Cell-free dna hydroxymethylation profiles in the evaluation of pancreatic lesions

Abstract

Disclosed herein are methods for identifying patients with pancreatic cancer and subjects at risk for developing pancreatic cancer, methods for monitoring a patient with an identified pancreatic lesion, methods for evaluating the effectiveness of a treatment used for a patient with pancreatic cancer, and methods for selecting a therapy for treating pancreatic cancer in a particular patient. The invention makes use of hydroxymethylation biomarkers, which in combination with one or more clinical parameters and optionally one or more additional types of biomarkers and/or patient-specific risk factors, exhibit a hydroxymethylation level that correlates with pancreatic cancer. Kits and other methods of use are also provided.

1 . A method for evaluating the risk that an identified pancreatic lesion in a patient is cancerous, the method comprising:
 (a) obtaining a cell-free DNA sample from the patient;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker; and   (e) calculating an index value representing the risk that the pancreatic lesion is cancerous from the comparison in step (d) combined with at least one additional parameter correlated with the risk that an individual has pancreatic cancer.   
     
     
         2 . The method of  claim 1 , wherein the plurality of additional parameters is selected from lesion size; lesion location; presence or absence of pancreatic inflammation; jaundice; presence or absence of other symptoms; patient age; weight; gender; ethnicity; family history; genetic mutations; diabetes; physical activity; diet; pro-inflammatory cytokine levels; and smoking status of the patient. 
     
     
         3 . The method of  claim 1 , wherein the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have not had a pancreatic lesion. 
     
     
         4 . The method of  claim 3 , wherein the patient has a risk factor for pancreatic cancer and the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have the risk factor. 
     
     
         5 . The method of  claim 4 , wherein the risk factor is pancreatitis and the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have been diagnosed with pancreatitis. 
     
     
         6 . The method of  claim 4 , wherein the pancreatic lesion was identified by imaging, and the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have had a pancreatic lesion identified on an imaging scan. 
     
     
         7 . The method of  claim 1 , wherein the cell-free DNA sample is extracted from a blood sample. 
     
     
         8 . The method of  claim 1 , wherein the cell-free DNA sample is extracted from pancreatic cyst fluid. 
     
     
         9 . The method of  claim 1 , further comprising generating a report indicating the index value calculated in (e). 
     
     
         10 . The method of  claim 6 , further comprising forwarding the report to a medical practitioner. 
     
     
         11 . The method of  claim 1 , wherein the pancreatic cancer is an exocrine pancreatic cancer. 
     
     
         12 . The method of  claim 11 , wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). 
     
     
         13 . The method of  claim 9 , wherein at least one hydroxymethylation biomarker exhibits an increase in hydroxymethylation level in the patient relative to the reference hydroxymethylation profile. 
     
     
         14 . The method of  claim 9 , wherein at least one hydroxymethylation biomarker exhibits a decrease in hydroxymethylation level in a patient relative to the reference hydroxymethylation profile. 
     
     
         15 . The method of  claim 1 , wherein the hydroxymethylation biomarkers comprise loci that are associated with one or more of the following genes: ADARB2-AS1, ANKRD36B, ASAH2B, ATG4B, ATP8B1, BOLA1, C11orf88, C17orf97, C1orf170, C3orf36, C8orf74, CAMSAP2, CCDC54, CCDC59, CKAP2, CLK2P, CRTC1, CSRP2, CYB5D1, DNAJC27, DYNAP, FAM166A, FAM188B, FAM196A, FAM86JP, FAT4, FBXO5, FGF2, FUT2, GAS2L2, GAS6, GGACT, GLRX5, GPX1, GPX5, HBD, HLA-A, HTR1F, IL36G, KANSL1, KCNH6, KCTD15, KLHL38, KLK2, KRT6B, LAMC1, LGALS14, LGALS8-AS1, LIFR, LINC00266-1, LINC00310, LOC100130452, LOC100130557, LOC100130894, LOC100288778, LOC100505633, LOC100505648, LOC100505738, LOC100652909, LOC389033, LOC90784, LRRC37A2, MED11, MRPL23-AS1, NAT8L, NEUROD1, NEUROG2, NMES, NOMO3, NPRL2, NXN, ODF3L1, ODF3L2, OSCP1, PARD6G, PGAM1, PLA2G2E, PLSCR4, PPAP2A, PPP1R15A, PPP1R3E, RASL10B, REXO1L1, RIMBP3, RNF126P1, RNU6-76, RPP25, RPS27, SH3PXD2B, SHISA4, SLC25A38, SLC4A1, SLCO5A1, SPDEF, SRSF6, STRA6, SYNM, TBCB, TDRD6, TEX26, TMEM253, TNFSF13B, TTC14, TUBA4A, UBB, VAMP8, VGLL2, WASH2P, WNT9B, XBP1, and ZNF789. 
     
     
         16 . The method of  claim 1 , wherein the hydroxymethylation biomarkers comprise loci that are associated with one or more of the following genes: GATA4, GATA6, PROX1, ONECUT2, YAP1, TEAD1, ONECUT2/ONECUT1-TCGA, IGF1, and IGF2. 
     
     
         17 . The method of  claim 2 , wherein the genetic mutations comprise a mutation in a gene selected from BRCA2, BRCA1, CDKN2A, ATM, STK11, PRSS1, MLH1, PALB2, KRAS, CDKN2A, TP53, SMAD4, and combinations thereof 
     
     
         18 . The method of  claim 1 , wherein step (b) comprises ligating adapters onto the DNA, functionalizing 5hmC residues in the DNA with an affinity tag that allows selective capture of tagged cfDNA, and removing the tagged cfDNA from the sample. 
     
     
         19 . The method of  claim 18 , wherein the affinity tag is comprised of a biotin moiety, and functionalizing the 5hmC residues comprises biotinylation. 
     
     
         20 . The method of  claim 19 , wherein the biotinylation is carried out by covalently attaching a chemoselective group to 5hmC residues and then reacting the chemoselective group with a functionalized biotin moiety. 
     
     
         21 . The method of  claim 20 , wherein the chemoselective group is UDP glucose-6-azide and the functionalized biotin moiety is an alkyne-functionalized biotin, such that the chemoselective group reacts with the functionalized biotin in a click chemistry reaction. 
     
     
         22 . The method of  claim 19 , wherein the affinity tagged cfDNA is captured with a solid support having a surface functionalized with a biotin-binding protein, to provide cfDNA bound to the solid support. 
     
     
         23 . The method of  claim 22 , wherein step (b) further comprises: amplifying the cfDNA without releasing the captured cfDNA from the support, to give a plurality of amplicons; sequencing the amplicons; and quantifying the nucleic acids that map to the reference loci from the sequence reads. 
     
     
         24 . The method of  claim 23 , wherein amplification comprises PCR. 
     
     
         25 . The method of  claim 18 , wherein the adapters additionally comprise at least one unique feature identifier (UFI) sequence. 
     
     
         26 . The method of  claim 25 , wherein the at least one UFI sequence includes a source identifier UFI sequence. 
     
     
         27 . The method of  claim 25 , wherein the at least one UFI sequence is a molecular UFI that enables molecular counting. 
     
     
         28 . The method of  claim 25 , wherein the at least one UFI sequence is a molecular UFI that enables molecular counting. 
     
     
         29 . A method for monitoring an identified pancreatic lesion in a patient, the method comprising:
 (a) obtaining an initial cell-free DNA sample from the patient;   (b) enriching for hydroxymethylated DNA in the initial sample;   (c) quantifying the nucleic acids in the enriched initial sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the enriched cell-free DNA in the initial sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker;   (e) generating an initial hydroxymethylation profile for the patient comprising the hydroxymethylation level of the enriched cell-free DNA in the initial sample, at each locus;   (f) repeating steps (a) through (c) at a later time with a subsequent cell-free DNA sample obtained from the patient;   (g) generating a subsequent hydroxymethylation profile for the patient comprising the hydroxymethylation level of the enriched cell-free DNA in the subsequent sample, at each locus; and   (h) comparing, at each locus, the hydroxymethylation level of the enriched cell-free DNA in the subsequent sample to the hydroxymethylation level of the enriched cell-free DNA in the initial sample, to ascertain a change in the pancreatic lesion.   
     
     
         30 . The method of  claim 29 , wherein steps (f) through (h) are repeated at selected time intervals throughout an extended monitoring period. 
     
     
         31 . The method of  claim 29 , wherein the change in the pancreatic lesion comprises an increase in size. 
     
     
         32 . The method of  claim 29 , wherein the change in the pancreatic lesion comprises a decrease in size. 
     
     
         33 . The method of  claim 29 , wherein the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have had a pancreatic lesion identified in an imaging scan. 
     
     
         34 . The method of  claim 29 , wherein the patient has a risk factor for pancreatic cancer and the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have the risk factor. 
     
     
         35 . The method of  claim 34 , wherein the risk factor is pancreatitis and the reference hydroxymethylation profile represents a composite of a plurality of hydroxymethylation profiles for individuals who have been diagnosed with pancreatitis. 
     
     
         36 . The method of  claim 29 , wherein the cell-free DNA sample is extracted from a blood sample. 
     
     
         37 . The method of  claim 29 , wherein the cell-free DNA sample is extracted from pancreatic cyst fluid. 
     
     
         38 . The method of  claim 29 , wherein the hydroxymethylation biomarkers comprise loci that are associated with one or more of the following genes: ADARB2-AS1, ANKRD36B, ASAH2B, ATG4B, ATP8B 1, BOLA1, C11orf88, C17orf97, C1orf170, C3orf36, C8orf74, CAMSAP2, CCDC54, CCDC59, CKAP2, CLK2P, CRTC1, CSRP2, CYB5D1, DNAJC27, DYNAP, FAM166A, FAM188B, FAM196A, FAM86JP, FAT4, FBXO5, FGF2, FUT2, GAS2L2, GAS6, GGACT, GLRX5, GPX1, GPX5, HBD, HLA-A, HTR1F, IL36G, KANSL1, KCNH6, KCTD15, KLHL38, KLK2, KRT6B, LAMC1, LGALS14, LGALS8-AS1, LIFR, LINC00266-1, LINC00310, LOC100130452, LOC100130557, LOC100130894, LOC100288778, LOC100505633, LOC100505648, LOC100505738, LOC100652909, LOC389033, LOC90784, LRRC37A2, MED11, MRPL23-AS1, NAT8L, NEUROD1, NEUROG2, NMES, NOMO3, NPRL2, NXN, ODF3L1, ODF3L2, OSCP1, PARD6G, PGAM1, PLA2G2E, PLSCR4, PPAP2A, PPP1R15A, PPP1R3E, RASL10B, REXO1L1, RIMBP3, RNF126P1, RNU6-76, RPP25, RPS27, SH3PXD2B, SHISA4, SLC25A38, SLC4A1, SLCO5A1, SPDEF, SRSF6, STRA6, SYNM, TBCB, TDRD6, TEX26, TMEM253, TNFSF13B, TTC14, TUBA4A, UBB, VAMP8, VGLL2, WASH2P, WNT9B, XBP1, and ZNF789. 
     
     
         39 . The method of  claim 38 , wherein the hydroxymethylation biomarkers additionally comprise loci that are associated with one or more of the following genes: GATA4, GATA6, PROX1, ONECUT2, YAP1, TEAD1, ONECUT2/ONECUT1-TCGA, IGF1, and IGF2. 
     
     
         40 . The method of  claim 29 , wherein step (b) comprises ligating adapters onto the DNA, functionalizing 5hmC residues in the DNA with an affinity tag that allows selective capture of tagged cfDNA, and removing the tagged cfDNA from the sample. 
     
     
         41 . The method of  claim 40 , wherein the affinity tag is comprised of biotin, and functionalizing the 5hmC residues comprises biotinylation. 
     
     
         42 . The method of  claim 41 , wherein the biotinylation is carried out by covalently attaching a chemoselective group to 5hmC residues and then reacting the chemoselective group with a functionalized biotin moiety. 
     
     
         43 . The method of  claim 42 , wherein the chemoselective group is UDP glucose-6-azide and the functionalized biotin moiety is an alkyne-functionalized biotin, such that the chemoselective group reacts with the functionalized biotin in a click chemistry reaction. 
     
     
         44 . The method of  claim 40 , wherein the affinity tagged cfDNA is captured with a solid support having a surface functionalized with a biotin-binding protein, to provide cfDNA bound to the solid support. 
     
     
         45 . The method of  claim 44 , wherein step (b) further comprises: amplifying the cfDNA without releasing the captured cfDNA from the support, to give a plurality of amplicons; sequencing the amplicons; and quantifying the nucleic acids that map to the reference loci from the sequence reads. 
     
     
         46 . The method of  claim 45 , wherein amplification comprises PCR. 
     
     
         47 . The method of  claim 40 , wherein the adapters additionally comprise at least one unique feature identifier (UFI) sequence. 
     
     
         48 . The method of  claim 47 , wherein the at least one UFI sequence includes a source identifier UFI sequence. 
     
     
         49 . The method of  claim 47 , wherein the at least one UFI sequence is a molecular UFI that enables molecular counting. 
     
     
         50 . The method of  claim 47 , wherein the at least one UFI sequence is a molecular UFI that enables molecular counting. 
     
     
         51 . A method for managing a patient with a pancreatic lesion identified in an imaging scan, the method comprising:
 (a) obtaining an initial cell-free DNA sample from the patient;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched initial sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the enriched cell-free DNA in the initial sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker;   (e) generating an initial hydroxymethylation profile for the patient comprising the hydroxymethylation level of the enriched cell-free DNA in the initial sample, at each locus;   (f) repeating steps (a) through (c) at a later time with a subsequent cell-free DNA sample obtained from the patient;   (g) generating a subsequent hydroxymethylation profile for the patient comprising the hydroxymethylation level of the enriched cell-free DNA in the subsequent sample, at each locus;   (h) comparing, at each locus, the hydroxymethylation level of the enriched cell-free DNA in the subsequent sample to the hydroxymethylation level of the enriched cell-free DNA in the initial sample, to ascertain a change in the pancreatic lesion; and   (i) based on the comparison in step (e), determining whether to treat the patient.   
     
     
         52 . The method of  claim 51 , where step (i) comprises determining that treatment is necessary. 
     
     
         53 . The method of  claim 52 , wherein the treatment is selected based on the change in the patient's hydroxymethylation profile at one or more of the selected loci. 
     
     
         54 . The method of  claim 53 , wherein treating the patient comprises radiation therapy, chemotherapy, surgical resection of the lesion, or a combination thereof. 
     
     
         55 . The method of  claim 51 , comprising repeating steps (a) through (h) are repeated at selected time intervals throughout an extended monitoring period. 
     
     
         56 . The method of  claim 51 , wherein the cell-free DNA sample is extracted from a blood sample. 
     
     
         57 . The method of  claim 51 , wherein the cell-free DNA sample is extracted from pancreatic cyst fluid. 
     
     
         58 . The method of  claim 51 , wherein the hydroxymethylation biomarkers comprise loci that are associated with one or more of the following genes: ADARB2-AS1, ANKRD36B, ASAH2B, ATG4B, ATP8B1, BOLA1, C11orf88, C17orf97, C1orf170, C3orf36, C8orf74, CAMSAP2, CCDC54, CCDC59, CKAP2, CLK2P, CRTC1, CSRP2, CYB5D1, DNAJC27, DYNAP, FAM166A, FAM188B, FAM196A, FAM86JP, FAT4, FBXOS, FGF2, FUT2, GAS2L2, GAS6, GGACT, GLRX5, GPX1, GPX5, HBD, HLA-A, HTR1F, IL36G, KANSL1, KCNH6, KCTD15, KLHL38, KLK2, KRT6B, LAMC1, LGALS14, LGALS8-AS1, LIFR, LINC00266-1, LINC00310, LOC100130452, LOC100130557, LOC100130894, LOC100288778, LOC100505633, LOC100505648, LOC100505738, LOC100652909, LOC389033, LOC90784, LRRC37A2, MED11, MRPL23-AS1, NAT8L, NEUROD1, NEUROG2, NMES, NOMO3, NPRL2, NXN, ODF3L1, ODF3L2, OSCP1, PARD6G, PGAM1, PLA2G2E, PLSCR4, PPAP2A, PPP1R15A, PPP1R3E, RASL10B, REXO1L1, RIMBP3, RNF126P1, RNU6-76, RPP25, RPS27, SH3PXD2B, SHISA4, SLC25A38, SLC4A1, SLCO5A1, SPDEF, SRSF6, STRA6, SYNM, TBCB, TDRD6, TEX26, TMEM253, TNFSF13B, TTC14, TUBA4A, UBB, VAMP8, VGLL2, WASH2P, WNT9B, XBP1, and ZNF789. 
     
     
         59 . The method of  claim 58 , wherein the hydroxymethylation biomarkers additionally comprise loci that are associated with one or more of the following genes: GATA4, GATA6, PROX1, ONECUT2, YAP1, TEAD1, ONECUT2/ONECUT1-TCGA, IGF1, and IGF2. 
     
     
         60 . A method for monitoring the effectiveness of treatment in a patient with a pancreatic lesion identified on an imaging scan, the method comprising:
 (a) obtaining an initial cell-free DNA sample from a patient who is being treated;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched initial sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the enriched cell-free DNA in the initial sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker;   (e) generating an initial hydroxymethylation profile for the patient comprising the hydroxymethylation level of the enriched cell-free DNA in the initial sample, at each locus;   (f) repeating steps (a) through (c) at a later time with a subsequent cell-free DNA sample obtained from the patient;   (g) generating a subsequent hydroxymethylation profile for the patient comprising the hydroxymethylation level of the enriched cell-free DNA in the subsequent sample, at each locus;   (h) comparing, at each locus, the hydroxymethylation level of the enriched cell-free DNA in the subsequent sample to the hydroxymethylation level of the enriched cell-free DNA in the initial sample, to ascertain a change in the pancreatic lesion; and   (i) if the comparison in step (e) evidences changes in the patient's hydroxymethylation profile that correlate with a progression toward cancer, changing the treatment protocol.   
     
     
         61 . The method of  claim 60 , wherein the cell-free DNA sample is extracted from a blood sample. 
     
     
         62 . The method of  claim 60 , wherein the cell-free DNA sample is extracted from pancreatic cyst fluid. 
     
     
         63 . The method of  claim 60 , wherein the hydroxymethylation biomarkers comprise loci that are associated with one or more of the following genes: ADARB2-AS1, ANKRD36B, ASAH2B, ATG4B, ATP8B1, BOLA1, C11orf88, C17orf97, C1orf170, C3orf36, C8orf74, CAMSAP2, CCDC54, CCDC59, CKAP2, CLK2P, CRTC1, CSRP2, CYB5D1, DNAJC27, DYNAP, FAM166A, FAM188B, FAM196A, FAM86JP, FAT4, FBXO5, FGF2, FUT2, GAS2L2, GAS6, GGACT, GLRX5, GPX1, GPX5, HBD, HLA-A, HTR1F, IL36G, KANSL1, KCNH6, KCTD15, KLHL38, KLK2, KRT6B, LAMC1, LGALS14, LGALS8-AS1, LIFR, LINC00266-1, LINC00310, LOC100130452, LOC100130557, LOC100130894, LOC100288778, LOC100505633, LOC100505648, LOC100505738, LOC100652909, LOC389033, LOC90784, LRRC37A2, MED11, MRPL23-AS1, NAT8L, NEUROD1, NEUROG2, NMES, NOMO3, NPRL2, NXN, ODF3L1, ODF3L2, OSCP1, PARD6G, PGAM1, PLA2G2E, PLSCR4, PPAP2A, PPP1R15A, PPP1R3E, RASL10B, REXO1L1, RIMBP3, RNF126P1, RNU6-76, RPP25, RPS27, SH3PXD2B, SHISA4, SLC25A38, SLC4A1, SLCO5A1, SPDEF, SRSF6, STRA6, SYNM, TBCB, TDRD6, TEX26, TMEM253, TNFSF13B, TTC14, TUBA4A, UBB, VAMP8, VGLL2, WASH2P, WNT9B, XBP1, and ZNF789. 
     
     
         64 . The method of  claim 63 , wherein the hydroxymethylation biomarkers additionally comprise loci that are associated with one or more of the following genes: GATA4, GATA6, PROX1, ONECUT2, YAP1, TEAD1, ONECUT2/ONECUT1-TCGA, IGF1, and IGF2. 
     
     
         65 . The method of  claim 60 , wherein the pancreatic cancer is an exocrine pancreatic cancer. 
     
     
         66 . The method of  claim 65 , wherein the pancreatic cancer is PDAC. 
     
     
         67 . A method for reducing the risk that a pancreatic lesion surgically removed from a patient is benign, comprising, prior to surgery,
 (a) obtaining a cell-free DNA sample from the patient;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker; and   (e) calculating an index value representing the risk that the pancreatic lesion is cancerous from the comparison in step (d) combined with at least one additional parameter correlated with the risk that an individual has pancreatic cancer; and   (f) carrying out surgical resection of the pancreatic lesion only if the index value is greater than a value corresponding to a low risk of cancer.   
     
     
         68 . The method of  claim 67 , wherein the cell-free DNA sample is extracted from a blood sample. 
     
     
         69 . The method of  claim 67 , wherein the cell-free DNA sample is extracted from pancreatic cyst fluid. 
     
     
         70 . The method of  claim 67 , wherein the hydroxymethylation biomarkers comprise loci that are associated with one or more of the following genes: ADARB2-AS1, ANKRD36B, ASAH2B, ATG4B, ATP8B1, BOLA1, C11orf88, C17orf97, C1orf170, C3orf36, C8orf74, CAMSAP2, CCDC54, CCDC59, CKAP2, CLK2P, CRTC1, CSRP2, CYB5D1, DNAJC27, DYNAP, FAM166A, FAM188B, FAM196A, FAM86JP, FAT4, FBXO5, FGF2, FUT2, GAS2L2, GAS6, GGACT, GLRX5, GPX1, GPX5, HBD, HLA-A, HTR1F, IL36G, KANSL1, KCNH6, KCTD15, KLHL38, KLK2, KRT6B, LAMC1, LGALS14, LGALS8-AS1, LIFR, LINC00266-1, LINC00310, LOC100130452, LOC100130557, LOC100130894, LOC100288778, LOC100505633, LOC100505648, LOC100505738, LOC100652909, LOC389033, LOC90784, LRRC37A2, MED11, MRPL23-AS1, NAT8L, NEUROD1, NEUROG2, NMES, NOMO3, NPRL2, NXN, ODF3L1, ODF3L2, OSCP1, PARD6G, PGAM1, PLA2G2E, PLSCR4, PPAP2A, PPP1R15A, PPP1R3E, RASL10B, REXO1L1, RIMBP3, RNF126P1, RNU6-76, RPP25, RPS27, SH3PXD2B, SHISA4, SLC25A38, SLC4A1, SLCO5A1, SPDEF, SRSF6, STRA6, SYNM, TBCB, TDRD6, TEX26, TMEM253, TNFSF13B, TTC14, TUBA4A, UBB, VAMP8, VGLL2, WASH2P, WNT9B, XBP1, and ZNF789. 
     
     
         71 . The method of  claim 70 , wherein the hydroxymethylation biomarkers additionally comprise loci that are associated with one or more of the following genes: GATA4, GATA6, PROX1, ONECUT2, YAP1, TEAD1, ONECUT2/ONECUT1-TCGA, IGF1, and IGF2. 
     
     
         72 . A method for evaluating the risk that a subject will develop pancreatic cancer, comprising:
 (a) obtaining a cell-free DNA sample from the subject;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker; and   (e) calculating an index value representing the subject's risk of developing pancreatic cancer from the comparison in step (d) combined with at least one additional parameter correlated with the risk that an individual will develop pancreatic cancer.   
     
     
         73 . A kit for carrying out the method of  claim 1 , comprising:
 at least one reagent for the determination of hydroxymethylation level at each of a plurality of hydroxymethylation biomarker loci in a cell-free DNA sample;   a solid support for capturing affinity-tagged 5hmC-containing cell-free DNA in the sample; and   written instructions for the use of the at least one reagent and the solid support in carrying out the method.   
     
     
         74 . The kit of  claim 73 , further including instructions for accessing and using software designed to perform modeling and prediction. 
     
     
         75 . A kit for carrying out the method of  claim 1 , comprising:
 a DNA β-glucosyl transferase;   UDP glucose modified with a chemoselective group;   a biotin moiety;   a solid support having a surface functionalized with a biotin-binding protein;   an adaptor comprising a molecular barcode; and   written instructions for carrying out the method.   
     
     
         76 . The kit of  claim 75 , further including instructions for accessing and using software designed to perform modeling and prediction. 
     
     
         77 . A method for determining the likelihood that an individual at risk for developing pancreatic cancer has pancreatic cancer, the method comprising:
 (a) obtaining a cell-free DNA sample from the patient;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker; and   (e) calculating an index value representing the likelihood that the individual has pancreatic cancer from the comparison in step (d).   
     
     
         78 . The method of  claim 77 , further including, prior to step (a), identifying the individual as being at risk for developing pancreatic cancer from one or more parameters selected from: an identified pancreatic lesion; pancreatic inflammation; jaundice; age; weight; gender; ethnicity; family history; genetic mutations; diabetes; physical activity; diet; pro-inflammatory cytokine levels; and cigarette smoking. 
     
     
         79 . The method of  claim 77 , further including determining the likelihood that the individual has at least one additional type of cancer. 
     
     
         80 . The method of  claim 79 , wherein the at least one additional type of cancer is selected from: bladder cancer; cancers of the blood and bone marrow; brain cancer; breast cancer; cervical cancer; colorectal cancer; esophageal cancer; liver cancer; lung cancer; ovarian cancer; prostate cancer; renal cancer; skin cancer; testicular cancer; thyroid cancer; and uterine cancer. 
     
     
         81 . In a multi-cancer test that determines the likelihood that an individual has pancreatic cancer and at least one additional type of cancer selected from colorectal, esophageal, lung, and liver cancer, the improvement which comprises determining the likelihood that the individual has pancreatic cancer by:
 (a) obtaining a cell-free DNA sample from the individual;   (b) enriching for hydroxymethylated DNA in the sample;   (c) quantifying the nucleic acids in the enriched sample that map to each of a plurality of selected loci in a reference hydroxymethylation profile, wherein each selected locus comprises a hydroxymethylation biomarker;   (d) comparing, at each locus, the hydroxymethylation level of the sample with the hydroxymethylation level in the reference profile, to ascertain differences in hydroxymethylation levels between the sample and the reference profile for each biomarker; and   (e) calculating an index value representing the likelihood that the individual has pancreatic cancer from the comparison in step (d).   
     
     
         82 . The improved multi-cancer test of  claim 81 , further including eliminating false positives for the at least one additional type of cancer prior to (a). 
     
     
         83 . The improved multi-cancer test of  claim 81 , further including eliminating false negatives for the at least one additional type of cancer prior to (a). 
     
     
         84 . A kit for carrying out the method of  claim 77 , comprising:
 at least one reagent for the determination of hydroxymethylation level at each of a plurality of hydroxymethylation biomarker loci in a cell-free DNA sample;   a solid support for capturing affinity-tagged 5hmC-containing cell-free DNA in the sample; and   written instructions for the use of the at least one reagent and the solid support in carrying out the method.   
     
     
         85 . The kit of  claim 84 , further including instructions for accessing and using software designed to perform modeling and prediction. 
     
     
         86 . A kit for carrying out the method of  claim 77 , comprising:
 a DNA β-glucosyl transferase;   UDP glucose modified with a chemoselective group;   a biotin moiety;   a solid support having a surface functionalized with a biotin-binding protein;   an adaptor comprising a molecular barcode; and   written instructions for carrying out the method.   
     
     
         87 . The kit of  claim 86 , further including instructions for accessing and using software designed to perform modeling and prediction.