Breast cancer detection

Abstract

To provide an improved method for detecting breast cancer.SOLUTION: Provided herein are techniques for breast cancer screening, employing a novel methylation marker, particularly methods, compositions, and related uses for detecting the presence of breast cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 592,828, filed November 30, 2017, the entire contents of which are incorporated herein by reference.

[0002] Provided herein is technology relating to breast cancer screening, particularly, but not limited to, methods, compositions, and related uses for detecting the presence of breast cancer. [Background technology]

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

[0004] Improved methods for detecting breast cancer are needed.

[0005] The present invention addresses these needs. Summary of the Invention

[0006] Methylated DNA has been investigated as a potential class of biomarker in tissues of most tumor types. 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 intriguing mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence their expression and therefore contribute to carcinogenesis. DNA methylation may be a more chemically and biologically robust diagnostic tool than RNA or protein expression (Laird (2010) Nat Rev Genet 11:191-203). Furthermore, in other cancers, such as sporadic colon cancer, methylation markers offer superior specificity and are more informative and sensitive than individual DNA mutations (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 mouse tissues in vivo and cell lines in vitro demonstrated that only approximately 0.3% of high-CpG-density promoters (HCPs, defined as having >7% CpG sequences within a 300-base pair region) were methylated, whereas low-CpG-density regions (LCPs, defined as having <5% CpG sequences within a 300-base pair region) tended to be highly methylated in dynamic tissue-specific patterns (Meissner et al. (2008) Nature 454:766-70). HCPs include promoters for ubiquitous housekeeping genes and highly regulated developmental genes. Among the HCP sites that were methylated at >50% 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 investigated as a potential class of biomarker in tissues of most tumor types. In a biologically intriguing mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence their expression and contribute to carcinogenesis. DNA methylation may be a more chemically and biologically robust diagnostic tool than RNA or protein expression. Furthermore, in other cancers, such as sporadic colon cancer, aberrant methylation markers offer more comprehensive information, higher sensitivity, and superior specificity than individual DNA mutations.

[0009] Several methods are available for discovering novel methylation markers. Microarray-based interrogation of CpG methylation is a rational, high-throughput approach, but this strategy is biased toward known regions of interest, primarily established tumor suppressor promoters. Alternative methods for genome-wide analysis of DNA methylation have been developed over the past decade. Three basic approaches exist. The first involves digestion of DNA with restriction enzymes that recognize specific methylation sites, followed by several possible analytical techniques to provide methylation data limited to the enzyme recognition sites or primers, which are then used to amplify the DNA in a quantification step (e.g., methylation-specific PCR, MSP). The second approach involves enriching the methylated fraction of genomic DNA using antibodies directed against methyl-cytosine or other methylation-specific binding domains, 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 a fragment. A third approach begins with bisulfite treatment of DNA to convert all unmethylated tyrosines to uracil, followed by restriction enzyme digestion and full sequencing of all fragments after ligation to adaptor ligands. The choice of restriction enzyme can enrich for fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during analysis.

[0010] RRBS provides CpG methylation status data at single-nucleotide resolution for 80-90% of all CpG islands and most tumor suppressor promoters at moderate to high read coverage. In cancer case-control studies, analysis of these reads leads to the identification of variably methylated regions (DMRs). Previous RRBS analysis of pancreatic cancer specimens discovered hundreds of DMRs, many of which were never associated with carcinogenesis and many of which were never annotated. Further validation studies on an independent set of tissue samples confirmed marker CpGs that were 100% sensitive and specific in terms of performance.

[0011] Provided herein is technology relating to breast cancer screening, particularly, but not limited to, methods, compositions, and related uses for detecting the presence of breast cancer.

[0012] In fact, 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 variably methylated regions (DMRs) to distinguish cancer from non-neoplastic control DNA in breast-derived DNA.

[0013] Such experiments have resulted in the enumeration and description of 375 novel DNA methylation markers that distinguish between breast cancer and benign breast tissue (see Tables 2 and 18, Examples I, II and III).

[0014] From these 375 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish between breast cancer tissue and benign breast tissue: ·ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CAL N1_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, CHS T2_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_113 8, 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, further experiments identified the following markers and / or panels of markers for detecting breast cancer in blood samples (e.g., plasma samples, whole blood samples, serum samples): 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, further experiments identified the following markers and / or panels of markers that can distinguish between triple-negative breast cancer tissue and benign breast tissue: ·ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr11.14926602-149271 48, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.767 34362-76734370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, M AX.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.chr11.14926602-1492 7148, 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 revealed that HER2 + The following markers and / or panels of markers have been identified that can distinguish between breast cancer tissue and benign breast tissue: ·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.chr8.14510413 2-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID 2_B、NID2_C、ODC1、OSR2_B、PAQR6、PCDH8、PIF1、PPARA、PPP2R5C、PRDM13_A、P RHOXNB, 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 (Chapter 4, the specifications I of this and). ·BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.9688 9013-96889128 NACAD TRIM67 ATP6V1B1 C17orf64 CHST2_B DLX4 DNM3_A EMX1_A IGF2BP3_A IGF2B P3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.1 24173030-124173395 MPZ ODC1 PLXNC1_A PRKCB LOC100132891 ITPRIPL1 ABLIM1 MAX.chr12.42739 06-4274012、MAX.chr19.46379903-46380197、ZSCAN12、BHLHE23_D、COL23A1、KCNK9、LINE、PLXNC1_A、RI C3, 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, GYP C_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, etc MAX.chr11.68622869-68622968(Chapter 11; ·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.chr5.42994866-42994 936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-6 8622968, 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 between luminal A breast cancer tissue and benign breast tissue: ·ARL5C, BHLHE23_C, BMP6, C10orf125, C17orf64, C19orf66, CAMKV, CD1D, CDH4_E, CDH4_F, CHST2_A, CRHBP, DLX6, DNM3_A, DN M3_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.chr1.4691393 1-46913950, MAX.chr11.68622869-68622968, MAX.chr12.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.7 3073682-73073814, MAX.chr20.1783841-1784054, MAX.chr21.47063802-47063851, MAX.chr4.8860002-8860038, MAX.chr 5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, M AX.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.chr15.96 889013-96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf125, C17orf64, CHST2_B, DNM3_A, EMX 1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.42 994866-42994936, MAX.chr8.124173030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, ST8SIA4, STX16_B UBTF, LOC100132891, ITPRIPL1, MAX.chr12.4273906-4274012, MAX.chr12.59990671-59990859, BHLH E23_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.chr12.4273906-42740 12, 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 between luminal B breast cancer tissue and 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.chr11.1 4926602-14927148, MAX.chr17.73073682-73073814, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.3071926 1-30719354, 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.chr11.14926602-1492 7148, MAX.chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, ITPRIPL1, KLF16, MAX.chr12.4273906-4 274012, 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.chr 11.68622869-68622968, 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, further experiments identified the following markers and / or panels of markers that can distinguish between BRCA1 breast cancer tissue and benign breast tissue: ·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, MA X.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.9688901 3-96889128 MAX.chr18.5629721-5629791 MAX.chr19.30719261-30719354 MAX.ch r22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77 268725, MAX.chr6.157556793-157556856, MAX.chr8.124173030-124173395, MN1, MP Z, 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 Figure 7, Table I, and others). ·BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, E MX1_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, further experiments identified the following markers and / or panels of markers that can distinguish between BRCA2 breast cancer tissue and benign breast tissue: ·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, further experiments identified the following markers and / or panels of markers that can distinguish between invasive breast cancer tissue and benign breast tissue: ·CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.7673436 2-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.chr4.8859602-885 9669, 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 panels of markers that can distinguish between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast tissue: 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 at 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 multiple methylated DNA markers and subsets thereof (e.g., 2, 3, 4, 5, 6, 7, or 8 marker sets) for use in breast cancer overall and various breast cancer types (e.g., triple-negative breast cancer, HER2 + Highly discriminates between breast cancers (luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer). Experiments applied selection filters to candidate markers to identify markers that provide high signal-to-noise ratios and low background levels, providing high specificity for breast cancer screening or diagnosis.

[0025] In some embodiments, the techniques involve 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 variably methylated regions (DMRs) as described herein, e.g., as provided in Tables 2 and 18. The methylation status is assessed in embodiments of the techniques. As such, the techniques provided herein are not limited by the method 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 methylation patterns at specific CpG sites are monitored by digestion of genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the region of interest (digestion-Southern). In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that involves digestion of genomic DNA with methylation-sensitive or methylation-dependent restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18:687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as a starting point for methylation analysis. 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).PCR techniques have been developed for the detection of genetic mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) and quantification of allele-specific expression (Szabo and Mann (1995) Genes Dev. 9:3097-3108, and Singer-Sam et al. (1992) PCR Methods Appl. 1:160-163). Such techniques use internal primers that anneal to the PCR-generated template and terminate immediately 5' to the single nucleotide being assayed. Methods using a "quantitative Ms-SNuPE assay" such as that described in U.S. Pat. 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 specific site (e.g., at a single nucleotide, at a specific region or locus, or at a longer sequence of interest, e.g., up to about 100 bp, 200 bp, 500 bp, 1000 bp of DNA or more) compared to the DNA population in a sample that contains that specific site. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using a calibrator. A known amount of DNA is then bisulfite treated, and the resulting methylation-specific sequence is determined using either real-time PCR or other exponential amplification, such as the QuARTS assay (e.g., as provided by U.S. Patent No. 8,361,720 and U.S. Patent Application Publication Nos. 2012 / 0122088 and 2012 / 0122106, which are incorporated herein by reference).

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

[0028] Also provided herein are compositions and kits for carrying out the methods. For example, in some embodiments, reagents (e.g., primers, probes) specific for one or more markers are provided, either singly or in sets (e.g., sets of primer pairs for amplifying multiple markers). Additional reagents for carrying out detection assays (e.g., enzymes, buffers, positive and negative controls for carrying out QuARTS, PCR, sequencing, bisulfite, or 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 carrying out the methods. Also provided are reaction mixtures containing the reagents. Also provided are master mix reagent sets containing multiple reagents that may be added to each other and / or to a test sample to complete a reaction mixture.

[0029] In some embodiments, the techniques described herein relate to programmable machines designed to perform a series of arithmetic or logical operations such as those provided by the methods described herein. For example, some embodiments of the techniques relate to (e.g., are implemented on) computer software and / or computer hardware. In one aspect, the techniques relate to computers that include some form of memory, elements for performing arithmetic and logical operations, and processing elements (e.g., microprocessors) 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 methylation status (e.g., of one or more DMRs, e.g., DMRs 1-375 as provided in Tables 2 and 18), comparing methylation status (e.g., of one or more DMRs, e.g., DMRs 1-375 as provided in Tables 2 and 18), generating a standard curve, determining Ct values, calculating the rate, 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 ROC curves and associated AUCs, sequence analysis, all as described herein or known in the art.

[0030] In some embodiments, the microprocessor or computer uses the methylation status data in an algorithm to predict the site of the cancer.

[0031] In some embodiments, a software or hardware component receives the results of multiple assays, determines a single value result, and reports it to a user indicating cancer risk based on the results of the multiple assays (e.g., determining the methylation status of multiple DMRs, e.g., 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 the results of multiple assays, e.g., determining the methylation status of multiple markers (e.g., multiple DMRs, e.g., as provided in Tables 2 and 18). In some embodiments, the methylation status of the DMRs defines a dimension and can have values ​​in a multidimensional space, and the coordinate defined by the methylation status of the multiple DMRs is a result, e.g., related to cancer risk, for reporting to a user, e.g.,

[0032] Some embodiments include storage media and memory components. The memory components (e.g., volatile and / or non-volatile memory) find use in storing instructions (e.g., process embodiments as provided herein) and / or data (e.g., workpieces such as methylation measurements, sequences, and their associated statistical descriptions). Some embodiments also relate to systems that include one or more of a CPU, a graphics card, and a user interface (e.g., including an output device such as a display and an input device such as a keyboard).

[0033] The programmable machines associated with the technology include conventional existing technologies as well as technologies under development or yet to be developed (e.g., quantum computers, chemical computers, DNA computers, optical computers, spintronics-based computers, etc.).

[0034] In some embodiments, the techniques involve wired (e.g., metal cable, fiber optic) or wireless transmission media for transmitting data. For example, some embodiments relate to data transmission over a network (e.g., a local area network (LAN), a wide area network (WAN), an ad-hoc network, the Internet, etc.). In some embodiments, the programmable machines reside on the network as peers, and in some embodiments, the programmable machines have 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 media, floppy disk, or the like.

[0036] In some embodiments, the technology provided herein involves multiple programmable devices operating in conjunction with one another to perform the methods described herein. For example, in some embodiments, multiple computers (e.g., connected by a network) may operate in parallel to collect and process data, e.g., in an implementation of cluster computing or grid computing or some other distributed computer architecture that relies on complete computers (with on-board CPUs, storage, power supplies, network interfaces, etc.) connected to a network (private, public, or the Internet) by traditional network interfaces such as Ethernet, fiber optics, etc., or by wireless networking technology.

[0037] For example, some embodiments provide a computer including a computer-readable medium. 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 processor, and may be any of a number of computer processors, such as those manufactured by Intel Corporation of Santa Clara, California and Motorola Corporation of Schaumburg, Illinois. Such a processor may include or be in communication 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 capable of providing 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, configurable processors, all optical media, all magnetic tapes or other magnetic media, or any other media from which a computer processor can read instructions. Also, various other forms of computer-readable media may transmit or carry instructions to a computer, including 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, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0039] In some embodiments, the computer is connected to a network. The computer may also include multiple 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, smartphones, pagers, digital tablets, laptop computers, Internet appliances, and other processor-based devices. Generally, computers relevant to aspects of the technology provided herein may be any type of processor-based platform running any operating system capable of supporting one or more programs, including the technology provided herein, such as Microsoft Windows, Linux, UNIX, Mac OS X, etc. Some embodiments include personal computers that run other application programs (e.g., applications). Applications may be stored in memory and may 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 a client device.

[0040] All such components, computers, and systems described herein as relating to the technology may be logical or virtual.

[0041] Accordingly, provided herein are methods for detecting breast cancer and / or various breast cancer types (e.g., triple-negative breast cancer, HER2, HER2+ ... +The present invention relates to a method for screening for breast cancer (e.g., breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), the method comprising assaying the methylation state of a marker in a sample (e.g., breast tissue) (e.g., a plasma sample) obtained from a subject, and identifying the subject as having breast cancer and / or a particular type of breast cancer if the methylation state of the marker differs from the methylation state of the marker assayed in a subject without breast cancer, wherein the marker comprises a base in a variably methylated region (DMR) selected from the group consisting of 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 state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state 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.chr11.68622869-6 8622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC10013289 1, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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.c hr20.4422, 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, when the sample obtained from the subject is a blood sample (e.g., plasma, serum, whole blood), the methylation status of one or more of the following markers differs from the methylation status of one or more markers assayed in a subject who does not have breast cancer, indicating that the subject has 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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.chr18.76734362-7673437 0, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has triple-negative breast cancer: ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr1 2.4273906-4274012, 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 state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the subject has: +These 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, IL1 5RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, L OC283999, LY6H, MAST1, MAX.chr1.158083198-158083476, MAX.chr1.22807 4764-228074977 MAX.chr1.46913931-46913950 MAX.chr10.130085265- 130085312. MAX.chr11.68622869-68622968. MAX.chr14.101176106-1011 76260. MAX.chr15.96889069-96889128. MAX.chr17.8230197-8230314 X.chr19.46379903-46380197; MAX.chr2.97193163-97193287; MAX.chr2.9 7193478-97193562; MAX.chr20.1784209-1784461; MAX.chr21.44782441- 44782498; MAX.chr22.23908718-23908782; MAX.chr5.145725410-145725 459 MAX.chr5.178957564-178957598 MAX.chr5.180101084-1 MAX.chr5.42952185-42952280 MAX.chr5.42994866-42994936 MAX.chr627064703-27064783, MAX.chr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID 2_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 state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the subject has: +Indicated to have 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 state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the subject has: +Indicated with 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.chr11.68622 869-68622968, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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, E TS1_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.chr1 2.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.73073682-73073814, MAX.chr20.1783841-1784054, MAX.chr21.4706380 2-47063851, MAX.chr4.8860002-8860038, MAX.chr5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-13068 6985, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has luminal A breast cancer: BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, SLC30A10, TR IM67, 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, BHLH E23_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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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.chr5.42994866-42994 936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX. chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, M AX.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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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.chr18.7673436 2-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, MAX.ch r22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-1457254 59, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has luminal B breast cancer: CALN1_A, LOC100132891, MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, DLX4, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, MAX.chr8.12417303 0-124173395, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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.chr5.42994866-4299 4936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, M AX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_C, A LOX5, 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, when the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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.chr11.1492660 2-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721- 5629791, MAX.chr19.30719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-17 8957598, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has BRCA1 breast cancer: BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr 15.96889013-96889128, NACAD, ATP6V1B1, BANK1, C17orf64, DLX4, EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.c hr11.14926602-14927148, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has 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, MA X.chr11.14926602-14926729, MAX.chr11.14926860-14927148, MAX.chr15.96889013-9 6889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.1 51145632-151145743, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has BRCA2 breast cancer: MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-1 4927148, 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, when the sample obtained from the subject is breast tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without breast cancer, the methylation state indicates that the subject has invasive breast cancer: CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-7 6734476, 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, distinguishing between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast cancer tissue, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in subjects without breast cancer: SCRT2_B, MPZ, MAX.chr8.124173030-124173395, ITPRIPL1, ITPRIPL1, DLX4, CALN1_A, and IGF2BP3_B (see Table 15, Example I).

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

[0064] In some embodiments, distinguishing between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast cancer tissue, where the sample obtained from the subject is breast tissue and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in subjects without breast cancer: 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 techniques may be used to treat breast cancer and / or various breast cancer types (e.g., triple-negative breast cancer, HER2 +The present invention relates to identifying and differentiating between breast cancers (e.g., 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, assaying 2 to 11 to 100 or 120 or 375 markers.

[0066] The techniques are not limited to the methylation state assessed. In some embodiments, assessing the methylation state of a marker in a sample comprises determining the methylation state of a single base. In some embodiments, assaying the methylation state of a marker in a sample comprises determining the degree of methylation at multiple bases. Further, in some embodiments, the methylation state of a marker comprises increased methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker comprises decreased methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker comprises 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 1 base. In some embodiments, the marker is in a high CpG density promoter.

[0068] The techniques are 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), a stool sample, or a urine sample.

[0069] Furthermore, the technique is not limited to the method used to determine the methylation status. In some embodiments, the assaying comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, the assaying comprises the use of methylation-specific oligonucleotides. In some embodiments, the technique determines the methylation status using massively parallel sequencing (e.g., next-generation sequencing), such as sequencing-by-synthesis, real-time (e.g., single-molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.

[0070] The technology provides reagents for detecting DMRs, for example, in some embodiments, a set of oligonucleotides is provided that includes the sequences provided by SEQ ID NOs: 1-422 (see Tables 10, 19, and 20). In some embodiments, oligonucleotides are provided that include sequences complementary to chromosomal regions that have bases in the DMR, e.g., oligonucleotides that are sensitive to the methylation status 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.chr5.1457 25410-145725459, 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).

[0072] The technology provides a panel of various markers used to identify breast cancer, for example, in some embodiments, the markers are 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, IGF2B The chromosomal regions with the following annotations are included: P3_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 annotated as 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.chr2 2.42679578-42679917, MAX.chr4.8859253-8859329, MAX.chr4.8859602-8859669, MAX.chr4.88 60002-8860038, MAX.chr5.145725410-145725459, MAX.chr6.157557371-157557657, MPZ, NKX2- 6, including chromosomal regions with annotations of 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).

[0075] The technology provides a panel of various markers used to identify triple-negative breast cancer, for example, in some embodiments, the markers are 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 The chromosomal regions with the following annotations are included: 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 a panel of various markers used to identify triple-negative breast cancer, for example, in some embodiments, the markers are ATP6V1B1, MAX.chr11.14926602-14927148, PRKCB, TRH_A, MPZ, GP5, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, MAX.chr12.4 The chromosomal regions with the following annotations are included: MAX.chr5.273906-4274012, 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 +Panels of various markers used to identify breast cancer are provided, 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, DNA JC6, 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, M AST1, 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.chr19.4637 9903-46380197, MAX.chr2.97193163-97193287, MAX.chr2.97193478-97193562, MAX.chr20.1784209-1784461, MAX.chr21.44782441-44782498, M AX.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.c hr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474 527, 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 +A panel of various markers used to identify breast cancer is provided, for example, in some embodiments, the markers include BHLHE23_C, CALN1_A, CD1D, CHST2_A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, NACAD, TRIM67, ATP6V1B1, C17orf64, CHST2_B, D LX4, DNM3_A, EMX1_A, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.chr5.4299 4866-42994936, 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, PLXNC 1_A, RIC3, ​​SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.ch r12.4273906-4274012, 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).

[0079] The technology is HER2 +Panels of various markers used to identify breast cancer are provided, 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.c hr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461 , LOC100132891, BHLHE23_C, ALOX5, MAX.chr19.46379903-46380197, ODC1, CHST2_A, MAX.chr 5.77268672-77268725, C17orf64, EMX1_A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, ABLIM1, BHLHE23_D, ZSCAN12, GRASP, C10orf125 (see Table 16B, Example II).

[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.chr4.8860002-886003 8, MAX.chr5.172234248-172234494, MAX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MA The chromosomal regions with the following annotations are included: X.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).

[0081] The technology provides a panel of various markers used to identify luminal A breast cancer, for example, in some embodiments, the markers include BHLHE23_C, CD1D, CHST2_A, FAM126A, FMN2, HOXA1_A, HOXA7_A, KCNH8, LOC100132891, MAX.chr15.96889013-96889128, SLC30A10, TRIM67, ATP6V1B1, BANK1, C10orf 125, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-1492 7148, 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.chr1 2.4273906-4274012, 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).

[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.chr2 0.1784209-1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chr19.46379903-46380197, OD The chromosomal regions with the following annotations are included: C1, 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.chr18.76734 423-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-426799 17, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.1 78957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B Includes chromosomal regions with annotations of 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, The chromosomal regions with the following annotations are included: 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).

[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.chr5.14572541 0-145725459, 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).

[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.chr11.14926 860-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.3 0719261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.c hr5.77268672-77268725, 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).

[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 The chromosomal regions with annotations are 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).

[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.chr2.23 8864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-1511 45743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B Includes chromosomal regions with annotations of TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64 (see Table 8, Example I).

[0089] The technology provides a panel of various markers used to identify BRCA2 breast cancer, for example, in some embodiments, the markers are MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LO Includes chromosomal regions with the following annotations: C100132891, 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 are CDH4_E, FLJ42875, GAD2, GRASP, ITPRIPL1, KCNA1, MAX.chr12.4273906-4274012, MAX.chr18.76734362-76734370, MAX.chr18.76734423-76734476, MAX.chr19.30719261-30719354, M Included are chromosomal regions with the following annotations: AX.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 ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (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 ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast tissue, for example, in some embodiments, the markers include SCRT2_B, ITPRIPL1, and the chromosomal region annotated as MAX.chr8.124173030-12417339 (100% sensitivity at 91% specificity) (see Table 15, Example I).

[0093] The technology provides a panel of various markers used to distinguish between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast tissue, for example, in some embodiments, the markers include chromosomal regions annotated as follows: 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] Kit embodiments are provided, for example, kits that include reagents that can modify DNA in a methylation-specific manner (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 DMRs 1-375 (from Tables 2 and 18) and has a methylation status associated with subjects without breast cancer. In some embodiments, the kit includes a bisulfite reagent and an oligonucleotide as described herein. In some embodiments, the kit includes reagents that can modify DNA in a methylation-specific manner (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 DMRs 1-375 (from Tables 2 and 18) and has a methylation status associated with subjects with breast cancer. Some kit embodiments include a sample collector for obtaining a sample from a subject (e.g., a stool sample, a breast tissue sample, a plasma sample, a serum sample, a whole blood sample), reagents capable of modifying DNA in a methylation-specific manner (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 compositions (e.g., reaction mixtures). In some embodiments, compositions are provided that include a nucleic acid containing a DMR and 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). Some embodiments provide compositions that include a nucleic acid containing a DMR and an oligonucleotide as described herein. Some embodiments provide compositions that include a nucleic acid containing a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide compositions that include a nucleic acid containing a DMR and a polymerase.

[0096] Additional related method embodiments involve detecting breast cancer and / or various breast cancer types (e.g., triple-negative breast cancer, HER2) in a sample (e.g., breast tissue sample, plasma sample, fecal sample) obtained from a subject. +For example, the method may include determining the methylation status of a marker in a sample containing a base in one or more DMRs selected from DMRs 1 to 375 (from Tables 2 and 18), comparing the methylation status of the marker in the subject sample with the methylation status of a marker in a subject sample containing a base in one or more DMRs selected from DMRs 1 to 375 (from Tables 2 and 18), and ... the subject sample with the methylation status of a marker in the subject sample. + and determining a confidence interval and / or p-value for the difference in 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 comprising a DMR with a reagent capable of modifying the nucleic acid in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent), e.g., to produce a nucleic acid modified in a methylation-specific manner; sequencing the nucleic acid modified in a methylation-specific manner to provide a nucleotide sequence of the nucleic acid modified in a methylation-specific manner; comparing the nucleotide sequence of the nucleic acid modified in a methylation-specific manner to a nucleotide sequence of a nucleic acid comprising a DMR from a subject without breast cancer and / or breast cancer type to identify differences between the two sequences; and, if differences are present, determining whether the subject has breast cancer (e.g., breast cancer and / or breast cancer type: triple-negative breast cancer, HER2 + The method provides a step of identifying a person as having breast cancer (e.g., luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer).

[0097] The technology provides a system for screening for breast cancer in a sample obtained from a subject. Exemplary embodiments of the system include, for example, detecting breast cancer and / or breast cancer types (e.g., triple-negative breast cancer, HER2-positive breast cancer, HER2-negative ... + The present invention also includes a system for screening for breast cancer (e.g., breast cancer, luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer), the system including an analysis component configured to determine the methylation status of a sample, a software component configured to compare the methylation status of the sample to the methylation status of a control or reference sample recorded in a database, and an alert component configured to alert a user of a breast cancer-associated methylation status. The alert, in some embodiments, is determined by a software component that receives results from multiple assays (e.g., multiple markers, e.g., determining the methylation status of DMRs, e.g., as provided in Tables 2 and 18), calculates a value or result, and reports based on the multiple results. Some embodiments provide a database of weighting parameters associated with each DMR provided herein for use in calculating a value or result and / or reporting an alert to a user (e.g., a doctor, nurse, clinician, etc.). In some embodiments, all of the results from the multiple 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 the multiple assays that is indicative of cancer risk in a 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 a database of breast cancer and / or specific breast cancer types (e.g., triple-negative breast cancer, HER2 +The present invention relates to a method for detecting breast cancer, including the detection of ...

[0099] In certain embodiments, methods are provided for 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 the human patient sample, assaying the methylation status of DNA methylation markers that include bases in a variably methylated region (DMR) selected from the group consisting of DMRs 1-375 from Table 2 and Table 18, and comparing the assayed methylation status of one or more DNA methylation markers with breast cancer and / or a particular breast cancer type (e.g., triple-negative breast cancer, HER2 + The method includes comparing the methylation level of one or more DNA methylation markers to a reference for human patients without breast cancer (e.g., 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 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 comprise assaying multiple DNA methylation markers. In some embodiments, such methods comprise assaying 2 to 11 DNA methylation markers. In some embodiments, such methods comprise assaying 12 to 120 DNA methylation markers. In some embodiments, such methods comprise assaying 2 to 375 DNA methylation markers. In some embodiments, such methods comprise assaying the methylation state of one or more DNA methylation markers in a sample, and comprising determining the methylation state of a single base. In some embodiments, such methods comprise assaying the methylation state of one or more DNA methylation markers in a sample, and comprising determining the degree of methylation at multiple bases. In some embodiments, such methods comprise assaying the methylation state of the forward strand or 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 in a high CpG density promoter.

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

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

[0105] In some embodiments, 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 Chromosomal regions having annotations selected from the group consisting of -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) comprise DNA methylation markers.

[0106] In some embodiments, 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, Chromosomal regions having annotations selected from the group consisting of 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) comprise DNA methylation markers.

[0107] In some embodiments, 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 (see Table 27, Example III) comprises a DNA methylation marker.

[0108] In some embodiments, 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.chr18.76734362-7673 4370, MAX.chr2.97193478-97193562, MAX.chr22.42679578-42679917, MAX.chr4.8859253-8 Chromosomal regions having annotations selected from the group consisting of: MAX.chr4.859329, 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) comprise DNA methylation markers.

[0109] In some embodiments, 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 Chromosomal regions having annotations selected from the group consisting of 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) contain DNA methylation markers.

[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) comprises a DNA methylation marker.

[0111] These include ABLIM1, AFAP1L1, AKR1B1, ALOX5, AMN, ARL5C, BANK1, and 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, IL1 5RA, INSM1, ITPKA_B, ITPRIPL1, KCNE3, KCNK17_B, LIME1, LOC100132891, L OC283999, LY6H, MAST1, MAX.chr1.158083198-158083476, MAX.chr1.22807 4764-228074977 MAX.chr1.46913931-46913950 MAX.chr10.130085265- 130085312. MAX.chr11.68622869-68622968. MAX.chr14.101176106-1011 76260. MAX.chr15.96889069-96889128. MAX.chr17.8230197-8230314 X.chr19.46379903-46380197; MAX.chr2.97193163-97193287; MAX.chr2.9 7193478-97193562; MAX.chr20.1784209-1784461; MAX.chr21.44782441- 44782498; MAX.chr22.23908718-23908782; MAX.chr5.145725410-145725 459 MAX.chr5.178957564-178957598 MAX.chr5.180101084-1 MAX.chr5.42952185-42952280 MAX.chr5.42994866-42994936 MAX.chr627064703-27064783, MAX.chr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474527, MCF2L2, MSX2P1, NACAD, NID 2_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 Chromosomal regions having annotations selected from the group consisting of 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, 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.chr8.124173030-1 24173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.ch r19.46379903-46380197, 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, MA Chromosomal regions having annotations selected from the group consisting of X.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, 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) comprise DNA methylation markers.

[0114] In some embodiments, 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, HA ND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931-4691395 0, 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,M AX.chr5.178957564-178957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MAX.chr8.688863-688924, MAX.chr9 Chromosomal regions having annotations selected from the group consisting of .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, 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, COL23 A1, KCNK9, OTX1, PLXNC1_A, HNF1B_B, MAST1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4 Chromosomal regions having annotations selected from the group consisting of: 274012, 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) comprise DNA methylation markers.

[0116] In some embodiments, 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.chr11.68622869-686229 68, 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) comprise DNA methylation markers.

[0117] In some embodiments, 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.chr19.30 719261-30719354, MAX.chr22.42679578-42679917, MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-1789 57598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B Chromosomal regions with annotations selected from the group consisting of SYNJ2, TMEM176A, TSHZ3, and VIPR2 (see Table 6, Example I) contain DNA methylation markers.

[0118] In some embodiments, 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. Chromosomal regions having annotations selected from the group consisting of 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) contain DNA methylation markers.

[0119] In some embodiments, 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.chr11.68622869-68622 968, 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) comprise DNA methylation markers.

[0120] In some embodiments, 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.chr15.96 889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.30719261-30719354, MA X.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.chr5.77268672 Chromosomal regions having annotations selected from the group consisting of: 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) comprise 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 Chromosomal regions having annotations 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) comprise DNA methylation markers.

[0122] In some embodiments, 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.chr11.14926860-149271 48, MAX.chr15.96889013-96889128, MAX.chr2.238864674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-151145743, MAX.ch r8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B Chromosomal regions with annotations selected from the group consisting of TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64 (see Table 8, Example I) contain DNA methylation markers.

[0123] In some embodiments, a chromosomal region 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 (see Table 11, Example I) comprises a DNA methylation marker.

[0124] In some embodiments, 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.chr4.88 Chromosomal regions having annotations selected from the group consisting of MAX.chr5.60002-8860038, 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, a chromosomal region 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) comprises a DNA methylation marker.

[0126] In some embodiments, a chromosomal region 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) comprises a DNA methylation marker.

[0127] In some embodiments, such methods comprise determining the methylation status of two DNA methylation markers. In some embodiments, such methods comprise determining the methylation status of a pair of DNA methylation markers provided in a row of Table 2 and / or Table 18.

[0128] In certain embodiments, the technology provides methods 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 involve determining the methylation status of DNA methylation markers in a sample containing bases in a DMR selected from the group consisting of DMRs 1-375 from Tables 2 and 18, and comparing the methylation status of the DNA methylation markers in the patient sample with breast cancer and / or specific breast cancer types (e.g., triple-negative breast cancer, HER2 + and determining a confidence interval and / or p-value for the difference in 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 produce a methylation-specifically modified nucleic acid; sequencing the methylation-specifically modified nucleic acid to provide a nucleotide sequence of the methylation-specifically modified nucleic acid; and comparing the nucleotide sequence of the methylation-specifically modified nucleic acid to the nucleotide sequence of a nucleic acid comprising a DMR from a subject without breast cancer to identify differences between the two sequences.

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

[0131] In some embodiments, such systems further comprise a component for isolating nucleic acids, hi some embodiments, such systems further comprise a component for collecting a sample.

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

[0133] In some embodiments, the database comprises nucleic acid sequences that contain DMRs. In some embodiments, the database comprises nucleic acid sequences from subjects that do not have breast cancer.

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

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

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

[0137] Additionally, as used herein, the term "or" is an inclusive "or" 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 additional unlisted factors to be based on unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "into" and "onto."

[0138] The transitional phrase "consisting essentially of," when used in the claims of this application, limits the scope of the claim to certain substances or steps and "those which 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" recited elements may contain unrecited contaminants at levels such that the contaminants, although present, do not alter the function of the recited composition when compared to a pure composition, i.e., a composition "consisting of" the recited ingredients.

[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. "Nucleic acid" includes, but is not limited to, single-stranded and double-stranded nucleic acids. As used herein, the term "nucleic acid" also includes DNA, as described above, containing one or more modified bases. Thus, DNA with a backbone modified for stability or other reasons is a "nucleic acid." As used herein, the term "nucleic acid" encompasses chemical forms of DNA characteristic of viruses and cells, including simple and complex cells, in addition to such chemically, enzymatically, or metabolically modified forms of nucleic acids.

[0140] The terms "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors and therefore on the ultimate function or use of the oligonucleotide. Oligonucleotides may be produced by any technique, including chemical synthesis, DNA replication, reverse transcription, or a combination 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 term "locus" or "region" of a nucleic acid refers to a small region of nucleic acid, e.g., a gene on a chromosome, a single nucleotide, a CpG island, and the like.

[0142] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial," in which only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands determines the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and 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 comprises coding sequences necessary for the production of RNA, or of a polypeptide or its precursor. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. The term "portion," when used in reference to a gene, refers to a fragment of that gene. Fragments can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "nucleotides comprising at least a portion of a gene" can include a fragment of a 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, regulatory, structural, and other sequences) located adjacent to the coding region at both the 5' and 3' ends, e.g., within a distance of approximately 1 kb on either end, such that a gene corresponds in length to the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), genomic forms or clones of a gene contain coding regions separated by non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA) and may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript and therefore are absent in the messenger RNA (mRNA) transcript, which functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

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

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

[0147] The term "allele" refers to genetic variation, including, but not limited to, variants and mutations, polymorphic and single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles may occur naturally in a population or may arise during the lifespan of any particular individual in a population.

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

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

[0150] The term "amplifying" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or portion of a polynucleotide, usually starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where 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 in a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810, incorporated herein by reference in its entirety) is a form of amplification. Additional types of amplification include allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611, incorporated herein by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408, incorporated herein by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594, incorporated herein by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671, each incorporated herein by reference in its entirety), inter-sequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, incorporated herein by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, 25:1854-1858 (1997), U.S. Patent No. 5,508,169, each of which is incorporated herein by reference in its entirety), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821-9826, each of which is incorporated herein 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 incorporated herein 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, each of which is incorporated herein by reference in its entirety), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15)7351-7367, which is incorporated herein 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, each of which is incorporated herein by reference in its entirety), reverse transcription PCR (see, e.g., Bustin, SA (2000) J. Molecular Endocrinology 25:169-193, each of which is incorporated herein by reference in its entirety), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don, et al., Nucleic 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). Polynucleotide amplification can also be achieved using digital PCR (e.g., 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, and U.S. 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 method of K.B. Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, which describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic or other DNA or RNA without cloning or purification. This process for amplifying a target sequence involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycles 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 the primers then anneal to their complementary sequences within the target molecule. After annealing, the primers are extended with a polymerase to form new pairs of 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 many "cycles") to obtain a highly concentrated amplified segment of the desired target sequence. The length of the amplified segment for the desired target sequence is determined by the relative positions of the primers with respect to each other; therefore, this length is a controllable parameter. Due to the repetitive aspect of the process, the method is referred to as a "polymerase chain reaction" ("PCR"). As the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified," and they are "PCR products" or "amplicons." Those skilled in the art will understand that the term "PCR" encompasses many variants of the originally described method, using, for example, real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single-primer and arbitrarily primed PCR, etc.

[0152] Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under the conditions in which they are used, will process only specific sequences of nucleic acid 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 join two oligonucleotides or polynucleotides if there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at 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 sequences defined by primer binding due to their ability to function at high temperatures, which therefore create thermodynamic conditions that favor primer hybridization with target sequences but not with 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, probe hybridization, structure-specific cleavage assays (e.g., INVADER assay (Hologic, Inc.)), and methods described in, for example, 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. Pat. No. 9,096,893, each of which is incorporated by reference herein in its entirety for all purposes), enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, each of which is incorporated by reference herein in its entirety), polymerase chain reaction (PCR) as described above, branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, each of which is incorporated by reference herein in its entirety), rolling circle replication (e.g., U.S. Pat. No. 6,210,884, , 6,183,960 and 6,235,502, the entireties of which are incorporated herein by reference), NASBA (e.g., U.S. Pat. No. 5,409,818, the entireties of which are incorporated herein by reference), molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, the entireties of which are incorporated herein by reference), E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170 and 6,063,573, the entireties of which are incorporated herein by reference), cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769 and 5,660,988, the entireties of which are incorporated herein by reference), Dade Examples of such hybridization methods include, but are not limited to, the 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, which are incorporated herein by reference in their entireties), the ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci. USA 88,189-93 (1991)), and sandwich hybridization (e.g., U.S. Patent No. 5,288,609, which is incorporated herein by reference in its entirety).

[0154] The term "amplifiable nucleic acid" refers to a nucleic acid that may be amplified by any amplification method. It is generally considered that an "amplifiable nucleic acid" will include a "sample template."

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

[0156] The term "primer" refers to an oligonucleotide, whether occurring naturally, e.g., as a nucleic acid fragment from a restriction digest, or produced synthetically, which can serve as a point of initiation of synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid template strand (e.g., in the presence of nucleotides and an inducing agent, such as DNA polymerase, and at a suitable temperature and pH). Primers are preferably single-stranded for maximum amplification efficiency, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to initiate synthesis of extension products in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and the use of the method.

[0157] The term "probe" refers to any oligonucleotide (e.g., a sequence of nucleotides), whether naturally occurring, as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification, that can hybridize to another oligonucleotide of interest. Probes can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). Any probe used in the present invention may, in some embodiments, be labeled with any "reporter molecule" and thereby be considered detectable by any detection system, including, but not limited to, enzymatic (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0158] The term "target," as used herein, refers to a nucleic acid that is sought to be sorted from other nucleic acids, e.g., by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, "target" refers to the region of nucleic acid bound by the primers used in the polymerase chain reaction, whereas when used in assays in which the target DNA is not amplified, e.g., in some embodiments of an invading cleavage assay, the target includes the site where the probe and invading oligonucleotide (e.g., an INVADER oligonucleotide) bind to form an invading cleavage structure, so that the presence of the target nucleic acid can be detected. A "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, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.

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

[0161] As used herein, a "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 a nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (e.g., the methylation status 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 genetic marker or DNA region as described herein) may indicate the methylation state of all bases in the sequence, or may indicate the methylation state of a subset of bases within the sequence (e.g., of one or more cytosines), or may indicate information about the local methylation density within the sequence, with or without providing information about the precise location within the sequence where methylation occurs.

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

[0165] Methylation status can optionally be expressed or indicated by a "methylation value" (e.g., representing a methylation frequency, rate, ratio, percent, etc.). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acid. Thus, a value, e.g., a methylation value, represents the methylation status and can thus be used as a quantitative indicator of methylation status across multiple copies of a locus. This is of particular use when it is desirable to compare the methylation status of sequences in a sample to a threshold or reference value.

[0166] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of instances in which a molecule or locus is methylated compared to the number of instances in which the molecule or locus is unmethylated.

[0167] As such, methylation state describes the methylation state of a nucleic acid (e.g., a genomic sequence). Furthermore, methylation state refers to characteristics of a nucleic acid segment at a particular genomic locus that are related to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated C across any particular region of the nucleic acid, and allelic differences in methylation due to, for example, differences in allelic origin. The terms "methylation state," "methylation profile," and "methylation status" also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated C across any particular region of the nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) in a nucleic acid sequence are methylated, it may be referred to as having "hypermethylation" or "increased methylation," whereas if a cytosine (C) residue(s) in a DNA sequence are unmethylated, it may be referred to as having "hypomethylation" or "decreased methylation." Similarly, if a cytosine (C) residue(s) in a nucleic acid sequence are methylated when compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to have hypermethylation or increased methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) in a DNA sequence are unmethylated when compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to have hypomethylation or decreased methylation compared to the other nucleic acid sequence. Furthermore, the term "methylation pattern," as used herein, refers to the collection of methylated and unmethylated nucleotides across a region of a nucleic acid. Two nucleic acids may have the same or similar methylation frequency or percent methylation, but may have different methylation patterns if the numbers of methylated and unmethylated nucleotides are the same or similar throughout the region but the positions of the methylated and unmethylated nucleotides are different.Sequences are said to have "variable methylation," or "differential methylation," or "differential methylation status" if they differ in the degree, frequency, or pattern of methylation (e.g., one has increased or decreased methylation compared to the other). The term "variable methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. It can also refer to the difference in the level or pattern between patients whose cancer recurs after surgery and those whose cancer does not recur. Variable methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, once precise cutoffs or predictive characteristics are defined.

[0168] Methylation state frequencies can be used to describe a population of individuals or a sample derived from a single individual. For example, a nucleotide locus with a 50% methylation state frequency is methylated in 50% of cases and unmethylated 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 a collection of nucleic acids. Thus, if the methylation in a first population or pool of nucleic acid molecules is different from the methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from the methylation state frequency 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 group of cells from a tissue sample is methylated or unmethylated 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. The 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 guanines and cytosines (also called CpG dinucleotide sequences), where the cytosine is located 5' to the guanine. Most 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, e.g., 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 total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency to expected frequency is 0.6; in some cases, a CpG island can be at least 500 base pairs in length, where the G:C content of the region is at least 55% and the ratio of observed CpG frequency to expected frequency is 0.65. The observed CpG frequency to expected frequency can be calculated by the method provided in Gardiner-Garden et al. (1987) J. Mol. Biol. 196: 261-281. For example, the observed CpG frequency relative to the expected frequency can be calculated by the formula R = (A x B) / (C x D), where R is the ratio of the observed CpG frequency to the expected 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. Methylation status is usually determined in CpG islands, for example, in promoter regions. However, it will be understood that other sequences in the human genome are prone to 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, a "methylation-specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule depending on 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 in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating nucleic acid molecules with such reagents can include contacting the nucleic acid molecule with the reagent, and, if desired, in combination with additional steps, to achieve the desired change in nucleotide sequence. Such methods can be applied in a manner that unmethylated nucleotides (e.g., each unmethylated cytosine) are modified to a different nucleotide. For example, in some embodiments, such reagents can deaminate unmethylated cytosine nucleotides to generate deoxyuracil residues. Examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents.

[0173] Alteration of a nucleic acid nucleotide sequence with 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 status of one or more CpG dinucleotide sequences within a sequence of a nucleic acid.

[0175] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to an art-recognized technique that uses CG-rich primers to allow global scanning of the genome, focusing on regions most likely to contain CpG dinucleotides, and is described in Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0176] The term "MethyLight™" refers to the art-recognized fluorescence-based real-time PCR technology 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 cover the CpG positions between or are covered by the 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 a methylation-specific blocking probe that covers the CpG positions between the amplification primers.

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

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

[0181] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to the art-recognized methylation assay 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, a "selected nucleotide" refers to one of the four nucleotides normally occurring in nucleic acid molecules (C, G, T, and A in DNA and C, G, U, and A in RNA) and can include methylated derivatives of normally occurring nucleotides (e.g., if C is a selected nucleotide, then both methylated and unmethylated C are included in the meaning of a selected nucleotide), whereas a selected methylated nucleotide specifically refers to a normally occurring methylated nucleotide and a selected unmethylated nucleotide specifically refers to a normally occurring unmethylated 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. For restriction enzymes that specifically cleave when the recognition site is unmethylated or hemimethylated (methylation-sensitive enzymes), cleavage will not occur (or will occur with significantly reduced efficiency) if the recognition site is methylated on one or both strands. For restriction enzymes that specifically cleave only when the recognition site is methylated (methylation-dependent enzymes), cleavage will not occur (or will occur with significantly reduced efficiency) if the recognition site is unmethylated. Methylation-specific restriction enzymes are preferred, and their recognition sequences contain a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). More preferred in some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at the C5 carbon atom.

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

[0186] As used herein, the "sensitivity" of a given marker (or set of markers used together) refers to the percentage of samples reporting DNA methylation values ​​above a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histologically confirmed neoplasm reporting a DNA methylation value above a threshold (e.g., a disease-associated range), and a false negative is defined as a histologically confirmed neoplasm reporting a DNA methylation value below a threshold (e.g., a disease-unassociated range). Thus, the sensitivity value reflects the probability that a DNA methylation measurement value 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 subjects with a clinical condition, will detect the presence of that clinical condition.

[0187] As used herein, the "specificity" of a given marker (or a set of markers used together) refers to the percentage of non-neoplastic samples reporting DNA methylation values ​​below a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample reporting a DNA methylation value below a threshold (e.g., a range not associated with disease), and a false positive is defined as a histologically confirmed non-neoplastic sample reporting a DNA methylation value above a threshold (e.g., a range associated with disease). Thus, the specificity value reflects the probability that a DNA methylation measurement value 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 patients without a clinical condition, will detect the absence of that clinical condition.

[0188] The term "AUC" as used herein 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 points of a diagnostic test. The ROC curve shows the trade-off between sensitivity and specificity, depending on the cut point selected (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 the best being 1, and a random test will have an 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] The term "neoplasm," as used herein, refers to any new, abnormal growth of tissue. Thus, a neoplasm can be a premalignant neoplasm or a malignant neoplasm.

[0190] The term "neoplasm-specific marker," as used herein, 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, a marker is a specific nucleic acid region (e.g., a gene, an intragenic region, a specific genetic locus, etc.). A region of a nucleic acid that is a marker may be referred to, for example, as a "marker gene," a "marker region," a "marker sequence," a "marker locus," etc.

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

[0192] The terms "precancerous" or "preneoplastic" and their cognates refer to any cell proliferative disorder that is undergoing malignant transformation.

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

[0194] As used herein, the application of a "diagnostic" test includes detecting or identifying a disease state or condition in a subject, determining the likelihood that a subject will suffer from a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to a therapy, determining the prognosis (or likelihood of progression or regression) of a subject with a disease or condition, and determining the effectiveness of a treatment for a subject with a disease or condition. For example, diagnostics can be used to detect the presence or likelihood of a subject suffering from a neoplasm or the likelihood that such a subject will respond favorably to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0195] The term "isolated," as used with reference to a nucleic acid, as with "isolated oligonucleotide," refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid exists in a form or setting that is 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 occur in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found on a host cell chromosome adjacent to adjacent genes, an RNA sequence, e.g., a particular mRNA sequence encoding a particular protein, found in a cell as a mixture containing many other mRNAs that encode multiple proteins, and the like. However, an isolated nucleic acid encoding a particular protein includes, by way of example, such a nucleic acid in a cell that normally expresses that protein, where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise adjacent to a nucleic acid sequence different from that in which it is 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 used to express a protein, the oligonucleotide will contain at least a sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may also contain both a sense and an antisense strand (i.e., the oligonucleotide may be double-stranded). The isolated nucleic acid may be combined with other nucleic acids or molecules after isolation from its natural or typical environment. For example, the isolated nucleic acid may be present in a host cell in which it is placed, for example, for heterologous expression.

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

[0197] The term "composition comprising" a given polynucleotide sequence or polypeptide refers broadly to any composition containing the given polynucleotide sequence or polypeptide. Compositions may include aqueous solutions containing salts (e.g., NaCl), detergents (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 specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental materials, such as surface materials, 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, the term "remote sample," when used in some contexts, refers to a sample that is collected indirectly from a site that is not the sample source of the cell, tissue, or organ. For example, if sample material derived from the pancreas is evaluated in a fecal sample (e.g., not derived from a sample taken directly from the breast), the sample is a remote sample.

[0200] As used herein, the term "patient" or "subject" refers to an organism undergoing various tests provided by the technology. The term "subject" includes animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In even more preferred embodiments, the subject is a human. Furthermore, with respect to diagnostic methods, preferred subjects are vertebrate subjects. 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, the technology enables the diagnosis of mammals, such as those mammals important because they are endangered, such as the Amur tiger, in addition to humans; those mammals of economic importance, such as animals raised on farms for human consumption; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores such as cats and dogs, swine, including pigs, boars, and wild boars, ruminants and / or ungulates, such as pinnipeds, for example, cattle, bulls, sheep, giraffes, deer, goats, bison, and camels, and horses. Accordingly, further provided are diagnostics and treatments for livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like. The subject matter disclosed herein further includes a system for diagnosing lung cancer in a subject. The system may be provided, for example, as a commercially available kit that can be used to screen for lung cancer risk or diagnose lung cancer in a subject from whom a biological sample has been collected. An exemplary system provided in accordance with the present technology comprises assessing 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 systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for conducting the assay, etc.) from one location to another. For example, a kit may include one or more enclosed containers (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system comprising two or more separate containers, each containing a portion of all the components of the kit. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains an oligonucleotide. The term "fragmented kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system that includes two or more separate containers, each containing a portion of all the components of the kit, is included in the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system that contains all 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 fragmented and composite 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 proliferation 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 group of cells (i.e., two or more cells), whether malignant or benign, resulting from excessive or abnormal cell division, as well as precancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cell growth, tumors can invade surrounding tissue and metastasize.

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

[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 HER2 negative.

[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 PR-negative.

[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, "HER2 + The term "breast cancer" refers to breast cancer in which at least a portion of the cancer cells are ER and PR negative, but HER2 positive.

[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 reduced expression of wild-type BRCA2.

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

[0211] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using computer system(s), including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information 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 about a genome, including, but not limited to, nucleic acid sequences, genes, percentage dimethylation, allele frequencies, RNA expression levels, protein expression, phenotypes associated with genotypes, etc. "Allele frequency information" refers to facts or data about allele frequencies, including, but not limited to, allele identity information, statistical correlations between the presence of alleles and traits in a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, the percentage likelihood of an allele being present in an individual with one or more particular traits, etc. 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, those skilled in the art will understand that the various embodiments may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily appreciate that the specific order in which the methods are presented and performed is exemplary; it is contemplated that the order can be changed and still remain within the spirit and scope of the various embodiments disclosed herein.

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

[0214] In fact, 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 variably methylated regions (DMRs) for distinguishing cancers in breast-derived DNA from non-tumorous control 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 distinguish 1) triple-negative breast cancer tissue from normal breast tissue, 2) HER2 +The assays can distinguish between breast cancer tissue and normal breast tissue, 3) between luminal A breast cancer tissue and normal breast tissue, 4) between luminal B breast cancer tissue and normal breast tissue, 5) between BRCA1 breast cancer tissue and normal breast tissue, 6) between BRCA2 breast cancer tissue and normal breast tissue, and 7) between invasive breast cancer tissue and normal breast tissue. Additionally, DMRs have been identified that can distinguish between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast cancer tissue. Additionally, DMRs have been identified that can distinguish between plasma from subjects with breast cancer and plasma from subjects without breast cancer.

[0215] Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

[0216] In certain aspects, the present 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., a stool sample, a breast tissue sample, a plasma sample) isolated from a subject, where a change in the methylation status of the marker indicates the presence, classification, or location of breast cancer. Certain embodiments are directed to the identification, determination, and / or classification of breast cancers, including breast cancers and various breast cancer types (e.g., triple-negative breast cancer, HER2-positive breast cancer, and HER2-negative breast cancer). + The present invention relates to markers containing variably methylated regions (DMRs, e.g., DMRs 1-375, see Tables 2 and 18) used in the diagnosis (e.g., screening) of breast cancer (e.g., luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer).

[0217] In addition to the embodiments provided herein and in which methylation analysis of at least one marker, region of a marker, or marker base comprising a DMR (e.g., a DMR, e.g., DMRs 1-375) listed in Table 2 and Table 18 is analyzed, the technology also provides panels of markers comprising at least one marker, region of a marker, or marker base comprising a DMR useful for detecting cancer, particularly breast cancer.

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

[0219] In some embodiments, the present technology enables the use of reagents that modify DNA in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) in combination with one or more methylation assays to determine the methylation status of CpG dinucleotide sequences within at least one marker comprising a DMR (e.g., DMRs 1-375, see Tables 2 and 18). Genomic CpG dinucleotides can be methylated or unmethylated (alternatively known as up- or down-methylation, respectively). However, the methods of the present invention are suitable for analyzing biological samples of heterogeneous nature, e.g., low concentrations of tumor cells, or biological material therefrom, within a background of a distant sample (e.g., blood, organ effluent, or feces). Thus, when analyzing the methylation status of CpG positions within such samples, quantitative assays for determining the level (e.g., percent, proportion, ratio, rate, or degree) of methylation at a particular CpG position may be used.

[0220] According to this technology, determining the methylation status of CpG dinucleotide sequences in markers containing DMRs is useful for both diagnosing and characterizing cancers such as breast cancer.

[0221] Marker Combination In some embodiments, the techniques involve assessing the methylation status of a combination of markers that include DMRs (e.g., DMR numbers 1-375) from Table 2 and Table 18. In some embodiments, assessing 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] Different cancers are predicted by different combinations of markers, for example, as identified by statistical techniques related to the specificity and sensitivity of the prediction. The present technology provides methods for identifying predictive and validated predictive combinations for several cancers.

[0223] Methods for assaying methylation status In certain embodiments, methods for analyzing nucleic acids for the presence of 5-methylcytosine include treating the DNA with reagents that modify 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] A frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method described by Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, expressly incorporated herein by reference in its entirety for all purposes, for the detection of 5-methylcytosine or its variations in DNA. 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; second, spontaneous deamination of the sulfonated reaction intermediate results in sulfonated uracil; and finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil base pairs with adenine (and therefore behaves like thymine), whereas 5-methylcytosine base pairs with guanine (and therefore behaves like cytosine). This allows for the differentiation of methylated from unmethylated cytosines, 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, for example, in U.S. Pat. No. 5,786,146, or assays involving sequence-specific probe cleavage, such as the QuARTS flap endonuclease assay (see, for example, Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation-specific technology" Clin Chem 56:A199, and U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392).

[0225] Some conventional techniques involve placing the DNA to be analyzed in an agarose matrix, thereby preventing DNA diffusion and renaturation (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 methylation status, demonstrating the usefulness 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 techniques typically involve amplifying short, specific fragments of known nucleic acids after bisulfite treatment, and then assaying the products to analyze individual cytosine positions by either sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or primer extension reaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31, WO95 / 00669, U.S. Patent No. 6,251,594). Some methods use enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-4). Hybridization detection has also been described in the art (Olek et al., WO99 / 28498). Additionally, the use of bisulfite techniques for methylation detection has been described for individual genes (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] Various methylation assay procedures can be used in conjunction with bisulfite treatment according to the present technique. These assays allow for the determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays include, among 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, genome 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 assessing methylation status, for example, as described by Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059 or embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0229] The 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 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). PCR amplification of the bisulfite-converted DNA is then performed using primers specific for the CpG island of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using a specific labeled hybridization probe. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR products, providing a linear method for quantitating 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™ analysis (e.g., as might be found in a typical COBRA™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, regions of genes, regions of markers, 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. Additionally, bisulfite conversion reagents may include DNA denaturing buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0231] Assays such as "MethyLight™" (fluorescence-based real-time PCR technology) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (methylation-sensitive single nucleotide primer extension) reaction (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP," Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Patent No. 5,786,146), and methylated CpG island amplification ("MCA," Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.

[0232] The "HeavyMethyl™" assay technology is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") that cover the CpG positions between or are covered by the 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 in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers. The HeavyMethyl™ assay may also be used in combination with methylation-specific amplification primers.

[0234] Typical reagents for HeavyMethyl™ analysis (e.g., as might 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, or bisulfite-treated DNA sequence or CpG island, etc.), blocking oligonucleotides, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0235] MSP (methylation-specific PCR) allows for the assessment of the methylation status of virtually any group of CpG sites within a CpG island, 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 with sodium bisulfite, which converts unmethylated cytosines, but not methylated cytosines, to uracil, and the product is then amplified with primers specific for methylated DNA versus unmethylated DNA. MSP requires only small amounts of DNA, is 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 (e.g., as might be found in a typical MSP-based kit) may include, but are not limited to, methylated and unmethylated PCR primers, optimized PCR buffers and deoxynucleotides, and specific probes for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.).

[0236] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) without the need for 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 into a mixed pool with methylation-dependent sequence differences in a sodium bisulfite reaction by standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, for example, using PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs both at the level of the amplification process and the fluorescence detection process.

[0237] The MethyLight™ assay is used as a quantitative test for methylation patterns in nucleic acid, e.g., genomic DNA samples, with sequence discrimination occurring at the level of probe hybridization. In the quantitative version, PCR reactions allow for methylation-specific amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for DNA input is provided by reactions in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing biased PCR pools with either control oligonucleotides that do not cover known methylation sites (e.g., fluorescent-based versions of HeavyMethyl™ and MSP technology) or oligonucleotides that cover potential methylation sites.

[0238] The MethyLight™ process can be 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 PCR reactions using a TaqMan® probe, e.g., MSP primers and / or HeavyMethyl blocker oligonucleotides and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific for relatively high GC content regions so that the probe melts during PCR cycles at a temperature approximately 10°C higher than the forward or reverse primer. This allows the TaqMan® probe to remain sufficiently hybridized during the PCR annealing / extension step. When Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach an annealed TaqMan® probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan® probe by digesting it and releasing a fluorescent reporter molecule for quantitative detection of the unquenched signal upon detection using a real-time fluorescence detection system.

[0239] Typical reagents for MethyLight™ analysis (e.g., as might be found in a typical MethyLight™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan® or Lightcycler® probes, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0240] The QM™ (Quantitative Methylation) Assay is an alternative quantitative test for methylation patterns in genomic DNA samples, where sequence discrimination is performed at the level of probe hybridization. In the quantitative version, PCR reactions allow unbiased amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for DNA input is provided by reactions in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing biased PCR pools with either control oligonucleotides that do not cover known methylation sites (fluorescence-based versions of HeavyMethyl™ and MSP technologies) or oligonucleotides that cover potential methylation sites.

[0241] The QM™ process can be used with any suitable probe during the amplification process, e.g., TaqMan® probes, Lightcycler® probes. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and TaqMan® probes. The TaqMan® probes are dual-labeled with fluorescent reporter and quencher molecules and are designed to be specific for relatively high GC content regions so that the probe melts during PCR cycles at a temperature approximately 10°C higher than the forward or reverse primers. 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 an annealed TaqMan® probe. The 5' to 3' endonuclease activity of Taq polymerase then displaces the TaqMan® probe by digesting it and releasing a fluorescent reporter molecule for quantitative detection of the unquenched signal upon detection using a real-time fluorescent detection system. Typical reagents for QM™ analysis (e.g., as might be found in a typical QM™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan® or Lightcycler® probes, optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0242] The Ms-SNuPE™ technology is a quantitative method for assessing differences in methylation at specific CpG sites based on bisulfite treatment of DNA followed by single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosines to uracil, while leaving 5-methylcytosines unchanged. Amplification of the desired target sequence is then performed using PCR primers specific to the bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG sites of interest. Small amounts of DNA can be analyzed (e.g., microdissected pathology sections), thereby avoiding the use of restriction enzymes to determine the methylation status at CpG sites.

[0243] Typical reagents for Ms-SNuPE™ analysis (e.g., as might be found in a typical Ms-SNuPE™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), optimized PCR buffers and deoxynucleotides, gel extraction kits, positive control primers, Ms-SNuPE™ primers for specific loci, reaction buffers (for the Ms-SNuPE reaction), and labeled nucleotides. Additionally, bisulfite conversion reagents may include DNA denaturing buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0244] Reduced representation bisulfite sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracils, followed by restriction enzyme digestion (e.g., with an enzyme that recognizes sites containing CG sequences, such as Mspl) and full sequencing of the fragments after ligation to an adaptor ligand. The choice of restriction enzyme enriches for fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple genetic locations during analysis. As such, RRBS reduces the complexity of the nucleic acid sample 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 for promoters, CpG islands, and other genomic features with frequent restriction enzyme cleavage sites in these regions, thus providing an assay for assessing the methylation status of one or more genomic loci.

[0245] A typical protocol for RRBS includes the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, inserting overhangs and A-tailing, ligating adapters, bisulfite conversion, and PCR (see, for example, Meissner et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7:133-6 and 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, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to assess methylation status. Three reactions occur sequentially in each QuARTS assay: the primary reaction involves amplification (reaction 1) and target probe cleavage (reaction 2), and the secondary reaction involves FRET cleavage and fluorescent signal generation (reaction 3). When a target nucleic acid is amplified with specific primers, a specific detection probe with a flap sequence loosely binds to the amplicon. The presence of a specific invading oligonucleotide at the target binding site causes a 5' nuclease, such as FEN-1 endonuclease, to cleave the gap between the detection probe and the flap sequence, thereby releasing the flap sequence. 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, resulting in cleavage between the FRET cassette fluorophore and quencher, generating a fluorescent signal. The cleavage reaction can cleave multiple probes per target, thus releasing multiple fluorophores per flap, providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes (see, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation-specific technology" Clin Chem 56:A199), and U.S. Patent 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, which, as disclosed herein, is useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences. Treatment methods are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated by reference in its entirety). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent, such as, but not limited to, n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, such as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or trihydroxybenzoic acid and its derivatives, such as gallic acid (see PCT / EP2004 / 011715, incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction involves treatment with ammonium bisulfite, for example, as described in WO2013 / 116375.

[0248] In some embodiments, fragments of the treated DNA are amplified using a set of primer oligonucleotides according to the invention (see, for example, Tables 10, 19, and 20) and an amplification enzyme. Amplification of several 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). 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 containing DMRs (e.g., DMRs 1-375, Table 2 and Table 18) may be detected by using methylation-specific primer oligonucleotides. This technique (MSP) is described in U.S. Patent No. 6,265,171 to Herman. The use of methylation-status-specific primers for the amplification of bisulfite-treated DNA allows for the discrimination between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to bisulfite-treated CpG dinucleotides. Thus, the primer sequence includes at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the C position in the CpG.

[0250] The fragments obtained by amplification can carry directly or indirectly detectable labels.In some embodiments, the label is a fluorescent label, a radionuclide, or a detachable molecular fragment with a typical mass that can be detected by mass spectrometer.When the label is a mass label, some embodiments provide that the labeled amplicon has a single positive or negative net charge, which allows good detectability in mass spectrometer.Detection can be performed and visualized, for example, by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or by using electrospray mass spectrometry (ESI).

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

[0252] In some embodiments, the markers described herein find use in a QUARTS assay performed on a fecal sample. In some embodiments, methods are provided for generating DNA samples, and in particular, for generating DNA samples that contain highly purified, low-abundance nucleic acids in small volumes (e.g., less than 100 microliters, less than 60 microliters) and that are substantially and / or effectively free of substances that inhibit assays used to test the DNA sample (e.g., PCR, INVADER, QuARTS assays, etc.). Such DNA samples find use in diagnostic assays that qualitatively detect the presence or quantitatively measure the activity, expression, or amount of genes, genetic variants (e.g., alleles), or genetic modifications (e.g., methylation) present in a sample obtained from a patient. For example, some cancers are correlated with the presence of specific mutant alleles or specific methylation states, and therefore, detecting and / or quantifying such mutant alleles or methylation states has predictive value in cancer diagnosis and treatment.

[0253] Many useful genetic markers are present in extremely small amounts in samples, and many of the events that produce such markers are rare.As a result, even highly sensitive detection methods such as PCR require a large amount of DNA to provide a sufficient amount of low-abundance targets to meet or replace the detection threshold of the assay.Furthermore, the presence of even small amounts of inhibitors impairs the accuracy and precision of these assays that aim to detect such low-abundance targets.Therefore, provided herein is a method for producing such DNA samples that provides the necessary volume and concentration control.

[0254] In some embodiments, the sample comprises blood, serum, plasma, or saliva. In some embodiments, the subject is a human. Such samples can be obtained by numerous means known in the art, e.g., as would be apparent to one skilled in the art. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to one skilled in the art, including, but not limited to, centrifugation and filtration. While non-invasive techniques are generally preferred for obtaining samples, obtaining samples such as tissue homogenates, tissue sections, and biopsies may still be preferred. The technique is not limited by the method used to prepare the sample and provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from a fecal sample, blood sample, or plasma sample using direct gene capture, e.g., as described in U.S. Pat. Nos. 8,808,990 and 9,169,511 and WO 2012 / 155072, or related methods.

[0255] The analysis of markers can be performed separately or simultaneously with additional markers in one test sample. For example, several markers can be combined in one test to efficiently process multiple samples and potentially provide higher accuracy of diagnosis and / or prognosis. In addition, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of a series of samples can allow for the identification of changes in the methylation status of markers over time. In addition to changes in methylation status, the lack of changes in methylation status can provide useful information about disease states, including, but not limited to, identifying the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and identifying the outcome of a subject, including the risk of future events.

[0256] Biomarker analysis 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 large numbers of test samples. Alternatively, single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example, in an outpatient or emergency room setting.

[0257] It is contemplated that embodiments of the technology may be provided in the form of a kit. The kit includes embodiments of the compositions, devices, apparatus, etc. described herein, as well as instructions for using the kit. Such instructions may describe suitable methods for preparing an analyte from a sample, e.g., collecting the sample and preparing nucleic acid from the sample. Individual components of the kit are packaged in suitable containers and packaging (e.g., vials, boxes, blister packs, ampoules, jars, bottles, tubes, etc.), and the components are packaged together in suitable containers (e.g., box(es)) for convenient storage, shipping, 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, validate, and / or ensure 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 a known concentration of the same or another nucleic acid for comparison, and, in some embodiments, a detection reagent (e.g., primers) 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 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 set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker that includes a DMR (e.g., DMRs 1-375, as provided in Tables 2 and 18); and 2) Detecting breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0259] 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 isolated from the following: 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.chr5.42994 866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-6862 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: 2968, 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; 2) Detecting breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[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., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) is subjected to PCR amplification 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_81 28, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7 764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, KLHDC7B_B, L AYN_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_ with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TBX1_B, TRH_A, and TRIM67_B; and 2) Detecting breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0261] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting 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 set of reagents that distinguish between methylated and 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 (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[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 subjected to amplification of the following: ABLIM1, AJAP1_B, ASCL2, ATP6V1B1, BANK1, CALN1_A, CALN1_B, CLIC6, DSCR6, FOXP4, GAD2, GCGR, GP5, GRASP, HBM, HNF1B_B, KLF16, MAGI2, MAX.chr1 1.14926602-14927148, MAX.chr12.4273906-4274012, MAX.chr17.73073682-73073814, MAX.chr18.767343 62-76734370, 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.chr6.1 57557371-157557657, MPZ, NKX2-6, PDX1, PLXNC1_A, PPARG, PRKCB, PTPRN2, RBFOX_A, SCRT2_A, SLC7A4, STAC2 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: STX16_B, STX16_A, STX16_B, TBX1, TRH_A, VSTM2B_A, ZBTB16, ZNF132, and ZSCAN23; and 2) Detecting triple-negative breast cancer (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0263] 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 isolated from a target gene, such as 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 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of 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., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0264] 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 bodily fluid such as blood or plasma or breast tissue) is isolated from the following: 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, S with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: CRT2_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., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0265] 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 subjected to PCR amplification of a gene encoding any of the following: 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, M AX.chr11.68622869-68622968, MAX.chr14.101176106-101176260, MAX.chr15.96889069-96889128, MAX.chr17.8230197-8230314, MAX.chr19.463 79903-46380197, 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.c hr7.152622607-152622638, MAX.chr8.145104132-145104218, MAX.chr9.136474504-136474 527, 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 contacting the chromosomal region with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of SYT5, TIMP2, TMEFF2, TNFRSF10D, TRH_B, TRIM67, TRIM71_C, USP44_A, USP44_B, UTF1, UTS2R, VSTM2B_A, VSTM2B_B, ZFP64, and ZNF132; and 2) HER2 + Detecting breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0266] 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 subjected to PCR amplification using the following PCR products: 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.chr8.1241 73030-124173395, MPZ, ODC1, PLXNC1_A, PRKCB, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr12.4273906-4274012, MAX.chr19.46 379903-46380197, ZSCAN12, BHLHE23_D, COL23A1, KCNK9, LAYN, PLXNC1_A, RIC3, ​​SCRT2_B, ALOX5, CDH4_E, HNF1B_B, TRH_A, MAST 1, ASCL2, MAX.chr20.1784209-1784461, RBFOX_A, MAX.chr12.4273906-4274012, GAS7, MAX.chr5.145725410-145725459, MAX.c with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of hr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, AJAP1_B, DSCR6, and MAX.chr11.68622869-68622968; and 2) HER2 + Detecting breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0267] 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 isolated from the following: ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.427 3906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-4299493 6, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.12417 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: MAX.chr19.3030-124173395, 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; and 2) HER2 + Detecting breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0268] 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 analyzed for the following: 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, GA S7, GYPC_A, GYPC_B, HAND2, HES5, HMGA2, HNF1B_B, IGF2BP3_A, IGF2BP3_B, KCNH8, KCNK17_A, KCNQ2, KLHDC7B, LOC100132891, MAX.chr1.46913931-4 6913950, MAX.chr11.68622869-68622968, MAX.chr12.4273906-4274012, MAX.chr12.59990591-59990895, MAX.chr17.73073682-73073814, MAX.ch r20.1783841-1784054, MAX.chr21.47063802-47063851, MAX.chr4.8860002-8860038, MAX.chr5.172234248-172234494, MAX.chr5.178957564-17 8957598, MAX.chr6.130686865-130686985, MAX.chr8.687688-687736, MAX.chr8.688863-688924, MAX.chr9.114010-114207, MPZ, NID2_A, NKX2-6, with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of 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., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[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) is subjected to PCR amplification using a PCR product 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, C10orf12 5, C17orf64, CHST2_B, DNM3_A, EMX1_A, GP5, IGF2BP3_A, IGF2BP3_B, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927 148, 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.chr5.14572541 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: MAX.chr.0-145725459, MAX.chr5.77268672-77268725, GYPC_B, DLX6, FBN1, OSR2_A, BEST4, DSCR6, MAX.chr11.68622869-68622968; 2) Detecting luminal A breast cancer (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0270] 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 isolated from one or more of the following: 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.chr5.429 94866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-6862296 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: MAX.chr8, 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 (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0271] 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 subjected to amplification of the following: 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.chr18.7673442 3-76734476, MAX.chr19.30719261-30719354, MAX.chr22.42679578-42679917 , MAX.chr4.8860002-8860038, MAX.chr5.145725410-145725459, MAX.chr5.17 8957564-178957598, MAX.chr5.77268672-77268725, MAX.chr8.124173128-124173268, MPZ, PPARA, PRMT1, RBFOX3_B, RYR2_A, SALL3, SCRT2_A, SPHK2, STX16_B contacting the chromosomal region with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of SYNJ2, TMEM176A, TSHZ3, and VIPR2; and 2) Detecting luminal B breast cancer (e.g., 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) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) is isolated from any of the following: 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.chr19.463799 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: 03-46380197, 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., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0273] 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 isolated from one or more of the following: ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, PLXNC1_A, MAX.ch r12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.429 94866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: 968, 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., 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) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) is subjected to amplification 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.chr11.1492686 0-14927148, MAX.chr15.96889013-96889128, MAX.chr18.5629721-5629791, MAX.chr19.307 19261-30719354, MAX.chr22.42679767-42679917, MAX.chr5.178957564-178957598, MAX.ch r5.77268672-77268725, 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 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides; 2) Detecting BRCA1 breast cancer (eg, 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) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma or breast tissue) is isolated from BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-8277527, MAX.chr15.96889013-96889128, NACAD, A TP6V1B1, 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, M with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of AX.chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6; and 2) Detecting BRCA1 breast cancer (eg, 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) nucleic acid obtained from a subject (e.g., genomic DNA isolated from a bodily fluid such as blood or plasma or breast tissue) is isolated from any of the following: 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, MA X.chr11.14926860-14927148, MAX.chr15.96889013-96889128, MAX.chr2.2388 64674-238864735, MAX.chr5.81148300-81148332, MAX.chr7.151145632-15114 5743, MAX.chr8.124173030-124173395, MAX.chr8.143533298-143533558, MERTK, MPZ, NID2_C, NTRK3, OLIG3_A, OLIG3_B, OSR2_C, PROM1, RGS17, SBNO2, STX16_B contacting the chromosomal region with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of TBKBP1, TLX1NB, VIPR2, VN1R2, VSNL1, and ZFP64; and 2) Detecting BRCA2 breast cancer (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater). 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 isolated from MAX.chr15.96889013-96889128, ATP6V1B1, C17orf64, ITPRIPL1, MAX.chr11.14926602-14927148, MAX.chr5.42994866-42994936, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: 903-46380197, COL23A1, LAYN, OTX1, TRH_A, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968; and 2) detecting BRCA2 breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater);

[0277] 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 bodily fluid such as blood or plasma or breast tissue) is isolated from the following: 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.chr4.8860002-8 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of: 860038, MAX.chr5.145725410-145725459, MAX.chr5.178957564-178957598, MAX.chr5.77268672-77268725, MPZ, NKX2-6, PRKCB, RBFOX3_B, SALL3, and VSTM2B_A; 2) detecting invasive breast cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater);

[0278] In some embodiments of the present technology, a method is provided that includes the following steps: 1) contacting 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 set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region 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 between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast tissue (e.g., 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 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 set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of SCRT2_B, ITPRIPL1, and MAX.chr8.124173030-12417339; and 2) A step of distinguishing between ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast tissue (e.g., 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 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 set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region 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 ductal carcinoma in situ high-grade (DCIS-HG) breast cancer tissue and ductal carcinoma in situ low-grade (DCIS-LG) breast tissue (e.g., 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 in a methylation-specific manner (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)ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274 012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-1 45725459, 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_81 28, 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, IPT RIPL1_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_57 42, 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 treated 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 nucleases, 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 DNA from a sample, 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.chr12.4273906-4274 012 CALN1_A ITPRIPL1 MAX.chr12.4273906-4274012 GYPC_B MAX. chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-1 45725459. MAX.chr11.68622869-68622968. MAX.chr8.124173030-12 4173395. MAX.chr20.1784209-1784461. LOC100132891. BHLHE23_D.M AX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268 725. C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1. (ii)ABLIM1_B、AJAP1_C、ALOX5_B、ASCL2_B、BANK1_B、BHLHE23_E、C10orf1 25_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_81 28 CHST2_8384 CHST2_9316 CHST2_9470 CLIC6_B CXCL12_B DLX4_B DNM 3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPT RIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LINE_B, LIME1_B, LMX1B_ D LOC100132891_B MAST1_B MAX.chr12.427.br MAX.chr20.4422 MPZ_57 42 MPZ_5554 MSX2P1_B ODC1_B OSR2_A OTX1_B PLXNC1_B PRKCB_7570S CRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B (iii) CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B, 2) measuring the amount of at least one reference marker in the DNA; and 3) calculating a value of the amount of at least one methylation marker gene measured in the DNA as a percentage of the amount of a reference marker gene measured in the DNA, the value representing the amount of at least one methylation marker DNA measured in the sample.

[0283] In some embodiments of the present technology, a method is provided that includes the following steps: 1) measuring the methylation level of CpG sites for one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with a bisulfite reagent that can modify DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent); 2) amplifying the modified genomic DNA using a set of primers for one or more selected genes; and 3) determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; 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.chr12.4273906-4274 012 CALN1_A ITPRIPL1 MAX.chr12.4273906-4274012 GYPC_B MAX. chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-1 45725459. MAX.chr11.68622869-68622968. MAX.chr8.124173030-12 4173395. MAX.chr20.1784209-1784461. LOC100132891. BHLHE23_D.M AX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268 725. C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1. (ii)ABLIM1_B、AJAP1_C、ALOX5_B、ASCL2_B、BANK1_B、BHLHE23_E、C10orf1 25_B, C17orf64_B, CALN1_1520, CALN_1B, CD1D_1058, CDH4_7890, CHST2_81 28 CHST2_8384 CHST2_9316 CHST2_9470 CLIC6_B CXCL12_B DLX4_B DNM 3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPT RIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LINE_B, LIME1_B, LMX1B_ D LOC100132891_B MAST1_B MAX.chr12.427.br MAX.chr20.4422 MPZ_57 42 MPZ_5554 MSX2P1_B ODC1_B OSR2_A OTX1_B PLXNC1_B PRKCB_7570S CRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B (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 in a methylation-specific manner (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.chr17.7307368 2-73073814, 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, T RH_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.chr12.4273906-4274 012 MAX.chr5.42994866-42994936 SCRT2_B MAX.chr5.145725410-1457 25459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4. (iv)ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, P.S RKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012 CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.429948 66-42994936 OSR2_A SCRT2_B MAX.chr5.145725410-145725459 MAX.chr11.6 8622869-68622968; MAX.chr8.124173030-124173395; MAX.chr20.1784209-17 84461 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.chr5.145725410-1457 25459, 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.chr11.686 22869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, ITPRIPL1, 2) amplifying the treated 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 nucleases, 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 DNA from a sample, wherein the one or more genes are selected from one of the following groups: (i)BHLHE23_C, CALN1_A, CD1D, HOXA7_A, LOC100132891, MAX.chr1.8277479-827 7527 MAX.chr15.96889013-96889128 NACAD ATP6V1B1 BANK1 C17orf64 DLX4 EMX1_A, FOXP4, GP5, ITPRIPL1, LMX1B_A, MAX.chr11.14926602-14927148, MAX.c hr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16_B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chr19.46379903-46380197, ZS CAN12, BHLHE23_D, CXCL12, KCNK9, OTX1, RIC3, ​​SCRT2_B, MAX.chr17.7307368 2-73073814 CDH4_E HNF1B_B TRH_A MAX.chr20.1784209-1784461 MAX.ch r5.145725410-145725459, 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, T RH_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.chr12.4273906-4274 012 MAX.chr5.42994866-42994936 SCRT2_B MAX.chr5.145725410-1457 25459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4. (iv)ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, P.S RKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012 CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.429948 66-42994936 OSR2_A SCRT2_B MAX.chr5.145725410-145725459 MAX.chr11.6 8622869-68622968; MAX.chr8.124173030-124173395; MAX.chr20.1784209-17 84461 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.chr5.145725410-1457 25459, 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.chr11.686 22869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, ITPRIPL1, 2) measuring the amount of at least one reference marker in the DNA; and 3) calculating a value of the amount of at least one methylation marker gene measured in the DNA as a percentage of the amount of a reference marker gene measured in the DNA, the value representing the amount of at least one methylation marker DNA measured in the sample.

[0286] In some embodiments of the present technology, a method is provided that includes the following steps: 1) measuring the methylation level of CpG sites for one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with a bisulfite reagent that can modify DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent); 2) amplifying the modified genomic DNA using a set of primers for one or more selected genes; and 3) determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; 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.chr17.7307368 2-73073814, 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, T RH_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.chr12.4273906-4274 012 MAX.chr5.42994866-42994936 SCRT2_B MAX.chr5.145725410-1457 25459, BHLHE23_D, MAX.chr5.77268672-77268725, EMX1_A, DSCR6, and DLX4. (iv)ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, P.S RKCB, TRH_A, MPZ, GP5, DNM3_A, TRIM67, PLXNC1_A, MAX.chr12.4273906-4274012 CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.429948 66-42994936 OSR2_A SCRT2_B MAX.chr5.145725410-145725459 MAX.chr11.6 8622869-68622968; MAX.chr8.124173030-124173395; MAX.chr20.1784209-17 84461 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.chr5.145725410-1457 25459, 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.chr11.686 22869-68622968, ITPRIPL1, MAX.chr5.145725410-145725459, BHLHE23_C, ITPRIPL1.

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

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

[0289] Technique is not limited to the method used to prepare sample and provide the nucleic acid for testing.For example, in some embodiments, DNA is separated from fecal sample, or from blood, or from plasma sample by direct gene capture, for example, as described in US Patent Application No. 61 / 485386, or related methods.

[0290] The genomic DNA sample is then treated with at least one reagent, or a series of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker that comprises a DMR (e.g., DMRs 1-375, 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 in terms of hybridization behavior, although in some embodiments the reagent may be a methylation-sensitive restriction enzyme.

[0291] In some embodiments, the genomic DNA sample is treated in such a way that cytosine bases that are not methylated at the 5' position are converted to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior. In some embodiments, this treatment is carried out with bisulfite (hydrogen sulfite) followed by alkaline hydrolysis.

[0292] The processed nucleic acid is then analyzed to determine the methylation status of the target gene sequence (at least one gene, genomic sequence, or nucleotide from a marker comprising a DMR, e.g., at least one DMR selected from DMRs 1-375, as provided in Tables 2 and 18). Methods of analysis may be selected from those listed herein, e.g., those known in the art, including QuARTS and MSP, as described herein.

[0293] Aberrant methylation, more specifically hypermethylation of markers that include DMRs (eg, DMRs 1-375, 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 comprises tissue and / or biological fluid obtained from a patient. In some embodiments, the sample comprises a secretion. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, a gastrointestinal biopsy sample, microdissected cells from a breast biopsy, and / or cells recovered from feces. In some embodiments, the sample comprises breast tissue. In some embodiments, the subject is human. The sample may include cells, secretions, or tissue from the breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the sample comprises cellular fluid, 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, e.g., as will be apparent to those skilled in the art. For example, urine and fecal samples are readily achievable, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, e.g., by using a needle and syringe. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to those skilled in the art, including, but not limited to, centrifugation and filtration. While it is generally preferred that non-invasive techniques be used to obtain samples, it may still be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsies.

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

[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 a person skilled in the art can estimate and even determine whether a subject suffers from a given disease or condition, or whether a subject is likely to develop a given disease or condition in the future. A person skilled in the art often makes a diagnosis based on one or more diagnostic indicators, such as biomarkers (e.g., DMR as disclosed herein), whose methylation status indicates the presence, severity, or absence of a condition.

[0298] Along with diagnosis, clinical cancer prognosis is related to determining the aggressiveness of cancer and the possibility 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 therapy, and in some cases, a less harsh therapy for the patient, can be selected.Evaluating cancer biomarkers (for example, determining methylation status) is useful for distinguishing between subjects who have a good prognosis and / or a low risk of developing cancer, and who will need no therapy or limited therapy, and subjects who are likely to develop cancer or have cancer recurrence and may benefit from more intensive treatment.

[0299] As such, "making a diagnosis" or "diagnosing," as used herein, further includes determining the risk of developing cancer or determining a prognosis, which may enable one to predict a clinical outcome (with or without medical treatment), select an appropriate treatment (or whether a treatment is effective), or monitor a current treatment and possibly modify the treatment based on an indication of a diagnostic biomarker (e.g., DMR) disclosed herein. Furthermore, in some embodiments of the presently disclosed subject matter, multiple determinations of biomarkers over time may be made to facilitate diagnosis and / or prognosis. Changes in biomarkers over time may be used to predict clinical outcomes, monitor the progression of breast cancer, and / or monitor the effectiveness of appropriate cancer-directed therapies. In such embodiments, one may expect to identify changes in the methylation status of one or more biomarkers (e.g., DMRs) disclosed herein (and optionally one or more additional biomarker(s), if monitored) in biological samples over time during the course of an effective therapy, for example.

[0300] The presently disclosed subject matter further provides, in some embodiments, a method for determining whether to initiate or continue cancer prevention or treatment in a subject. In some embodiments, the method includes providing a series of biological samples from a subject over a period of time, analyzing the series of biological samples to determine the methylation status of at least one biomarker disclosed herein in each 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 a biomarker over a period of time can be used to predict the risk of developing cancer, predict clinical outcome, determine whether to initiate or continue cancer prevention or therapy, and whether 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 a time after the start of treatment. Methylation status can be measured in each of the samples taken at different time points, and qualitative and / or quantitative differences noted. Changes in the methylation status of biomarker levels from the different samples can be correlated with breast cancer risk, prognosis, determining the effectiveness of treatment, 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 disease at an early stage, e.g., before disease symptoms appear, hi some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at a clinical stage.

[0302] As previously mentioned, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and the change in the marker over time can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined a first time and again a second time. In such embodiments, an increase in a marker from the first to the second time can be diagnostic of a particular cancer type or severity, or a given prognosis. Similarly, a decrease in a marker from the first to the second time can indicate a particular cancer type or severity, or a given prognosis. Furthermore, the degree of change in one or more markers can be related to the severity of the cancer and future adverse events. Those skilled in the art will understand that, in certain embodiments, comparative measurements of the same biomarkers can be made at multiple time points, but also that a given biomarker can be measured at one time point and a second biomarker at a second time point, and comparison of these markers can provide diagnostic information. As used herein, the phrase "determining prognosis" refers to a method by which a person skilled 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 likely or unlikely to occur based on the methylation status of a biomarker (e.g., DMR). Instead, those skilled in the art will understand that the term "prognosis" refers to the probability that a certain course or outcome will occur, i.e., the increased probability that a course or outcome is likely to occur in a subject who exhibits a given condition compared to those individuals who do not exhibit the condition. For example, an individual who does not exhibit a condition (e.g., has a normal methylation status of one or more DMRs) may have a very low probability of a given outcome (e.g., suffering from breast cancer).

[0303] In some embodiments, statistical analysis correlates prognostic indicators with predisposition to adverse outcomes. For example, in some embodiments, a methylation status that differs from that in a normal control sample obtained from a patient without cancer may indicate that the subject is more likely to suffer from cancer than a subject with a methylation status more similar to that in the control sample, as determined by the level of statistical significance. In addition, changes in methylation status from baseline (e.g., "normal") levels may reflect the subject's prognosis, and the degree of change in methylation status may be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and / or p-value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, incorporated herein by reference in its entirety. Exemplary confidence intervals of the present 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 status of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be established, and the degree of change in the methylation status of the biomarker in a biological sample can be simply compared to the threshold degree of change in methylation status. Preferred threshold changes in the methylation status of the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a "nomogram" can be established, whereby the methylation status of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to a property associated with a given outcome. Those skilled in the art are familiar with the use of such nomograms and relate the two values, understanding that because individual sample measurements, rather than population averages, are referenced, the uncertainty in this measurement is the same as the uncertainty in the marker concentration.

[0305] In some embodiments, a control sample is analyzed simultaneously with the biological sample, so that results obtained from the biological sample can be compared with results obtained from the control sample. It is further contemplated that a calibration curve may be provided to which assay results for a biological sample can be compared. Such a calibration curve displays the methylation status of the biomarkers according to assay units, e.g., fluorescent signal intensity if a fluorescent label is used. Using samples from multiple donors, the calibration curve may define "at-risk" levels of one or more biomarkers in tissue from donors with metaplasia or 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 upon identifying an aberrant methylation status of one or more DMRs provided herein in a biological sample obtained from the subject. In other embodiments of the method, detecting an aberrant methylation status of one or more of such biomarkers in a biological sample obtained from the subject results in the subject being identified as having cancer.

[0306] The analysis of markers can be performed separately or simultaneously with additional markers in one test sample. For example, several markers can be combined in one test to efficiently process multiple samples and potentially provide higher accuracy of diagnosis and / or prognosis. In addition, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of a series of samples can allow for the identification of changes in the methylation status of markers over time. In addition to changes in methylation status, the lack of changes in methylation status can provide useful information about disease states, including, but not limited to, identifying the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and identifying the outcome of a subject, including the risk of future events.

[0307] Biomarker analysis 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 large numbers of test samples. Alternatively, single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example, in an outpatient or emergency room setting.

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

[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, diagnosing a subject as having or at risk of developing breast cancer involves a certain threshold measurement, e.g., indicating that the methylation state of one or more biomarkers in a biological sample is 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, in which 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 one of skill in the art, based in part on the embodiment of the method being performed, the desired specificity, etc.

[0310] Furthermore, with respect to diagnostic methods, preferred subjects are vertebrate subjects. 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, the present technology enables the diagnosis of mammals, such as those mammals important because they are endangered in addition to humans, such as the Amur tiger, those mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores, such as cats and dogs; swine, including pigs, boars, and wild boars; ruminants and / or ungulates, such as cows, bulls, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, further provided are diagnostics and treatments for livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like.

[0311] The presently disclosed subject matter is directed to treating breast cancer and / or specific breast cancer types (e.g., triple-negative breast cancer, HER2 + The present technology further includes systems for diagnosing breast cancer (e.g., luminal A breast cancer, luminal B breast cancer, BRCA1 breast cancer, BRCA2 breast cancer). The systems may be provided, for example, as commercial kits that can be used to screen for breast cancer risk or diagnose breast cancer in a subject from whom a biological sample has been collected. Exemplary systems provided in accordance with the present technology include assessing the methylation status of DMRs as provided in Table 2 and Table 18. [Example]

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

[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 methylation marker discovery 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 or prophylactic mastectomy), 9 histologically normal breasts (prophylactic mastectomy) from germline BRCA carriers, and 18 normal buffy coats). Tumor and breast tissue sections were reviewed by an expert GI pathologist to confirm the diagnosis and estimate abnormal cellularity. Sections were then microdissected. Genomic DNA was purified using a 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] Tissue samples were then subjected to bisulfite conversion (twice) using a modified 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 10 mM each deoxyribonucleotide triphosphate (dNTP), 5 μL of primer cocktail (approximately 5 μM), 15 μL of template (sample), 1 μL of PfuTurbo Cx Hot Start (Agilent, Santa Clara, CA), and 22.75 μL of water. The temperature and time were 95°C for 5 minutes, 98°C for 30 seconds, 16 cycles of 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, 72°C for 5 minutes, and a 4°C hold. Samples were purified with SPRI beads and then tested on a Bioanalyzer 2100 (Agilent) to assess the DNA size distribution of the enrichment. 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 cutoff was 0.7x-1.1x the sample volume. Samples were combined (equimolar) into 4-plex libraries based on a randomization scheme and tested for final size and concentration validation using the Bioanalyzer and qPCR (KAPA Library Quantification Kit - KAPA Biosystems, Cape Town, South Africa).

[0317] Tissue samples were loaded onto single-read flow cells with randomized lane assignment, and sequencing was performed on an Illumina HiSeq 2000 platform by the Next Generation Sequencing Core at the Mayo Clinic Medical Genome Facility. Reads were unidirectional 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 tissue yields a large number of discriminatory DMRs, many of which have not been previously identified. Comparing the methylation of breast cancer tissue samples with normal breast tissue, 327 methylated regions were identified that distinguished breast cancer tissue from normal breast tissue (see Table 2). (The genomic coordinates of the regions listed in Table 2 are based on the Human Feb. 2009 (GRCh37 / hg19) Assembly.) Table 3 lists the 48 methylated regions that distinguished triple-negative breast cancer tissue from normal breast tissue. Table 4 lists the HER2 + Table 5 shows 122 methylation regions that distinguished breast cancer tissue from normal breast tissue. Table 6 shows 39 methylation regions that distinguished luminal A 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] TIFF0007757338000003.tif252169TIFF0007757338000004.tif252169TIFF0007757338000005.tif252169TIFF0007757338000006.tif252169TIFF0007757338000007.tif252169TIFF0007757338000008.tif252169TIFF0007757338000009.tif252169TIFF0007757338000010.tif252169TIFF0007757338000011.tif252169TIFF0007757338000012.tif252169TIFF0007757338000013.tif252169TIFF0007757338000014.tif252169TIFF0007757338000015.tif252169TIFF0007757338000016.tif252169TIFF0007757338000017.tif252169TIFF0007757338000018.tif252169TIFF0007757338000019.tif97169

[0320]

Table 3

[0321]

Table 4

[0322]

Table 5

[0323]

Table 6

[0324]

Table 7

[0325]

Table 8

[0326] [Table 9] TIFF0007757338000055.tif252169TIFF0007757338000056.tif50169

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

[0328] qMSP primers were designed for each marker region using Methprimer software (Li LC and Dahiya R. Bioinformatics. 2002 Nov;18(11):1427-31). They were synthesized by IDT (Integrated DNA Technologies). Assays were tested and optimized (using a Roche LightCycler 480) on dilutions of bisulfite-converted, extensively methylated DNA, along with converted, unmethylated DNA, and converted and unconverted leukocyte DNA negative controls (10 ng / ea). Assays that proceeded were required to demonstrate a linear regression curve and a negative control value less than 5-fold below the minimum standard (1.6 genome 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 all 127 designs), 80 high-performing MSP assays met the QC criteria and were applied to the samples. MSP primer sequences, each containing 2-8 CpGs, were designed to provide a simple means of assessing methylation in samples and, as such, were biased toward amplification efficiency by targeting the most discriminatory CpGs, which required extensive optimization time.

[0329] DNA was purified as described in the Discovery RRBS section and quantified using picogreen absorbance (Tecan / Invitrogen). 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 two markers (as well as standards and controls), for a total of 40 plates. The 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 the LightCycler 480 SYBR I Master Mix (Roche) and 0.5 μmol primers and analyzed by absolute quantification, typically with a fit point of 18%. All parameters (noise band, threshold, etc.) were pre-specified in the automated macro to avoid user subjectivity. The raw data, expressed as genome copy numbers, were normalized to DNA (β-actin) input. Results were logistically analyzed using JMP and presented as AUC values. 12 comparisons were performed: each breast cancer subtype versus normal breast, and each subtype versus buffy coat. In addition, methylation fold change ratios (mFCRs) were calculated using both the mean and median methylation ratios for each comparison (FCR = cancer (methylated copies / β-actin copies) / normal (methylated copies / β-actin copies)). Both of these performance metrics are important for assessing the potential of markers in clinical blood-based tests.

[0330] >90% of the markers tested yielded excellent performance in both AUC and FCR categories, with many AUCs above 0.90, cancer vs. normal tissue FCRs >10, and cancer vs. buffy coat FCRs >50.

[0331] Table 11 shows the area under the curve for the 80 identified methylation regions that distinguish basal / 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 the 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 the 80 identified methylated regions that distinguish basal / 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 the 80 identified methylated 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] For high-grade vs. low-grade DCIS, the AUCs of the 16 markers tested ranged from 0.57 to 0.92. Several combinations of two markers achieved 95% sensitivity with 91% specificity (with only one false positive) (Table 15). A three-marker combination (SCRT2_B, ITPRIPL1, MAX.chr8.124173030-124173395) had 100% sensitivity with 91% specificity.

[0336] [Table 10] TIFF0007757338000058.tif252169TIFF0007757338000059.tif252169TIFF0007757338000060.tif252169TIFF0007757338000061.tif252169TIFF0007757338000062.tif252169TIFF0007757338000063.tif252169TIFF0007757338000064.tif252169TIFF0007757338000065.tif252169TIFF0007757338000066.tif252169TIFF0007757338000067.tif252169TIFF0007757338000068.tif252169TIFF0007757338000069.tif252169TIFF0007757338000070.tif252169TIFF0007757338000071.tif252169TIFF0007757338000072.tif252169TIFF0007757338000073.tif252169TIFF0007757338000074.tif252169TIFF0007757338000075.tif252169TIFF0007757338000076.tif127169

[0337]

Table 11

[0338]

Table 12

[0339] [Table 13] TIFF0007757338000092.tif252169TIFF0007757338000093.tif252169TIFF0007757338000094.tif252169 TIFF0007757338000095.tif252169TIFF0007757338000096.tif252169TIFF0007757338000097.tif121169

[0340] [Table 14] TIFF0007757338000099.tif252169TIFF0007757338000100.tif252169TIFF0007757338000101.tif252169 TIFF0007757338000102.tif252169TIFF0007757338000103.tif252169TIFF0007757338000104.tif103169

[0341] [Table 15] TIFF0007757338000106.tif80169

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

[0343] Independent tissue samples (fresh-frozen) were selected from the Mayo Clinic Rochester Cancer Registry and reviewed by an expert pathologist to confirm accurate classification and guide microdissection. Cases included 29 triple-negative / basal-like, 34 HER2, 36 luminal A, and 25 luminal B invasive breast cancers. Five BRCA1 and six BRCA2 cancers, 21 DCIS with HGD, and 12 DCIS with LGD were also included. 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 a list of 80 MDMs tested in discovery samples (see Example I and Tables 11-15).

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

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

[0347] Results were analyzed logistically using JMP10 (SAS, Cary NC). Cases were compared separately with 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 most AUCs and methylation fold changes (FCs) exceeding 0.90 and 50, respectively. The results are shown in Table 16A (triple negative) and Table 16B (HER2 + ), Table 16C (Luminous A), Table 16D (Luminous D), and Table 16E (Overall). Here, MDMs were ranked by AUC (comparing overall cases to buffy coat samples). This is an important metric for potential applications in plasma, since the majority of cell-free DNA (cfDNA) is derived from white blood cells. Any MDM that does not highly differentiate epithelial-derived cancers from white blood cell DNA will fail in a blood test format, regardless of its performance in tissue. 41 of the 55 MDMs had a cancer-to-buffy coat AUC greater than 0.9, and 3 achieved perfect discrimination (AUC=1). Tables 16A, 16B, 16C, 16D, and 16E also list AUC, FC, p-value, and % cancer methylation as other important metrics in assessing and demonstrating the superiority of these MDMs.

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

[0350] [Table 16A] TIFF0007757338000108.tif252169TIFF0007757338000109.tif252169TIFF0007757338000110.tif127169

[0351] [Table 16B] TIFF0007757338000112.tif252169TIFF0007757338000113.tif252169TIFF0007757338000114.tif193169

[0352] [Table 16C] TIFF0007757338000116.tif252169TIFF0007757338000117.tif252169TIFF0007757338000118.tif211169

[0353] [Table 16D] TIFF0007757338000120.tif252169TIFF0007757338000121.tif252169TIFF0007757338000122.tif223169

[0354] [Table 16E] TIFF0007757338000124.tif252169 TIFF0007757338000125.tif252169TIFF0007757338000126.tif252169TIFF0007757338000127.tif186169

[0355] [Table 17]

[0356] Example III. This example describes the identification of breast tissue 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. Initially, 58 markers were identified, and target-enriched long-probe quantitative amplification signal assays were designed and compiled (see, e.g., WO 2017 / 075061 and U.S. Patent Application No. 15,841,006 for general techniques). (Table 18 shows methylation regions that distinguish breast cancer tissue from normal breast tissue.) (Tables 19 and 20 show primer and probe sequences for the markers listed in Table 18). After design screening and redesign, 56 markers (see Table 21) were selected and tested on tissues in triplicate using assays. The assays were split equally between FAM and HEX reporting and replicated in triplicate with the reference assay B3GALT6 reporting Quasar670.

[0358] [Table 18] TIFF0007757338000130.tif252169TIFF0007757338000131.tif252169TIFF0007757338000132.tif36169

[0359] [Table 19] TIFF0007757338000134.tif252169TIFF0007757338000135.tif252169TIFF0007757338000136.tif252169TIFF0007757338000137.tif163169

[0360] [Table 20] TIFF0007757338000139.tif253169TIFF0007757338000140.tif252169TIFF0007757338000141.tif86169

[0361] [Table 21] TIFF0007757338000143.tif88169

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

[0363] [Table 22] TIFF0007757338000145.tif249169TIFF0007757338000146.tif200169

[0364] Based on the results of the tissue testing, a set of 28 markers was selected and tested on a set of plasma samples collected from breast cancer patients and normal controls. The 28 markers were divided into two pools of 14 markers depending on the 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] Testing of Pool 7 markers 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. Testing of Pool 8 markers 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 Pool 7 and Pool 8 testing, a collection of 14 assays was selected for further testing (shown in Table 25).

[0368] [Table 25]

[0369] Testing of the Pool 9 markers 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 methylated regions of the Pool 9 markers. Table 27 shows the demonstrated sensitivity and 90% specificity of the Pool 9 markers. Tables 28 and 29 show primer and probe information for the Pool 9 markers. The collection of four markers (FAM59B, ITPRIPL1, TRH_A, and C17orf64_B) showed a sensitivity of 74% at a specificity of 90%. The resulting ROC curve showed an AUC of 0.884.

[0370] [Table 26]

[0371] [Table 27]

[0372] [Table 28] TIFF0007757338000153.tif97169

[0373] [Table 29]

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

Claims

1. 1. A method of screening for breast 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 breast 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 breast cancer; The method, wherein the at least one DMR is derived from RYR2_A and / or RYR2_B and TRH_A and / or TRH_B.

2. 2. The method of claim 1, wherein determining the methylation level of at least one DMR comprises determining the presence or absence of methylation at one or more CpG sites.

3. 3. The method of 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. 2. The method of claim 1, wherein determining the methylation level of at least one DMR comprises determining a methylation frequency.

5. 10. The method of claim 1, wherein determining the methylation level of at least one DMR comprises determining a methylation pattern.

6. 2. 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. 2. The method of claim 1, wherein 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. 10. The method of 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. 9. The method of claim 8, wherein the control sample is from a subject who does not have breast cancer.

10. 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 of claim 10, wherein the tissue sample is a breast tissue sample.

12. The method of claim 1 , wherein the at least one DMR comprises TRH_A.

13. The method of claim 1 , wherein the at least one DMR comprises TRH_B.

14. The method of claim 1, wherein the at least one DMR includes RYR2_A.

15. The method of claim 1, wherein the at least one DMR includes RYR2_B.

16. 10. The method of claim 1, wherein the at least one DMR further comprises CD1D.

17. 17. The method of claim 16, wherein the at least one DMR comprises RYR2_A, TRH_A, and CD1D.

18. 17. The method of claim 16, wherein the at least one DMR comprises RYR2_A, TRH_B, and CD1D.

19. The method described in claim 16, wherein the at least one DMR includes RYR2_B, TRH_A and CD1D.

20. The method described in claim 16, wherein the at least one DMR includes RYR2_B, TRH_B and CD1D.

21. 17. The method of claim 16, wherein the at least one DMR comprises RYR2_A, TRH_A, TRH_B, and CD1D.

22. The method of claim 16, wherein the at least one DMR includes RYR2_B, TRH_A, TRH_B, and CD1D.

23. The method of claim 16, wherein the at least one DMR includes RYR2_A, RYR2_B, TRH_A, and CD1D.

24. The method of claim 16, wherein the at least one DMR includes RYR2_A, RYR2_B, TRH_B, and CD1D.

25. The method of claim 16, wherein the at least one DMR includes RYR2_A, RYR2_B, TRH_A, TRH_B, and CD1D.

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

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

28. 28. The method of claim 27, wherein the set of primers specific for TRH_A is capable of binding to the amplicon bounded by SEQ ID NOs: 245 and 246.

29. The method of claim 16, 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 specific to CD1D, and the set of primers specific to CD1D can bind to the amplicon bounded by SEQ ID NOs: 33 and 34.

30. 28. The method of claim 27, wherein the set of primers specific for TRH_A is capable of binding to at least a portion of the chromosomal region chr3:129693484-129693575, and / or the set of primers specific for TRH_B is capable of binding to at least a portion of the chromosomal region chr3:129694457-129694501.

31. The method of claim 16, 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 specific to CD1D, and wherein the set of primers specific to CD1D can bind to at least a portion of the chromosomal region chr1:158150864-158151129.

32. The method of claim 27, wherein the set of primers specific to RYR2_A can bind to at least a portion of the chromosomal region chr1:237205369-237205428, and / or the set of primers specific to RYR2_B can bind to at least a portion of the chromosomal region chr1:237205619-237205640.

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