences). Many of the cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific CpG dinucleotide-rich genomic regions known as CpG islands (see, for example, Antequera et al. (1990) Cell 62:503-514).

[0171] As used herein, "CpG island" refers to a G:C rich region of genomic DNA that contains an increased number of CpG dinucleotides compared to the genomic DNA as a whole. CpG islands can be at least 100, 200, or more base pairs in length, in which case the G:C content of the region is at least 50% and the ratio of the observed CpG frequency to the predicted frequency is 0.6. In some cases, CpG islands can be at least 500 base pairs in length, in which case the G:C content of the region is at least 55% and the ratio of the observed CpG frequency to the predicted frequency is 0.65. The ratio of the observed CpG frequency to the predicted frequency can be calculated by the method provided by Gardiner-Garden et al (1987) J. Mol. Biol. 196:261-281. For example, the ratio of the observed CpG frequency to the predicted frequency can be calculated by the formula R = (A × B) / (C × D), where R is the ratio of the observed CpG frequency to the predicted frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. The methylation state is usually determined in CpG islands, for example, in the promoter region. However, other sequences in the human genome are understood to have a tendency for DNA methylation such as CpA and CpT (see 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) Biochem. Biophys. Res. Commun. 145:888-894).

[0172] As used herein, the term "methylation-specific reagent" refers to a reagent that modifies nucleotides of a nucleic acid molecule according to the methylation state of the nucleic acid molecule, or a methylation-specific reagent refers to a compound or composition or other agent that can change the nucleotide sequence of a nucleic acid molecule by a method 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, if desired, in combination with additional steps, to achieve a desired change in the nucleotide sequence. Such a method may be applied in a manner in which unmethylated nucleotides (e.g., each unmethylated cytosine) are modified to a different nucleotide. For example, in some embodiments, such a reagent may deaminate unmethylated cytosine nucleotides to produce deoxyuracil residues. Examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents.

[0173] Changes in the nucleotide sequence of a nucleic acid by a methylation-specific reagent can also result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide.

[0174] The term "methylation assay" refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within the sequence of a nucleic acid.

[0175] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to a technique recognized in the art that enables a genome-wide scan using CG-rich primers and focuses on regions most likely to contain CpG dinucleotides, as described in Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0176] The term "MethyLight™" refers to a fluorescence-based real-time PCR technique recognized in the art as described by Eads et al. (1999) Cancer Res. 59:2302-2306.

[0177] The term "HeavyMethyl™" refers to an assay in which methylation-specific blocking probes (also referred to herein as blockers) that span CpG positions between amplification primers, or are spanned by amplification primers, enable methylation-specific selective amplification of a nucleic acid sample.

[0178] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay in which the MethyLight™ assay is combined with methylation-specific blocking probes that span CpG positions between amplification primers.

[0179] The term "Ms-SNuPE" (methylation-sensitive single nucleotide primer extension) refers to an assay recognized in the art as described by Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0180] The term "MSP" (methylation-specific PCR) refers to a methylation assay recognized in the art as described by Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826 and U.S. Patent No. 5,786,146.

[0181] The term "COBRA" (combined bisulfite restriction analysis) refers to a methylation assay recognized in the art as described by Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0182] The term "MCA" (methylated CpG island amplification) refers to the methylation assay described by Toyota et al. (1999) Cancer Res. 59:2307-12 and in WO00 / 26401A1.

[0183] As used herein, "selected nucleotide" refers to one of the four nucleotides (C, G, T, and A for DNA and C, G, U, and A for RNA) that are normally present in a nucleic acid molecule and may include methylated derivatives of the normally present nucleotides (e.g., if C is the selected nucleotide, both methylated C and non-methylated C are included in the meaning of the selected nucleotide), whereas "selected methylated nucleotide" specifically refers to the normally present methylated nucleotide, and "selected non-methylated nucleotide" specifically refers to the normally present non-methylated nucleotide.

[0184] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cleaves when the recognition site is not methylated or is hemimethylated (a methylation-sensitive enzyme), cleavage will not occur (or will occur with significantly reduced efficiency) when the recognition site is methylated on one or both strands. In the case of a restriction enzyme that specifically cleaves only when the recognition site is methylated (a methylation-dependent enzyme), cleavage will not occur (or will occur with significantly reduced efficiency) when the recognition site is not methylated. Methylation-specific restriction enzymes are preferred, and their recognition sequences contain CG dinucleotides (e.g., recognition sequences such as CGCG or CCCGGG). Even more preferred in some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at carbon atom C5.

[0185] As used herein, "different nucleotide" refers to a nucleotide that is chemically different from the selected nucleotide, and as a result, typically, the different nucleotide has Watson-Crick base pairing properties different from those of the selected nucleotide, such that the normally occurring nucleotide complementary to the selected nucleotide is not the same as the normally occurring nucleotide complementary to the different nucleotide. For example, if C is the selected nucleotide, U or T can be a different nucleotide, as illustrated by the complementarity of C to G and U or T to A. As used herein, a nucleotide complementary to the selected nucleotide, or to a different nucleotide, refers to a nucleotide that base pairs with the selected nucleotide or the different nucleotide with higher affinity than base pairing of a complementary nucleotide to three of the four normally occurring nucleotides under high stringency conditions. An example of complementarity is the Watson-Crick base pairing of DNA (e.g., A-T and C-G) and RNA (e.g., A-U and C-G). Thus, for example, if C is the selected nucleotide, G is the nucleotide complementary to the selected nucleotide because G base pairs with C with higher affinity than G base pairs with G, A, or T under high stringency conditions.

[0186] As used herein, the “sensitivity” of a given marker (or set of markers used together) refers to the percentage of samples that report DNA methylation values exceeding a threshold that distinguishes neoplastic from non-neoplastic samples. In some embodiments, positive is defined as a histologically confirmed neoplasm that reports a DNA methylation value exceeding a threshold (e.g., a range associated with the disease), and false negative is defined as a histologically confirmed neoplasm that reports a DNA methylation value below a threshold (e.g., a range not associated with the disease). Thus, the value of sensitivity reflects the probability that a DNA methylation measurement of a given marker obtained from a known diseased sample will fall within the range of disease-associated measurements. As defined herein, the clinical relevance of a calculated sensitivity value represents an estimate of the probability that a given marker, when applied to a subject with a clinical condition, will detect the presence of that clinical condition.

[0187] As used herein, the “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 from non-neoplastic samples. In some embodiments, negative is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value below a threshold (e.g., a range not associated with the disease), and false positive is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value exceeding a threshold (e.g., a range associated with the disease). Thus, the value of specificity reflects the probability that a DNA methylation measurement of a given marker obtained from a known non-neoplastic sample will fall within the range of non-disease-associated measurements. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a given marker, when applied to a patient without a clinical condition, will detect the absence of that clinical condition.

[0188] As used herein, the term "AUC" is an abbreviation for "area under the curve". In particular, AUC refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for different possible cut-off points of a diagnostic test. The ROC curve shows the trade-off between sensitivity and specificity that depends on the selected cut-off point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is an indicator of the accuracy of a diagnostic test (the larger the area, the better, with a maximum of 1, and a random test having a ROC curve located on the diagonal with an area of 0.5. See J.P. Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0189] As used herein, the term "neoplasm" refers to any new abnormal growth of tissue. Thus, a neoplasm can be a pre-malignant or malignant neoplasm.

[0190] As used herein, the term "neoplasm-specific marker" refers to any biological material or element that can 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 intronic region, a specific locus, etc.). The nucleic acid region that is a marker may be referred to, for example, as a "marker gene", "marker region", "marker sequence", "marker locus", etc.

[0191] As used herein, the term "adenoma" refers to a benign tumor of glandular origin. Although these growths are benign, over time they can progress and become malignant.

[0192] The terms "preneoplastic" or "pre-tumorous" and their synonyms refer to any cell proliferative disorder that has undergone malignant transformation.

[0193] A "site" such as a neoplasm, adenoma, cancer, etc. is a tissue, organ, cell type, anatomical region, body part, etc. in the body of a subject in which the neoplasm, adenoma, cancer, etc. is located.

[0194] As used herein, the application of a "diagnostic" test includes detecting or identifying a subject's disease state or condition, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject having a disease or condition will respond to a therapy, determining the prognosis (or the likelihood of progression or regression) of a subject having a disease or condition, and determining the effect of a treatment on a subject having a disease or condition. For example, a diagnosis can be used to detect the presence or likelihood of a subject having a neoplasm or the likelihood that such a subject will respond favorably to a compound (e.g., a medicament, e.g., a drug) or other treatment.

[0195] The term "isolated", when used with respect to a nucleic acid, as is the case with an "isolated oligonucleotide", refers to a nucleic acid sequence that has been identified and separated from at least one contaminating nucleic acid with which it is ordinarily associated in its natural source. An isolated nucleic acid exists in a form or context different from that in which it is found in nature. In contrast, non-isolated nucleic acids such as DNA and RNA are found in the state in which they exist in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found on the host cell chromosome in proximity to an adjacent gene, an RNA sequence, e.g., a particular mRNA sequence encoding a specific protein, found in the cell as part of a mixture containing a number of other mRNAs encoding a plurality of proteins. However, examples of an isolated nucleic acid encoding a particular protein include such a nucleic acid in a cell that ordinarily expresses the protein, where the nucleic acid is present at a chromosomal location different from that of its natural cell, or alternatively, is adjacent to a nucleic acid sequence different from that found in nature. An isolated nucleic acid or oligonucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is utilized for protein expression, the oligonucleotide will contain at least the sense strand or coding strand (i.e., the oligonucleotide may be single-stranded), but may also contain both the sense strand and the antisense strand (i.e., the oligonucleotide may be double-stranded). An isolated nucleic acid may be combined with other nucleic acids or molecules after isolation from its natural or typical environment. For example, an isolated nucleic acid may be present, for example, in a host cell in which it has been placed for heterologous expression.

[0196] The term "purified" refers to a molecule, such as a nucleic acid or amino acid sequence, that has been removed from its natural environment, isolated, or separated, i.e., either a nucleic acid or an amino acid sequence. Thus, an "isolated nucleic acid sequence" may be a purified nucleic acid sequence. A "substantially purified" molecule contains at least 60% less, preferably at least 75% less, more preferably at least 90% less of other components with which they are naturally associated. As used herein, the terms "purified" or "purifying" also refer to the removal of contaminants from a sample. Removal of contaminating proteins increases the percentage of the polypeptide or nucleic acid of interest in the sample. In another example, a recombinant polypeptide is expressed in a plant, bacterium, yeast, or mammalian host cell, and the polypeptide is purified by removal of host cell proteins, thereby increasing the percentage of the recombinant polypeptide in the sample.

[0197] The term "composition comprising" a given polynucleotide sequence or polypeptide broadly refers to any composition containing the given polynucleotide sequence or polypeptide. The composition may include an aqueous solution containing salts (e.g., NaCl), surfactants (e.g., SDS), and other components (e.g., Denhardt's solution, milk powder, salmon sperm DNA, etc.).

[0198] The term "sample" is used in its broadest sense. In one sense, it can refer to animal cells or tissues. In another sense, it refers to biological samples and environmental samples in addition to specimens or cultures obtained from any source. Biological samples may be obtained from plants or animals (including humans) and include fluids, solids, tissues, and gases. Environmental samples include environmental substances such as surface substances, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0199] As used herein, "remote sample" refers to a sample that is indirectly collected from a site that is not the source of the sample of cells, tissue, or organ, when used in some contexts. For example, when a sample substance derived from the pancreas is evaluated in a fecal sample (e.g., not derived from a sample directly taken from the breast), the sample is a remote sample.

[0200] As used herein, the terms "patient" or "subject" refer to an organism that undergoes various tests provided by the technology. The term "subject" includes mammals, including animals, preferably humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Further with regard to diagnostic methods, the preferred subject is a vertebrate subject. Preferred vertebrates are warm-blooded animals, and preferred warm-blooded vertebrates are mammals. Preferred mammals are most preferably humans. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, by the present technology, mammals, such as, in addition to humans, those mammals that are critically endangered, such as the Amur tiger, those economically important mammals that are farmed for human consumption, and / or as pets, or animals kept in zoos, such as those socially important animals for humans, can be diagnosed. Examples of such animals include carnivores such as cats and dogs, pigs (including swine such as pigs, boars, and wild boars), ruminants and / or ungulates such as cows, bulls, sheep, giraffes, deer, goats, bison, and camels, pinnipeds, and horses, but are not limited thereto. Thus, further provided are the diagnosis and treatment of livestock, including but not limited to domesticated pigs, ruminants, ungulates, horses (including racehorses), etc. The subject matter disclosed herein further includes a system for diagnosing lung cancer in a subject. The system can be provided, for example, as a commercially available kit that screens for the risk of lung cancer in a subject from whom a biological sample has been collected or can be used to diagnose lung cancer. Exemplary systems provided in accordance with the present technology include evaluating the methylation status of the markers described herein.

[0201] 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 suitable containers) and / or support materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosed containers (e.g., boxes) containing the relevant reaction reagents and / or support materials. As used herein, the term "subdivided kit" refers to a delivery system that includes containers, each of which contains a portion of the total components of the kit, in two or more separate containers. The containers may be delivered to the intended recipient together or separately. For example, the first container may contain an enzyme for use in the assay, while the second container may contain oligonucleotides. The term "subdivided kit" is intended to include, but is not limited to, kits that contain analyte-specific reagents (ASRs) that are regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system that includes containers, each of which contains a portion of the total components of the kit, in two or more separate containers is included in the term "subdivided kit". In contrast, a "combined kit" refers to a delivery system that contains all of the components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both subdivided and combined kits.

[0202] As used herein, the term "breast cancer" generally refers to the uncontrolled growth of breast tissue, and more specifically, to a condition characterized by the abnormally rapid growth of abnormal cells in one or both breasts of a subject. The abnormal cells are often referred to as malignant or "neoplastic cells", which are transformed cells that can form solid tumors. The term "tumor" refers to an abnormal mass or collection of cells (i.e., two or more cells) resulting from excessive or abnormal cell division, whether malignant or benign, as well as pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cell growth, the tumor can invade surrounding tissues and metastasize.

[0203] As used herein, the term "HER2 + breast cancer" refers to breast cancer in which at least a portion of the cancer cells express an elevated level of the HER2 protein (HER2 (derived from human epidermal growth factor receptor 2) or HER2 / neu) that promotes rapid cell growth.

[0204] As used herein, the term "luminal A breast cancer" refers to breast cancer in which at least a portion of the cancer cells are estrogen receptor (ER) positive and progesterone receptor (PR) positive, but negative for HER2.

[0205] As used herein, the term "luminal B breast cancer" refers to breast cancer in which at least a portion of the cancer cells are ER positive, HER2 positive, and negative for PR.

[0206] As used herein, the term "triple-negative breast cancer" refers to breast cancer in which at least a portion of the cancer cells are negative for ER, HER2, and PR.

[0207] As used herein, the term "HER2 + breast cancer" refers to breast cancer in which at least a portion of the cancer cells are negative for ER and PR, but positive for HER2.

[0208] As used herein, the term "BRCA1 breast cancer" refers to breast cancer in which at least a portion of the cancer cells are characterized by a mutation in the BRCA1 gene and / or have reduced expression of wild-type BRCA1.

[0209] As used herein, the term "BRCA2 breast cancer" refers to breast cancer in which at least a portion of the cancer cells are characterized by a mutation in the BRCA2 gene and / or have reduced expression of wild-type BRCA2.

[0210] As used herein, the term "ductal carcinoma in situ" (DCIS) refers to a non-invasive cancer in which abnormal cells are found inside the milk ducts of the breast. "Low-grade" DCIS refers to DCIS that has a nuclear atypia of 1 or a low mitotic rate. "High-grade" DCIS refers to DCIS that has a nuclear atypia of 3 or a high mitotic rate. "Invasive" DCIS refers to ductal breast cancer that has metastasized to non-ductal tissue.

[0211] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using a computer system(s), including but not limited to the Internet, the term refers to any data stored in any form (e.g., analog, digital, optical, etc.). As used herein, the term "information related to a subject" refers to facts or data about a subject (e.g., a human, plant, or animal). The term "genomic information" refers to information related to the genome, including but not limited to nucleic acid sequences, genes, percentage methylation, allele frequencies, RNA expression levels, protein expression, phenotypes related to genotypes, etc. "Allele frequency information" refers to facts or data related to allele frequencies, including but not limited to allele identification information, statistical correlations between alleles and the presence of traits in a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, and the percentage likelihood of alleles present in individuals having one or more specific traits.

DETAILED DESCRIPTION OF THE INVENTION

[0212] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, one of ordinary skill in the art will understand that these various embodiments may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form. Further, one of ordinary skill in the art can readily understand that the specific order in which the methods are presented and performed is exemplary, and the order can be changed while still remaining within the spirit and scope of the various embodiments disclosed herein.

[0213] Provided herein are technologies related to breast cancer screening, and in particular, methods, compositions, and related uses for detecting the presence of breast cancer and / or certain breast cancer types (e.g., triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), but not limited thereto. As the technology is described herein, the headings of the sections used are for organizational purposes only and should not be construed in any way as limiting the subject matter.

[0214] Indeed, as described in Examples I, II, and III, experiments conducted during the process of identifying embodiments of the present invention identified a novel set of 375 variable methylation regions (DMRs) to distinguish cancerous from non-tumorous control DNA and breast-derived DNA. From these 375 novel DNA methylation markers, further experiments identified markers that can distinguish different breast cancer types from normal breast tissue. For example, distinct sets of DMRs were identified that 1) distinguish triple-negative breast cancer tissue from normal breast tissue, and 2) HER2 +It is possible to distinguish breast cancer tissue from normal breast tissue, 3) luminal A breast cancer tissue from normal breast tissue, 4) luminal B breast cancer tissue from normal breast tissue, 5) BRCA1 breast cancer tissue from normal breast tissue, 6) BRCA2 breast cancer tissue from normal breast tissue, and 7) invasive breast cancer tissue from normal breast tissue. Additionally, DMRs have been identified, and these can distinguish non-invasive ductal carcinoma high-grade (DCIS-HG) breast cancer tissue from non-invasive ductal carcinoma low-grade (DCIS-LG) breast tissue. Additionally, DMRs have been identified, and these can distinguish the plasma of subjects with breast cancer from the plasma of subjects without breast cancer.

[0215] The disclosure herein refers to certain exemplary embodiments, which are presented by way of example and not limitation.

[0216] In certain aspects, the technology provides compositions and methods for identifying, determining, and / or classifying cancers such as breast cancer. The methods include determining the methylation status of at least one methylation marker in a biological sample (e.g., fecal sample, breast tissue sample, plasma sample) isolated from a subject, wherein a change in the methylation status of the marker indicates the presence, classification, or location of breast cancer. Certain embodiments relate to markers that include variable methylation regions (DMRs, e.g., DMR1-375, see Tables 2 and 18) for use in the diagnosis (e.g., screening) of breast cancer and various breast cancer types (e.g., triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer).

[0217] In addition to embodiments in which the methylation analysis of at least one marker, marker region, or marker base that includes a DMR (e.g., DMR, e.g., DMR1-375) provided herein and listed in Tables 2 and 18 is analyzed, the technology also provides a panel of markers that includes at least one marker, marker region, or marker base that includes a DMR useful for the detection of cancer, particularly breast cancer.

[0218] Some embodiments of the technology are based on an analysis of at least one marker, a region of a marker, or the CpG methylation status of the bases of a marker, including DMR.

[0219] In some embodiments, the technology enables the use of reagents (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) that modify DNA by methylation-specific methods in combination with one or more methylation assays, to determine the methylation status of CpG dinucleotide sequences within at least one marker including DMR (e.g., see DMR1-375, Tables 2 and 18). Genomic CpG dinucleotides can be methylated or unmethylated (or are known as hypermethylation and hypomethylation, respectively). However, the methods of the present invention are suitable for the analysis of heterogeneous properties, such as biological samples with low concentrations of tumor cells, or biological substances derived therefrom, within the background of remote samples (e.g., blood, organ effluents, or feces). Thus, when analyzing the methylation status of CpG positions within such samples, a quantitative assay may be used to determine the level (e.g., percentage, proportion, ratio, rate, or degree) of methylation at a specific CpG position.

[0220] According to the technology, determination of the methylation status of CpG dinucleotide sequences within markers including DMR is useful for both the diagnosis and characterization of cancers such as breast cancer.

[0221] Combination of markers In some embodiments, the technology relates to evaluating the methylation status of combinations of markers including DMR (e.g., DMR numbers 1-375) from Tables 2 and 18. In some embodiments, evaluating the methylation status of two or more markers improves the specificity and / or sensitivity of screening or diagnosis for identifying a neoplasm (e.g., breast cancer) in a subject.

[0222] Various cancers are predicted by various combinations of markers, such as those identified by statistical techniques related to the specificity and sensitivity of prediction. The present technology provides a method for identifying predictive combinations and validated predictive combinations for several cancers.

[0223] Method for assaying methylation status In certain embodiments, a method for analyzing nucleic acids for the presence of 5-methylcytosine includes treating the DNA with a reagent that modifies the DNA in a methylation-specific manner. Examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents.

[0224] Frequently used methods for analyzing nucleic acids for the presence of 5-methylcytosine are based on the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosine in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, which is hereby expressly incorporated by reference in its entirety for all purposes) or their diversity. The bisulfite method for mapping 5-methylcytosine is based on the observation that cytosine reacts with hydrogen sulfite ions (also known as bisulfite) rather than 5-methylcytosine. The reaction is typically carried out in the following steps: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, spontaneous deamination of the sulfonated reaction intermediate results in sulfonated uracil. Finally, sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil base pairs with adenine (and thus behaves like thymine), while 5-methylcytosine base pairs with guanine (and thus behaves like cytosine). This allows for the discrimination between methylated and non-methylated cytosine, for example, by using bisulfite genomic 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 involving 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 technology” Clin Chem 56:A199, and see also U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392).

[0225] Some prior art relates to methods that involve placing the DNA to be analyzed in an agarose matrix, thereby preventing diffusion and reannealing of the DNA (bisulfite reacts only 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 individual cells for their methylation status and demonstrate the utility and sensitivity of the method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0226] Bisulfite technology typically involves amplifying short specific fragments of known nucleic acids after bisulfite treatment, and then assaying the products either by 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 use 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). Additionally, the use of bisulfite technology for methylation detection with respect to individual genes has been 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).

[0227] A variety of methylation assay procedures can be used in combination with bisulfite treatment according to this technique. These assays enable determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays include, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific restriction enzymes, e.g., methylation-sensitive or methylation-dependent enzymes.

[0228] For example, genomic sequencing has been simplified for the analysis of methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). Furthermore, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA finds use in the assessment of methylation status, as described, for example, by Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059 or as embodied by a method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0229] COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in the PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). Next, PCR amplification of bisulfite-converted DNA is carried out using primers specific for the CpG island of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using a specific labeled hybridization probe. The methylation level in the original DNA sample is represented by the relative amounts of digested and undigested PCR products, by a linear quantitative method over a wide range of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0230] Typical reagents for COBRA (trademark) analysis (such as those that may be found in a typical COBRA (trademark)-based kit) may include, but are not limited to, PCR primers for specific loci (such as specific genes, markers, DMRs, 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. Further, bisulfite conversion reagents may include DNA denaturation buffer, sulfonation buffer, DNA recovery reagents or kits (such as precipitation, ultrafiltration, affinity column), desulfonation buffer, and DNA recovery components.

[0231] Assays, such as "MethyLight" (trademark) (fluorescence-based real-time PCR technology) (Eads et al., Cancer Res. 59:2302 - 2306, 1999), Ms-SNuPE (trademark) (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 methylation CpG island amplification ("MCA", Toyota et al., Cancer Res. 59:2307 - 12, 1999), etc. are used alone or in combination with one or more of these methods.

[0232] The "HeavyMethyl" (trademark) assay technology is a quantitative method for evaluating methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") that cover the CpG positions between amplification primers or are covered by amplification primers enable methylation-specific selective amplification of nucleic acid samples.

[0233] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay, where the MethyLight™ assay is combined with methylation-specific blocking probes that span the CpG positions between amplification primers. The HeavyMethyl™ assay may also be used in combination with methylation-specific amplification primers.

[0234] Typical reagents for HeavyMethyl™ analysis (such as may be found in typical MethyLight™-based kits) may include, but are not limited to, PCR primers for a particular locus (such as a particular gene, marker, region of a gene, region of a marker, bisulfite-treated DNA sequence, CpG island, or bisulfite-treated DNA sequence or CpG island, etc.), blocking oligonucleotides, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0235] MSP (methylation-specific PCR) enables the evaluation of the methylation status of substantially any of the bases of CpG sites within CpG islands, independent of the use of methylation-sensitive 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 by sodium bisulfite, which converts unmethylated cytosine, but not methylated cytosine, to uracil, and subsequently the product is amplified with primers specific for methylated DNA as compared to unmethylated DNA. Only a small amount of DNA is required for MSP, and it is highly sensitive to 0.1% methylated alleles of a given CpG island locus and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents for MSP analysis (such as may be found in a typical MSP-based kit) may include, but are not limited to, methylation and unmethylated PCR primers for a specific locus (such as a specific gene, marker, region of a gene, region of a marker, bisulfite-treated DNA sequence, CpG island, etc.), optimized PCR buffer and deoxynucleotides, and specific probes.

[0236] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) that does not require 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 that is converted by a sodium bisulfite reaction by standard procedures into a mixed pool with methylation-dependent sequence differences (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction using, for example, PCR primers that overlap known CpG dinucleotides. The sequence discrimination is performed at both the level of the amplification process and the level of the fluorescence detection process.

[0237] The MethyLight™ assay is used as a quantitative test of methylation patterns in nucleic acids, e.g., genomic DNA samples, and sequence discrimination is done at the level of probe hybridization. In the quantitative version, methylation-specific amplification is made possible by a PCR reaction in the presence of a fluorescent probe overlapping a specific putative methylation site. A bias-free control for DNA input is provided by a reaction where neither the primers nor the probe overlap any CpG dinucleotide. Alternatively, a qualitative test of genomic methylation is achieved by probing a biased PCR pool with either a control oligonucleotide that does not cover known methylation sites (e.g., the HeavyMethyl™ and fluorescent-based versions of the MSP technology) or an oligonucleotide that covers potent methylation sites.

[0238] The MethyLight™ process is used with any suitable probe (e.g., “TaqMan®” probe, Lightcycler® probe, 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, for example, MSP primers and / or HeavyMethyl blocker oligonucleotides and TaqMan® probe. The TaqMan® probe is double-labeled with fluorescent “reporter” and “quencher” molecules and is designed to be specific to a relatively high GC-containing region such that the probe melts during the PCR cycle at a temperature approximately 10° C. higher than the forward or reverse primer. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. When Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the finally annealed TaqMan® probe. Then, the 5’ to 3’ endonuclease activity of Taq polymerase will digest the TaqMan® probe to release the fluorescent reporter molecule, thereby displacing the probe to quantitatively detect the signal not quenched at the time of detection using a real-time fluorescence detection system.

[0239] Typical reagents for MethyLight™ analysis (e.g., such as may be 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, region of a gene, region of a marker, bisulfite-treated DNA sequence, CpG island, etc.), TaqMan® or Lightcycler® probe, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0240] The QM (trademark) (Quantitative Methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, and sequence discrimination is performed at the level of probe hybridization. In the quantitative version, PCR reactions enable unbiased amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for DNA input is provided by a reaction where neither the primers nor the probes overlap any arbitrary CpG dinucleotide. Alternatively, qualitative testing of genomic methylation is achieved by probing a biased PCR pool with either a control oligonucleotide that does not cover known methylation sites (HeavyMethyl (trademark) and the fluorescence-based version of MSP technology) or an oligonucleotide that covers potential methylation sites.

[0241] The QM (trademark) process can be used with any suitable probe during the amplification process, for example, "TaqMan (registered trademark)" probes, Lightcycler (registered trademark) probes. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and TaqMan (registered trademark) probes. TaqMan (registered trademark) probes are double-labeled with fluorescent "reporter" and "quencher" molecules and are designed to be specific for relatively high GC-containing regions such that the probe melts during the PCR cycle at a temperature approximately 10°C higher than the forward or reverse primer. This allows the TaqMan (registered trademark) probe to continue to hybridize fully during the PCR annealing / extension step. When Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the finally annealed TaqMan (registered trademark) probe. Then, the 5' to 3' endonuclease activity of Taq polymerase will replace the TaqMan (registered trademark) probe by digesting it to release the fluorescent reporter molecule in order to quantitatively detect the signal that is not quenched during detection using a real-time fluorescence detection system. Typical reagents for QM (trademark) analysis (such as may be found in a typical QM (trademark)-based kit) may include, but are not limited to, PCR primers for specific loci (such as specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan (registered trademark) or Lightcycler (registered trademark) probes, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0242] The Ms-SNuPE (trademark) technique is a quantitative method for evaluating methylation differences 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 leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then carried out using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site of interest. A small amount of DNA can be analyzed (e.g., microdissected pathological sections), thereby avoiding the use of restriction enzymes to determine the methylation status at the CpG site.

[0243] Typical reagents for Ms-SNuPE (trademark) analysis (such as may be found in a typical Ms-SNuPE (trademark)-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 deoxynucleotides, a gel extraction kit, positive control primers, Ms-SNuPE (trademark) primers for specific loci, reaction buffer (for the Ms-SNuPE reaction), and labeled nucleotides. Further, bisulfite conversion reagents may include DNA denaturation buffer, sulfonation buffer, DNA recovery reagent or kit (e.g., precipitation, ultrafiltration, affinity column), desulfonation buffer, and DNA recovery components.

[0244] Reduced Representation Bisulfite Sequencing (RRBS) starts with bisulfite treatment of nucleic acids, converting all unmethylated cytosines to uracil, followed by restriction enzyme digestion (e.g., by an enzyme that recognizes sites containing CG sequences such as Mspl) and complete sequencing of the fragments after ligation to adapter ligands. By the choice of restriction enzyme, fragments of CpG-dense regions are enriched and the number of redundant sequences that may map to multiple genetic loci during analysis is reduced. As such, 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 generated by restriction enzyme digestion contain DNA methylation information for at least one CpG dinucleotide. As such, RRBS enriches samples of promoters, CpG islands, and other genomic features with frequently occurring restriction enzyme cleavage sites in these regions, and thus provides an assay for assessing the methylation status of one or more genomic loci.

[0245] A typical protocol for RRBS includes steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, inserting overhangs and A-tailing, ligating an adapter, bisulfite conversion step, as well as a PCR step. See, 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. 33:5868-77.

[0246] In some embodiments, the Quantitative Allele-Specific Real-Time Target and Signal Amplification (QuARTS) assay is used to evaluate methylation status. In each QuARTS assay, three reactions occur sequentially, with the primary reactions including amplification (Reaction 1) and target probe cleavage (Reaction 2), and the secondary reaction including FRET cleavage and fluorescence signal generation (Reaction 3). When the target nucleic acid is amplified with specific primers, a specific detection probe with a flap sequence binds loosely to the amplicon. The presence of a specific invading oligonucleotide at the target binding site causes the flap sequence to be released by cleavage between the detection probe and the flap sequence by a 5' nuclease, such as FEN-1 endonuclease. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence functions as an invading oligonucleotide on the FRET cassette, causing cleavage between the FRET cassette fluorophore and quencher and generating a fluorescence signal. The cleavage reaction can cleave multiple probes for each target, and thus release multiple fluorophores for each flap, providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. For example, see Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56:A199), as well as U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, each of which is incorporated herein by reference for all purposes.

[0247] The term "bisulfite reagent" refers to a reagent containing bisulfite, disulfite, hydrogen sulfite, or a combination thereof, and as disclosed herein, is useful for distinguishing methylated CpG dinucleotide sequences from unmethylated CpG dinucleotide sequences. Methods of treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each incorporated by reference in its entirety). In some embodiments, 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, but not limited to, a chroman derivative such as 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid or trihydroxybenzoic acid and derivatives thereof such as gallic acid (see PCT / EP2004 / 011715, incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction includes treatment with ammonium bisulfite as described, for example, in WO2013 / 116375.

[0248] In some embodiments, the fragments of treated DNA are amplified using a set of primer oligonucleotides according to the invention (see, for example, Tables 10, 19, and 20) as well as an amplification enzyme. Amplification of some DNA segments can be carried out simultaneously in one and the same reaction vessel. Typically, amplification is carried out using the polymerase chain reaction (PCR). The amplicons are typically 100 to 2000 base pairs in length.

[0249] In another embodiment of the method, the methylation status of CpG positions within or near markers that include DMRs (e.g., DMR1-375, Tables 2 and 18) may be detected by use of methylation-specific primer oligonucleotides. This technique (MSP) is described in Herman U.S. Patent No. 6,265,171. Use of methylation status-specific primers for amplification of bisulfite-treated DNA allows discrimination between methylated and unmethylated nucleic acids. An MSP primer pair contains at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Thus, the primer sequences include at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the position of the C in the CpG.

[0250] Fragments obtained by amplification may carry a directly or indirectly detectable label. In some embodiments, the label is a fluorescent label, a radionuclide, or a removable molecular fragment having a typical mass detectable by a mass spectrometer. Where the label is a mass label, some embodiments result in the labeled amplicon having a single positive or negative net charge, allowing good detectability in a mass spectrometer. Detection may be performed and visualized, for example, by matrix-assisted laser desorption ionization mass spectrometry (MALDI) or using electrospray ionization mass spectrometry (ESI).

[0251] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments include isolation of nucleic acids as described in U.S. Patent Application No. 13 / 470,251, "Isolation of Nucleic Acids", which is hereby incorporated by reference in its entirety.

[0252] In some embodiments, the markers described herein find use in the QUARTS assay performed on fecal samples. In some embodiments, provided are methods of generating a DNA sample and, in particular, methods of generating a DNA sample that is substantially and / or effectively free of substances that inhibit assays (e.g., PCR, INVADER, QuARTS assay, etc.) used to test the DNA sample and that contain a small amount (e.g., less than 100 microliters, less than 60 microliters) of highly purified low copy number nucleic acids. Such DNA samples find use in diagnostic assays that 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 that quantitatively measure their activity, expression, or amount. For example, some cancers correlate with the presence of specific variant alleles or specific methylation states, and thus, detecting and / or quantifying such variant alleles or methylation states has predictive value in cancer diagnosis and treatment.

[0253] Many useful genetic markers are present in very small amounts in a sample, and many of the events that generate such markers are rare. As a result, even highly sensitive detection methods such as PCR require large amounts of DNA to sufficiently provide a low copy number target that meets or replaces the assay's detection threshold. Furthermore, the presence of inhibitory substances, even in small amounts, compromises the accuracy and precision of these assays aimed at detecting such low copy number targets. Accordingly, provided herein are methods that provide the necessary control of volume and concentration for generating such DNA samples.

[0254] In some embodiments, the sample includes blood, serum, plasma, or saliva. In some embodiments, the subject is human. Such samples can be obtained by a number of means known in the art, for example, apparent to those of ordinary skill in the art. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to those of ordinary skill in the art, including, but not limited to, centrifugation and filtration. Although non-invasive techniques are generally preferred for obtaining samples, it may still be preferred in some cases to obtain samples such as tissue homogenates, tissue sections, and biopsy materials. The techniques are not limited to methods used to prepare the sample and to provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from fecal samples, or from blood, or from plasma samples using direct gene capture, such as that detailed in U.S. Patent Nos. 8,808,990 and 9,169,511, and WO2012 / 155072, or related methods.

[0255] Analysis of the marker can be performed separately or simultaneously with additional markers within one test sample. For example, some markers can be combined in one test for efficient processing of multiple samples and for the potential to provide higher accuracy in diagnosis and / or prognosis. In addition, those of ordinary skill in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing on a series of samples can enable identification of changes over time in the methylation state of the marker. In addition to changes in the methylation state, the absence of changes in the methylation state can provide useful information about the outcome of the subject, including, but not limited to, identifying the approximate time from onset of an event, the presence and amount of recoverable tissue, the validity of drug therapy, the effectiveness of various therapies, and the risk of future events.

[0256] The analysis of biomarkers can be carried out in various physical forms. For example, the use of microtiter plates or automation can be used to facilitate the processing of a large number of test samples. Alternatively, a single sample format can be developed in a timely manner to facilitate immediate treatment and diagnosis, for example, in the context of external transportation or emergency room situations.

[0257] Embodiments of the technology are contemplated to be provided in the form of a kit. The kit includes the compositions, devices, apparatuses, etc. described herein, and embodiments of instructions regarding the use of the kit. Such instructions describe appropriate methods for preparing an analyte from a sample, for example, methods for collecting a sample and preparing nucleic acids from the sample. The individual components of the kit are packaged in suitable containers and packaging (e.g., vials, boxes, blister packs, ampoules, bottles, flasks, tubes, etc.), and the components are packaged together in a suitable container (e.g., box(es)) for convenient storage, transportation, and / or use by the user of the kit. It is understood that liquid components (e.g., buffers) may be provided in lyophilized form to be reconstituted by the user. The kit may include controls or references to evaluate, verify, and / or guarantee 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 or another nucleic acid of known concentration for comparison, and in some embodiments, a detection reagent (e.g., primer) specific for the control nucleic acid. The kit is suitable for use in a clinical setting and, in some embodiments, for use in the user's home. The components of the kit, in some embodiments, provide the functionality of a system for preparing a nucleic acid solution from a sample. In some embodiments, certain components of the system are provided by the user.

[0258] Method In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker containing DMR (e.g., DMR1 - 375 as provided in Tables 2 and 18), and 2) Detecting breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0259] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having annotations selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602 - 14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906 - 4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906 - 4274012, GYPC_B, MAX.chr5.42994866 - 42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410 - 145725459, MAX.chr11.68622869 - 68622968, MAX.chr8.124173030 - 124173395, MAX.chr20.1784209 - 1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903 - 46380197, CHST2_B, MAX.chr5.77268672 - 77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1, and 2) Detect breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0260] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Nucleic acid obtained from a subject (e.g., isolated from a body fluid such as blood or plasma or breast tissue, e.g., genomic DNA) is contacted with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, KLHDC7B_B, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr17.73073682 - 73073814, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TBX1_B, TRH_A, and TRIM67_B, and 2) Detect breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0261] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting a nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B, and 2) detecting breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0262] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) is contacted with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr11.14926602-14927148, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, MAX.chr4.8859253-8859329, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr6.157557371-157557657, MPZ, NKX2-6, PDX1, PLXNC1_A, PPARG, PRKCB, PTPRN2, RBFOX_A, SCRT2_A, SLC7A4, STAC2_B, STX16_A, STX16_B, TBX1, TRH_A, VSTM2B_A, ZBTB16, ZNF132, and ZSCAN23, and 2) Detecting triple-negative breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0263] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue), with at least one reagent or series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of CALN1_A, LOC100132891, NACAD, TRIM67, ATP6V1B1, DLX4, GP5, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ST8SIA4, STX16_B ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, KCNK9, SCRT2_B, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, and DSCR6), and 2) detecting triple-negative breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0264] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4273906-4274012, MAX.chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4), and 2) detecting triple-negative breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0265] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Nucleic acids obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma, or breast tissue), such as ABLIM1, AFAP1L1, AKR1B1, ALOX5, AMN, ARL5C, BANK1, BCAT1, BEGAIN, BEST4, BHLHE23_B, BHLHE23_C, C17orf64, C1QL2, C7orf52, CALN1_B, CAV2, CD8A, CDH4_A, CDH4_B, CDH4_C, CDH4_D, CDH4_E, CDH4_F, CHST2_B, CLIP4, CR1, DLK1, DNAJC6, DNM3_A, EMX1_A, ESPN, FABP5, FAM150A, FLJ42875, GLP1R, GNG4, GYPC_A, HAND2, HES5, HNF1B_A, HNF1B_B, HOXA1_A, HOXA1_B, HOXA7_A, HOXA7_B, HOXA7_C, HOXD9, IGF2BP3_A, IGF2BP3_B, IGSF9B_A, IL15RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, LOC283999, LY6H, MAST1, MAX.chr1.158083198 - 158083476, MAX.chr1.228074764 - 228074977, MAX.chr1.46913931 - 46913950, MAX.chr10.130085265 - 130085312, MAX.chr11.68622869 - 68622968, MAX.chr14.101176106 - 101176260, MAX.chr15.96889069 - 96889128, MAX.chr17.8230197 - 8230314, MAX.chr19.46379903 - 46380197, MAX.chr2.97193163 - 97193287, MAX.chr2.97193478 - 97193562, MAX.chr20.1784209 - 1784461, MAX.chr21.44782441 - 44782498, MAX.chr22.23908718 - 23908782, MAX.chr5.145725410 - 145725459, MAX.chr5.178957564 - 178957598, MAX.chr5.180101084 - 180101094, MAX.chr5.At least one marker selected from chromosomal regions having an annotation selected from the group consisting of 42952185-42952280, MAX.chr5.42994866-42994936, MAX.chr6.27064703-27064783, MAX.chr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID2_B, NID2_C, ODC1, OSR2_B, PAQR6, PCDH8, PIF1, PPARA, PPP2R5C, PRDM13_A, PRHOXNB, PRKCB, RBFOX3_A, RBFOX3_B, RFX8, SNCA, STAC2_A, STAC2_B, STX16_B SYT5, TIMP2, TMEFF2, TNFRSF10D, TRH_B, TRIM67, TRIM71_C, USP44_A, USP44_B, UTF1, UTS2R, VSTM2B_A, VSTM2B_B, ZFP64, and ZNF132, contacting with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides, and. 2) HER2 + Detecting breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0266] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non-methylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, NACAD, TRIM67, ATP6V1B1, C17orf64, CHST2_B, DLX4, DNM3_A, EMX1_A, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, COL23A1, KCNK9, LAYN, PLXNC1_A, RIC3, SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, and MAX.chr11.68622869-68622968; and 2) HER2 + detecting breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0267] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Nucleic acids obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue), in at least one marker selected from chromosomal regions having annotations selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, C10orf125, contacting with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non-methylated CpG dinucleotides, and 2) HER2 + Detecting breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0268] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non-methylated CpG dinucleotides within at least one marker selected from chromosomal regions having annotations selected from the group consisting of ARL5C, BHLHE23_C, BMP6, C10orf125, C17orf64, C19orf66, CAMKV, CD1D, CDH4_E, CDH4_F, CHST2_A, CRHBP, DLX6, DNM3_A, DNM3_B, DNM3_C, ESYT3, ETS1_A, ETS1_B, FAM126A, FAM189A1, FAM20A, FAM59B, FBN1, FLRT2, FMN2, FOXP4, GAS7, GYPC_A, GYPC_B, HAND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931-46913950, MAX.chr11.68622869-68622968, MAX.chr12.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.73073682-73073814, MAX.chr20.1783841-1784054, MAX.chr21.47063802-47063851, MAX.chr4.8860002-8860038, MAX.chr5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MAX.chr8.688863-688924, MAX.chr9.114010-114207, MPZ, NID2_A, NKX2-6, ODC1, OSR2_A, POU4F1, PRDM13_B, PRKCB, RASGRF2, RIPPLY2, SLC30A10, ST8SIA4, SYN2, TRIM71_A, TRIM71_B, TRIM71_C, UBTF, ULBP1, USP44_B, and VSTM2B_A, and 2) Detecting luminal A breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0269] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue), in at least one marker selected from chromosomal regions having an annotation selected from the group consisting of BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013 - 96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf125, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602 - 14927148, MAX.chr5.42994866 - 42994936, MAX.chr8.124173030 - 124173395, MPZ, ODC1, PLXNC1_A, PRKCB, ST8SIA4, STX16_B UBTF, LOC100132891, ITPRIPL1, MAX.chr12.4273906 - 4274012, MAX.chr12.59990671 - 59990859, BHLHE23_D, COL23A1, KCNK9, OTX1, PLXNC1_A, HNF1B_B, MAST1, ASCL2, MAX.chr20.1784209 - 1784461, RBFOX_A, MAX.chr12.4273906 - 4274012, GAS7, MAX.chr5.145725410 - 145725459, MAX.chr5.77268672 - 77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, DSCR6, MAX.chr11.68622869 - 68622968, contacting with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non - methylated CpG dinucleotides, and 2) Detecting luminal A breast cancer (obtained, for example, with a sensitivity of 80% or more and a specificity of 80% or more).

[0270] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Nucleic acid obtained from a subject (isolated from a body fluid such as blood or plasma or breast tissue, for example, genomic DNA) is contacted with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having annotations selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125, and 2) Detecting luminal A breast cancer (obtained, for example, with a sensitivity of 80% or more and a specificity of 80% or more).

[0271] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non-methylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of ACCN1, AJAP1_A, AJAP1_B, BEST4, CALN1_B, CBLN1_B, CDH4_E, DLX4, FOXP4, IGSF9B_B, ITPRIPL1, KCNA1, KLF16, LMX1B_A, MAST1, MAX.chr11.14926602-14927148, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B SYNJ2, TMEM176A, TSHZ3, and VIPR2; and 2) detecting luminal B breast cancer (e.g., obtained with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0272] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting a nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non-methylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of CALN1_A, LOC100132891, MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, DLX4, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ITPRIPL1, KLF16, MAX.chr12.4273906-4274012, MAX.chr19.46379903-46380197, BHLHE23_D, HNF1B_B, TRH_A, ASCL2, MAX.chr20.1784209-1784461, MAX.chr12.4273906-4274012, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, AJAP1_B, and DSCR6, and 2) Detecting luminal B breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0273] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D; and 2) detecting luminal B breast cancer (e.g., obtained with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0274] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a bodily fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of C10orf93, C20orf195_A, C20orf195_B, CALN1_B, CBLN1_A, CBLN1_B, CCDC61, CCND2_A, CCND2_B, CCND2_C, EMX1_B, FAM150B, GRASP, HBM, ITPRIPL1, KCNK17_A, KIAA1949, LOC100131176, MAST1, MAX.chr1.8277285-8277316, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.30719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MAX.chr6.157556793-157556856, MAX.chr8.124173030-124173395, MN1, MPZ, NR2F6, PDXK_A, PDXK_B, PTPRM, RYR2_B, SERPINB9_A, SERPINB9_B, SLC8A3, STX16_B TEPP, TOX, VIPR2, VSTM2B_A, ZNF486, ZNF626, and ZNF671, and 2) detecting BRCA1 breast cancer (e.g., obtained with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0275] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting at least one reagent or series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6; and 2) detecting BRCA1 breast cancer (e.g., obtained with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0276] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting the nucleic acid obtained from the subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from non-methylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of ANTXR2, B3GNT5, BHLHE23_C, BMP4, CHRNA7, EPHA4, FAM171A1, FAM20A, FMNL2, FSCN1, GSTP1, HBM, IGFBP5, IL17REL, ITGA9, ITPRIPL1, KIRREL2, LRRC34, MAX.chr1.239549742-239549886, MAX.chr1.8277479-8277527, MAX.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-151145743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64, and 2) Detecting BRCA2 breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more). In some embodiments of the present technology, a method is provided that includes the following steps: 1) The nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) is selected within at least one marker having an annotation selected from the group consisting of MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, contacting with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides, and 2) Detecting BRCA2 breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0277] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting a nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr4.8859602-8859669, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MPZ, NKX2-6, PRKCB, RBFOX3_B, SALL3, and VSTM2B_A, and 2) Detecting invasive breast cancer (e.g., obtained with a sensitivity of 80% or more and a specificity of 80% or more).

[0278] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting a nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) with at least one reagent or a series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of SCRT2_B, MPZ, MAX.chr8.124173030-124173395, ITPRIPL1, ITPRIPL1, DLX4, CALN1_A, and IGF2BP3_B, and 2) A step of distinguishing high-grade ductal carcinoma in situ (DCIS-HG) breast cancer tissue from low-grade ductal carcinoma in situ (DCIS-LG) breast tissue (obtained, for example, with a sensitivity of 80% or more and a specificity of 80% or more).

[0279] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting a nucleic acid obtained from a subject (for example, a body fluid such as blood or plasma or genomic DNA isolated from breast tissue) with at least one reagent or series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of SCRT2_B, ITPRIPL1, and MAX.chr8.124173030-12417339, and 2) A step of distinguishing high-grade ductal carcinoma in situ (DCIS-HG) breast cancer tissue from low-grade ductal carcinoma in situ (DCIS-LG) breast tissue (obtained, for example, with a sensitivity of 100% or more and a specificity of 91% or more).

[0280] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Contacting a nucleic acid obtained from a subject (for example, a body fluid such as blood or plasma or genomic DNA isolated from breast tissue) with at least one reagent or series of reagents that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker selected from chromosomal regions having an annotation selected from the group consisting of DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, and ITPRIPL1, and 2) A step of distinguishing between high-grade ductal carcinoma in situ (DCIS-HG) breast cancer tissue and low-grade ductal carcinoma in situ (DCIS-LG) breast tissue (obtained, for example, with a sensitivity of 80% or more and a specificity of 80% or more).

[0281] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Measuring the methylation level of one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with a reagent that modifies DNA by a methylation-specific technique (for example, where the reagent is a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme), wherein the one or more genes are selected from one of the following groups: (i) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1, (ii)ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B, and (iii)CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B, 2) Amplifying the processed genomic DNA using a set of primers for one or more of the selected genes, and 3) Determining the methylation level of one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture.

[0282] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Measuring the amount of at least one methylation marker gene in the DNA from the sample, wherein one or more genes are selected from one of the following groups: (i) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1, (ii) ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B, and (iii) CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B, 2) A step of measuring the amount of at least one reference marker in DNA, and 3) A step of calculating the value of the amount of at least one methylated marker gene measured in DNA as a percentage of the amount of the reference marker gene measured in DNA, the value indicating the amount of at least one methylated marker DNA measured in the sample.

[0283] In some embodiments of the present technology, a method including the following steps is provided: 1) By treating genomic DNA in a biological sample of a human individual with a bisulfite reagent (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) capable of modifying DNA by a methylation-specific technique, a step of measuring the methylation level of CpG sites related to one or more genes in the biological sample of the human individual, 2) A step of amplifying the modified genomic DNA using a set of primers for one or more selected genes, and 3) A step of determining the methylation level of CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR, A step in which one or more genes are selected from one of the following groups: (i) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1, (ii) ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B, and (iii) CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B.

[0284] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Measuring the methylation level of one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with a reagent that modifies DNA by a methylation-specific technique (e.g., where the reagent is a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme), wherein the one or more genes are selected from one of the following groups: (i) BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6, (ii) MAX.chr15.96889013 - 96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602 - 14927148, MAX.chr5.42994866 - 42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903 - 46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410 - 145725459, and MAX.chr11.68622869 - 68622968, (iii) ATP6V1B1, MAX.chr11.14926602 - 14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906 - 4274012, CALN1_A, MAX.chr12.4273906 - 4274012, MAX.chr5.42994866 - 42994936, SCRT2_B, MAX.chr5.145725410 - 145725459, BHLHE23_D, MAX.chr5.77268672 - 77268725, EMX1_A, DSCR6, and DLX4, (iv) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, and C10orf125, (v)ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125, (vi) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D, and (vii) DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, ITPRIPL1, 2) Amplifying the processed genomic DNA using a set of primers for one or more selected genes, and 3) Determining the methylation level of one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture.

[0285] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Measuring the amount of at least one methylation marker gene in the DNA from a sample, wherein one or more genes are selected from one of the following groups: (i) BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6, (ii) MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, and MAX.chr11.68622869-68622968, (iii) ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4273906-4274012, MAX.chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4, (iv) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, and C10orf125, (v)ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125, (vi) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D, and (vii) DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030-124173395, OSR2_A, MAX.chr11.68622869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, ITPRIPL1, 2) A step of measuring the amount of at least one reference marker in the DNA, and 3) A step of calculating the value of the amount of at least one methylated marker gene measured in the DNA as a percentage of the amount of the reference marker gene measured in the DNA, the value indicating the amount of at least one methylated marker DNA measured in the sample.

[0286] In some embodiments of the present technology, a method is provided that includes the following steps: 1) Treating genomic DNA in a biological sample with a bisulfite reagent (e.g., methylation-sensitive restriction enzyme, methylation-dependent restriction enzyme, and bisulfite reagent) capable of modifying DNA by a methylation-specific method to measure the methylation level of CpG sites related to one or more genes in the biological sample of a human individual; 2) Amplifying the modified genomic DNA using a set of primers for one or more selected genes, and 3) Determining the methylation level of CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR, wherein One or more genes are selected from one of the following groups: (i) BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZSCAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, SCRT2_B, MAX.chr17.73073682-73073814, CDH4_E, HNF1B_B, TRH_A, MAX.chr20.1784209-1784461, MAX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6; (ii) MAX.chr15.96889013 - 96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602 - 14927148, MAX.chr5.42994866 - 42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903 - 46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410 - 145725459, and MAX.chr11.68622869 - 68622968, (iii) ATP6V1B1, MAX.chr11.14926602 - 14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906 - 4274012, CALN1_A, MAX.chr12.4273906 - 4274012, MAX.chr5.42994866 - 42994936, SCRT2_B, MAX.chr5.145725410 - 145725459, BHLHE23_D, MAX.chr5.77268672 - 77268725, EMX1_A, DSCR6, and DLX4, (iv) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, and C10orf125, (v)ATP6V1B1, LMX1B_A, BANK1, OTX1, ST8SIA4, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr5.77268672-77268725, EMX1_A, CHST2_B, ITPRIPL1, IGF2BP3_B, CDH4_E, ABLIM1, SLC30A10, C10orf125, (vi) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602 - 14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906 - 4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906 - 4274012, GYPC_B, MAX.chr5.42994866 - 42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410 - 145725459, MAX.chr11.68622869 - 68622968, MAX.chr8.124173030 - 124173395, MAX.chr20.1784209 - 1784461, LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903 - 46380197, CHST2_B, MAX.chr5.77268672 - 77268725, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, CDH4_E, DLX4, ABLIM1, BHLHE23_D, and (vii) DSCR6, SCRT2_B, MPZ, MAX.chr8.124173030 - 124173395, OSR2_A, MAX.chr11.68622869 - 68622968, ITPRIPL1, MAX.chr5.145725410 - 145725459, BHLHE23_C, ITPRIPL1.

[0287] Preferably, the sensitivity of such a method is from about 70% to about 100%, or from about 80% to about 90%, or from about 80% to about 85%. Preferably, the specificity is from about 70% to about 100%, or from about 80% to about 90%, or from about 80% to about 85%.

[0288] Genomic DNA may be isolated by any means, including the use of commercially available kits. Briefly, if the DNA of interest is encapsulated by a cell membrane, the biological sample must be disrupted and lysed by enzymatic, chemical, or mechanical means. Next, proteins and other contaminants may be removed from the DNA solution, for example, by digestion with proteinase K. Next, the genomic DNA is recovered from the solution. This may be carried out by various methods, including salting out, organic extraction, or binding of the DNA to a solid support. The choice of method will be influenced by several factors, including time, cost, and the amount of DNA required. All clinical sample types containing neoplastic or preneoplastic substances are suitable for use in this method, for example, cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, feces, breast tissue, colonic effluents, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0289] The technology is not limited to the methods used to prepare the sample and to provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from a fecal sample, or from blood, or from a plasma sample using direct gene capture, for example, as detailed in U.S. Patent Application No. 61 / 485386, or related methods.

[0290] Next, the genomic DNA sample is treated with at least one reagent, or a series of reagents, that distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides within at least one marker containing a DMR (e.g., DMR1-375, as provided in Tables 2 and 18). In some embodiments, the reagent converts cytosine bases that are not methylated at the 5' position to uracil, thymine, or another base that differs from cytosine with respect to hybridization behavior. However, in some embodiments, the reagent may be a methylation-sensitive restriction enzyme.

[0291] In some embodiments, genomic DNA samples are processed by a technique such that cytosine bases that are not methylated at the 5'-position are converted to uracil, thymine, or another base that is different from cytosine with respect to hybridization behavior. In some embodiments, this processing is carried out with bisulfite (hydrogen sulfite, disulfite), followed by alkaline hydrolysis.

[0292] The processed nucleic acid is then analyzed to determine the methylation status of a target gene sequence (DMR, e.g., at least one gene, genomic sequence, or nucleotide from a marker comprising at least one DMR selected from DMR1 - 375, such as those provided in Tables 2 and 18). The method of analysis may be selected from those known in the art, including those listed herein, e.g., QuARTS and MSP as described herein.

[0293] Aberrant methylation, more specifically, hypermethylation of markers containing DMRs (e.g., DMR1 - 375 as provided in Tables 2 and 18) is associated with breast cancer.

[0294] The technology relates to the analysis of any sample associated with breast cancer. For example, in some embodiments, the sample includes tissue and / or biological fluid obtained from a patient. In some embodiments, the sample includes secretions. In some embodiments, the sample includes blood, serum, plasma, gastric secretions, pancreatic juice, gastrointestinal biopsy samples, microdissected cells from breast biopsies, and / or cells recovered from feces. In some embodiments, the sample includes breast tissue. In some embodiments, the subject is human. Samples may include cells, secretions, or tissues from the breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyp, gallbladder, anus, and / or peritoneum. In some embodiments, the sample includes cytosol, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample. In some embodiments, the sample is a breast tissue sample.

[0295] Such samples can be obtained by a number of means known in the art, for example, readily apparent to one of ordinary skill in the art. For example, urine and fecal samples are readily achievable, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, for example, by using needles and syringes. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to those of ordinary skill in the art, including, but not limited to, techniques such as centrifugation and filtration. Although non-invasive techniques are generally preferred for obtaining samples, it may still be preferred in some cases to obtain samples such as tissue homogenates, tissue sections, and biopsy materials.

[0296] In some embodiments, the technology relates to a method of treating a patient (e.g., a patient having breast cancer, having early-stage breast cancer, or at risk of developing breast cancer) (e.g., a patient having one or more of triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), the method comprising determining the methylation status of one or more DMRs as provided herein and administering treatment to the patient based on the results of determining the methylation status. The treatment may be administration of a pharmaceutical compound, administration of a vaccine, performance of surgery, imaging of the patient, performance of another test. Preferably, the use is in a method of clinical screening, a method of prognostic evaluation, a method of monitoring the results of therapy, a method of identifying the patients most likely to respond to a particular therapeutic treatment, a method of imaging a patient or subject, and a method of drug screening and development.

[0297] In some embodiments of the present technology, a method for diagnosing breast cancer in a subject is provided. The terms "diagnosing" and "diagnosis" as used herein refer to a method by which one of ordinary skill in the art can infer and further determine whether a subject is suffering from a given disease or condition, or is likely to develop a given disease or condition in the future. One of ordinary skill in the art will often make a diagnosis based on one or more diagnostic indicators, such as a biomarker (e.g., a DMR as disclosed herein), the methylation state of which indicates the presence, severity, or absence of the condition.

[0298] Along with diagnosis, clinical cancer prognosis relates to determining the aggressiveness of the cancer and the likelihood of tumor recurrence, and planning the most effective therapy. If a more accurate prognosis can be made or the potential risk of developing cancer can be evaluated, appropriate therapies, and in some cases, less aggressive therapies for the patient can be selected. Evaluation of cancer biomarkers (e.g., determining methylation status) is useful for separating subjects who have a favorable prognosis and / or a low risk of developing cancer and who will require no therapy or only limited therapy from subjects who are likely to develop or recur with cancer and who may benefit from more intensive treatment.

[0299] As such, "performing a diagnosis" or "diagnosing", as used herein, further includes determining the risk of developing cancer or determining a prognosis, which may enable predicting a clinical outcome (regardless of the presence or absence of medical treatment), selecting an appropriate treatment (or whether the treatment is effective), or monitoring the current treatment and possibly changing the treatment, based on an indication of a diagnostic biomarker (e.g., DMR) disclosed herein. Further, in some embodiments of the subject matter disclosed herein, multiple determinations of biomarkers over time may be made to facilitate diagnosis and / or prognosis prediction. Changes in biomarkers over time may be used to predict a clinical outcome, monitor the progression of breast cancer, and / or monitor the effectiveness of an appropriate therapy for cancer. In such embodiments, for example, it may be expected that changes in the methylation status of one or more biomarkers (e.g., DMR) disclosed herein (and, if monitored, optionally one or more additional biomarkers) in a biological sample will be confirmed over time during the course of an effective therapy.

[0300] The subject matter disclosed herein, in some embodiments, further provides a method for determining whether to initiate or continue the prevention or treatment of cancer in a subject. In some embodiments, the method includes providing a series of biological samples from the 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 of the biological samples, and comparing any measurable changes in the methylation status of one or more of the biomarkers in each of the biological samples. Any changes in the methylation status of the biomarker over a period of time can be used to predict the risk of developing cancer, to predict clinical outcomes, to determine whether to initiate or continue cancer prevention or therapy, and to determine whether the current therapy is effectively treating the cancer. For example, a first time point can be selected before the start of treatment, and a second time point can be selected at some point after the start of treatment. The methylation status can be measured in each of the samples taken at different time points, noting qualitative and / or quantitative differences. Changes in the methylation status of biomarker levels from different samples can correlate with breast cancer risk, prognosis, determination of treatment efficacy, and / or cancer progression in the subject.

[0301] In preferred embodiments, the methods and compositions of the invention are for the treatment or diagnosis of a disease at an early stage, e.g., before the symptoms of the disease appear. In some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of a disease in a clinical stage.

[0302] As already described, 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 diagnosis or prognosis. For example, a diagnostic marker can be determined initially and then again a second time. In such embodiments, an increase in the marker from the first to the second determination can diagnose a particular cancer type or severity, or a given prognosis. Similarly, a decrease in the marker from the first to the second determination can indicate a particular cancer type or severity, or a given prognosis. Further, the degree of change for one or more markers can be related to cancer severity and future adverse events. One of ordinary skill in the art will understand that in certain embodiments, comparative measurements of the same biomarker can be made at multiple time points, but it is also possible to measure a given biomarker at one time point and a second biomarker at a second time point, and diagnostic information can be provided by comparing these markers. As used herein, the phrase "determining a prognosis" refers to a way by which one of ordinary skill in the art can predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or the ability to predict that a given course or outcome is highly, or lowly, likely to occur based on the methylation status of a biomarker (e.g., a DMR). Instead, one of ordinary skill in the art will understand that the term "prognosis" refers to the probability that a particular course or outcome will occur, i.e., an increase in the probability that a course or outcome will occur in a subject showing a given condition as compared to those individuals not showing the condition. For example, in individuals not showing a condition (e.g., having a normal methylation status of one or more DMRs), the likelihood of a given outcome (e.g., developing breast cancer) may be very low.

[0303] In some embodiments, statistical analysis correlates prognostic indicators with risk factors for adverse outcomes. For example, in some embodiments, methylation states that differ from those in normal control samples obtained from patients without cancer may indicate that a subject is more likely to develop cancer than subjects having a similar level of methylation state in the control samples, as determined by a level of statistical significance. Additionally, changes in methylation state from baseline (e.g., “normal”) levels may reflect the prognosis of a subject, and the degree of change in methylation state may be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations to determine confidence intervals and / or p-values. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is hereby incorporated by reference in its entirety. Exemplary confidence intervals for the subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, while exemplary p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0304] In other embodiments, a threshold degree of change in the methylation state of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein may be established, and the degree of change in the methylation state of the biomarker in a biological sample is simply compared to the threshold degree of change in the methylation state. Preferred threshold changes in the methylation state of the biomarker 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 still other embodiments, a “nomogram” may be established, whereby the methylation state of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to the property associated with a given outcome. One of ordinary skill in the art is familiar with the use of such nomograms and understands that, since individual sample measurements rather than population averages are referenced, the uncertainty of this measurement is the same as the uncertainty of the marker concentration, and correlates the two numerical values.

[0305] In some embodiments, the control sample is analyzed concurrently with the biological sample, such that the results obtained from the biological sample can be compared to the results obtained from the control sample. Additionally, a calibration curve may be provided and it is contemplated that the assay results of the biological sample may be compared thereto. Such a calibration curve presents the methylation status of the biomarker according to the assay unit, e.g., the fluorescence signal intensity if a fluorescent label is used. When using samples taken from multiple donors, the calibration curve may define the "at risk" levels of one or more biomarkers in tissue taken from donors with metaplasia or from donors with breast cancer, in addition to the control methylation status of one or more biomarkers in normal tissue. In certain embodiments of the method, a subject is identified as having metaplasia when identifying an abnormal methylation status of one or more of the DMRs provided herein in a biological sample obtained from the subject. In other embodiments of the method, detecting an abnormal methylation status of one or more of such biomarkers in a biological sample obtained from a subject results in the subject being identified as having cancer.

[0306] Analysis of the markers can be performed separately or concurrently with additional markers within a single test sample. For example, some markers can be combined in one test for efficient processing of multiple samples and for the potential to provide higher accuracy in diagnosis and / or prognosis. Additionally, one of ordinary skill in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing on a series of samples may enable identification of changes over time in the methylation status of the markers. In addition to changes in the methylation status, the lack of change in the methylation status can provide useful information about the subject's outcome, including, but not limited to, identifying the disease state, e.g., the approximate time from onset of the event, the presence and amount of recoverable tissue, the validity of drug therapy, the effectiveness of various therapies, and the risk of future events.

[0307] The analysis of biomarkers can be performed in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of a large number of test samples. Alternatively, a single sample format can be developed in a timely manner to facilitate immediate treatment and diagnosis in, for example, an outpatient transport or emergency room situation.

[0308] In some embodiments, a subject is diagnosed with breast cancer if there is a measurable difference in the methylation state of at least one biomarker in a sample when compared to a control methylation state. Conversely, if no change in the methylation state is identified in a biological sample, the subject can be identified as not having breast cancer, having no cancer risk, or having a low cancer risk. In this regard, subjects with cancer or a risk thereof can be distinguished from subjects with little or substantially no cancer or a risk thereof. Those subjects at risk of developing breast cancer can be placed on a more intensive and / or regular screening schedule, including endoscopic surveillance. On the other hand, those subjects with a low or substantially no risk may avoid being subjected to additional breast cancer tests (e.g., invasive procedures) until it is shown that the risk of breast cancer has manifested in those subjects at the time of future screening, e.g., screening performed in accordance with the present technique.

[0309] As described above, depending on the embodiment of the method of the present technology, detecting a change in the methylation state of one or more biomarkers can be a qualitative determination or it can be a quantitative determination. As such, the step of diagnosing a subject having or at risk of developing breast cancer involves a particular threshold measurement being made, e.g., the methylation state of one or more biomarkers in a biological sample being different from a predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state for the biomarker. 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 the method, the predetermined methylation state is based on and / or identified by a calibration curve. In other embodiments of the method, the predetermined methylation state is a specific state or range of states. As such, the predetermined methylation state can be selected, within acceptable limits that will be apparent to those skilled in the art, based in part on the embodiment of the method being performed and the desired specificity, etc.

[0310] Furthermore, with regard to the diagnostic method, preferred subjects are vertebrate subjects. Preferred vertebrates are warm-blooded animals, and preferred warm-blooded vertebrates are mammals. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Accordingly, veterinary therapeutic uses are provided herein. As such, by the present technology, in addition to mammals such as humans, those mammals that are critically endangered, such as the Amur tiger, those mammals that are economically important, such as animals raised on farms for human consumption, and / or as pets, or animals raised in zoos, etc., which are socially important animals for humans, can be diagnosed. Examples of such animals include carnivores such as cats and dogs, pigs (including pigs, boars, and wild boars), ruminants and / or ungulates, such as cows, bulls, sheep, giraffes, deer, goats, bison, and camels, and horses, but are not limited thereto. Accordingly, further provided are the diagnosis and treatment of domesticated pigs, ruminants, ungulates, horses (including racehorses), etc., but not limited thereto.

[0311] The subject matter disclosed herein further includes a system for diagnosing breast cancer and / or specific breast cancer types (e.g., triple-negative breast cancer, HER2 + breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer) in a subject. The system can be provided, for example, as a commercially available kit that can be used to screen for the risk of breast cancer in a subject from whom a biological sample has been collected or to diagnose breast cancer. An exemplary system provided in accordance with the present technology includes evaluating the methylation status of DMRs as provided in Tables 2 and 18.

Examples

[0312] Example I. This example describes the discovery of breast cancer-specific markers and the verification of tissues.

[0313] Table 1 shows the number of tissue samples for each subtype of breast cancer used in the discovery of breast cancer-specific markers.

[0314]

Table 1

[0315] For the discovery of methylation markers by RRBS, frozen tissue samples were obtained from 72 invasive breast cancer cases (18 luminal A, 18 luminal B, 18 basal-like / triple negative, and 18 HER2+), 15 invasive breast cancers from patients with BRCA germline mutations (6 BRCA1, 9 BRCA2), and 45 controls (18 normal breasts (reduction mammoplasty or prophylactic mastectomy), 9 histologically normal breasts from germline BRCA carriers (prophylactic mastectomy), and 18 normal buffy coats). Tumor and breast tissue sections were reexamined by a specialized GI pathologist to confirm the diagnosis and estimate abnormal cellularity. The sections were then microdissected. Genomic DNA was purified using the QiaAmp Mini kit (Qiagen, Valencia CA). DNA (300 ng) was fragmented by digestion with 10 units of MspI. The digested fragments were end-repaired and A-tailed with 5 units of Klenow fragment (3'-5' exo-), and ligated overnight to methylated TruSeq adapters (Illumina, San Diego CA) containing barcode sequences (to associate each fragment with its sample ID). The reaction was purified using AMPure XP SPRI beads / buffer (Beckman Coulter, Brea CA).

[0316] Subsequently, bisulfite conversion was performed on tissue samples (twice) using the improved EpiTect protocol (Qiagen). Optimal enrichment Ct was determined using qPCR (LightCycler480 - Roche, Mannheim Germany). The following conditions were used for the final enrichment PCR: Each 50 μL reaction contained 5 μL of 10× buffer, 1.25 μL of each 10 mM deoxyribonucleotide triphosphate (dNTP), 5 μL of primer cocktail (approx. 5 μM), 15 μL of template (sample), 1 μL of PfuTurbo Cx HotStart (Agilent, Santa Clara CA), and 22.75 μL of water, and the temperature and time were 95°C - 5 minutes, 98°C - 30 seconds, 16 cycles of 98°C - 10 seconds, 65°C - 30 seconds, 72°C - 30 seconds, 72°C - 5 minutes, and hold at 4°C, respectively. Samples were purified with SPRI beads and then tested on a Bioanalyzer 2100 (Agilent) to evaluate the enriched DNA size distribution. Size selection of 160 - 520 bp fragments (40 - 400 bp inserts) was performed using AMPure XP SPRI beads / buffer (Beckman Coulter, Brea CA). The buffer cut-off was 0.7× - 1.1× sample volume. Samples were combined (equimolar) into a 4-plex library based on a randomization scheme and tested using a Bioanalyzer for final size and concentration verification, as well as qPCR (KAPA Library Quantification Kit - KAPA Biosystems, Cape Town South Africa) was used for testing.

[0317] The tissue samples were loaded onto a single-read flow cell with randomized lane assignment, and sequencing was performed on the Illumina HiSeq 2000 platform by the Next Generation Sequencing Core at the Mayo Clinic Medical Genome Facility. The reads were single-directional for 101 cycles. The standard Illumina pipeline was run for primary analysis. SAAP-RRBS (Streamlined Analysis and Annotation Pipeline for Reduced Representation Bisulfite Sequencing) was used for quality scoring, sequence alignment, annotation, and methylation extraction.

[0318] Breast cancer tissues resulted in numerous distinct DMRs, many of which had not been previously identified. Comparing the methylation of breast cancer tissue samples with normal breast tissue, 327 methylation regions were identified that distinguished breast cancer tissue from normal breast tissue (see Table 2) (the genomic coordinates of the regions shown in Table 2 are based on the Human Feb.2009 (GRCh37 / hg19) Assembly). Table 3 shows 48 methylation regions that distinguished triple-negative breast cancer tissue from normal breast tissue. Table 4 shows + 122 methylation regions that distinguished breast cancer tissue from normal breast tissue. Table 5 shows 75 methylation regions that distinguished luminal A breast cancer tissue from normal breast tissue. Table 6 shows 39 methylation regions that distinguished luminal B breast cancer tissue from normal breast tissue. Table 7 shows 49 methylation regions that distinguished BRCA1 breast cancer tissue from normal breast tissue. Table 8 shows 45 methylation regions that distinguished BRCA2 breast cancer tissue from normal breast tissue. Table 9 shows 21 methylation regions that distinguished invasive breast cancer tissue from normal breast tissue.

[0319]

Table 2

[0320]

Table 3

[0321]

Table 4

[0322]

Table 5

[0323]

Table 6

[0324]

Table 7

[0325]

Table 8

[0326]

Table 9

[0327] Next, SYBR Green methylation-specific PCR (qMSP) was performed on the discovery samples to confirm the accuracy and reproducibility of the candidate DMRs shown in Table 2. In addition, 16 marker subsets were run on frozen low- and high-grade DCIS samples to test their applicability (22 high-grade / CIS / P3 DCIS (ductal carcinoma in situ, non-invasive), 11 low-grade / P1 DCIS).

[0328] qMSP primers were designed for each of the marker regions using Methprimer software (Li LC and Dahiya R. Bioinformatics. 2002 Nov;18(11):1427-31). They were synthesized by IDT (Integrated DNA Technologies). The assays were tested and optimized (using Roche LightCycler480) with dilutions of bisulfite-converted highly methylated DNA, converted unmethylated DNA, and converted and unconverted leukocyte DNA negative controls (10 ng / ea). The assays being advanced needed to demonstrate a linear regression curve and negative control values less than 5-fold below the minimum threshold (1.6 genomic copies). Some of the more promising DMRs that failed the assay or control were redesigned. Of the 127 total designs (Table 10 shows forward and reverse primer sequence information for the 127 total designs), 80 high-performance MSP assays met the QC criteria and were applied to samples. The MSP primer sequences each contained 2-8 CpGs and were designed to provide a simple means of assessing methylation in samples and, as such, weighted amplification efficiency to target the most discriminative CpGs, which would require a long optimization time.

[0329] DNA was purified as described in the discovery RRBS section and quantified using picogreen absorbance (Tecan / Invitrogen). The 2 μg of sample DNA was then treated with sodium bisulfite and purified using the Zymo EZ-96 Methylation kit (Zymo Research). The eluted material was amplified on a Roche 480 LightCycler using a 384 well block. Each plate could accommodate 2 markers (as well as standards and controls), and there were 40 plates in total. 80 MSP assays had different optimal amplification profiles (Tm = 60 °C, 65 °C, or 70 °C) and were grouped accordingly. 20 μL reactions were run for 50 cycles using LightCycler480 SYBR I master mix (Roche) and 0.5 μmol of primers and were generally analyzed by absolute quantification with a Fit Point of 18%. All parameters (noise band, threshold, etc.) were pre-specified with an automated macro to avoid user subjectivity. Raw data expressed as genomic copy number was normalized based on the DNA (β-actin) input. Results were analyzed logistically using JMP and presented as AUC values. 12 comparisons were performed: each breast cancer subtype against normal breast, and each subtype against buffy coat. Additionally, the methylation fold change rate (mFCR) was calculated for each comparison using both the mean and median methylation rates (FCR = cancer (methylated copy / β-actin copy) / normal (methylated copy / β-actin copy)). Both of these performance metrics are important for assessing the potential of markers in clinical blood-based tests.

[0330] >90% of the markers tested resulted in excellent performance in both the AUC and FCR categories, with a number of AUCs exceeding 0.90, cancer vs normal tissue FCR > 10, and cancer vs buffy coat FCR > 50.

[0331] Table 11 shows the area under the curve for 80 identified methylation regions that distinguish basal cell / triple negative breast tissue, HER2+ breast tissue, luminal A breast tissue, luminal B breast tissue, BRCA1 breast tissue, and BRCA2 breast tissue compared to normal breast tissue.

[0332] Table 12 shows the area under the curve for 80 identified methylation regions that distinguish basal cell / triple negative breast tissue, HER2+ breast tissue, luminal A breast tissue, luminal B breast tissue, BRCA1 breast tissue, and BRCA2 breast tissue compared to normal buffy coat.

[0333] Table 13 shows the methylation fold change for 80 identified methylation regions that distinguish basal cell / triple negative breast tissue, HER2+ breast tissue, luminal A breast tissue, luminal B breast tissue, BRCA1 breast tissue, and BRCA2 breast tissue compared to normal breast tissue.

[0334] Table 14 shows the methylation fold change for 80 identified methylation regions that distinguish basal cell / triple negative breast tissue, HER2+ breast tissue, luminal A breast tissue, luminal B breast tissue, BRCA1 breast tissue, and BRCA2 breast tissue compared to normal buffy coat.

[0335] In the comparison of high-grade vs. low-grade DCIS, the AUC of the 16 markers tested ranged from 0.57 to 0.92. Combinations of some of the 2 markers achieved 95% sensitivity with 91% specificity (only 1 false positive) (Table 15). The combination of 3 markers (SCRT2_B, ITPRIPL1, MAX.chr8.124173030-124173395) had 100% sensitivity with 91% specificity.

[0336]

Table 10

[0337]

Table 11

[0338]

Table 12

[0339]

Table 13

[0340]

Table 14

[0341]

Table 15

[0342] Example II. This example describes the validation of tissue of breast cancer specific markers.

[0343] Independent tissue samples (fresh frozen) were selected from the cancer registry system of Mayo Clinic Rochester, reexamined by a specialized pathologist to confirm accurate classification, and microdissection was guided. The cases included 29 triple negative / basal-like, 34 HER2 type, 36 luminal A, and 25 luminal B invasive breast cancers. It also included 5 BRCA1 and 6 BRCA2 cancers, 21 DCIS w / HGD and 12 DCIS w / LGD. The controls included 27 age-matched normal breast tissues and 18 buffy coat samples from healthy women.

[0344] Fifty-five methylated DNA markers (MDMs) were selected from the list of 80 MDMs tested in the discovery samples (see Examples I and Tables 11 - 15).

[0345] Genomic DNA was prepared using the QIAamp DNA Mini Kit (Qiagen, Valencia CA) and bisulfite converted using the EZ-96 DNA Methylation kit (Zymo Research, Irvine CA). Amplification primers were designed from marker sequences using Methprimer software (University of California, San Francisco CA) and commercially synthesized (IDT, Coralville IA). The assay was optimized by rigorous testing with bisulfite-converted (methylated and unmethylated genomic DNA) as well as unconverted controls by SYBR Green qPCR (Roche). Assays that cross-reacted with negative controls were either redesigned or discarded. Melting curve analysis was utilized to ensure that specific amplification was occurring.

[0346] qMSP was performed using a LightCycler480 instrument with 2 μL of converted DNA in a total reaction volume of 25 μL. Standards were obtained from serially diluted and extensively methylated DNA (Zymo Research). Raw marker copies were normalized to CpG-spanning β-actin, i.e., the marker for total genomic DNA.

[0347] Results were analyzed logistically using JMP10 (SAS, Cary NC). Cases were compared separately to normal breast controls and normal buffy coat samples. Methylation rates and absolute differences were calculated for each of the MDMs.

[0348] MDM performance in independent samples was excellent, with many AUCs and methylation fold change (FC) rates being greater than 0.90 and greater than 50, respectively. Results are presented in Table 16A (triple negative), Table 16B (HER2 +) Provide to Table 16C (Luminal A), Table 16D (Luminal D), and Table 16E (Overall). In this specification, MDMs were ranked by AUC (comparing the whole cases with buffy coat samples). This is an important measure for possible use in plasma because the majority of cell-free DNA (cfDNA) is derived from white blood cells. Any MDM that does not highly distinguish epithelial-derived cancers from white blood cell DNA will fail in a blood test format regardless of its performance in tissues. 41 out of 55 MDMs have a cancer vs. buffy coat AUC exceeding 0.9, and 3 achieved perfect discrimination (AUC = 1). Table 16A, Table 16B, Table 16C, Table 16D, and Table 16E also list % cancer methylation as other important measures when evaluating and demonstrating the excellence of these MDMs, including AUC, FC, p-value.

[0349] Table 17 highlights the top 10 MDMs for distinguishing DCIS HGD from DCIS LGD.

[0350]

Table 16A

[0351]

Table 16B

[0352]

Table 16C

[0353]

Table 16D

[0354]

Table 16E

[0355]

Table 17

[0356] Example III. This example describes the identification of breast tissue markers and plasma markers for detecting breast cancer.

[0357] Candidate methylation markers for detecting breast cancer were identified by RRBS of breast cancer and normal breast tissue samples. First, 58 markers were identified and a targeted enrichment long probe quantitative amplification signal assay was designed and organized (see, for example, WO2017 / 075061 and US Patent Application No. 15,841,006 for general techniques) (Table 18 shows the methylated regions that distinguish breast cancer tissue from normal breast tissue) (Tables 19 and 20 show the primer and probe sequences of the markers shown in Table 18). After design screening and redesign, 56 markers (see Table 21) were selected and the assay was performed in triplicate and tested on tissues. The assay was equally divided between FAM and HEX reports and repeated three times with the reference assay of B3GALT6 reporting Quasar670.

[0358]

Table 18

[0359]

Table 19

[0360]

Table 20

[0361]

Table 21

[0362] A collection of 38 standard breast cancer samples including 6 BRCA carriers and 113 breast cancer tissue samples including luminal A and B, HER2+, BRCA1+, BRCA2+, triple negative and DCIS diversity was tested for the presence of 56 methylation markers. The 56 markers showed a sensitivity range of approximately 15% - 92% with 95% specificity. Table 22 shows the markers demonstrating a sensitivity of 25% or more with 95% specificity. A 5 - marker panel (SPHK2, c17orf64_B, DLX4_B, MPZ_5742, ITPRIPL1_1138) showed a sensitivity of 96% with 100% specificity. The resulting ROC curve had an AUC of 0.995.

[0363]

Table 22

[0364] Based on the results of the tissue tests, 28 marker sets were selected and tested in a set of plasma samples collected from breast cancer patients and normal controls. The 28 markers were divided into 14 pairs of two pools by a number of markers tested. The markers in the two pools are shown in Tables 23 and 24 below.

[0365]

Table 23

[0366]

Table 24

[0367] The pool 7 marker test was performed on a collection of EDTA plasma samples consisting of 85 breast cancer samples (33 stage I, 33 stage II, 18 stage III, and 1 stage IV) and 100 healthy normal controls. The pool 8 marker test was performed on a similar collection of EDTA plasma samples consisting of 85 breast cancer samples (34 stage I, 32 stage II, 18 stage III and 1 stage IV) and 100 healthy normal controls. Based on the results of the pool 7 and pool 8 tests, a collection of 14 assays was selected for further testing (shown in Table 25).

[0368]

Table 25

[0369] The pool 9 marker test was performed on a collection of LBgard (Biomatrica, San Diego, CA) plasma samples consisting of 42 breast cancer samples (1 stage I, 16 stage II, 14 stage III, and 11 stage IV) and 84 healthy normal controls. Table 26 shows the identified methylation regions of the pool 9 markers. Table 27 shows the indicated sensitivity and 90% specificity of the pool 9 markers. Tables 28 and 29 show the primer information and probe information of the pool 9 markers. The collection of 4 markers (FAM59B, ITPRIPL1, TRH_A, and C17orf64_B) showed 74% sensitivity with 90% specificity. The resulting ROC curve showed an AUC of 0.884.

[0370]

Table 26

[0371]

Table 27

[0372]

Table 28

[0373]

Table 29

[0374] All publications and patents mentioned in the above specification are hereby incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. While the technology has been described in connection with specific exemplary embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications to the described methods for practicing the invention which are obvious to those skilled in the pharmacological, biochemical, medical science, or related fields are intended to be within the scope of the following claims.

Claims

1. A method for screening for cancer in a sample obtained from a subject, comprising: determining the methylation level of at least one variably methylated region (DMR) in the sample obtained from the subject by treating the sample with a reagent that modifies DNA in a methylation-specific manner; identifying the subject as having cancer if the methylation level of the at least one DMR is elevated compared to the methylation level of one or more corresponding DMRs assayed in subjects without cancer; The method, wherein the at least one DMR is from FAIM2, PRKCB, EMX1, ZNF671, CAPN2, PPP2R5C, CD1D and / or NTRK3.

2. The method described in claim 1, wherein the step of determining the methylation level of at least one DMR includes a step of determining the presence or absence of methylation at one or more CpG sites.

3. The method described in claim 2, wherein the one or more CpG sites are present in a coding region, a non-coding region and / or a regulatory region of a gene.

4. The method described in claim 1, wherein the step of determining the methylation level of at least one DMR includes a step of determining a methylation frequency.

5. The method described in claim 1, wherein the step of determining the methylation level of at least one DMR includes a step of determining a methylation pattern.

6. The method of claim 1, wherein the reagent that modifies DNA in a methylation-specific manner comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent.

7. The method of claim 1, wherein the step of determining the methylation level of at least one DMR comprises performing at least one of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR flap assay, and bisulfite genomic sequencing PCR.

8. The method described in claim 1, wherein the at least one DMR comprises an increase in the percentage of methylation or an increase in the ratio of hypermethylation relative to a control sample.

9. The method described in claim 8, wherein the control sample is derived from a subject who does not have cancer.

10. The method of claim 1, wherein the sample is a blood sample, a fecal sample, a urine sample or a tissue sample.

11. The method described in claim 10, wherein the tissue sample is a breast tissue sample.

12. The method described in claim 1, wherein the at least one DMR is derived from FAIM2.

13. The method of claim 1, wherein the at least one DMR is derived from PRKCB.

14. The method described in claim 1, wherein at least one DMR is derived from EMX1.

15. The method of claim 1, wherein the at least one DMR is derived from ZNF671.

16. The method described in claim 1, wherein at least one DMR is derived from CAPN2.

17. The method described in claim 1, wherein at least one DMR is derived from PPP2R5C.

18. The method described in claim 1, wherein at least one DMR is derived from CD1D.

19. The method of claim 1, wherein the at least one DMR is derived from NTRK3.

20. The method described in claim 1, wherein the cancer is breast cancer.

21. The method of claim 1, further comprising determining the methylation level of the at least one DMR from a reference gene.

22. The method of claim 1, wherein the step of determining the methylation level of at least one DMR comprises amplifying at least a portion of the DMR using a set of primers.