Patent Publication Number: US-2021171617-A1

Title: Dna methylation markers for noninvasive detection of cancer and uses thereof

Description:
FIELD OF THE INVENTION 
     The present invention relates to DNA methylation signatures in human DNA, particularly in the field of molecular diagnos.ics. 
     BACKGROUND OF THE INVENTION 
     Cancer has become a major killer of humans. Early detection of cancer can significantly improve cure rates and reduce the horrific personal and financial cost to the patients their families and the health care system For example. Hepatocellular Carcinoma (HCC) is the fifth most common cancer world-wide (El-Serag, 2011). It is particularly prevalent in Asia, and its occurrence is highest in areas where hepatitis B is prevalent, indicating a possible causal relationship (Flores &amp; Marrero, 2014). Follow up of high-risk populations such as chronic hepatitis patients and early diagnosis of transitions from chronic hepatitis to HCC wculd improve cure rates. The survival rate of hepatocellular carcinoma is currently extremely low because it is almost always diagnosed at the late stages. Liver cancer could be effectively treated with cure rates of &gt;80% if diagnosed early. Advances in imaging have improved noninvasive detection of HCC (Tan, Low, &amp; Thng, 2011; Valente et al., 2014). However, current diagnostic methods, which include imaging and immunoassays with single proteins such as alpha- fetoprotein often fail to diagnose HCC early (Flores &amp; Marrero, 2014) These challenges are not limited to HCC but common to other cancers as well. For example, early detection of breast cancer and colorectal cancer could dramatically reduce morbidity and mortality and the cost to the public health system and insurance companies. Moreover, certain cancers such as pancreatic cancers are detected almost invariably late resulting in virtually certain mortality. Advances in imaging have improved early detection of Cancers, however high-resolution imaging such as MRI is expensive, requires highly trained personnel and is unavailable in many locations. It has not evolved yet to a method of screening of wide populations. To have an impact on reducing morbidity and mortality from cancer it is necessary to develop a noninvasive, robust but nevertheless low-cost method that could be used in wide geographic areas for routine screening of the population. The main challenge is that solid tumors hide in internal organs and evolve long before they exhibit clinical symptoms. It is however possible to obtain tumor material noninvasively. 
     It is widely established by now that tumor DNA is shed into the system and could be found in plasma (Warton &amp; Samimi, 2015) and possibly other secreted body fluids such as urine and saliva, as well as feces. By measuring molecular characteristics of tumor DNA, it is possible to determine that the DNA found in body fluids originated in the tumor (Zhai et al., 2012). Although tumor cells develop mutations that could distinguish tumor DNA from normal cells DNA, the number of possible mutations is vast and common mutations don&#39;t occur in all tumors (Dominguez-Vigil, Moreno-Martinez, Wang, Roehrl, &amp; Barrera-Saldana, 2018). 
     DNA methylation, a covalent modification of DNA, which is a primary mechanism of epigenetic regulation of genome function is ubiquitously altered in tumors (Aguirre-Ghiso, 2007; Baylin et al., 2001; Ehrlich, 2002; Issa et al., 1993). DNA methylation profiles of tumors are potentially robust tools for tumor classification, prognosis and prediction of response to chemotherapy (Stefansson et al., 2014). The major drawback for using tumor DNA methylation in early diagnosis is that it requires invasive procedures and anatomical visualization of the suspected tumor. Circulating tumor cells are a noninvasive source of tumor DNA and are used for measuring DNA methylation in tumor suppressor genes (Radpour et al., 2011). Hypomethylation of HCC DNA is detectable in patients&#39; blood (Ramzy, Omran, Hamad, Shaker, &amp; Abboud, 2011) and genome wide bisulfite sequencing was recently applied to detect hypomethylated DNA in plasma from HCC patients (Chan et al., 2013). However, this source is limited, particularly at early stages of cancer and the DNA methylation profiles are confounded by host DNA methylation profiles. Genome wide bisulfite sequencing is a relatively costly procedure and requires significant bioinformatics analysis which makes it unfeasible as a screening tool. The challenge is therefore to delineate a small number of CGs that could robustly differentiate tumor DNA from nontumor DNA and develop a high throughput low cost assay that will enable the screening of wide populations in broad and diverse geographic areas. More recently several groups have performed comparative analysis of genome wide DNA methylation maps of cancer and normal DNA and blood DNA (Zhai et al., 2012). However, the main challenge with these approaches is that they have not taken into account cell free DNA from other tissues that is found in blood at different levels that are unanticipated a priori. Contaminating DNA from another tissue that has a similar methylation profile as the cancer tissue could lead to false positives. In addition, past approaches have quantitatively compared DNA methylation in normal and cancer tissues. This quantitative difference is diluted when tumor DNA is mixed with different and unknown amounts of DNA from other untransformed tissues, which can cause false negatives. These deficiencies in current methods necessitate a different approach that is disclosed in the present inventive subject matter. 
     Further publications dealing with the use of systems and methods for detecting cancer are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 June; 16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 February 25; 22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5′ region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97 46705, WO 95 15373 and WO 45560.
     Aguirre-Ghiso, J. A. (2007). Models, mechanisms and clinical evidence for cancer dormancy.  Nat Rev Cancer,  7(11), 834-846. doi:10.1038/nrc2256   Baylin, S. B., Esteller, M., Rountree, M. R., Bachman, K. E., Schuebel, K., &amp; Herman, J. G. (2001). Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer.  Hum Mol Genet,  10(7), 687-692.   Breitbach, S., Tug, S., Helmig, S., Zahn, D., Kubiak, T., Michal, M., . . . Simon, P. (2014). Direct quantification of cell-free, circulating DNA from unpurified plasma.  PLoS One,  9(3), e87838. doi:10.1371/journal.pone.0087838   Chan, K. C., Jiang, P., Chan, C. W., Sun, K., Wong, J., Hui, E. P., . . . Lo, Y. M. (2013). Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing.  Proc Natl Acad Sci USA,  110(47), 18761-18768. doi:10.1073/pnas.1313995110   Dominguez-Vigil, I. G., Moreno-Martinez, A. K., Wang, J. Y., Roehrl, M. H. A., &amp; Barrera-Saldana, H. A. (2018). The dawn of the liquid biopsy in the fight against cancer.  Oncotarget,  9(2), 2912-2922. doi:10.18632/oncotarget.23131   Ehrlich, M. (2002). DNA methylation in cancer: too much, but also too little.  Oncogene,  21(35), 5400-5413.   El-Serag, H. B. (2011). Hepatocellular carcinoma.  N Engl J Med,  365(12), 1118-1127. doi:10.1056/NEJMra1001683   Flores, A., &amp; Marrero, J. A. (2014). Emerging trends in hepatocellular carcinoma: focus on diagnosis and therapeutics.  Cin Med Insights Oncol,  8, 71-76. doi:10.4137/CMO.S9926   Issa, J. P., Vertino, P. M., Wu, J., Sazawal, S., Celano, P., Nelkin, B. D., . . . Baylin, S. B. (1993). Increased cytosine DNA-methyltransferase activity during colon cancer progression.  J Natl Cancer Inst,  85(15), 1235-1240.   Luczak, M. W., &amp; Jagodzinski, P. P. (2006). The role of DNA methylation in cancer development.  Folia Histochem Cytobiol,  44(3), 143-154.   Radpour, R., Barekati, Z., Kohler, C., Lv, Q., Burki, N., Diesch, C., . . . Zhong, X. Y. (2011). Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer.  PLoS One,  6(1), e16080. doi:10.1371/journal.pone.0016080   Ramzy, II, Omran, D. A., Hamad, O., Shaker, O., &amp; Abboud, A. (2011). Evaluation of serum LINE-1 hypomethylation as a prognostic marker for hepatocellular carcinoma.  Arab J Gastroenterol,  12(3), 139-142. doi:10.1016/j.ajg.2011.07.002   Stefansson, O. A., Moran, S., Gomez, A., Sayols, S., Arribas-Jorba, C., Sandoval, J., . . . Esteller, M. (2014). A DNA methylation-based definition of biologically distinct breast cancer subtypes.  Mol Oncol . doi:10.1016/j.molonc.2014.10.012   Tan, C. H., Low, S. C., &amp; Thng, C. H. (2011). APASL and AASLD Consensus Guidelines on Imaging Diagnosis of Hepatocellular Carcinoma: A Review.  Int J Hepatol,  2011, 519783. doi:10.4061/2011/519783   Valente, S., Liu, Y., Schnekenburger, M., Zwergel, C., Cosconati, S., Gros, C., . . . Mai, A. (2014). Selective non-nucleoside inhibitors of human DNA methyltransferases active in cancer including in cancer stem cells.  J Med Chem,  57(3), 701-713. doi:10.1021/jm4012627   Warton, K., &amp; Samimi, G. (2015). Methylation of cell-free circulating DNA in the diagnosis of cancer.  Front Mol Biosci,  2, 13. doi:10.3389/fmolb.2015.00013   Xu, R. H., Wei, W., Krawczyk, M., Wang, W., Luo, H., Flagg, K., . . . Zhang, K. (2017). Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma.  Nat Mater,  16(11), 1155-1161. doi:10.1038/nmat4997   Zhai, R., Zhao, Y., Su, L., Cassidy, L., Liu, G., &amp; Christiani, D. C. (2012). Genome-wide DNA methylation profiling of cell-free serum DNA in esophageal adenocarcinoma and Barrett esophagus.  Neoplasia,  14(1), 29-33.   

     SUMMARY OF THE INVENTION 
     Embodiments of the claimed subject matter show that cancer is associated with a set of “categorically” distinct DNA methylation signatures that are different from any normal tissue and blood cell DNA methylation profiles. These sites create a binary differentiation between cancer and other tissues, whereby these sites are only methylated in cancer and fully unmethylated in other cancers. Thus, it is possible using deep next generation sequencing to detect even a few molecules of cancer cells on the background of normal cell DNA profile of methylation. Embodiments of the inventive subject matter enable detection of cell free tumor DNA even on a high background of cell free DNA from other tissues and are thus particularly suitable for early detection of cancer using cell free (CF) DNA extracted from body fluids, for example saliva, plasma, urine, feces etc. Embodiments also allow for early detection of cancer in tissue smears such as pup smears as well as biopsies and needle biopsies. Previous analyses in the prior art only compared normal and cancer cells from the same tissue and blood and derived sites that are quantitatively different in their DNA methylation level (Xu et al., 2017). However, sites discovered by such prior art analyses can&#39;t detect CF tumor DNA when it is mixed with other tissue CF DNA (see  FIG. 2  for ctDNA markers for HCC from Sun Yat Sen University Cancer hospital). One embodiment of the present claimed subject matter reveals a unique set of sites that are unmethylated in all tissues but methylated in specific cancers. Another embodiment reveals a method to discover categorically distinct methylation sites in cancers, other tissues and other diseases called the “binary-categorical differentiation (BCD) method” using different sources of genome wide DNA methylation data derived by next generation sequencing, MeDIP arrays, MeDIP sequencing, and the like. One embodiment reveals a combination of “Categorical” DNA methylation sites for detection of a. hepatocellular carcinoma (HCC), b. lung cancer, c. prostate cancer, d. breast cancer, e. colorectal cancer, f. head and neck squamous cell carcinoma (HNSC). g. pancreatic cancer, h. brain cancer (glioblastoma), i. gastric cancer j. ovarian cancer, k. cervical cancer, 1. esophageal carcinoma m. bladder cancer, n. renal cancer, o. testicular cancer, p. common solid tumors, q. blood cancer profiles in a discovery set of genome wide data. Another embodiment also reveals a combination of “Categorical” DNA methylation sites that differentiate tumors by their tissue of origin. This embodiment differentiates the assay from prior art methods for detecting methylated CF DNA which have low tissue specificity. Embodiments validate the polygenic DNA methylation assays for detection of cancer in DNA methylation data from hundreds of patients as well as the tissue of origins of the tumors with high sensitivity and specificity. The present invention discloses a method that accurately measures DNA methylation in a polygenic set of CG IDs in hundreds of people concurrently, by sequential amplification with target specific primers followed by barcoding primers and multiplexed sequencing in a single next generation Miseq sequencing reaction, data extraction and quantification of methylation from a small volume of body fluids such as plasma, saliva or urine. Another embodiments of the inventive subject matter also discloses measurement of methylation of the said DNA methylation CG IDs using pyrosequencing assays or methylation specific PCR. Another embodiment discloses the calculation of either a “categorical” or a polygenic weighted methylation score that differentiates people with cancer from healthy people. Another embodiment discloses a novel process leading from plasma, urine, feces, tissue biopsy or tissue swabs to prediction of cancer in a person with no other clinical evidence for cancer. Another embodiment could be used by any person skilled in the art to detect cancer as well as other diseases that involve cell death and release of CF DNA such Alzheimer disease and other neurodegenerative diseases for neurons, heart disease for heart muscle cells. The DNA methylation markers (CG IDs) described in the embodiments will be utilized for a. noninvasive early detection of cancer in otherwise “healthy” people through routine “checkup”; b. monitoring “high risk” people such as chronic hepatitis patients who are at high risk for HCC or smokers who are at high risk for lung cancer; c. monitoring response to therapy in patients undergoing cancer treatment to detect recurrence or metastasis. 
     Embodiments demonstrate the utility detecting cancer of unknown samples using polygenic or categorical scores based on the DNA methylation measurement methods disclosed herein. The disclosed embodiments could be used by any person skilled in the art to detect cancer in body fluids, feces, urine and tissues of any cancer or diseased tissue using any method for methylation analysis that are available to those skilled in the art such as for example next generation bisulfite sequencing, Illumina Epic microarrays, capture sequencing, methylated DNA Immunoprecipitation (MeDIP) methylation specific PCR and any methylation measurements that becomes available. 
     Embodiments also disclose the potential for discovery of new “polygenic” categorical DNA methylation markers for other cancers and diseases using any method available to people skilled in the art for genome wide sequencing such as next generation bisulfite sequencing, MeDip sequencing, ion torrent sequencing, Epic microarrays etc. followed by binary-categorical differentiation (BCD) method of analysis for discovering specific and sensitive markers that will be used for noninvasive detection of disease Embodiments of the present inventive subject matter include: 
     In a first aspect, embodiments provide polygenic DNA methylation markers of cancer in cell free DNA in body fluids such as plasma for early detection of cancer, said polygenic DNA methylation markers set being derived using “binary-categorical differentiation (BCD) analysis” as disclosed herein on genome wide DNA methylation derived by mapping methods such as Illumina 450K or EPIC arrays, genome wide bisulfite sequencing, methylated DNA Immunoprecipitation (MeDIP) sequencing or hybridization with oligonucleotide arrays. 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below (or a short subset of this list such as the example listed below Table 1) for early detection of Liver cancer hepatocellular carcinoma (HCC) using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 1               Liver_detect                                                        cg00370303   cg10900437   cg02012576   cg16460359   cg04035559   cg17419241       cg00931619   cg11223367   cg03768777   cg16977570   cg04085025   cg18607529       cg05040544   cg19289599   cg06233293   cg24804544   cg26523670   cg09992116       cg05739190   cg24599205                    
Subset for detect:
 
cg02012576, cg03768777, cg24804544, cg05739190
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example shown below Table 2) for specifying the origin of the cancer as HCC and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 2               Liver_spec                                                        cg12137206   cg14126493   cg06105778   cg22076972   cg13341720   cg03705926       cg09363194   cg07036412   cg02702614   cg10181419   cg05876864   cg11068343       cg17167468   cg15375239   cg00026222   cg17283781   cg16147221   cg26386472       cg14570307   cg06207432   cg07610192   cg03422204   cg11684022   cg23693289       cg21107197   cg04920951   cg20385508   cg25296314   cg20707679   cg26703661       cg00456086   cg05009389   cg19388016   cg08460435   cg04739306   cg04221886       cg26797073   cg04109768   cg05337743   cg00483503   cg18668780   cg10604002       cg27650175   cg05684891   cg26026416   cg00177496   cg14221460   cg16551483       cg13438961   cg24432073   cg21059834   cg23305567   cg04809136   cg21105227                    
Subset for specificity:
 
cg14126493
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 3) for early detection of lung cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 3               Lung_detect                                                        cg04223424   cg06530490   cg08944430   cg09463882   cg11017065   cg12405785       cg16405026   cg21410080   cg23141355   cg25024074                    
Subset for detect:
 
cg04223424, cg23141355
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 4) for specifying the origin of the cancer as lung cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 4               Lung_spec                                                        cg05917792   cg02682457   cg23407396   cg23601095   cg23141355   cg15386964       cg02578368   cg24631970   cg27487839   cg16405026       cg23141355   cg06530490   cg04223424   cg25470077   cg07138603   cg23460835       cg20678442   cg15436096                    
Subset for spec:
 
cg05917732, cg25470077
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below (or a short subset of this list such as the example listed below Table 5) for early detection of prostate cancer as well as for specifying the origin of the cancer as prostate cancer and discriminating from other 16 common solid tumor cancers using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 5               Prostate_detect_spec                                                cg14283569   cg18085998   cg13303553   cg17929627                    
Subset for detect_spec:
 
cg14283569
 
[it is a subset of the 4 listed in the table above]
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 6) for early detection of breast cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 6               Breast_detect                                                        cg13031251   cg19595750   cg16842053   cg18565473   cg09734791   cg25694349       cg09695735   cg03637878   cg26261793   cg18800085   cg16132520   cg05617413       cg17228900   cg06945936   cg08406370   cg24427504   cg26937500   cg11297107       cg02215070   cg14140647   cg05377226   cg07070305   cg24899571   cg07844931                    
Subset for detect:
 
cg13031251, cg09734791, cg09695735, cg03637878
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 7) for specifying the origin of the cancer as breast cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 7               Breast_Spec                                                    cg00467244   cg04194674   cg06879394   cg10720997   cg19966212       cg00722320   cg04751811   cg06998282   cg11498607   cg24525457       cg01308827   cg06282270   cg08066035   cg14862207   cg26228266       cg03113878   cg06405186   cg08296680   cg17945153   cg20180843                    
Subset for spec:
 
cg03113878, cg20180843
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 8) for early detection of colorectal cancer (CRC) as well as for specifying the origin of the cancer as colorectal cancer and discriminating from other 16 common solid tumor cancers using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 8               CRC_detect_spec                                                        cg08808128   cg03788131   cg09854653   cg21627760   cg08169901   cg07494047       cg01566242   cg13788592   cg24102266   cg17716617   cg16733654                    
Subset for detect-spec
 
cg09854653, cg01566242
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 9) for early detection of pancreatic cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 9               Pancreas_detect                                                        cg11017065   cg23833588   cg25024074   cg21277995   cg22286978   cg06241792       cg11591516   cg10096177   cg12035092   cg03611007   cg17996329   cg13807970       cg16678602   cg15386964   cg01423964   cg07900968   cg19118812   cg06728579       cg16232979   cg08406370                    
Subset for detect:
 
cg25024074, cg15386964, cg16232979
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 10) for specifying the origin of the cancer as pancreatic cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 10               Pancreas_Spec                                                        cg01237565   cg08182975   cg15323936   cg19102272   cg01311909   cg26927232       cg22290704   cg15832577   cg29983577   cg26466027   cg09796911   cg15850155       cg15510118   cg25591377   cg16165258   cg25595541   cg18151519   cg19749445       cg14870128   cg13441142                    
Subset for spec:
 
cg01237565, cg08182975, cg20983577, cg25591377
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 11) for early detection of brain cancer (glioblastoma) as well as for specifying the origin of the cancer as brain cancer (glioblastoma) and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid CF DNA. 
                         TABLE 11               Brain_detect-spec                                                        cg06887260   cg19116006   cg10938374   cg03663746   cg25568243   cg27449131       cg24917627   cg04134305   cg09797645   cg02892595   cg13231951   cg26269703       cg09183941   cg16842053   cg26551897   cg26988692   cg07849581   cg25026703       cg06798642   cg22963915   cg04245373   cg25533556   cg24917627   cg27659841       cg04692993   cg06045408   cg06887260   cg11345323   cg17167076   cg17526812                                 cg19929355   cg22513169   cg22865720                    
Subset for spec-detect
 
     Cg19929355 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 12) for early detection of stomach (gastric) cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 12               Stomach_detect                                                        cg04125371   cg05377226   cg05611779   cg06241792   cg07900968   cg09734791       cg11017065   cg12510981   cg13807970   cg15760257   cg18323466   cg19118812       cg19419279   cg19769760   cg20334243   cg26261793                    
Subset for detect:
 
cg05611779, cg09734791, cg15760257
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example shown below Table 13) for specifying the origin of the cancer as gastric cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 13               Stomach_Spec                                                        cg17187167   cg07350470   cg05000488   cg25612145   cg09851120   cg00904726       cg04911739   cg18192294   cg11861709   cg19452853   cg02706110   cg03768513       cg05611779   cg06981182   cg06118999   cg04812509   cg10131095   cg05339066       cg19235339                    
Subset for spec:
 
cg05611779, cg19235339
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example shown below Table 14) for early detection of ovarian cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 14               Ovarian_detect                                                        cg24339193   cg04008429   cg06849719   cg22694153   cg11252337   cg15804105       cg12479674   cg06961071   cg21210985   cg01556502   cg02537149   cg23983315       cg03597143   cg27209395                    
Subset for detect:
 
cg24339193, cg22694153, cg11252337, cg21210985
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 15) for specifying the origin of the cancer as ovarian cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 15               Ovarian_spec                                                        cg00895834   cg01159194   cg01961086   cg02649698   cg03345116   cg03456771       cg09392827   cg10178270   cg10581012   cg13459217   cg15701612   cg17130982       cg05901462   cg07068768   cg08389588   cg09173621   cg19276014   cg19846609       cg18476766   cg19129687                    
Subset for spec:
 
cg07068768, cg19846609
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example shown below Table 16) for early detection of cervical cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 16               Cervix_detect                                                        cg00578154   cg13644629   cg01423964   cg15745619   cg04522671   cg21621906       cg00757182   cg14962363   cg01601746   cg17228900   cg07126167   cg22806837       cg08134106   cg22922289   cg09260640   cg24625128   cg11259628   cg27420520       cg08535260   cg23141355   cg09734791   cg25024074                    
Subset for detect:
 
cg00757182, cg01601746
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 17) for specifying the origin of the cancer as cervical cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 17               Cervix_spec                                                        cg00829990   cg02401399   cg04996219   cg07066594   cg07195011   cg07576142       cg09260640   cg12961842   cg13668618   cg18543270                    
Subset for spec:
 
cg07066594, cg09260640, cg12961842
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 18) for early detection of head and neck squamous cell carcinoma (HNSC) carcinoma using plasma CF DNA or other body fluid CF DNA. 
                         TABLE 18               HNSC_detect                                                        cg01613638   cg02776314   cg03280624   cg04524120   cg05151803   cg07746323       cg07900968   cg08406370   cg11108676   cg12083965   cg15397448   cg17428324                                 cg18403606   cg20334243   cg26770917   cg27009208   cg27420520                    
Subset for detect:
 
cg07900968, cg20334243, cg27420520
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 19) for specifying the origin of the cancer as head and neck squamous cell carcinoma (HNSC) and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 19               HNSC_spec                                                        cg01217080   cg03522799   cg06672120   cg09136346   cg10155875   cg18006328       cg18443253   cg19287220                    
Subset for spec:
 
cg18006328, cg19287220
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or in a short subset of this list (such as the example listed below Table 20) for early detection of esophageal carcinoma using plasma CF DNA or other body fluid CF DNA. 
                         TABLE 20               Esophagus_detect                                                        cg03280624   cg03735888   cg06530490   cg06963053   cg08944430   cg09734791       cg11017065   cg12035092   cg18344922   cg19118812   cg20334243   cg22128431                                 cg23141355   cg24740531   cg27009208   cg27420520                    
Subset for detect:
 
cg03280624, cg03735888, cg09734791, cg27420520
 
     In one embodiment, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 21) for specifying the origin of the cancer as esophageal carcinoma and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 21               Esophagus_spec                                                        cg00532449   cg02763101   cg04743654   cg08055087   cg08932440   cg09556952       cg10608333   cg12473285   cg12966367   cg17579667   cg17949440   cg18723937       cg21554552   cg22647407   cg23286646   cg23730575   cg27569446                    
Subset for spec:
 
Cg09556952, cg12473285
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 22) for early detection of bladder cancer using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 22               Bladder_detect                                                        cg01423964   cg04223424   cg01556502   cg23141355   cg10723962   cg09734791       cg25024074   cg21039778   cg19113641   cg04342821   cg09008417   cg05873285                    
Subset for detect:
 
cg04223424, cg10723962, cg25024074
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (as shown in the example listed below Table 23) for specifying the origin of the cancer as bladder cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid DNA. 
                     TABLE 23               Bladder_spec                                                        cg14773260   cg08827322   cg05085809   cg13544006   cg02293936   cg10153335       cg05039004   cg14718568   cg07790170   cg12995090   cg16495265   cg01508045       cg04933208   cg03231163   cg06708937   cg00675569   cg06312813   cg14126688       cg12984729   cg02384661   cg08307030   cg26884027   cg20540209   cg04355159       cg08857479   cg23395715   cg22006640   cg26279336   cg19898108   cg17446010       cg12911122                    
Subset for spec:
 
cg13544006
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as the example listed below Table 24) for early detection of renal (kidney) cancer for specifying the origin of the cancer as renal cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 24               Kidney_detect_spec                                                        cg08884571   cg00011225   cg14535274   cg05507490   cg10367244   cg07190763       cg02971546   cg19990785   cg03084949   cg08765317   cg02820958   cg23946709       cg26642667                    
Subset for detect spec:
 
cg08884571, cg00011225, cg00011225
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list (such as shown in the example listed below Table 25) for early detection of testicular cancer for specifying the origin of the cancer as testicular cancer and discriminating from other 10 common solid tumor cancers using plasma CF DNA or other body fluid CF DNA. 
                         TABLE 25               Testicular_detect_spec                                                            cg14531093   cg11777290   cg13283877   cg03966099   cg26939375   cg26297530       cg16157016   cg14002772   cg09890687   cg05134918   cg09719850   cg06456125       cg17978367   cg20100671   cg26627956   cg19789755   cg14393609   cg21634331       cg02039634   cg25159927   cg01895439                    
Subset for detect and spec:
 
cg14531093, cg25159927
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs in the list below or a short subset of this list such (as shown in the example listed below Table 26) for early detection of one of 13 most common solid tumors using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 26               Pan-cancer_detect                                                        cg01423964   cg04223424   cg06530490   cg06543087   cg07900968   cg08406370       cg08848774   cg09734791   cg10723962   cg11017065   cg15759056   cg15760257       cg16405026   cg17228900   cg21277995   cg22286978   cg23141355   cg24427504       cg25024074   cg27420520                    
Subset for detect:
 
cg10723962, cg15759056, cg24427504, cg25024074
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs delineated by the BCD method on genome wide DNA methylation data as shown in Table 27 (or a short subset of this combination as shown below Table 27) for early detection of blood cancers such as AML, CLL, etc. using white blood cells, plasma CF DNA or other body fluid CF DNA. 
                     TABLE 27               AML_detect-spec                                                        cg00594866   cg05608159   cg18658397   cg18780412   cg20430388   cg22828045       cg25375340                    
Subset for detect-spec:
 
cg18658397, cg18780412, cg20439288, cg22828045, cg25375340
 
     In other embodiments, the polygenic DNA methylation markers are a combination of CG IDs shown in the list below (or a short subset of this list shown in the example listed below Table 28) for early detection of Melanoma for specifying the origin of the cancer as melanoma and discriminating from other 16 common solid tumor cancers using plasma CF DNA or other body fluid CF DNA. 
                     TABLE 28               Melanoma_detect-spec                                                        cg00325866   cg01228636   cg04616691   cg04824711   cg05569109   cg06019853       cg08611163   cg10830649   cg12593303   cg15307891   cg18866529   cg19530886       cg19634213   cg20146030   cg21755725   cg22280705   cg24217704   cg24678095       cg26127345   cg27084903                    
Subset for detect-spec:
 
cg15307891, cg18866529, cg27084903
 
     In another aspect of the inventive subject matter, there is provided a kit and a process for detecting cancer, comprising means and reagents for detecting DNA methylation measurements of polygenic DNA methylation markers. 
     In one embodiment, a kit is provided for detecting hepatocellular carcinoma comprising means and reagents for DNA methylation measurements of the CG IDs of table 1 and 2. 
     In another embodiment, a kit is provided for detecting lung cancer comprising means and reagents for DNA methylation measurements of the CG IDs of table 3 and 4. 
     In another embodiment, a kit is provided for detecting prostate cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 5. 
     In another embodiment, a kit is provided for detecting breast cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 6 and 7. 
     In another embodiment, a kit is provided for detecting colorectal cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 8. 
     In another embodiment, a kit is provided for detecting pancreatic cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 9 and 10. 
     In yet another embodiment, a kit is provided for detecting brain cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 11. 
     In another embodiment, a kit is provided for detecting gastric cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 12 and 13. 
     In another embodiment, a kit is provided for detecting ovarian cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 14 and 15. 
     In another embodiment, a kit is provided for detecting cervical cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 16 and 17. 
     In another embodiment, a kit is provided for detecting head and neck squamous carcinoma (HNSC) comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 18 and 19. 
     In another embodiment, a kit is provided for detecting esophageal carcinoma comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 20 and 21. 
     In another embodiment, a kit is provided for detecting bladder cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 22 and 23. 
     In another embodiment, a kit is provided for detecting renal cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 24. 
     In another embodiment, a kit is provided for detecting testicular cancer comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 25 
     In other embodiments, a kit is provided for detecting one of 13 common cancers (bladder, brain, breast, cervical, colorectal, esophageal, HNSC, HCC (liver), lung, ovarian, pancreatic, prostate, stomach) comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 26. 
     In another embodiment, a kit is provided for detecting blood cancers such as AML and CLL comprising means and reagents for detecting DNA methylation measurements of the CG IDs detected by the BCD method that are specific for different subtypes of blood cancer Table 27 
     In another embodiment, a kit is provided for detecting melanoma comprising means and reagents for detecting DNA methylation measurements of the CG IDs of table 28. 
     In another embodiment, DNA pyrosequencing methylation assays are used for predicting HCC in body fluids such as plasma CF DNA by using CG IDs listed above, for example by using the below disclosed primers and standard conditions of pyrosequencing reactions: 
     
       
         
           
               
               
            
               
                   
                 cg02012576 
               
               
                   
                 Forward: 
               
               
                   
                 GGTAGTTAGGAAGTTTAGAGGTTGTAGTA 
               
               
                   
               
               
                   
                 Reverse (biotinylated): 
               
               
                   
                 ACCACTACCCCAACCCAACCCTA 
               
               
                   
               
               
                   
                 Sequence: 
               
               
                   
                 GGTTTTAGGATGTTTG 
               
               
                   
               
               
                   
                 cg03768777 (VASH2) 
               
               
                   
                 Forward: 
               
               
                   
                 AGAATAATATTAGAGAATGGGATATGGAA 
               
               
                   
               
               
                   
                 Reverse (biotinylated): 
               
               
                   
                 ACAACTCCAAAATCCTACCT 
               
               
                   
               
               
                   
                 Sequence: 
               
               
                   
                 GAATGGGATATGGAATGA 
               
               
                   
               
               
                   
                 cg05739190 (CCNJ) 
               
               
                   
                 Forward: 
               
               
                   
                 GTTTAGGAGTTGGGTTTTAGTTGAG 
               
               
                   
               
               
                   
                 Reverse (biotinylated): 
               
               
                   
                 ACCCCACCCTAACTCCCTTACC 
               
               
                   
               
               
                   
                 Sequence: 
               
               
                   
                 TGGGTTTTAGTTGAGG 
               
               
                   
               
               
                   
                 cg24804544 (GRID2IP) 
               
               
                   
                 Forward(biotinylated): 
               
               
                   
                 GGGTAGGGGAGGGTTTTGAAATA 
               
               
                   
               
               
                   
                 Reverse: 
               
               
                   
                 TAACCCCCCCTCCAACCTCATTC 
               
               
                   
               
               
                   
                 Sequence: 
               
               
                   
                 CACCCAACTTCTCAAT 
               
            
           
         
       
     
     The specificity of the tissue of origin of the cancer is determined by measuring the DNA methylation of the following CGID cg02012576 (HPX) 
     
       
         
           
               
               
            
               
                   
                 Forward(biotinylated): 
               
               
                   
                 ATTTTTATGGGTATTAGTTTTAGGGAGAA 
               
               
                   
               
               
                   
                 Reverse (biotinylated): 
               
               
                   
                 CCAAAACTATCCTATAACCTCTACAACTCA 
               
               
                   
               
               
                   
                 Sequence: 
               
               
                   
                 ACCATTACCACCCCT 
               
            
           
         
       
     
     In another embodiment, a polygenic multiplexed amplicon bisulfite sequencing DNA methylation assay is used for predicting cancer in body fluids such as plasma CF DNA by using CG IDs listed above. For example, predicting prostate cancer using the below disclosed primers and standard conditions that involve bisulfite conversion, sequential amplification with target specific primers (PCR 1) followed by barcoding primers (PCR 2) and multiplexed sequencing in a single next generation MiSeq sequencer (Illumina), demultiplexing using Illumina software, data extraction and quantification of methylation using standard methods for methylation analysis such as Methylkit, followed by calculation of the weighted DNA methylation score and prediction of cancer from a small volume of body fluids such as plasma, saliva or urine. 
     The steps to detect prostate cancer the first PCR is performed as follows:
 
For CGID cg02879662
 
                            Forward primer:           5′ACACTCTTTCCCTACACgACgCTCTTCCgA                   TCTNNNNNGGTAGGAGTTTTGGGAATTGG3′                   Reverse primer:           5′gTgACTggAgTTCAgACgTgTgCTCT                   TCCgATCTCCACCCCTACAATCCCTAA3′                   For CGID cg16232979           Forward primer:           5′ACACTCTTTCCCTACACgACgCTCTTCCgAT                   CTNNNNNYGGTTTYGGGTTTYGTATT3′                   Reverse primer:           5′gTgACTggAgTTCAgACgTgTgCTCTTCCgA                   TCTACRCAAAAATATAAATCRACRATC3′            
To test that the cancer specifically originates in the prostate the first PCR is performed as follows:
 
                            For CGID: cg14041701 and cg14498227           Forward primer:           5′ACACTCTTTCCCTACACgACgCTCTTCCgATCTNNNNN                   GTTTTGYGTTTYGGATTTGGGTT3′                   Reverse primer:           5′gTgACTggAgTTCAgACgTgTgCTCTTCCgATCTCATAAA                   CAACACCTTTAAATAAACACTAAA3′            
To barcode the samples, use a second PCR reaction with the following primers:
 
     
       
         
           
               
               
            
               
                   
                 Forward primer: 
               
               
                   
                 5′AATgATACggCgACCACCgAgATCT 
               
               
                   
               
               
                   
                 ACACTCTTTCCCTACACgAC3′ 
               
               
                   
               
               
                   
                 Barcoding primer (reverse): 
               
               
                   
                 5′CAAgCAgAAgACggCATACgAgATAGTCAT 
               
               
                   
               
               
                   
                 CGgTgACTggAgTTCAgACgTg3′ 
               
               
                   
               
               
                   
                 (red bases are the index; 200 
               
               
                   
                 variations of this index are used) 
               
            
           
         
       
     
     In other embodiments, Receiver operating characteristics (ROC) assays are used for detecting cancer by defining a threshold value between cancer and normal using weighted DNA methylation measurements of CG IDs. Samples above/below threshold will be classified as cancer. For example, CGIDs listed above for detecting HCC: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
             
            
               
                   
                 cg02012576 
                 cg03768777 
                 cg05739190 
                 cg24804544. 
               
               
                   
               
            
           
         
       
     
     In another embodiment, hierarchical Clustering analysis assays are used for predicting cancer by using measurements of methylation of CG IDs listed above. 
     In another aspect of the inventive subject matter, methods for identifying DNA methylation markers for detecting cancer and other disease comprise the step of performing statistical analysis with the “binary-categorical differentiation (BCD)” method previously disclosed regarding DNA methylation measurements obtained from clinical samples. 
     In another embodiment, the method includes performing statistical analysis and the “binary-categorical differentiation (BCD)” method on DNA methylation measurements obtained from samples, with DNA methylation measurements obtained by performing Illumina Beadchip 450K or EPIC array of DNA extracted from at least one sample. 
     In another embodiment, the DNA methylation measurements are obtained by performing DNA pyrosequencing of DNA extracted from a sample followed by mass spectrometry based (Epityper™), PCR based methylation assays and targeted amplification of a region spanning the target CG IDs disclosed herein from bisulfite converted DNA followed by barcoding in a second set of amplification and indexed-multiplexed sequencing on an Illumina next generation sequencer. 
     In other embodiments, the statistical analysis includes Receiver operating characteristics (ROC) assays. 
     In other embodiments, the statistical analysis includes hierarchical clustering analysis assays. 
     Definitions 
     As used herein, the term “CG” refers to a di-nucleotide sequence in DNA containing cytosine and guanosine bases. These di-nucleotide sequences could become methylated in human and other animals&#39; DNA. The CG ID reveals its position in the human genome as defined by the Illumina 450K manifest (The annotation of the CGs listed herein is publicly available at https://bioconductor.org/packages/release/data/annotation/html/IlluminaHumanMethylation405_db.html and installed as an R package IlluminaHumanMethylation450k.db IlluminaHumanMethlation450k.db: Illumina Human Methylation 450 k annotation data. R package version 2.0.9.). 
     As used herein, the term “beta-value” refers to an estimation of methylation level at a CG ID position derived by normalization and quantification of Illumina 450K arrays using the ratio of intensities between methylated and unmethylated probes using the formula beta value=methylated C intensity/(methylated C intensity+unmethylated C intensity) between 0 and 1 with 0 being fully unmethylated and 1 being fully methylated. 
     As used herein, the term “penalized regression” refers to a statistical method aimed at identifying the smallest number of predictors required to predict an outcome out of a larger list of biomarkers as implemented for example in the R statistical package “penalized” as described in Goeman, J. J., L1 penalized estimation in the Cox proportional hazards model. Biometrical Journal 52(1), 70-84. 
     As used herein, the term “clustering” refers to the grouping of a set of objects in such a way that objects in the same group (called a cluster) are more similar (in some sense or another) to each other than to those in other groups (clusters). 
     As used herein, the term “Hierarchical clustering” refers to a statistical method that builds a hierarchy of “clusters” based on how similar (close) or dissimilar (distant) are the clusters from each other as described for example in Kaufman, L.; Rousseeuw, P. J. (1990).  Finding Groups in Data: An Introduction to Cluster Analysis  (1 ed.). New York: John Wiley. ISBN 0-471-87876-6. 
     As used herein, the term “Receiver operating characteristics (ROC) assay” refers to a statistical method that creates a graphical plot that illustrates the performance of a predictor. The true positive rate of prediction is plotted against the false positive rate at various threshold settings for the predictor (i.e. different % of methylation) as described for example in Hanley, James A.; McNeil, Barbara J. (1982). “The Meaning and Use of the Area under a Receiver Operating Characteristic (ROC) Curve”. Radiology 143 (1): 29-36. 
     As used herein, the term “multivariate or polygenic linear regression” refers to a statistical method that estimates the relationship between multiple “independent variables” or “predictors” such as percentage of methylation in CG IDs, and a “dependent variable” such as cancer. This method determines the “weight” or coefficient of each CG IDs in predicting the “outcome” (dependent variable such as cancer) when several “independent variables” such as CG IDs are included in the model. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . shows a shortlist of completely unmethylated sites across blood samples and normal tissues in hundreds of individuals. Illustration A. shows CG IDs across 17 tissues that are unmethylated in all individuals (&lt;0.1) in Illumina450K genome wide methylation arrays (GSE50192) were overlapped with unmethylated CG IDs in genome wide DNA methylation arrays of blood samples from 312 individuals (GSE61496) to generate a list of 33477 CG IDs. B illustrates shortlisting the most robustly unmethylated CG IDs the list of 33477 CG IDs from A was overlapped with unmethylated CG IDs in DNA methylation arrays of blood samples from 656 individuals (females and male) aged from 19 to 101 years (GSE40279). Combined, these analyses generated a list of high confidence 28754 CG IDs that are unmethylated across tissues and blood samples of many individuals across all age spans. These 28754 positions were used for discovery of sites that are categorically methylated in cancer but not in other tissues using the “binary-categorical differentiation (BCD)” method disclosed by the present inventive subject matter. 
         FIG. 2  is an illustration showing the Lack of Tissue specificity of current Circulating DNA markers for HCC. The illustrated heatmap shows 10 CG IDs shortlisted in Xu et al., (Xu et al., 2017) as biomarkers of HCC and methylation levels for these sites in other normal tissues. Several of the CG IDs proposed as specific biomarkers of HCC are methylated in other tissues are well and show varying levels of methylation in blood DNA. (blue 0 methylation dark red 100% methylation) 
         FIG. 3  is an illustration showing the specificity of HCC DNA methylation markers discovered using the BCD method for cancer DNA. The illustrated heatmap shows 4 CG IDs selected as HCC DNA methylation markers by the BCD method described herein. Methylation levels are categorically different between cancer (HCC) and normal tissues and blood, whereby the sites are unmethylated in all individuals in blood and other tissues and measurably methylated in HCC. 
         FIG. 4  is an illustration showing the lack of cancer tissue of origin specificity of current DNA methylation markers for colorectal cancer and comparison with the “detect-spec” method according to embodiments of the inventive subject matter. Illustration A shows the CG sites in Sept9 gene as included in the “Epi-colon” CF DNA methylation marker for colorectal cancer (sold by Epigenomics Inc.) which can be used to detect many other cancers utilizing methylation data from the TCGA collection of cancer DNA methylation data and thus lack specificity for colorectal cancer (HKG-Colon (HKG-epiCRC), blue). The markers disclosed in the present inventive subject matter for detection of colorectal cancer (Table 9) discovered using the BCD method (HKG-Colon orange) (Table 10) are highly specific for colorectal cancer when tested against other common solid tumors cancers. Illustrations B and C are scatter-plots of DNA methylation values for tumor DNA from different individuals with different cancers using either the HKG-Colon (HKG-epiCRC) (B) or Epi-Colon (C) DNA methylation markers. Of note are the tight and categorical differences in DNA methylation between colorectal cancer and other cancers using the HKG-epiCRC markers (B) versus the scattered heterogenous profile of Epi-Colon markers (C). 
         FIG. 5  is an illustration showing a discovery of a polygenic DNA methylation marker for early detection of liver cancer (HCC). Illustration A shows a table which lists the source and number of patients whose methylation data was used for the discovery of a set of 4 CGIDs for detection of HCC according to an embodiment using the BCD method (Table 1) and CG IDs for determining the specific cancer of origin (Table 2). Illustration B at the bottom left panel of  FIG. 5  (Detect) shows the combined methylation score for these CG IDs (Table 1) for each of the tested people listed from 1-145 (79 normal and 66 HCC). The polygenic score categorically differentiates between people with HCC and normal liver tissue. Illustration C at the bottom right panel shows the methylation score for the 1 CGID detecting the specific tumor origin (Table 2) using data on 8 different tumors (Table 2). The markers categorically differentiate between cancers from other origins and HCC. 
         FIG. 6  is an illustration of a validation of a polygenic DNA methylation marker for HCC (spec) using DNA methylation data from GSE76269 (n=227). Illustration A is a ROC plot showing area under the curve for the HCC DNA methylation markers using 227 liver cancer patient DNA methylation data and 10 normal. Illustration B of  FIG. 6  shows the sensitivity, specificity and accuracy of HCC detection. Illustration C shows the prediction rate of detection of HCC in the validation dataset. 
         FIG. 7  is an illustration of a validation of the polygenic HKG-epiLiver-detect and spec markers accuracy and specificity for HCC versus other cancers in TCGA methylation data (n=4166). Illustration A of  FIG. 7  shows a detection rate of the HKG-Liver detect/spec markers DNA methylation data of patients with different cancers. Note the almost perfect specificity for HCC. B. ROC plot of the HKG-Liver-detect markers specificity and sensitivity for HCC in 4166 patient DNA methylation data from TCGA. C. Sensitivity and specificity to HCC versus cancers from other origins. 
         FIG. 8  is an illustration of a discovery of a polygenic DNA methylation marker for lung cancer. Illustration A of  FIG. 8  is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of lung cancer disclosed in embodiments using the BCD method (Table 3) as well as CG IDs for determining the specific cancer tissue of origin (Table 4). Illustration B at the bottom left panel of  FIG. 8  (Detect) shows the combined methylation score for these CG IDs (Table 3) for each of the tested people listed from 1-20 (10 normal and 10 lung cancer). The polygenic score categorically differentiates between people with lung cancer and normal tissue. Illustration C at the bottom right panel of  FIG. 8  shows the methylation score for the CGIDs (Table 4) detecting the specific tumor origin using data from people who have 8 different tumors (n=80). In these embodiments, the markers categorically differentiate between cancers from other origins and lung cancer. 
         FIG. 9  is an illustration of the validation of the polygenic HKG-epiLung-detect and spec markers accuracy and specificity for HCC versus other cancers in TCGA methylation data (n=4166). Illustration A of  FIG. 9  shows a detection rate of the HKG-epiLung detect/spec markers using DNA methylation data of patients with different cancers. Of note is the specificity for lung cancer. Illustration B of  FIG. 9  shows a ROC plot of the HKG-Lung-detect markers specificity and sensitivity for lung cancer on 4166 patient DNA methylation data from TCGA. Illustration C of  FIG. 9  shows a sensitivity and specificity to lung cancer versus cancers from other origins. 
         FIG. 10  is an illustration of the discovery of a polygenic DNA methylation marker for prostate cancer. Illustration A of  FIG. 10  is a table listing the source and number of a patient whose methylation data was used for discovery of a set of CGIDs for detection of prostate cancer disclosed in embodiments using the BCD method (Table 5) and CGIDs for determining the specific cancer tissue of origin (Table 6). Illustration B at the bottom left panel of  FIG. 10  (Detect) shows the combined methylation score for these CGIDs (Table 5) for each of the tested people listed from 1-15 (5 normal and 10 prostate cancer). The polygenic score categorically differentiates between people with prostate cancer and normal. Illustration C at the bottom right panel of  FIG. 10  shows the methylation score for the CGs detecting the specific tumor tissue of origin (Table 6) using data from people who have 8 different tumors (n=80). In these embodiments, the markers categorically differentiate between cancers from other origins and prostate cancer. 
         FIG. 11  is an illustration of a validation of the polygenic HKG-epiProstate-detect and spec markers accuracy and specificity for prostate versus other cancers in TCGA methylation data (n=4166). Illustration A of  FIG. 11  shows the detection rate of the HKG-Prostate detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for prostate cancer. Illustration B of  FIG. 11  is a ROC plot of the HKG-Prostate-detect markers specificity and sensitivity for lung cancer using DNA methylation data from 4166 patients in TCGA. Illustration C of  FIG. 11  shows the sensitivity and specificity to prostate versus cancers from other origins. 
         FIG. 12  is an illustration of a discovery of a polygenic DNA methylation marker for breast cancer. Illustration A of  FIG. 12  is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGs for detection of breast cancer disclosed in embodiments using the BCD method (Table 7) and CGIDs for determining the specific cancer of origin (Table 8). Illustration B at the bottom left panel of  FIG. 12  (Detect) shows the combined methylation score for these CGIDs (Table 7) for each of the tested people listed from 1-27 (17 normal and 10 breast cancer). The polygenic score categorically differentiates between people with breast cancer and normal breast tissue. Illustration C at the bottom right panel of  FIG. 12  shows the methylation score for the CGIDs detecting the specific origin of the tumor (Table 8) using data from people who have 8 different tumors (n=80). In these embodiments, the markers categorically differentiate between cancers from other origins and breast cancer. 
         FIG. 13  is an illustration of a HKG-epiBreast-detect polygenic DNA methylation marker detect noninvasive as well as invasive breast cancer in validation cohort GSE60185 (n=285). Illustration A of  FIG. 13  is a ROC plot showing area under the curve for the breast cancer polygenic DNA methylation marker using 239 breast cancer patient DNA methylation data, 17 mamoplastic surgery patients with no breast cancer and 29 adjacent tissues. The sensitivity, specificity and accuracy for all breast cancer are listed in B and the prediction rate of DCIS (ductal carcinoma in situ), invasive and mixed breast cancer samples are shown in Illustration C of  FIG. 13 . Of note is that the breast cancer markers detect even very early breast cancers (DCIS). 
         FIG. 14  is an illustration of a validation of the polygenic HKG-epiBreast-detect and spec markers accuracy and specificity for breast versus other cancers in TCGA methylation data (n=4166). Illustration A of  FIG. 14  shows a detection rate of the HKG-epiBreast detect/spec markers in DNA methylation data from patients with different cancers. Of note is the specificity for breast cancer. Illustration B of  FIG. 14  is a ROC plot of the HKG-Breast-detect markers specificity and sensitivity for detecting breast cancer using DNA methylation data from 4166 patients in TCGA. Illustration C of  FIG. 14  shows the sensitivity and specificity to breast versus cancers from other origins. 
         FIG. 15  is an illustration of a discovery of a polygenic DNA methylation marker for colorectal cancer (CRC). Illustration A of  FIG. 15  is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of colorectal cancer disclosed in embodiments using the BCD method (Table 9) and CGIDs for determining the specific cancer of origin (Table 10). Illustration B at the bottom left panel of  FIG. 15  (Detect) shows the combined methylation score for these CGIDs for each of the tested people listed from 1-75 (25 normal and 50 colorectal cancer). The polygenic score categorically differentiates between people with cancer and normal tissue. Illustration C at the bottom right panel of  FIG. 15  shows the methylation score for the CGIDs detecting the specific origin of the tumor using DNA methylation data from people who have 8 different tumors (n=80). In these embodiments, the markers categorically differentiate between cancers from other origins and colorectal cancer. 
         FIG. 16  is an illustration validation of the polygenic HKG-epiCRC-detect and spec markers accuracy and specificity for CRC versus other cancers using TCGA DNA methylation data set (n=4166). Illustration A of  FIG. 16  is a detection rate of the HKG-epiCRC detect/spec markers using DNA methylation data of patients with different cancers. Of note is the specificity for colorectal cancer. Illustration B of  FIG. 16  is a ROC plot of the HKG-epiColon-detect markers specificity and sensitivity for colorectal cancer using DNA methylation data from 4166 patients in TCGA. Illustration C of  FIG. 16  shows sensitivity and specificity to colorectal cancer versus cancers from other origins. 
         FIG. 17  illustrates a discovery of a polygenic DNA methylation marker for pancreatic cancer. Illustration A of  FIG. 17  is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of pancreatic cancer disclosed in the present invention using the BCD method (Table 11) and CGIDs for determining the specific cancer of origin (Table 12). Illustration B at the bottom left panel of  FIG. 17  (Detect) shows the combined methylation score for these CGIDs (Table 11) for each of the tested people listed from 1-32 (12 normal and 20 pancreatic cancer). The polygenic score categorically differentiates between people with pancreatic cancer and normal tissue. Illustration C at the bottom right panel of  FIG. 17  shows the methylation score for the CGIDs detecting the specific origin of the tumor (Table 12) using data from people who have 10 different tumors (n=100). In these embodiments, the markers categorically differentiate between cancers from other origins and pancreatic cancer. 
         FIG. 18  is an illustration of a validation of the polygenic HKG-epiPancreas-detect and spec markers accuracy and specificity for pancreatic cancer versus other cancers in TCGA methylation data (n=4854). Illustration A of  FIG. 18  is a detection rate of the HKG-epiPancreas detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for pancreatic cancer. Illustration B of  FIG. 18  is a ROC plot of the HKG-epiPancreas-detect markers specificity and sensitivity for pancreatic cancer using DNA methylation data for 4854 patients in TCGA. Illustration C is the sensitivity and specificity to pancreatic cancer versus cancers from other origins. 
         FIG. 19  is an illustration of a discovery of a polygenic DNA methylation marker for brain cancer (glioblastoma). Illustration A of  FIG. 19  is a table listing the source and number of patient whose methylation data was used for discovery of a set of CGIDs for detection of brain cancer disclosed in the present invention using the BCD method (Table 13) and CGIDs for determining the specific origin of the cancer (Table 13). Illustration B at the bottom left panel (Detect/spec) shows the combined methylation score for these CGIDs (Table 13) for each of the tested people listed from 1-16 (6 normal and 10 brain cancer). The polygenic score categorically differentiates between people with brain cancer, 110 other cancers, normal tissues. 
         FIG. 20  is an illustration of a validation of the polygenic HKG-epiBrain-detect and spec markers accuracy and specificity for breast versus other cancers in TCGA methylation data (n=4854). Illustration A is a detection rate of the HKG-epiBrain detect/spec markers using DNA methylation data from patients with different cancers. Note the specificity for brain cancer. Illustration B is a ROC plot of the specificity and sensitivity of the HKG-epiBrain-detect markers for brain cancer using DNA methylation data from 4854 patients in TCGA. Illustration C shows a sensitivity and specificity to brain cancer in TCGA data set (n=695). 
         FIG. 21  is an illustration of a discovery of a polygenic DNA methylation marker for detection of gastric (stomach) cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of gastric cancer disclosed in the present invention using the BCD method (Table 14) and CGIDs for determining the specific origin of the cancer (Table 15). Illustration B at the bottom left panel of  FIG. 21  (Detect) shows the combined methylation score for these CGIDs (Table 14) for each of the tested people listed from 1-28 (14 normal and 20 gastric cancer). The polygenic score categorically differentiates between people with gastric cancer and normal tissue. Illustration C at the bottom right panel of  FIG. 21  (Spec) shows the polygenic methylation scores for people who have 10 different tumors (n=100). In these embodiments, the markers categorically differentiate between cancers from other origins and gastric cancer. 
         FIG. 22  is an illustration of a validation of the polygenic HKG-Stomach-detect and spec markers accuracy and specificity for gastric cancer versus other cancers in TCGA methylation data (n=4817). Illustration A is a detection rate of the HKG-epiStomach detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for gastric cancer. Illustration B is a ROC plot specificity and sensitivity of the HKG-epiStomach-detect spec 1 markers for stomach (gastric cancer) using DNA methylation data from 4420 patients in TCGA. Illustration C is a ROC plot of specificity and sensitivity the HKG-epiStomach-spec 1 markers for gastric cancer using DNA methylation data from 4854 patients in TCGA. Of note is that there is a significant cross reactivity with colorectal and esophageal cancer which attests to a shared origin. 
         FIG. 23  is an illustration of a discovery of a polygenic DNA methylation marker for ovarian cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of ovarian cancer disclosed in the present invention using the BCD method (Table 16) and CGIDs for determining the specific cancer of origin (Table 17). Illustration B at the bottom left panel of  FIG. 23  (Detect) shows the combined methylation score for these CGIDs for each of the tested people listed from 1-15 (5 normal and 10 ovarian cancer). The polygenic score categorically differentiates between people with ovarian cancer and normal tissue. Illustration C at the bottom right panel of  FIG. 23  shows the methylation score for the CGIDs detecting the specific tumor origin using data from people who have 11 different tumors (n=110). In these embodiments, the markers categorically differentiate between cancers from other origins and ovarian cancer. 
         FIG. 24  is an illustration of a validation of the polygenic HKG-epiOvarian-detect and spec markers accuracy and specificity for cervical versus other cancers in TCGA methylation data (n=6522). Illustration A is a detection rate of the HKG-epiOvarian detect/spec markers using DNA methylation data from patients with different cancers. Note the specificity for ovarian cancer. Illustration B is a ROC plot specificity and sensitivity of the HKG-epiOvarian-detect and spec markers for ovarian cancer on DNA methylation data from 4723 patients in TCGA. Illustration C shows the sensitivity and specificity to ovarian cancer. 
         FIG. 25  is an illustration of a discovery of a polygenic DNA methylation marker for cervical cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of cervical cancer disclosed in the present invention using the BCD method (Table 18) and CGs for determining the specific cancer of origin (Table 19). Illustration B at the bottom left panel of  FIG. 25  (Detect) shows the combined methylation score for these CGIDs (Table 18) for each of the tested people listed from 1-30 (20 normal and 10 ovarian cancer). The polygenic score categorically differentiates between cervical cancer and normal tissue. Illustration C at the bottom right panel of  FIG. 25  shows the methylation score for the CG IDs detecting the specific origin of the tumor (Table 19) using data from people who have 8 different tumors (n=80). In these embodiments, the markers categorically differentiate between cancers from other origins and cervical cancer, note however some measurable detection of colorectal cancer. 
         FIG. 26  is an illustration of a validation of the polygenic HKG-Cervix-detect and spec markers accuracy and specificity for cervix versus other cancers in TCGA methylation data (n=6522). Illustration A shows a detection rate of the HKG-Cervix detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for cervical cancer. Illustration B is a ROC plot of specificity and sensitivity of the HKG-Cervix-detect spec markers for cervical cancer using DNA methylation data from 4420 patients in TCGA. Illustration C shows a sensitivity and specificity to cervical cancer. 
         FIG. 27  is an illustration of a discovery of a polygenic DNA methylation marker for Head and Neck squamous cell Carcinoma (HNSC). Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CG IDs for detection of HNSC disclosed in the present invention using the BCD method (Table 20) and CGs for determining the specific cancer of origin (Table 21). Illustration B at the bottom left panel of  FIG. 27  shows the combined methylation score for these CG IDs (Table 20) for each of the tested people listed from 1-140 (10 cancer, 10 normal and 120 other cancers). Illustration C shows the polygenic score which categorically differentiates between HNSC and normal tissue samples in the embodiments as well as categorically differentiating between cancers from other origins and HNSC. 
         FIG. 28  is an illustration of a validation of the polygenic HKG-epiHNSC-detect/spec markers accuracy and specificity for HNSC versus other cancers in TCGA methylation data (n=4166). Illustration A is a detection rate of the HKG-epiHNSC detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for HNSC. Illustration B is a ROC plot of the specificity and sensitivity of HKG-epiHNSC-detect markers for HNSC on DNA methylation data from 4166 patients in TCGA. Illustration C shows the sensitivity and specificity for HNSC versus cancers from other origins. 
         FIG. 29  is an illustration of a discovery of a polygenic DNA methylation marker for esophageal cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of esophageal cancer disclosed in embodiments using the BCD method (Table 22) and CGIDs for determining the specific origin of the cancer (Table 23). Illustration B at the bottom left panel of  FIG. 29  shows the combined methylation score for these CGIDs (Table 22) for each of the tested people listed from 1-15 (6 normal, 10 cancer). Illustration C shows the polygenic score categorically differentiating between esophageal cancer and normal tissue in the embodiments as well as categorically differentiating between cancers from other origins and esophageal cancer listed from 1-220 (20 cancer, 190 other cancer and 10 healthy blood). 
         FIG. 30  is an illustration of a validation of the polygenic HKG-epiEsophageal—detect/spec markers accuracy and specificity for esophageal cancer versus other cancers in TCGA methylation data (n=7102). Illustration A shows a detection rate of the HKG-epiEsophageal detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for esophageal cancer. Illustration B is a ROC plot of the specificity and sensitivity of the HKG-epiEsophageal-detect markers for HNSC on 4166 patient DNA methylation data in TCGA. Illustration C shows sensitivity and specificity to esophageal cancer versus cancers from other origins. 
         FIG. 31  is an illustration of a discovery of a polygenic DNA methylation marker for bladder cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of bladder cancer disclosed in embodiments using the BCD method (Table 24) and CGIDs for determining the specific origin of the cancer (Table 25). Illustration B at the bottom left panel of  FIG. 31  (Detect) shows the combined methylation score for these CGIDs (Table 24) for each of the tested people listed from 1-15 (5 normal and 10 bladder cancer). Illustration C at the bottom right panel of  FIG. 31  shows the methylation score for the CGIDs (Table 25) detecting the specific origin of the tumor using data from people who have 13 different tumors (n=130). In these embodiments, the markers differentiate between cancers from other origins and bladder cancer. Also of note are some measurable detection of colorectal cancer with these markers. 
         FIG. 32  is an illustration of a validation of accuracy and specificity of the polygenic HKG-epiBladder-detect and spec markers for bladder cancer versus other cancers in TCGA (n=4723). Illustration A shows a detection rate of the HKG-epiBladder spec (A) and detect markers (B) on DNA methylation data of patients with different cancers (A) and bladder cancer (B). Illustration C is a ROC plot of the specificity and sensitivity of HKG-epiBladder spec markers for bladder cancer using DNA methylation data from 4420 patients in TCGA. Illustration D is a ROC plot of the specificity and sensitivity of the HKG-epiBladder detect markers for bladder cancer (n=440). 
         FIG. 33  is an illustration of a discovery of a polygenic DNA methylation marker for kidney cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of kidney cancer disclosed in embodiments using the BCD (hypo) method and for determining the specific origin of the cancer (Table 26). Illustration B at the bottom left panel of  FIG. 33  (Detect/spec) shows the combined methylation score for these CGIDs (Table 26) for each of the tested people listed from 1-226 (180 other cancers, 10 healthy blood, 6 normal kidney, 30 renal cancer). In these embodiments, the polygenic score categorically differentiates between kidney cancer, other cancers and normal blood. 
         FIG. 34  is an illustration of a validation of the accuracy and specificity of polygenic HKG-epiKidney-detect and spec markers for kidney cancer versus other cancers and normal tissues using TCGA DNA methylation data (n=7102). Illustration A is the detection rate of the HKG-epiKidney detect/spec markers using DNA methylation data from different cancers. Of note is the specificity for kidney cancer. Illustration B is a ROC plot of the specificity and sensitivity of HKG-Cervix-detect spec markers for renal cancer using DNA methylation data from 6367 cancers in TCGA. Illustration C is a sensitivity and specificity to renal (kidney) cancer. Of additional note is a crossover with brain, HCC and testicular cancer. 
         FIG. 35  is an illustration of a discovery of a polygenic DNA methylation marker for testicular cancer. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of testicular cancer disclosed in embodiments using the BCD (hypo) method and for determining the specific cancer of origin (Table 27). Illustration B at the bottom left panel of  FIG. 35  (Detect/spec) shows the combined methylation score for these CG IDs (Table 27) for each of the tested people listed from 1-226 (10 testicular cancer, 180 other cancers, 10 normal blood). In these embodiments, the polygenic score categorically differentiates between testicular cancer and normal blood and other cancers. 
         FIG. 36  is an illustration of a validation of the accuracy and specificity of the polygenic HKG-epiTestis-detect and spec markers for testicular cancer versus other normal tissues and cancers in TCGA methylation data (n=7102). Illustration A shows the detection rate of the HKG-epiTesstis detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for Testis cancer. Illustration B is a ROC plot of the specificity and sensitivity of HKG-epiTestis-detect spec markers for testicular cancer using DNA methylation data from 6367 patients in TCGA. Illustration C is the sensitivity and specificity to testicular cancer. 
         FIG. 37  is an illustration of a discovery of a Pan cancer polygenic DNA methylation marker for 13 common cancers. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of 13 common cancers (Table 28) (bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer CRC, esophageal cancer, liver cancer, lung cancer. ovarian cancer, pancreatic cancer, prostate cancer, and stomach cancer) disclosed in embodiments using the BCD method (Table 28). Illustration B shows the combined methylation score for these CGIDs for each of the tested people listed from 1-310 (180 cancer and 10 normal). In these embodiments, the polygenic score differentiates between cancers and normal tissue. 
         FIG. 38  is an illustration of a validation of the polygenic HKG epiPancancer markers accuracy and specificity in TCGA methylation data (n=7102). Illustration A shows Methylation scores calculated using the epiPancancer polygenic DNA methylation markers in patients with 13 different cancers using TGCA data. Illustration B is a ROC plot of the specificity and sensitivity of HKG-epiPancancer detect and spec markers using DNA methylation data from for all cancers from 4878 patients in TCGA. Illustration C is a ROC plot of the epiPancancer polygenic markers describing specificity and sensitivity for detection of 13 common cancers. Illustration D shows the overall sensitivity and specificity of the pan cancer markers for detecting cancer. In these embodiments, one or more colors are used, for example orange (weighted methylation score) and blue (detection of one BCD marker per sample is scored as a positive cancer). 
         FIG. 39  is an illustration of a discovery of polygenic DNA methylation marker for Melanoma. Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of melanoma (Table 45) disclosed in embodiments using the BCD method (Table 45). Illustration B is the combined methylation score for these CGIDs for each of the tested people listed from 1-220 (other cancers and normal blood) and 10 patients with melanoma. In these embodiments, the polygenic score differentiates between melanoma, other cancers and normal tissue. 
         FIG. 40  is an illustration of a validation of the accuracy and specificity of the polygenic HKG-epiMelanoma-detect and spec markers for melanoma versus other normal tissues and cancers in TCGA methylation data (n=7102). Illustration A shows the detection rate of the HKG-epiMelanoma detect/spec markers using DNA methylation data from patients with different cancers. Of note is the specificity for melanoma (with overlapping detection of liver cancer brain and cancer and prostate cancer). Illustration B is a ROC plot of the specificity and sensitivity of HKG-Melanoma-detect spec markers for melanoma using DNA methylation data from 6367 patients in TCGA. Illustration C shows the sensitivity and specificity to melanoma. 
         FIG. 41  is an illustration of a discovery of polygenic DNA methylation marker for blood cancers (Acute Myeloid Leukemia (AML). Illustration A is a table listing the source and number of patients whose methylation data was used for discovery of a set of CGIDs for detection of blood cancer AML (Table 46) disclosed embodiments using the BCD method (Table 46). Illustration B is the combined methylation score for these CGIDs for each of the tested people listed from 1-10 (normal blood) and 10 patients with AML. In these embodiments, the polygenic score differentiates between AML and normal blood. 
         FIG. 42  is an illustration of a validation of the accuracy and specificity of the polygenic HKG-epiAML-detect and spec markers for AML in GSE86409 (n=79) and in TCGA (n−140) versus normal blood in GSE40279 and GSE61496 (n=968). Illustration A shows a detection rate of the HKG-epiAML detect/spec markers using DNA methylation data from patients with AML and healthy blood. Of note is the specificity for melanoma (with overlapping detection of liver cancer brain and cancer and prostate cancer). Illustration B is a ROC plot of the specificity and sensitivity of HKG-AML-detect spec markers for AML using DNA methylation data from GSE86409 (n=79), TCGA (n-140) GSE40279 and GSE61496 (n=968). Illustration C shows the sensitivity and specificity to AML. 
         FIG. 43  is an illustration of a validation that the primers selected for detecting different cancers exhibit BCD properties ˜0 methylation in plasma derived from normal people (each sample is a mixture of plasma from normal patients). The first PCR1 reaction targeting the specific CGs was performed using sequence targeted primers. Following a second PCR, the amplified fragments were purified and subjected to next generation sequencing. DNA methylation was quantified in each of the indicated CG ID positions. 
         FIG. 44  is an illustration of a validation that the indicated primers selected for detecting different cancers exhibit BCD properties ˜0 methylation in plasma derived from normal people (each sample is a mixture of plasma from normal patients). 
         FIG. 45  is an illustration of a primer design for multiplex amplification and sequencing. The first PCR reaction targets the specific regions of interest, note PCR1 primer has complementary sequence to second PCR2 primers. The second set of primers introduces the index for each patient as well as the reverse and forward sequencing primers. 
         FIG. 46  is an illustration of an optimization of PCR conditions for detecting prostate cancer. A multiplex PCR1 reaction using varying primer concentrations as indicated DNA for the three markers of prostate cancer HIF3A 232 bp, TPM4 213 bp, and CTTN 199 bp is shown on the right panel. 
         FIG. 47 . is an illustration of a bioinformatics workflow for determining DNA methylation levels. PCR2 products are combined, quantified and purified and subjected to next generation sequencing on a Miseq Illumina sequencer. Sequence is demultiplexed, FASTQ files are generated for each patient and analyzed with the workflow shown in the scheme. DNA methylation scores are calculated for each patient. 
     
    
    
     DETAILED DESCRIPTION 
     All illustrations of the drawings are for the purpose of describing selected embodiments and are not intended to limit the scope of the claimed subject matter. 
     Embodiment 1. “Discovery of Categorically Unmethylated CGIDs Across Hundreds of Individuals in Normal Tissues and Blood DNA 
     Cell free DNA originating in tumors is known to be found in body fluids such as plasma, urine and in feces. It is also established that DNA methylation profiles of CF tumor DNA are similar to tumor DNA (Dominguez-Vigil et al., 2018). A vast body of data has established that tumor DNA is differentially methylated compared to normal tissues (Luczak &amp; Jagodzinski, 2006). Therefore, many groups have tried to delineate by logistic regressions CGID positions in DNA (CG IDs in the Illumina 450K manifest) that are differentially methylated between cancerous and its normal tissue of origin for example, liver cancer versus adjacent liver tissue. However, since these methods measure quantitative differences between cancer and untransformed tissue rather than categorical qualitative differences, these quantitative differences between tumor and normal tissue would be diluted and erased by CF DNA from normal tissue, leading to false negatives and reduced sensitivity. In addition, other tissues that were not included in the analysis might have a DNA methylation profile similar to tumor DNA and since most studies only compare the tumor DNA to its untransformed counterparts and not to other tissues, this could lead to false positives. Varying and unpredictable quantities of DNA from different tissues have been detected in CF DNA (Breitbach et al., 2014) and thus the measured DNA methylation reflects a composite of unknown and unpredictable mixture of tissue DNA from different sources and tumor DNA. Thousands of tumor samples have been subjected to genome wide DNA methylation analysis using Illumina 450K arrays and are found in the public domain (TCGA). Examining the profiles of methylation of many normal tissues as well as cancer tissues, the inventors noticed that there is a significant group of CGs in the genome that are completely unmethylated in all normal tissues but methylated in DNA from tumors. A subset of these sites is unmethylated across numerous individuals whose DNA methylation was profiled in the public domain. The inventors also noticed that in many cancers these robustly unmethylated sites become methylated in cancer. Thus, creating a qualitative “categorical difference” between tumor DNA and all other DNA that might be found in blood. Using deep next generation sequencing even few methylated molecules could be easily identified on a background of completely unmethylated copies. 
     Data Bases; Illumina 450K DNA Methylation Data 
     We used publicly available data bases of normalized beta values of methylation for ˜450,000 CG across the human genome from a large number of individuals deposited either in the Gene expression Omnibus (GEO) https://www.ncbi.nlm.gov/geo/ or The Cancer Genome Atlas TCGA https://cancergenome.nih.gov/ public data bases. We used the following databases to derive the list of robustly unmethylated CG IDs in many normal tissues and blood DNA: GSE50192, GSE50192, GSE40279. 
     DNA from white blood cells is one of the main sources of CF DNA in plasma. The inventors first generated a list of 47981 CGIDS that are unmethylated in all individuals in 17 different somatic human tissues using Illumina 450K data in GSE50192 and the logical COUNTIF and IF functions in Excel: 
     NmCGID x =COUNTIF (betaCGID x n 1 :n i ,“&gt;0.1”)
 
umCGID x =IF(NmCGID x =0, TRUE, FALSE)
 
NmCGIDx=number of normal subjects that have the CGIDx methylated.
 
umCGIDx=unmethylated CGIDx in all subjects
 
betaCGIDx=the methylation values for a given CGIDx
 
x=any CGID on the Illumina 450 k array
 
n 1 =the first subject in the array,
 
n i =the last subject in the array.
 
     The inventors then generated a list of 68260 unmethylated CGIDs (UMCGIDs) in blood DNA from 312 individuals using the same criteria. The inventors then overlapped the list of 47981 and 68260 CG IDS and obtained a list of 33477 CG IDs that are unmethylated in both blood and somatic tissues across all individuals ( FIG. 1A ). To increase the robustness of this list of unmethylated CG IDs the inventors delineated a list of 60,379 CG IDs unmethylated CGIDs in Illumina 450K arrays of whole blood DNA from 656 individuals males and females aged from 19 to 101 years (GSE40279). These are robustly unmethylated sites in blood that are sex and age independent across hundreds of individuals. This list of 60,379 CG IDs was overlapped with the list of 33,477 CG IDs that are unmethylated both in somatic tissues and blood to generate a final list of 28,754 CG IDs which were used for discovery of categorical methylation markers for cancer. This list includes CG ID positions that are robustly unmethylated across tissues and individuals. 
     To identify DNA methylation positions that are categorically different between cancer and normal tissues the inventors examined whether any of these 28754 CG IDs are methylated in different cancers. The inventors noticed following examination of tumor DNA methylation data that methylation of a subset of these 28754 CG IDs is common in tumor DNA from individual patients. However, not all individuals have the same position methylated. Thus, a combination of CG IDs is required to detect cancer with high specificity. The inventors therefore discovered a polygenic combination of CG IDs for detection of cancers. 
     The inventors used 10 to 50 DNA methylation profiles from the public domain from either TCGA or GEO as a “discovery set” to discover a polygenic set of CGIDs whose methylation state is “categorically” different between tumor and normal tissues that could detect cancer with highest sensitivity and specificity. These CGIDs were then tested on hundreds of TCGA and GEO tumor DNA methylation array data as a “validation set” to validate the sensitivity and specificity of the polygenic DNA methylation markers for detecting cancer as disclosed in Embodiment 2. 
     Embodiment 2: Binary-Categorical Differentiation (BCD)” Method for Detecting Cancer in Cell Free DNA 
     The following publicly available data bases of normalized beta values of methylation for ˜450,000 CGs (CG IDs) across the human genome were used to derive the list of cancer specific DNA methylation markers: 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 liver cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Normal Liver 
                 GSE61258 
                 Discovery 
                 79 
                 Detect 
               
               
                 HCC 
                 TCGA 
                 Discovery 
                 50 
                 Detect 
               
               
                 HCC 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-HCC cancers 
                 TCGA 
                 Discovery 
                 80 
                 Spec 
               
               
                 HCC 
                 GSE75041 
                 Validatio   
                 66 
                 Detect 
               
               
                 HCC 
                 GSE76269 
                 Validatio   
                 227 
                 Detect 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Detect 
               
               
                 Bladder cancer 
                 TCGA 
                 Validatio   
                 439 
                 Spec 
               
               
                 Brain cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast cancer 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 Cervix cancer 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 CRC cancer 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA cancer 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 Kidney cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Ovarian cancer 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PANC cancer 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 PRAD cancer 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach cancer 
                 TCGA 
                 Validatio   
                 397 
                 Spec 
               
               
                 Testis cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal Liver 
                 GSE76269 
                 Validatio   
                 10 
                 Spec 
               
               
                 Normal Liver 
                 GSE69852 
                 Validatio   
                 6 
                 Spec 
               
               
                 Normal Liver 
                 GSE75041 
                 Validatio   
                 10 
                 Spec 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 lung cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Lung Cancer 
                 GSE63704 
                 Discovery 
                 10 
                 Detect 
               
               
                 Normal Lung 
                 GSE66836 
                 Discovery 
                 10 
                 Detect 
               
               
                 Lung Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-lung cancers 
                 TCGA 
                 Discovery 
                 80 
                 Spec 
               
               
                 Lung Cancer 
                 GSE66836 
                 Validatio   
                 164 
                 Detect 
               
               
                 Lung Cancer 
                 GSE63704 
                 Validatio   
                 17 
                 Detect 
               
               
                 Lung Cancer 
                 GSE76269 
                 Validatio   
                 56 
                 Detect 
               
               
                 Lung Cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validatio   
                 439 
                 Detect 
               
               
                 Brain Cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validatio   
                 396 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal Lung 
                 GSE63704 
                 Validatio   
                 112 
                 Detect 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 prostate cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Control 
                 GSE52955 
                 Discovery 
                 5 
                 Detect 
               
               
                 PRAD 
                 TCGA 
                 Discovery 
                 10 
                 Detect 
               
               
                 PRAD 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-PRAD 
                 TCGA 
                 Discovery 
                 80 
                 Spec 
               
               
                 PRAD 
                 GSE73549 
                 Validatio   
                 77 
                 Detect 
               
               
                 PRAD 
                 GSE52955 
                 Validatio   
                 25 
                 Detect 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Bladder 
                 TCGA 
                 Validatio   
                 439 
                 Spec 
               
               
                 Brain 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 Cervix 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Ovarian 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 Stomach 
                 TCGA 
                 Validatio   
                 397 
                 Spec 
               
               
                 Testis 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal Prostat   
                 GSE73549 
                 Validatio   
                 15 
                 Detect 
               
               
                 Normal Prostat   
                 GSE52955 
                 Validatio   
                 5 
                 Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 breast cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Healthy 
                 GSE60185 
                 Discovery 
                 17 
                 Detect 
               
               
                 Breast Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Detect 
               
               
                 Breast Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-breast cancers 
                 TCGA 
                 Discovery 
                 80 
                 Spec 
               
               
                 Breast Cancer 
                 GSE60185 
                 Validation 
                 239 
                 Detect 
               
               
                 Breast Cancer 
                 GSE75067 
                 Validation 
                 188 
                 Detect 
               
               
                 Breast Cancer 
                 TCGA 
                 Validation 
                 891 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validation 
                 439 
                 Spec 
               
               
                 brain Cancer 
                 TCGA 
                 Validation 
                 689 
                 Spec 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validation 
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validation 
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validation 
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validation 
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validation 
                 871 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validation 
                 919 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validation 
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validation 
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validation 
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validation 
                 396 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validation 
                 156 
                 Spec 
               
               
                 Healthy breast tissu   
                 GSE10196   
                 Validation 
                 121 
                 Detect 
               
               
                 Healthy breast tissu   
                 GSE60185 
                 Validation 
                 17 
                 Detect 
               
               
                 Normal adjasent tiss   
                 GSE60185 
                 Validation 
                 29 
                 Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validation 
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validation 
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 colorectal cancer CRC 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Control 
                 GSE6550 
                 Discovery 
                 25 
                 Detect 
               
               
                 CRC 
                 TCGA 
                 Discovery 
                 50 
                 Detect 
               
               
                 CRC 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-HCC 
                 TCGA 
                 Discovery 
                 80 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 260 
                 Detect 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 459 
                 Detect 
               
               
                 Bladder 
                 TCGA 
                 Validatio   
                 439 
                 Spec 
               
               
                 Brain 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 Cervix 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Ovarian 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach 
                 TCGA 
                 Validatio   
                 397 
                 Spec 
               
               
                 Testis 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal Colorec   
                 GSE6550 
                 Validatio   
                 8 
                 Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Pancreatic cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Healthy 
                 GSE53051 
                 Discovery 
                 12 
                 Detect 
               
               
                 Pancreatic Canc   
                 TCGA 
                 Discovery 
                 20 
                 Detect 
               
               
                 Pancreatic Canc   
                 TCGA 
                 Discovery 
                 20 
                 Spec 
               
               
                 Non-pancreatic 
                 TCGA 
                 Discovery 
                 100 
                 Spec 
               
               
                 Pancreatic Canc   
                 E-MTAB-   
                 Validatio   
                 24 
                 Detect 
               
               
                 Pancreatic Canc   
                 TCGA 
                 Validatio   
                 195 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validatio   
                 439 
                 Spec 
               
               
                 brain Cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validatio   
                 396 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal Blood 
                 GSE40179 
                 Validatio   
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 Brain cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spec 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Brain Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec-Detect 
               
               
                 Non-brain canc   
                 TCGA 
                 Discovery 
                 158 
                 Spec-Detect 
               
               
                 Brain Cancer 
                 GSE36278 
                 Validatio   
                 136 
                 Spec-Detect 
               
               
                 Brain Cancer 
                 GSE58298 
                 Validatio   
                 40 
                 Spec-Detect 
               
               
                 Brain Cancer 
                 GSE58218 
                 Validatio   
                 228 
                 Spec-Detect 
               
               
                 Brain Cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec-Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validatio   
                 439 
                 Spec-Detect 
               
               
                 Breast Cancer 
                 TCGA 
                 Validatio   
                 891 
                 Spec-Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validatio   
                 312 
                 Spec-Detect 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec-Detect 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec-Detect 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec-Detect 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec-Detect 
               
               
                 Lung Cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec-Detect 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validatio   
                 10 
                 Spec-Detect 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec-Detect 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec-Detect 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validatio   
                 396 
                 Spec-Detect 
               
               
                 Testis Cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec-Detect 
               
               
                 Brain Cancer 
                 GSE36278 
                 Validatio   
                 6 
                 Spec-Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec-Detect 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec-Detect 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Stomach cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Healthy 
                 GSE99553 
                 Discovery 
                 14 
                 Detect 
               
               
                 Stomach Cancer 
                 TCGA 
                 Discovery 
                 20 
                 Detect 
               
               
                 Stomach Cancer 
                 TCGA 
                 Discovery 
                 7 
                 Spec 
               
               
                 Non-stomach cancer 
                 TCGA 
                 Discovery 
                 100 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validation 
                 396 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validation 
                 439 
                 Spec 
               
               
                 brain Cancer 
                 TCGA 
                 Validation 
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validation 
                 891 
                 Spec 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validation 
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validation 
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validation 
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validation 
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validation 
                 871 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validation 
                 919 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validation 
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validation 
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validation 
                 553 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validation 
                 156 
                 Spec 
               
               
                 Normal Tissue 
                 GSE99553 
                 Validation 
                 42 
                 Detect 
               
               
                 Normal Blood 
                 GSE40179 
                 Validation 
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validation 
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Ovarian cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Healthy 
                 GSE65821 
                 Discovery 
                 6 
                 Detect 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Detect 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-ovarian cancers 
                 TCGA 
                 Discovery 
                 110 
                 Spec 
               
               
                 Ovarian Cancer 
                 GSE65821 
                 Validatio   
                 113 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validatio   
                 439 
                 Spec 
               
               
                 brain Cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validatio   
                 891 
                 Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validatio   
                 396 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Healthy ovarian tissue 
                 GSE87621 
                 Validatio   
                 9 
                 Detect 
               
               
                 Healthy ovarian tissue 
                 GSE74845 
                 Validatio   
                 216 
                 Detect 
               
               
                 Healthy ovarian tissue 
                 GSE81228 
                 Validatio   
                 10 
                 Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Cervical cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Healthy 
                 GSE46306 
                 Discovery 
                 20 
                 Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-cervical cancers 
                 TCGA 
                 Discovery 
                 110 
                 Spec 
               
               
                 Cervical Cancer 
                 GSE68339 
                 Validatio   
                 270 
                 Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validatio   
                 312 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validatio   
                 439 
                 Spec 
               
               
                 brain Cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validatio   
                 396 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 HNSC 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HNSC 
                 TCGA 
                 Discovery 
                 20 
                 Detect 
               
               
                 Control 
                 GSE75537 
                 Discovery 
                 10 
                 Detect 
               
               
                 HNSC 
                 TCGA 
                 Discovery 
                 20 
                 Spec 
               
               
                 Non-HNSC 
                 TCGA 
                 Discovery 
                 190 
                 Spec 
               
               
                 Healthy 
                 GSE40279 
                 Discovery 
                 10 
                 Spec 
               
               
                 HNSC 
                 TCGA 
                 Validation 
                 580 
                 Detect 
               
               
                 Control 
                 GSE52068 
                 Validation 
                 24 
                 Detect 
               
               
                 HNSC 
                 GSE75537 
                 Validation 
                 54 
                 Detect 
               
               
                 HNSC 
                 GSE79556 
                 Validation 
                 83 
                 Detect 
               
               
                 Control 
                 GSE75537 
                 Validation 
                 54 
                 Detect 
               
               
                 HNSC 
                 GSE52068 
                 Validation 
                 24 
                 Detect 
               
               
                 Bladder 
                 TCGA 
                 Validation 
                 439 
                 Spec 
               
               
                 Brain 
                 TCGA 
                 Validation 
                 689 
                 Spec 
               
               
                 Breast 
                 TCGA 
                 Validation 
                 891 
                 Spec 
               
               
                 Cervix 
                 TCGA 
                 Validation 
                 312 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validation 
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validation 
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validation 
                 430 
                 Spec 
               
               
                 Kidney 
                 TCGA 
                 Validation 
                 871 
                 Spec 
               
               
                 Lung 
                 TCGA 
                 Validation 
                 919 
                 Spec 
               
               
                 Ovarian 
                 TCGA 
                 Validation 
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validation 
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validation 
                 553 
                 Spec 
               
               
                 Stomach 
                 TCGA 
                 Validation 
                 396 
                 Spec 
               
               
                 Testis 
                 TCGA 
                 Validation 
                 156 
                 Spec 
               
               
                 Normal Blood 
                 GSE40279 
                 Validation 
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE52068 
                 Validation 
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Esophageal cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Esophageal Cancer 
                 IGSE52826 
                 Discovery 
                 6 
                 Detect 
               
               
                 ESCA 
                 TCGA 
                 Discovery 
                 10 
                 Detect 
               
               
                 Non-esophageal canc   
                 TCGA 
                 Discovery 
                 190 
                 Spec 
               
               
                 Healthy blood 
                 GSE40279 
                 Discovery 
                 10 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Discovery 
                 20 
                 Spec 
               
               
                 Esophageal Cancer 
                 TCGA 
                 Validation 
                 202 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validation 
                 439 
                 Spec 
               
               
                 Brain Cancer 
                 TCGA 
                 Validation 
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validation 
                 891 
                 Spec 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validation 
                 312 
                 Spec 
               
               
                 CRC Cancer 
                 TCGA 
                 Validation 
                 459 
                 Spec 
               
               
                 Renal Cancer 
                 TCGA 
                 Validation 
                 871 
                 Spec 
               
               
                 HNSC 
                 TCGA 
                 Validation 
                 580 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validation 
                 430 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validation 
                 919 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validation 
                 10 
                 Spec 
               
               
                 Pancreatic Cancer 
                 TCGA 
                 Validation 
                 195 
                 Spec 
               
               
                 Prostate Cancer 
                 TCGA 
                 Validation 
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validation 
                 397 
                 Spec 
               
               
                 Testis 
                 TCGA 
                 Validation 
                 156 
                 Spec 
               
               
                 Normal Blood 
                 GSE40279 
                 Validation 
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validation 
                 312 
                 Spec 
               
               
                 Normal tissues 
                 Mix 
                 Validation 
                 115 
                 Spec 
               
               
                 Normal tissues 
                 GSE85566 
                 Validation 
                 189 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 41 
               
             
            
               
                   
               
               
                 Bladder cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spe   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Bladder Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Detect 
               
               
                 Normal bladder 
                 GSE52955 
                 Discovery 
                 5 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec 
               
               
                 Non-bladder cancers 
                 TCGA 
                 Discovery 
                 180 
                 Spec 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validatio   
                 439 
                 Detect 
               
               
                 Brain Cancer 
                 TCGA 
                 Validatio   
                 689 
                 Spec 
               
               
                 Breast Cancer 
                 TCGA 
                 Validatio   
                 891 
                 Spec 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validatio   
                 312 
                 Spec 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validatio   
                 871 
                 Spec 
               
               
                 Lung Cancer 
                 TCGA 
                 Validatio   
                 919 
                 Spec 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validatio   
                 10 
                 Spec 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validatio   
                 396 
                 Spec 
               
               
                 Testis Cancer 
                 TCGA 
                 Validatio   
                 156 
                 Spec 
               
               
                 Normal tissues 
                 GSE50192 
                 Validatio   
                 70 
                 Spec 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 42 
               
             
            
               
                   
               
               
                 Kidney cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spec 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Control 
                 GSE52955 
                 Discovery 
                 6 
                 Spec-Detect 
               
               
                 Kidney Cancer 
                 TCGA 
                 Discovery 
                 30 
                 Spec-Detect 
               
               
                 Healthy blood 
                 GSE40279 
                 Discovery 
                 10 
                 Spec-Detect 
               
               
                 Non-kidney can   
                 TCGA 
                 Discovery 
                 180 
                 Spec-Detect 
               
               
                 Kidney 
                 TCGA 
                 Validatio   
                 871 
                 Spec-Detect 
               
               
                 Bladder 
                 TCGA 
                 Validatio   
                 439 
                 Spec-Detect 
               
               
                 Brain 
                 TCGA 
                 Validatio   
                 689 
                 Spec-Detect 
               
               
                 Breast 
                 TCGA 
                 Validatio   
                 891 
                 Spec-Detect 
               
               
                 Cervix 
                 TCGA 
                 Validatio   
                 312 
                 Spec-Detect 
               
               
                 CRC 
                 TCGA 
                 Validatio   
                 459 
                 Spec-Detect 
               
               
                 ESCA 
                 TCGA 
                 Validatio   
                 202 
                 Spec-Detect 
               
               
                 HNSC 
                 TCGA 
                 Validatio   
                 580 
                 Spec-Detect 
               
               
                 HCC 
                 TCGA 
                 Validatio   
                 430 
                 Spec-Detect 
               
               
                 Lung 
                 TCGA 
                 Validatio   
                 919 
                 Spec-Detect 
               
               
                 Ovarian 
                 TCGA 
                 Validatio   
                 10 
                 Spec-Detect 
               
               
                 PANC 
                 TCGA 
                 Validatio   
                 195 
                 Spec-Detect 
               
               
                 PRAD 
                 TCGA 
                 Validatio   
                 553 
                 Spec-Detect 
               
               
                 Stomach 
                 TCGA 
                 Validatio   
                 397 
                 Spec-Detect 
               
               
                 Testis 
                 TCGA 
                 Validatio   
                 156 
                 Spec-Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validatio   
                 656 
                 Spec-Detect 
               
               
                 Normal Blood 
                 GSE61496 
                 Validatio   
                 312 
                 Spec-Detect 
               
               
                 Normal tissues 
                 Mix 
                 Validatio   
                 189 
                 Spec-Detect 
               
               
                 Normal tissues 
                 GSE85566 
                 Validatio   
                 115 
                 Spec-Detect 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 Testicular cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spec 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Testicular Cancer 
                 TCGA 
                 Discovery 
                 10 
                 Spec-Detect 
               
               
                 Healthy blood 
                 GSE40279 
                 Discovery 
                 10 
                 Spec-Detect 
               
               
                 Non-testis cancers 
                 TCGA 
                 Discovery 
                 170 
                 Spec-Detect 
               
               
                 Testis Cancer 
                 TCGA 
                 Validation 
                 156 
                 Spec-Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validation 
                 439 
                 Spec-Detect 
               
               
                 Brain Cancer 
                 TCGA 
                 Validation 
                 689 
                 Spec-Detect 
               
               
                 Breast Cancer 
                 TCGA 
                 Validation 
                 891 
                 Spec-Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validation 
                 312 
                 Spec-Detect 
               
               
                 CRC 
                 TCGA 
                 Validation 
                 459 
                 Spec-Detect 
               
               
                 ESCA 
                 TCGA 
                 Validation 
                 202 
                 Spec-Detect 
               
               
                 HCC 
                 TCGA 
                 Validation 
                 430 
                 Spec-Detect 
               
               
                 Kidney Cancer 
                 TCGA 
                 Validation 
                 871 
                 Spec-Detect 
               
               
                 Lung Cancer 
                 TCGA 
                 Validation 
                 919 
                 Spec-Detect 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validation 
                 10 
                 Spec-Detect 
               
               
                 PANC 
                 TCGA 
                 Validation 
                 195 
                 Spec-Detect 
               
               
                 PRAD 
                 TCGA 
                 Validation 
                 553 
                 Spec-Detect 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validation 
                 396 
                 Spec-Detect 
               
               
                 Normal tissues 
                 GSE50192 
                 Validation 
                 70 
                 Spec-Detect 
               
               
                 Normal tissues 
                 Mix 
                 Validation 
                 119 
                 Spec-Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validation 
                 656 
                 Spec-Detect 
               
               
                 Normal Blood 
                 GSE61496 
                 Validation 
                 312 
                 Spec-Detect 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 44 
               
             
            
               
                   
               
               
                 Pan-cancer 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Status 
                 Source 
                 Cohort 
                 N 
                 Detect/Spec 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Healthy Blood 
                 GSE40279 
                 Discovery 
                 10 
                 Detect 
               
               
                 TCGA cancers 
                 TCGA 
                 Discovery 
                 170 
                 Detect 
               
               
                 Bladder Cancer 
                 TCGA 
                 Validation 
                 439 
                 Detect 
               
               
                 Brain Cancer 
                 TCGA 
                 Validation 
                 689 
                 Detect 
               
               
                 Breast Cancer 
                 TCGA 
                 Validation 
                 891 
                 Detect 
               
               
                 Cervical Cancer 
                 TCGA 
                 Validation 
                 312 
                 Detect 
               
               
                 CRC Cancer 
                 TCGA 
                 Validation 
                 459 
                 Detect 
               
               
                 Esophagus Cancer 
                 TCGA 
                 Validation 
                 202 
                 Detect 
               
               
                 HNSC Cancer 
                 TCGA 
                 Validation 
                 580 
                 Detect 
               
               
                 HCC Cancer 
                 TCGA 
                 Validation 
                 430 
                 Detect 
               
               
                 Lung Cancer 
                 TCGA 
                 Validation 
                 919 
                 Detect 
               
               
                 Ovarian Cancer 
                 TCGA 
                 Validation 
                 10 
                 Detect 
               
               
                 Pancreatic Cancer 
                 TCGA 
                 Validation 
                 195 
                 Detect 
               
               
                 Prostate Cancer 
                 TCGA 
                 Validation 
                 553 
                 Detect 
               
               
                 Stomach Cancer 
                 TCGA 
                 Validation 
                 397 
                 Detect 
               
               
                 Normal Blood 
                 GSE40279 
                 Validation 
                 656 
                 Detect 
               
               
                 Normal Blood 
                 GSE61496 
                 Validation 
                 312 
                 Detect 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 45 
               
             
            
               
                   
               
               
                 Melanoma 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Statu   
                 Source 
                 Cohort 
                 N 
                 Detect/Spec 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Bladder Can   
                 TCGA 
                 Validation 
                 439 
                 Detect/Spec 
               
               
                 Brain Cancer 
                 TCGA 
                 Validation 
                 689 
                 Detect/Spec 
               
               
                 Breast Cance   
                 TCGA 
                 Validation 
                 891 
                 Detect/Spec 
               
               
                 Cervical Can   
                 TCGA 
                 Validation 
                 312 
                 Detect/Spec 
               
               
                 CRC Cancer 
                 TCGA 
                 Validation 
                 459 
                 Detect/Spec 
               
               
                 Esophagus C   
                 TCGA 
                 Validation 
                 202 
                 Detect/Spec 
               
               
                 HNSC Cancer 
                 TCGA 
                 Validation 
                 580 
                 Detect/Spec 
               
               
                 HCC Cancer 
                 TCGA 
                 Validation 
                 430 
                 Detect/Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 46 
               
             
            
               
                   
               
               
                 AML 
               
            
           
           
               
               
               
               
               
            
               
                 Disease Statu   
                 Source 
                 Cohort 
                 N 
                 Detect/Spec 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Normal Bloo   
                 GSE40279 
                 Validation 
                 656 
                 Detect/Spec 
               
               
                 Normal Bloo   
                 GSE61496 
                 Validation 
                 312 
                 Detect/Spec 
               
               
                 AML 
                 GSE86409 
                 Validation 
                 79 
                 Detect/Spec 
               
               
                 AML 
                 TCGA 
                 Validation 
                 140 
                 Detect/Spec 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     BCD Method 
     The following are the steps of the Binary Categorical Differentiation method (BCD) invented by the inventors to discover polygenic DNA methylation markers for early prediction of different cancers. 
     We filtered the 28,754 CGIDs that are robustly unmethylated in normal tissues. 
     For the discovery cohort we delineated within the list of 28,754 CG IDs that are robustly unmethylated in normal tissues, CGIDs that are categorically methylated in a particular cancer and are not methylated in unaffected tissue and normal tissues using the COUNTIF and IF functions in Microsoft excel. 
     NmcCGIDx=COUNTIF (betaCGIDxCancer n 1 :n i ,“&gt;0.2”)
 
NmnCGIDx=COUNTIF (betaCGIDxNormal n 1 :n i ,“&gt;0.1”)
 
     DMCGIDx=IF((AND(NmcCGIDx&gt;0, NmnCGIDx=0)),“TRUE”,“FALSE”) 
     DM CGIDx were sorted from highest to lowest number
 
Up to 20 top TRUE DM CGIDx positions were selected
 
NmcCGID x =Number of cancer patients with methylated CGIDx
 
Nmn=number of normal adjacent or similar tissue samples with methylated CGIDx
 
betaCGIDx=level of methylation of CGIDx
 
n=patient from 1 to i
 
DM=differentially methylated CGIDx
 
     The inventors noticed that testicular and kidney cancer exhibit pervasive lack of methylation at CG IDs that are highly methylated in all tissues. We therefore used a modification of the BCD method to discover categorically differentially methylated CG ID positions for testicular and kidney (renal) cancer which we term “BCDhypo”; categorically unmethylated in cancer and methylated in normal tissue. The following steps were used for discovery of Differentially hypomethylated CGID positions in testicular and kidney cancer. 
     For the discovery cohort we delineated hypomethylated CGIDs in testis or kidney that are fully methylated in normal tissues using the COUNTIF and IF functions in excel. 
     NucCGIDx=COUNTIF (betaCGIDxCancer n i :n i ,“&lt;0.2”)
 
NunCGIDx=COUNTIF (betaCGIDxNormal n i :n i ,“&lt;0.9”)
 
     DHMCGIDx=IF((AND(NucCGIDx&gt;0, NunCGIDx=0)),“TRUE”,“FALSE”) 
     DHM CGID positions were sorted from highest to lowest number
 
20 top TRUE DHM sites were selected and subjected to penalized regression analysis
 
NucCGIDx=Number of cancer patients with unmethylated CGID X
 
NunCGIDx=number of normal tissue samples with unmethylated CGID X
 
n=patient from 1 to i
 
DHM=differentially hypomethylated CGID
 
     The inventors then performed on the top 20 DM (or DHM) CGIDx a penalized regression using the package penalized in R to delineate the minimal combination of CGIDx that predicts cancer at highest sensitivity and specificity. The polygenic combination of CGIDx was further tested in a multivariate linear regression equation to determine the regression coefficient between methylation levels of these CGIDs in the polygenic combination and cancer. The model was used to calculate a methylation score for each patient for a typical cancer. 
         Ms=α+Σ   i=1   n β i   CG   i  
 
     Ms=methylation score, α=intercept, β i =coefficient for CG ID i , CG i =methylation level per CG in combination. 1 to i=number of CGs in combination. 
     Embodiment 3. Discovery of a Polygenic DNA Methylation Marker for Liver Cancer (HCC) 
     The inventors used normalized Illumina 450K DNA methylation data from GSE61258 (normal liver) and 66 randomly selected samples from the TCGA HCC collection of HCC DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDx that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect HCC with high sensitivity and specificity in the training cohort ( FIG. 5B , Table 1) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 8 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between HCC and other tumors ( FIG. 5C , Table 2) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 4. Utility of HCC Polygenic DNA Methylation Markers for Detecting HCC 
     The inventors then demonstrated that the weighted HCC DNA methylation score detected HCC in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from GSE76269 for 227 HCC patients for the CGIDs in Table 1. Using this method, 95% of the HCC samples were detected as HCC ( FIG. 6C ). A ROC curve presented in  FIG. 6A  reveals the specificity (1) and sensitivity (0.96) of this methylation score for detecting cancer. The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for detecting HCC and differentiating between HCC and other cancers using a “validation cohort” with methylation data from GSE75041 and TCGA for HCC and 8 other types of cancer. A ROC curve presented in  FIG. 7B  reveals the specificity (0.97) and sensitivity (0.95) of this methylation score for differentiating HCC from other normal tissue and other cancers. These DNA methylation markers and the calculated methylation score could be used for screening and early detection of cancer in people at risk as well as the general healthy population using different biomaterial from people such as tissue, feces, saliva, plasma and urine. 
     Embodiment 5. Discovery of a Polygenic DNA Methylation Marker for Lung Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 10 people from GSE61258 (normal lung) and 10 randomly selected samples from the TCGA lung cancer collection of lung cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect lung cancer with high sensitivity and specificity (Samples included both adenocarcinoma and squamous cell carcinoma) in the training cohort ( FIG. 8B , Table 3) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 8 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between lung cancer and other tumors ( FIG. 8C , Table 4) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 6. Utility of Lung Polygenic DNA Methylation Markers for Detecting Lung Cancer 
     The inventors then demonstrated that the weighted lung cancer DNA methylation score and threshold developed in embodiment 3 (detect) detects lung cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from GSE66836, GSE63704, GSE76269 and 919 lung cancer patients from TCGA. Using this method 96% of the lung cancer samples were detected as lung cancer ( FIG. 9A ). The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for differentiating between lung cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for lung cancer and 8 other types of cancer ( FIG. 9A ). A ROC curve presented in  FIG. 9B  reveals the specificity (0.96) and sensitivity (0.84) of this methylation score for detecting lung cancer from other normal tissue and other cancers ( FIG. 9C ). These DNA methylation markers and the calculated methylation score could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 7. Discovery of a Polygenic DNA Methylation Marker for Prostate Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 5 people from GSE52955 (normal prostate) and 10 randomly selected samples from the TCGA prostate cancer collection of prostate cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect prostate cancer with high sensitivity and specificity in the training cohort ( FIG. 10 b   , Table 5) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 8 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between prostate cancer and other tumors ( FIG. 10C , Table 6) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 8. Utility of Prostate Cancer Polygenic DNA Methylation Markers for Detecting Prostate Cancer 
     The inventors then demonstrated that the weighted prostate cancer DNA methylation score and threshold developed in embodiment 3 (detect) detects prostate cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from GSE73549, GSE2955, and 430 prostate cancer patients from TCGA. Using this method 99% of the prostate cancer samples were detected as prostate cancer ( FIG. 11A ). The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for differentiating between prostate cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for prostate cancer and 8 other types of cancer ( FIG. 11A ). A ROC curve presented in  FIG. 11B  reveals the specificity (0.99) and sensitivity (0.98) of this methylation score for detecting prostate cancer from other normal tissue and other cancers ( FIG. 11C ). These DNA methylation markers and the calculated methylation score could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 9. Discovery of a Polygenic DNA Methylation Marker for Breast Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 17 people from GSE60185(normal breast) and 10 randomly selected samples from the TCGA breast cancer collection of breast cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect breast cancer with high sensitivity and specificity in the training cohort ( FIG. 12B , Table 7) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 8 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between breast cancer and other tumors ( FIG. 12C , Table 8) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 10. Utility of Breast Cancer Polygenic DNA Methylation Markers for Detecting Breast Cancer 
     The inventors then demonstrated that the weighted breast cancer DNA methylation score and threshold developed in embodiment 9 (detect) detects breast cancer in a “validation cohort” that included 891 breast cancer patients using normalized Illumina 450K DNA methylation beta values from GSE60185, GSE75067, and from TCGA. Using this method 91% of the breast cancer samples were detected as breast cancer ( FIG. 13A ) and both DCIS and invasive cancers were detected. The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for differentiating between breast cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for breast cancer and 8 other types of cancer ( FIG. 14A ). A ROC curve presented in  FIG. 14B  reveals the specificity (0.89) and sensitivity (0.87) of this methylation score for differentiating breast cancer from other normal tissue and other cancers ( FIG. 14C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of breast cancer in women at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 11. Discovery of a Polygenic DNA Methylation Marker for Colorectal Cancer (CRC) 
     The inventors used normalized Illumina 450K DNA methylation data for 25 people from GSE(32146) (normal) and 50 randomly selected samples from the TCGA colorectal cancer collection of colorectal cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect colorectal cancer with high sensitivity and specificity in the training cohort ( FIG. 15B , Table 9) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 8 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between colorectal cancer and other tumors ( FIG. 15C , Table 10) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 12. Utility of Colorectal Cancer Polygenic DNA Methylation Markers for Detecting Colorectal Cancer 
     The inventors then demonstrated that the weighted colorectal cancer DNA methylation score and threshold developed in embodiment 11 (detect) detects colorectal cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from GSE69550 and 459 colorectal cancer patients from TCGA. Using this method 98% of the colorectal cancer samples were detected as colorectal cancer ( FIG. 16A ). The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for differentiating between colorectal cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for colorectal cancer and 8 other types of cancer ( FIG. 16A ). A ROC curve presented in  FIG. 16B  reveals the specificity (0.96) and sensitivity (0.98) of this methylation score for detecting colorectal cancer from other normal tissues and other cancers ( FIG. 16C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk for CRC as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 13. Discovery of a Polygenic DNA Methylation Marker for Pancreatic Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 12 people from GSE53051 (normal) and 20 randomly selected samples from the TCGA collection of pancreatic cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect pancreatic cancer with high sensitivity and specificity in the training cohort ( FIG. 17B , Table 11) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 100 randomly selected DNA methylation samples from TCGA representing 10 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between pancreatic cancer and other tumors ( FIG. 17C , Table 12) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 14. Utility of Pancreatic Cancer Polygenic DNA Methylation Markers for Detecting Pancreatic Cancer 
     The inventors then demonstrated that the weighted pancreatic cancer DNA methylation score and threshold developed in embodiment 13 (detect) detects pancreatic cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 891 pancreatic cancer patients from TCGA. Using this method 86% of the pancreatic cancer samples were detected as pancreatic cancer ( FIG. 18A ). The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for differentiating between pancreatic cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for pancreatic cancer and 9 other types of cancer ( FIG. 18A ). A ROC curve presented in  FIG. 18B  reveals the specificity (0.93) and sensitivity (0.86) of this methylation score for detecting pancreatic cancer and differentiating it from other normal tissue and other cancers ( FIG. 18C ). These DNA methylation markers and the calculated methylation scores from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 15. Discovery of a Polygenic DNA Methylation Marker for Brain Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 10 people from GSE65820 (normal) and 10 randomly selected samples from the TCGA collection of brain cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a set of binary categorical differentially methylated CGIDs that detect brain cancer with high sensitivity and specificity in the training cohort ( FIG. 19B , Table 13) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 110 randomly selected DNA methylation samples from TCGA representing 11 different tumor types. The inventors used this training cohort and discovered that the detect CGID also differentiates between brain cancer and other tumors ( FIG. 19C , Table 13) (detect-spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 16. Utility of Brain Cancer Polygenic DNA Methylation Markers for Detecting Brain Cancer 
     The inventors then demonstrated that the weighted brain cancer DNA methylation score and threshold developed in embodiment 15 (detect) detects brain cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 689 brain cancer patients from TCGA, 40 patients from GSE58298 and 136 patients from GSE36278. Using this method 91-97% of the brain cancer samples were detected as brain cancer ( FIG. 20A ). The inventors then demonstrated the utility of the same CGIDs for differentiating between brain cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for brain cancer and 9 other types of cancer ( FIG. 20A ). A ROC curve presented in  FIG. 22B  reveals the specificity (1) and sensitivity (0.97) of this methylation score for detecting brain cancer from other normal tissue and other cancers ( FIG. 20C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 17. Discovery of a Polygenic DNA Methylation Marker for Stomach Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 18 people from GSE99553 (normal) and 10 randomly selected samples from the TCGA collection of stomach cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect stomach cancer with high sensitivity and specificity in the training cohort ( FIG. 21B , Table 14) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 100 randomly selected DNA methylation samples from TCGA representing 11 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between stomach cancer and other tumors ( FIG. 21C , Table 15) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 18. Utility of Stomach Cancer Polygenic DNA Methylation Markers for Detecting Stomach Cancer 
     The inventors then demonstrated that the weighted stomach cancer DNA methylation score and threshold developed in embodiment 17 (detect) detects stomach cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 397 stomach cancer patients from TCGA. Using this method, 88% of the stomach cancer samples were detected as stomach cancer ( FIG. 23A ). The inventors then demonstrated the utility of the combined spec and detect DNA methylation scores for differentiating between stomach cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for stomach cancer and 10 other types of cancer ( FIG. 23A ). A ROC curve presented in  FIG. 22B  reveals the specificity (0.9 for) and sensitivity (0.9) of this methylation score for detecting stomach cancer from other normal tissues and other cancers ( FIG. 22C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for screening and early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 19. Discovery of a Polygenic DNA Methylation Marker for Ovarian Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 5 people from GSE65820 (normal) and 10 randomly selected samples from the TCGA collection of ovarian cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect ovarian cancer with high sensitivity and specificity in the training cohort ( FIG. 23B , Table 16) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 100 randomly selected DNA methylation samples from TCGA representing 10 different tumor types and blood. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between ovarian cancer and other tumors ( FIG. 2C , Table 17) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 20. Utility of Ovarian Cancer Polygenic DNA Methylation Markers for Detecting Ovarian Cancer 
     The inventors then demonstrated that the weighted ovarian cancer DNA methylation score and threshold developed in embodiment 19 (detect) detects ovarian cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 114 ovarian cancer patients from TCGA. Using this method 86% of the ovarian cancer samples were detected as ovarian cancer ( FIG. 24A ). The inventors then demonstrated the utility of the spec DNA methylation scores for differentiating between ovarian cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for ovarian cancer and 9 other types of cancer ( FIG. 24A ). A ROC curve presented in  FIG. 24B  reveals the specificity (0.99) and sensitivity (1) of this methylation score for differentiating ovarian cancer from other normal tissue and other cancers ( FIG. 24C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 21. Discovery of a Polygenic DNA Methylation Marker for Cervical Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 20 people from GSE46306 (normal) and 10 randomly selected samples from the TCGA collection of cervix cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect cervical cancer with high sensitivity and specificity in the training cohort ( FIG. 25B , Table 18) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 8 different tumor types and blood. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between cervical cancer and other tumors ( FIG. 25C , Table 19) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 22. Utility of Cervical Cancer Polygenic DNA Methylation Markers for Detecting Cervical Cancer 
     The inventors then demonstrated that the weighted cervical cancer DNA methylation score and threshold developed in embodiment 21 (detect) detects cervical cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 313 cervical cancer patients from TCGA. Using this method 91% of the cervical cancer samples were detected as cervical cancer ( FIG. 26A ). The inventors then demonstrated the utility of the spec DNA methylation scores for differentiating between cervical cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for cervical cancer and 9 other types of cancer ( FIG. 26A ). A ROC curve presented in  FIG. 26B  reveals the specificity (0.9) and sensitivity (0.9) of this methylation score for detecting cervical cancer and differentiating it from other normal tissue and other cancers ( FIG. 26C ). These DNA methylation markers and the calculated methylation scores from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 23. Discovery of a Polygenic DNA Methylation Marker for Head and Neck Squamous Carcinoma (HNSC) 
     The inventors used normalized Illumina 450K DNA methylation data for 10 people from GSE(52068) (normal) and 10 randomly selected samples from the TCGA cancer collection of HNSC DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect HNSC with high sensitivity and specificity in the training cohort ( FIG. 27B , Table 20) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 12 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between HNSC and other tumors ( FIG. 27C , Table 21) (spec). 
     Embodiment 24. Utility of Head and Neck Squamous Carcinoma (HNSC) Polygenic DNA Methylation Markers for Detecting HNSC 
     The inventors then demonstrated that the weighted HNSC DNA methylation score and threshold developed in embodiment 23 (detect) detects HNSC in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from GSE52068 and. Using this method 88%-96% of the HNSC samples were detected ( FIG. 28A ). The inventors then demonstrated the utility of the DNA methylation detect scores for differentiating between HNSC and other cancers using a “validation cohort” with methylation data from GSE and TCGA for HNSC and 12 other types of cancer ( FIG. 28A ). A ROC curve presented in  FIG. 28B  reveals the specificity (0.86) and sensitivity (0.88) of this methylation score for differentiating HNSC from other normal tissue and other cancers ( FIG. 28C ). The markers also detect several other cancers (at relatively high sensitivity and is thus of limited specificity for these cancers). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 25. Discovery of a Polygenic DNA Methylation Marker for ESOPHAGEAL CANCER 
     The inventors used normalized Illumina 450K DNA methylation data for 10 people from GSE(52068) (normal) and 10 randomly selected samples from the TCGA cancer collection of esophageal cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect esophageal cancer with high sensitivity and specificity in the training cohort ( FIG. 29B , Table 22) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 12 different tumor types. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between esophageal cancer and other tumors ( FIG. 29C , Table 23) (spec). 
     Embodiment 26. Utility of ESOPHAGEAL CANCER Polygenic DNA Methylation Markers for Detecting ESOPHAGEAL CANCER 
     The inventors then demonstrated that the weighted esophageal cancer DNA methylation score and threshold developed in embodiment 25 (detect) detects esophageal cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from GSE52068 and. Using this method 88%-96% of the esophageal cancer samples were detected ( FIG. 30A ). The inventors then demonstrated the utility of the detect DNA methylation scores for differentiating between esophageal cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for esophageal cancer and 12 other types of cancer ( FIG. 30A ). A ROC curve presented in  FIG. 30B  reveals the specificity (0.86) and sensitivity (0.88) of this methylation score for differentiating esophageal cancer from other normal tissue and other cancers ( FIG. 30C ). The markers also detect several other cancers (at relatively high sensitivity and are thus of limited specificity for these cancers). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 27. Discovery of a Polygenic DNA Methylation Marker for Bladder Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for 5 people from GSE52955 (normal) and 10 randomly selected samples from the TCGA collection of bladder cancer DNA methylation data as a “training” cohort. The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detects bladder cancer with high sensitivity and specificity in the training cohort ( FIG. 31B , Table 24) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. The inventors then generated a “training cohort” from 80 randomly selected DNA methylation samples from TCGA representing 13 different tumor types and normal blood. The inventors used this training cohort to discover a polygenic set of differentially methylated CGIDs between bladder cancer and other tumors ( FIG. 31C , Table 25) (spec). A weighted DNA methylation score was developed for the CGIDs as described in embodiment 2. 
     Embodiment 28. Utility of Bladder Cancer Polygenic DNA Methylation Markers for Detecting Bladder Cancer 
     The inventors then demonstrated that the weighted bladder cancer DNA methylation score and threshold developed in embodiment 27 (detect) detects bladder cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 439 bladder cancer patients from TCGA. Using this method 96% of the bladder cancer samples were detected as bladder cancer ( FIG. 32B ). The inventors then demonstrated the utility of the spec DNA methylation scores for differentiating between bladder cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for bladder cancer and 13 other types of cancer ( FIG. 32B ). A ROC curve presented in  FIG. 32C  reveals the specificity (0.86) and sensitivity (0.88) of this methylation score for detecting bladder cancer from other normal tissue and other cancers ( FIG. 32C ). There is cross detection at reasonably high rates however of stomach, pancreatic esophageal and colorectal cancer. These DNA methylation markers and the calculated methylation scores from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 29. Discovery of a Polygenic DNA Methylation Marker for Kidney Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for kidney (renal) cancer from 10 people from GSE52955 (normal) and 10 randomly selected samples per cancer from 13 cancers in the TCGA dataset as a “training” cohort as well as normal tissues and blood (GSE40279, GSE 52955). The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that are robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD hypo method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect kidney cancer with high sensitivity and specificity in the training cohort and are specific for kidney cancer against other cancers “Detect-Spec” ( FIG. 33B , Table 26) (detect-spec). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. 
     Embodiment 30. Utility of Kidney Cancer Polygenic DNA Methylation Markers for Detecting Kidney Cancer 
     The inventors demonstrated that the weighted kidney cancer DNA methylation score and threshold developed in embodiment 27 (“Detect-Spec”) detects kidney cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 871 kidney cancer patients from TCGA and differentiate kidney cancer from other cancers. Using this method 90% of the kidney cancer samples were detected as kidney cancer ( FIG. 34A ). The inventors then demonstrated the utility of the “Detect-Spec” DNA methylation scores for differentiating between kidney cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for kidney cancer and 13 other types of cancer ( FIG. 34A ). A ROC curve presented in  FIG. 34B  reveals the specificity (0.87) and sensitivity (0.91) of this methylation score for detecting kidney cancer from other normal tissue and other cancers ( FIG. 34C ) (high crossover with HCC, brain and testis). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of kidney cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 31. Discovery of a Polygenic DNA Methylation Marker for Testicular Cancer 
     The inventors used normalized Illumina 450K DNA methylation data for testicular cancer from 10 people from GSE46306 (normal) and 10 randomly selected samples per cancer from 13 cancers in the TCGA dataset as a “training” cohort as well as normal tissues and blood (GSE40279, GSE 61496). The inventors first shortlisted in the “training cohort” dataset 28754 CGIDs that were discovered in embodiment 1 as sites that robustly unmethylated across normal tissues and blood samples. The inventors then used the BCD hypo method described in embodiment 2 to discover a polygenic set of binary categorical differentially methylated CGIDs that detect testicular cancer with high sensitivity and specificity in the training cohort and are specific for testicular cancer against other cancers “Detect-Spec” ( FIG. 35B , Table 27) (detect-spec). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. 
     Embodiment 32. Utility of Testicular Cancer Polygenic DNA Methylation Markers for Detecting Testis Cancer 
     The inventors then demonstrated that the weighted testicular cancer DNA methylation score and threshold developed in embodiment 31 (“Detect-Spec”) detects testicular cancer in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 156 testicular cancer patients from TCGA and differentiate testicular cancer from other cancer. Using this method 96% of the testicular cancer samples were detected as testicular cancer ( FIG. 36A ). The inventors then demonstrated the utility of the “Detect-Spec” DNA methylation scores for differentiating between testicular cancer and other cancers using a “validation cohort” with methylation data from GSE and TCGA for testicular cancer and 13 other types of cancer ( FIG. 36A ). A ROC curve presented in  FIG. 36B  reveals the specificity (0.97) and sensitivity (0.96) of this methylation score for detecting testicular cancer from other normal tissue and other cancers ( FIG. 36C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 33. Discovery of a Polygenic Pan-Cancer DNA Methylation Marker for 13 Common Solid Tumors 
     The inventors used normalized Illumina 450K DNA methylation data for 10 randomly selected samples per cancer from 13 cancers (bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, HNSC, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer) in the TCGA dataset as a “training” cohort as well as normal tissues and blood from TCGA an GEO. The inventors then performed a penalized regression on the combined list of CGIDs for detection of 10 different cancer listed in tables x-y and shortlisted CGIDs that detect any of the 10 common cancers at high sensitivity and specificity ( FIG. 37B , Table 28) (detect). A weighted DNA methylation score and a threshold value for cancer was developed for the CGIDs as described in embodiment 2. 
     Embodiment 34. Utility of Pan-Cancer Polygenic DNA Methylation Markers for Detecting Cancer 
     The inventors then demonstrated that the weighted cancer DNA methylation score and threshold developed in embodiment 33 (“Detect”) detects 13 common cancers (bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, HNSC, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer) in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 3644 cancer patients from TCGA from other normal tissues. Using this method 90-95% of cancer samples were detected ( FIG. 38A ). A ROC curve presented in  FIG. 38B  reveals the specificity (0.99) and sensitivity (0.95) of this methylation score for detecting 13 cancers from other normal tissue ( FIG. 38C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of cancer in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 35. Discovery of a Polygenic DNA Methylation Marker for Detection of Melanoma 
     The inventors used normalized Illumina 450K DNA methylation data for 10 randomly selected melanoma samples and 220 samples from other cancers (bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, HNSC, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer) and normal blood in the TCGA and GEO datasets as a “training” cohort. The inventors then performed a penalized regression on the combined list of CGIDs for detection of melanoma and shortlisted CGIDs that detect melanoma at high sensitivity and specificity ( FIG. 39 , Table 28) (detect-spec). A weighted DNA methylation score and a threshold value for melanoma was developed for the CGIDs as described in embodiment 2. 
     Embodiment 36. Utility of Melanoma Polygenic DNA Methylation Marker for Detecting Melanoma 
     The inventors then demonstrated that the weighted melanoma DNA methylation score and threshold developed in embodiment 35 (“Detect-spec”) detects melanoma in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 475 melanoma patients from TCGA from other cancer and normal tissues. Using this method 98% of melanoma samples were detected ( FIG. 40A ). A ROC curve presented in  FIG. 40B  reveals the specificity (0.98) and sensitivity (0.95) of this methylation score for detecting melanoma from other normal tissue and other cancers ( FIG. 40C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of melanoma in people at risk as well as the general healthy population using different biomaterial from the patient from tissues, feces, saliva, plasma and urine. 
     Embodiment 37. Discovery of a Polygenic DNA Methylation Marker for Detection of Acute Myeloid Leukemia (AML) 
     The inventors used normalized Illumina 450K DNA methylation data for 10 randomly selected AML samples and 10 normal blood samples in the GEO datasets as a “training” cohort. The inventors then performed a penalized regression on the combined list of CGIDs for detection of AML and shortlisted CGIDs that detect melanoma at high sensitivity and specificity ( FIG. 41 , Table 27) (detect-spec). A weighted DNA methylation score and a threshold value for melanoma was developed for the CGIDs as described in embodiment 2. 
     Embodiment 38. Utility of Acute Myeloid Leukemia (AML) Polygenic DNA Methylation Markers for Detecting AML in Blood DNA 
     The inventors then demonstrated that the weighted melanoma DNA methylation score and threshold developed in embodiment 37 (“Detect-spec”) detects AML in a “validation cohort” that included normalized Illumina 450K DNA methylation beta values from 79 AML patients from GEO and 140 patients from TGCA and normal blood. Using this method 100% of AIL samples were detected ( FIG. 42A ). A ROC curve presented in  FIG. 42B  reveals the specificity (1) and sensitivity (1) of this methylation score for detecting AML from blood ( FIG. 42C ). These DNA methylation markers and the calculated methylation score from the methylation values could be used for early detection of AML in people at risk as well as the general healthy population using blood DNA. 
     Embodiment 39. Bisulfite Conversion, Multiplex Amplification and Next Generation Sequencing and Calculation of a Methylation Score for Predicting Prostate Cancer 
     Blood was collected in 9-ml tubes containing K3-EDTA and processed within 1 h. Fresh blood samples were centrifuged at 1000 g for 10 min at 4° C. The supernatant was carefully transferred to a Falcon tube without disturbing the cellular layer and centrifuged again for 10 min for complete removal of any residual cells and frozen at −80 C. Plasma samples are thawed, and DNA is extracted by several available methods and commercially available kits for plasma DNA extraction such as Qiagen kit for plasma DNA or EZ DNA direct extraction method. DNA is purified using commercially available methods such as on AMPure XP magnetic beads and purified DNA is treated with sodium bisulfite using for example the EZ DNA bisulfite treatment kit. A library of targeted sequences is generated by two step PCR reactions ( FIG. 40 ). The first PCR reaction targets the specific CGIDs from table 5 and 6, note PCR1 primer has complementary sequence to second PCR2 primers ( FIG. 40 ). The inventors used human bisulfite converted genomic DNA from HEK293 cells to amplify concurrently three sequences of DNA that contain CGIDs that detect prostate cancer from HIF3A (232 base pairs region), TPM4 (213 base pairs region), and CTTN (199 base pairs region) in a multiplexed PCR reaction using the following primers in a standard Taq polymerase reaction: For CGID cg02879662; 
                            5′ACACTCTTTCCCTACACgACgCTCTT                   CCgATCTNNNNNGGTAGGAGTTTTGGG                   forward primer:           AATTGG3′                   and           reverse primer:           5′gTgACTggAgTTCAgACgTgTgCTCTT                   CCgATCTCCACCCCTACAATCCCTAA3′                   For CGID cg16232979;           forward primer:           5′ACACTCTTTCCCTACACgACgCTCTTCC                   gATCTNNNNNYGGTTTYGGGTTTYGTATT3′                   and reverse primer:           5′gTgACTggAgTTCAgACgTgTgCTCTTCCg                   ATCTACRCAAAAATATAAATCRACRATC3′                   ForCGID: cg14041701 and           cg14498227; forward primer:           5′ACACTCTTTCCCTACACgACgCTCTTCCgA                   TCTNNNNNGTTTTGYGTTTYGGATTTGGGTT3′                   and reverse primer:           5’gTgACTggAgTTCAgToACgTgTgCTCTT                   CCgATCTCATAAACAACACCTTTAAATAAA                   CACTAAA3′.            
The amplified fragments were fractionated on an agarose gel
 
To barcode the samples, we use a second PCR reaction with the following primers:
 
                            Forward primer:           5′AATgATACggCgACCACCgAgATCTACACT                   CTTTCCCTACACgAC3′                           Barcoding primer (reverse):           5′CAAgCAgAAgACggCATACgAgAT AGTCAT                       CG gTgACTggAgTTCAgACgTg3′            
(bold bases are the index; 200 variations of this index are used. The second set of primers introduces the index for each patient as well as the reverse and forward sequencing primers. A multiplex PCR1 reaction for the three markers of prostate cancer HIF3A 232 bp, TPM4 213 bp, and CTTN 199 bp is shown on the right panel using varying primer concentrations as indicated in  FIG. 41 .
 
     Embodiment 40: Utility of the Method of Bisulfite Conversion, Multiplex Amplification and Next Generation Sequencing and Calculation of a Methylation Score for Predicting Cancer 
     The inventors demonstrate that embodiment 35 could be used for high throughput prediction of prostate cancer and other cancers using plasma samples from hundreds of patients at the same time. Indexed amplification of highly predictive CG IDs and a streamlined method for calculating a methylation score that is indicative of cancer could be used for early detection of prostate cancer and any other cancer. 
     Embodiment 41. Demonstration that the Biomarkers Selected Exhibit True BCD Properties are Totally Hypomethylated in Plasma from Healthy People 
     Plasma DNA was extracted from plasma prepared from 40 healthy individuals and was subjected to targeted amplification with cancer specific primers for the following cancers: liver, prostate, lung ( FIG. 43 ) and stomach, pan cancer and CRC ( FIG. 44 ) followed by barcoding using a second set of amplifications (PCR 2) and next generation sequencing as described in embodiments 39 and 40. All CGs exhibited very low levels of methylation in plasma from healthy people ( FIGS. 43 and 44 ). 
     Embodiment 42. Bioinformatics Work-Flow for Determining DNA Methylation Levels 
     PCR2 products are combined quantified and purified and subjected to next generation sequencing on a Miseq Illumina sequencer. Sequence is demultiplexed using Illumina software for indexed sequencing, FASTQ files are generated for each patient. A Perl text editing script https://www.activestate.com/activeperl/downloads is use to count Ts and Cs in the FASTQ files per patient per CG IDS and quantify the fraction of methylated Cs in a CG ID in a patient by dividing the number of C/C+T. (see scheme in  FIG. 42 ). The output CSV file is used for calculating the methylation score (Ms) for each patient using the equation: Ms=α+Σ i=1   n β i CG i  when α=intercept β=coefficient for CG ID i, CG=methylation level per CG in the combination of CG IDs from 1 to n. n=number of CGs in combination. MS=methylation score. 
     Applications of the Inventive Subject Matter 
     The applications of the inventive subject matter are in the field of molecular diagnostics and early prediction of cancer in general. Any person skilled in the art could use the present inventive subject matter to derive similar noninvasive biomarkers for early prediction of other cancers and other diseases that are accompanied by cell death and shedding of cell free DNA into the system such as neurological diseases, diabetes, heart disease such as cirrhosis and damage to heart tissue in cardiovascular disease. The present inventive subject matter provides a path to finding exquisite methylation markers of specific cell types and tissues using the BCD and BCDhypo method. Also disclosed are methods and biomarkers for early prediction of a wide range of cancers that could be used by anyone skilled in the art to detect cancer early and dramatically enhance survival rates and cure from cancer. The methods disclosed by the present invention could be used by anyone skilled in the art for routine yearly screening of healthy populations, to identify people who are starting to develop cancer and to treat them immediately and prevent the dire personal social and economic consequences of cancer mortality and morbidity, as well as for monitoring “high risk” people and monitoring response to therapy in patients undergoing treatment to detect recurrence or metastasis. Adoption of the present invention described here for routine health care management by health providers and health check-up facilities will have a vast impact on reducing the burden of cancer as well as health care costs. 
     The fact that the inventive subject matter includes a number of different dependent claims does not mean that one cannot use a combination of these claims for predicting cancer. The embodiments disclosed herein for measuring and statistically analyzing and predicting cancer should not be considered limiting. Various other modifications will be apparent to those skilled in the art to measure DNA methylation in cancer patients such as Illumina EPIC arrays, capture array sequencing, next generation sequencing, methylation specific PCR, epityper, restriction enzyme-based analyses and other methods found in the public domain. Similarly, there are numerous statistical methods in the public domain in addition to those listed here to use the inventive subject matter for prediction of cancer in patient samples. 
     Although the inventive subject matter has been explained in relation to its embodiments including one or more preferred embodiments, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the claimed subject matter.