Table S1. Overview of the 19 investigated candidate methylation biomarkers for breast cancer.

BC= breast cancer

Gene / Full name / Genomic location / Function encoded protein / Reference / Profiling platform/ Methylation assay / Material used in methylation analysis (N) / Hypermethylation validated in BC patients (N) / Methylation percentages, sensitivity / If not validated in BC, validated in other cancer patients / Hypermethylation validated in nipple/ductal fluid BC patients
ALX1 / ALX homeobox protein1 / 12q21.31 / In rodents role in survival of forebrain and possible development of cervix; in humans unknown / [11] / Illumina Infinium HumanMethylation27 array / BC patient tissue (103), healthy breast tissue (21) / Yes / No / NA / No
AKR1B1 / Aldo-keto reductase family 1, member B1 (aldose reductase) / 7q35 / Member of the aldo-keto reductase superfamily and catalyzes the reduction of certain aldehydes / [11, 12] / Illumina Infinium HumanMethylation27 array; cMethDNA / BC patient tissue (103), healthy breast tissue (21); sera recurrent stage 4 BC (24) and healthy women (28) / Primary tumors (82); sera recurrent stage 4 BC (33) and healthy women (27) / In 10 gene panel sensitivity 91% and specificity 96% in detecting metastatic BC in serum / NA / No
ARHGEF7 / Rho guanine nucleotide exchange factor (GEF) 7 / 13q34 / Member of cytoplasmic proteins that activate Ras/like family of Rho proteins by and exchange of bound GDP to GTP; forms complex with Rac1 and recruits Rac1 to membrane ruffles and focal adhesions / [18, 12] / Infinium HumanMethylation27 Beadchip Kit; cMethDNA / BC tissue (39); sera recurrent stage 4 BC (24) and healthy women (28) / BC tissue (132); sera recurrent stage 4 BC (33) and healthy women (27) / Sensitivity 100% , specificity 85% in BC; in 10 gene panel sensitivity 91% and specificity 96% in detecting metastatic BC in serum / NA / No
FZD10 / Frizzled family receptor 10 / 12q24.33 / Member of frizzled gene family that encode 7-transmembrane domain proteins that are receptors for the Wingless type MMTV integration site family of signaling proteins; most frizzled receptors are coupled to the beta-catenin canonical signaling pathway / [19] / Quantitative Multiplex Methylation-Specific PCR (QM-MSP) / Random fine needle aspiration healthy women (136) / No / No / No / No
GHSR / Growth hormone secretagogue receptor / 3q26.31 / Member of G-protein coupled receptor family with possible role in energy homeostasis; transcript 1a encodes functional protein that is the receptor for the Ghrelin ligand; transcript 1b is not a receptor for Ghrelin / [20] / Oligonucleotide arrays capable of screening methylation patterns of selected CGIs generated using Geniom One microarray technology / BC samples (31) / BC samples (26) / AUC 0.9933 in BC tissue (screening and validation set) with sensitivity of 89.3-100% and specificity of 83.3-100% / NA / No
GPX7 / Glutathione peroxidase 7 / 1p32.3 / Protection against oxidative stress and DNA damage / [11, 12] / Illumina Infinium HumanMethylation27 array; cMethDNA / BC patient tissue (103), normal breast tissue (21); sera recurrent stage 4 BC (24) and healthy women (28) / Sera recurrent stage 4 BC (33) and healthy women (27) / In 10 gene panel sensitivity 91% and specificity 96% in detecting metastatic BC in serum / NA / No
GREM1 / Gremlin 1 / 15q13.3 / Member of the BMP (bone morphogenic protein) antagonist family, CAN (cerberus and dan) subfamily; by antagonizing BMP it may play a role in organogenesis, body patterning, and tissue differentiation / [21] / Whole human transcriptome microarray screen / BC cell lines (4) different metastatic potential / BC samples (127) / In early stage BC sensitivity 69.3% and specificity 91.9% / NA / No
GSTP1 / Glutathione S-transferase pi 1 / 11q13.2 / Member of family glutathione S-transferases (GSTs) that consists of enzymes that play a role in detoxification by catalyzing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione / [22] / MethyLight assay; Illumina Infinium HumanMethylation27 array / Invasive BC tissue (20); sera metastatic BC patients (6) and healthy women (6) / IDC tissue (30), DCIS (35) / In DCIS 31% methylated, in invasive BC 28% methylated (screening + validation set) / NA / [23]
HOXD1 / Homeobox D1 / 2q31.1 / Member of the Antp homeobox family and encodes a protein with a homeobox DNA-binding domain; nuclear protein that is involved in differentiation and limb development / [21, 24] / Whole human transcriptome microarray screen; combination methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays / BC cell lines (4) different metastatic potential; low grade in situ and invasive BC (10) vs. healthy samples (10) / BC samples (120); invasive (75) and in situ (13) BC samples / In early stage BC sensitivity 64.2% and specificity 95.7%; frequency methylation in situ lesions 67%, in invasive lesions 72-88% / NA / No
KL / Klotho / 13q13.1 / Type-I membrane protein, related to beta-glucosidases / [21] / Whole human transcriptome microarray screen / BC cell lines (4) of different metastatic potential / BC samples (30) / Methylation frequency ~10% in BC samples / NA / No
LHX2 / LIM homeobox 2 / 9q33.3 / Member of large protein family that carry LIM domain; functions possibly as transcriptional regulator / [25] / MeDIA in combination with high-resolution CpG microarray analysis / Stage 1 BC samples (3) / BC samples (39) / Methylation frequency stage I-III BC patients 43.6% / NA / No
MAL / Mal, T cell differentiation protein / 2q11.1 / Member of MAL family proteolipids; role in formation, stabilization and maintenance glycosphingolipid-enriched membrane microdomains; localized to endoplasmic reticulum T-cells and candidate linker protein in T-cell signal transduction; localized in compact myelin of cells nervous system and implicated in myelin biogenesis and/or function / [26] / Bisulfite sequencing / BC cell lines (14) / BC samples (36) / Methylation frequency 69% BC samples / NA / No
MGMT / O-6-methylguanine-DNA methyltransferase / 10q26.3 / Repairs alkylated guanine in DNA / [27-29] / Mehylation-Specific PCR(MSP); pyrosequencing; MSP / Tripe negative BC (92); BC tissue ranging from DCIS to invasive tumors stage I to IV (238); basal-like BC (26) / Yes [30-32] / Methylation frequency 30.2% in BRCA1 mutated, 63.6% wild-type, and 58.3% unknown mutational status; methylation frequency 3.9% DCIS, 11.6% stage I, 10% stage II, 22.7& stage III, and 27.7% stage 4 BC; methylation frequency 27% basal-like BC. / NA / No
NDRG2 / NMYC downstrean-regulated gene (NDRG)family member 2 / 14q11.2 / Member of N-myc downregulated gene family belonging to alpha/beta hydrolase superfamily; cytoplasmic protein that may play role in neurite outgrowth / [21] / Whole human transcriptome microarray screen / BC cell lines (4) of different metastatic potential / BC samples (127) / In early stage BC sensitivity 88.9% and specificity 93.9% / NA / No
RASGRF2 / Ras protein-specific guanine nucleotide-releasing factor 2 / 5q14.1 / Calcium-regulated nucleotide exchange factor activating both RAS and RAC1 through exchange of bound GDP for GTP, thereby coordinating signaling distinct mitogen-activated protein kinase pathways / [18, 12] / Infinium HumanMethylation27 Beadchip Kit; cMethDNA / BC tissue (39); sera recurrent stage 4 BC (24) and healthy women (28) / BC samples (132);sera recurrent stage 4 BC (33) and healthy women (27) / Sensitivity 94% , specificity 85% in BC; in 10 gene panel sensitivity 91% and specificity 96% in detecting metastatic BC in serum / NA / No
SFRP1 / Secreted frizzled-related protein 1 / 8p11.21 / Member of SFRPfamily that contains cysteine-rich domain homologous to putative Wnt-binding site of Frizzled proteins and act as soluble modulators of Wnt signaling; epigenetic silencing plays role in carcinogenesis / [21, 22] / Whole human transcriptome microarray screen; MethyLight assay / BC cell lines (4) of different metastatic potential; invasive BC tissue (20) / BC samples (30); invasive BC tissue (30), DCIS (35) / Methylation frequency ~27% in BC samples; in DCIS 26% methylated, in invasive BC 46% (screening + validation set) / NA / No
SFRP2 / Secreted frizzled-related protein 2 / 4q31.3 / Member of SFRPfamily that contains a cysteine-rich domain homologous to putative Wnt-binding site of Frizzled proteins and act as soluble modulators of Wnt signaling; epigenetic silencing plays role in carcinogenesis / [20] / Oligonucleotide arrays capable of screening methylation patterns of selected CGIs generated using Geniom One microarray technology / BC samples (31) / BC samples (26) / AUC 0.9418 in BC tissue (screening + validation set) / NA / No
TM6SF1 / Transmembrane 6 superfamily member 1 / 15q25.2 / Unknown / [12] / cMethDNA / Sera recurrent stage 4 BC (24) and healthy women (28) / Sera recurrent stage 4 BC (33) and healthy women (27) / In 10 gene panel sensitivity 91% and specificity 96% in detecting metastatic BC in serum / NA / No
TMEFF2 / Transmembrane protein with EGF-like and two follistatin-like domains / 2q32.2 / May be survival factor for hippocampal and mesencephalic neurons; shedded form up-regulates cancer cell proliferation / [22, 11, 12] / MethyLight assay; Illumina Infinium HumanMethylation27 array; cMethDNA / Invasive BC tissue (20); BC patient tissue (103), healthy breast tissue (21); sera recurrent stage 4 BC (24) and healthy women (28) / IDC tissue (30), DCIS (35); primary tumors (82); sera recurrent stage 4 BC (33) and healthy women (27) / Methylation frequency DCIS 86%, invasive BC 76% (screening + validation set); in 10 gene panel sensitivity 91% and specificity 96% in detecting metastatic BC in serum / NA / No