Methods for early prediction, treatment response, recurrence and prognosis monitoring of pancreatic cancer

Abstract

The present invention discloses a novel set of epigenetic biomarkers for early prediction, treatment response, recurrence, and prognosis monitoring of pancreatic cancer. Aberrant gene methylation can be detected in tumor tissue and plasma samples from pancreatic cancer patients, but not in healthy individuals. The present disclosure also discloses primers and probes used therein.

Description

[Technical Field]

[0001] The present disclosure relates to genetic biomarkers for predicting the risk or susceptibility of pancreatic cancer, and / or the prognosis and aggressiveness of pancreatic cancer. In particular, the present disclosure detects pancreatic cancer, predicts the risk or susceptibility of pancreatic cancer, and / or the prognosis and aggressiveness of pancreatic cancer by assessing the methylation of genetic biomarkers. [Background technology]

[0002] Cancer is a group of diseases involving abnormal cell growth that can infiltrate and metastasize to other parts of the body and is a leading cause of death worldwide.

[0003] Methylated DNA has been investigated as a promising biomarker in tissues of most tumor types. DNA methyltransferases often add methyl groups to cytosine-phosphate-guanine (CpG) island sites in DNA as an epigenetic control of gene expression.

[0004] US20080311570A1 provides a cancer screening method comprising the following steps: (1) providing a test specimen; (2) detecting the methylation status of a CpG sequence in at least one target gene (the target genes consist of SOX1, PAX1, LMX1A, NKX6-1, WT1, and ONECUT1) in the genomic DNA of the test specimen; and (3) determining whether the specimen contains cancer or a cancerous pathological change based on the presence or absence of the methylation status in the target gene. WO2016071477A1 relates to evaluating the response of cancer patients to treatment by analyzing CpG methylation in the shox2 gene. EP2828405B1 provides a method for detecting colorectal neoplasia by evaluating multiple gene markers in blood or plasma and stool.

[0005] However, current techniques for detecting pancreatic cancer are not satisfactory. Summary of the Invention

[0006] The present disclosure relates to one or more novel epigenetic biomarkers for early prediction, treatment response, recurrence, and prognosis monitoring of pancreatic cancer. Abnormal gene methylation can be detected in tumor tissue and plasma samples from cancer patients, but not in healthy individuals. The present disclosure also discloses primers and probes used therein. <Pancreatic cancer detection>

[0007] In one embodiment, the present disclosure provides a method for detecting methylation status in a subject in need thereof, detecting a predisposition to or incidence of pancreatic cancer, or predicting treatment response, prognosis, or recurrence of pancreatic cancer in a subject, the method comprising: (a) providing a biological sample containing DNA from a subject, wherein the DNA comprises a target gene ZFP30 or a fragment thereof; (b) assaying the methylation status of one or more epigenetic biomarkers having CpG sites in the target gene ZFP30 or a fragment thereof; (c) identifying the target gene ZFP30 or a fragment thereof in the biological sample having a methylation level higher than that of a healthy individual as hypermethylated, wherein hypermethylation of the target gene ZFP30 or a fragment thereof indicates pancreatic cancer, or a predisposition to pancreatic cancer, or the incidence, poor treatment response, poor prognosis, or recurrence of pancreatic cancer.

[0008] In some embodiments, the methylation status is detected by polymerase chain reaction (PCR), nucleic acid sequencing (such as bisulfite sequencing or pyrosequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, microarrays, or combinations thereof. In certain embodiments, the methylation status is detected by PCR, such as methylation-specific PCR (MSP), real-time methylation-specific PCR, quantitative methylation-specific PCR (QMSP), methylation-sensitive DNA restriction enzyme analysis, bisulfite genomic sequencing PCR, or PCR using methylated DNA-specific binding proteins.

[0009] In some embodiments, the step of assaying the methylation status in (b) comprises extracting genomic DNA from a biological sample of a subject suspected of or afflicted with pancreatic cancer, amplifying the genomic DNA with a primer pair specific for ZFP30, and measuring the methylation level of one or more CpG sites of ZFP30 by polymerase chain reaction (PCR). In further embodiments, the step of assaying the methylation status in (b) further comprises treating the extract obtained after extracting the genomic DNA from the biological sample with bisulfite and amplifying the bisulfite-treated genomic DNA.

[0010] In some embodiments, the step of (b) assaying the methylation status comprises assaying the methylation status of two, three, four, five, six, seven or more epigenetic biomarkers having CpG sites in the target gene ZFP30 or a fragment thereof.

[0011] Particular embodiments of ZFP30-specific primer pairs used to amplify genomic DNA or bisulfite-treated genomic DNA have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences of SEQ ID NOs: 1 and 2.

[0012] In some embodiments, a ZFP30 methylation-specific probe is used to assay the methylation level of the target gene ZFP30 or a fragment thereof. In some embodiments, the ZFP30 methylation-specific probe has a sequence that is at least 85% identical to SEQ ID NO: 9. In some embodiments, the ZFP30 methylation-specific probe has a sequence that is about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 9. In one embodiment, the ZFP30 methylation-specific probe has the sequence of SEQ ID NO: 9.

[0013] In one embodiment, the one, two or more epigenetic biomarkers having a CpG site are located at Chr19 37692300-37692450 of the target gene ZFP30.

[0014] In one embodiment, the present disclosure provides a method for detecting methylation status in a subject in need thereof, detecting a predisposition to or incidence of pancreatic cancer, or predicting treatment response, prognosis, or recurrence of pancreatic cancer in a subject, the method comprising: (ai) providing a biological sample containing DNA from a subject, wherein the DNA comprises the target gene FBXL7 or a fragment thereof; (bi) assaying the methylation status of one or more epigenetic biomarkers having CpG sites in the target gene FBXL7 or a fragment thereof; (c) identifying the target gene FBXL7 or a fragment thereof in the biological sample having a methylation level higher than that of a healthy individual as hypermethylated, wherein hypermethylation of the target gene FBXL7 or a fragment thereof indicates pancreatic cancer, or a predisposition to pancreatic cancer, or the incidence, poor treatment response, poor prognosis or recurrence of pancreatic cancer.

[0015] In some embodiments, the methylation status is detected by polymerase chain reaction (PCR), nucleic acid sequencing (such as bisulfite sequencing or pyrosequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, microarrays, or combinations thereof. In certain embodiments, the methylation status is detected by PCR, such as methylation-specific PCR (MSP), real-time methylation-specific PCR, quantitative methylation-specific PCR (QMSP), methylation-sensitive DNA restriction enzyme analysis, bisulfite genomic sequencing PCR, or PCR using methylated DNA-specific binding proteins.

[0016] In some embodiments, the step of assaying the methylation status of (bi) comprises extracting genomic DNA from a biological sample of a subject suspected of or afflicted with pancreatic cancer, amplifying the genomic DNA with a primer pair specific to FBXL7, and measuring the methylation level of one or more CpG sites in FBXL7 by polymerase chain reaction (PCR). In further embodiments, the step of assaying the methylation status of (bi) further comprises treating the extract obtained after extracting the genomic DNA from the biological sample with bisulfite and amplifying the bisulfite-treated genomic DNA.

[0017] In some embodiments, the step of assaying the methylation status of (bi) comprises assaying the methylation status of two, three, four, five, six, seven or more epigenetic biomarkers having CpG sites in the target gene FBXL7 or a fragment thereof.

[0018] Particular embodiments of FBXL7-specific primer pairs used to amplify genomic DNA or bisulfite-treated genomic DNA have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences of SEQ ID NOs: 3 and 4.

[0019] In some embodiments, an FBXL7 methylation-specific probe is used to assay the methylation level of the target gene FBXL7 or a fragment thereof. In some embodiments, the FBXL7 methylation-specific probe has a sequence having at least 85% identity to SEQ ID NO: 10. In some embodiments, the FBXL7 methylation-specific probe has a sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 10. In one embodiment, the FBXL7 methylation-specific probe has the sequence of SEQ ID NO: 10.

[0020] In one embodiment, the one, two, three or more epigenetic biomarkers having CpG sites are located at Chr5 15500544-15500694 of the target gene FBXL7.

[0021] In a further embodiment, the present disclosure provides a method for detecting methylation status in a subject in need thereof, detecting a predisposition to or incidence of pancreatic cancer, or predicting treatment response, prognosis, or recurrence of pancreatic cancer in a subject, the method comprising: (aii) providing a biological sample containing DNA from a subject, wherein the DNA comprises a target gene ZFP30 or a fragment thereof, and a target gene FBXL7 or a fragment thereof; (bii) assaying the methylation status of one or more epigenetic biomarkers having a CpG site in the target gene ZFP30 or a fragment thereof, and the methylation status of one or more epigenetic biomarkers having a CpG site in the target gene FBXL7 or a fragment thereof; (cii) identifying target genes ZFP30 and FBXL7 or fragments thereof in a biological sample having a methylation level higher than that of a healthy individual as hypermethylated, wherein hypermethylation of target genes ZFP30 and FBXL7 or fragments thereof indicates pancreatic cancer, or a predisposition to pancreatic cancer, or the incidence, poor treatment response, poor prognosis, or recurrence of pancreatic cancer.

[0022] In some embodiments, the methylation status is detected by polymerase chain reaction (PCR), nucleic acid sequencing (such as bisulfite sequencing or pyrosequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, microarrays, or combinations thereof. In certain embodiments, the methylation status is detected by PCR, such as methylation-specific PCR (MSP), real-time methylation-specific PCR, quantitative methylation-specific PCR (QMSP), methylation-sensitive DNA restriction enzyme analysis, bisulfite genomic sequencing PCR, or PCR using methylated DNA-specific binding proteins.

[0023] In some embodiments, the step of assaying the methylation status in (bii) comprises extracting genomic DNA from a biological sample of a subject suspected of or afflicted with pancreatic cancer, amplifying the genomic DNA with primers consisting of a pair of primers specific for ZFP30 and a pair of primers specific for FBXL7, and measuring the methylation level of one or more CpG sites in ZFP30 and one or more CpG sites in FBXL7 by polymerase chain reaction (PCR). In further embodiments, the step of assaying the methylation status in (bii) further comprises treating the extract obtained after extracting the genomic DNA from the biological sample with bisulfite and amplifying the bisulfite-treated genomic DNA.

[0024] Particular embodiments of ZFP30-specific primer pairs used to amplify genomic DNA or bisulfite-treated genomic DNA have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences of SEQ ID NOs: 1 and 2. Particular embodiments of FBXL7-specific primer pairs used to amplify genomic DNA or bisulfite-treated genomic DNA have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences of SEQ ID NOs: 3 and 4.

[0025] In some embodiments, a ZFP30 methylation-specific probe and an FBXL7 methylation-specific probe are used to assay the methylation levels of the target gene ZFP30 or a fragment thereof and the target gene FBXL7 or a fragment thereof. In some embodiments, the ZFP30 methylation-specific probe has a sequence that is at least 85% identical to SEQ ID NO:9. In some embodiments, the ZFP30 methylation-specific probe has a sequence that is about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:9. In one embodiment, the ZFP30 methylation-specific probe has the sequence of SEQ ID NO:9. In some embodiments, the FBXL7 methylation-specific probe has a sequence that is at least 85% identical to SEQ ID NO:10. In some embodiments, the FBXL7 methylation-specific probe has a sequence that is about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 10. In one embodiment, the FBXL7 methylation-specific probe has the sequence of SEQ ID NO: 10.

[0026] In some embodiments, the step of assaying the methylation status of (bii) comprises assaying the methylation status of two or more epigenetic biomarkers having CpG sites in the target gene ZFP30 or a fragment thereof, and the methylation status of two, three or more epigenetic biomarkers having CpG sites in the target gene FBXL7 or a fragment thereof.

[0027] In one embodiment, the one, two or more epigenetic biomarkers having a CpG site are located at Chr19 37692300-37692450 of the target gene ZFP30.

[0028] In one embodiment, the one, two, three or more epigenetic biomarkers having CpG sites are located at Chr5 15500544-15500694 of the target gene FBXL7.

[0029] In some embodiments, in method (a), (ai), or (aii), the DNA further comprises one or more of the target genes CNTN4 and KLRG2 or fragments thereof; (biii) further comprises assaying the methylation status of one or more epigenetic biomarkers of the target genes CNTN4 and KLRG2 or fragments thereof; (ciii) further comprises identifying one or more of the target genes CNTN4 and KLRG2 or fragments thereof in the biological sample as hypermethylated, the target genes having a methylation level higher than the methylation level of the control gene compared to a healthy individual, wherein hypermethylation of the target genes ZFP30, FBXL7, and one or more of CNTN4 and KLRG2 indicates pancreatic cancer, or a predisposition to pancreatic cancer, or incidence, poor treatment response, poor prognosis, or recurrence of pancreatic cancer.

[0030] In some embodiments, the methylation status is detected by polymerase chain reaction (PCR), nucleic acid sequencing (such as bisulfite sequencing or pyrosequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, microarrays, or combinations thereof. In certain embodiments, the methylation status is detected by PCR, such as methylation-specific PCR (MSP), real-time methylation-specific PCR, quantitative methylation-specific PCR (QMSP), methylation-sensitive DNA restriction enzyme analysis, bisulfite genomic sequencing PCR, or PCR using methylated DNA-specific binding proteins.

[0031] In some embodiments, the step of assaying the methylation status in (biii) comprises extracting genomic DNA from a biological sample of a subject suspected of or afflicted with pancreatic cancer, amplifying the genomic DNA with primers consisting of a CNTN4-specific primer pair and / or a KLRG2-specific primer pair, and measuring the methylation level of one or more CpG sites in CNTN4 and / or the methylation level of one or more CpG sites in KLRG2 by polymerase chain reaction (PCR). In further embodiments, the step of assaying the methylation status in (biii) further comprises treating the extract obtained after extracting the genomic DNA from the biological sample with bisulfite and amplifying the bisulfite-treated genomic DNA.

[0032] Particular embodiments of CNTN4-specific primer pairs used to amplify genomic DNA or bisulfite-treated genomic DNA have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences of SEQ ID NOs:5 and 6.

[0033] Particular embodiments of KLRG2-specific primer pairs used to amplify genomic DNA or bisulfite-treated genomic DNA have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences of SEQ ID NOs: 7 and 8.

[0034] In some embodiments, a CNTN4 methylation-specific probe, a KLRG2 methylation-specific probe, or a combination thereof is used to assay the methylation level of one or more of the target gene CNTN4 and KLRG2 DNA or fragments thereof and a control DNA sequence in a biological sample.

[0035] In some embodiments, the CNTN4 methylation-specific probe has a sequence that is at least 85% identical to SEQ ID NO: 11. In some embodiments, the CNTN4 methylation-specific probe has a sequence that is about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 11. In one embodiment, the CNTN4 methylation-specific probe has the sequence of SEQ ID NO: 11.

[0036] In some embodiments, the KLRG2 methylation-specific probe has a sequence having at least 85% identity to SEQ ID NO: 12. In some embodiments, the KLRG2 methylation-specific probe has a sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 12. In one embodiment, the KLRG2 methylation-specific probe has the sequence of SEQ ID NO: 12.

[0037] In some embodiments, the biological sample described herein is a tissue sample, a pancreatic juice sample, a pancreatic cyst fluid sample, a stool sample, a cell sample, a blood sample, a urine sample, a serum sample, or a plasma sample.

[0038] In some embodiments, a kit for detecting a methylation status in a subject in need of detection of pancreatic cancer, or for detecting a predisposition to, or incidence of, pancreatic cancer, or for predicting treatment response, prognosis, or recurrence of pancreatic cancer in a subject, comprises a primer pair having the sequences of SEQ ID NO: 1 and 2 and / or a probe having the sequence of SEQ ID NO: 9 for assaying the methylation status of an epigenetic biomarker in the target gene ZFP30 or a fragment thereof.

[0039] In some embodiments, the kit further comprises a primer pair having the sequences of SEQ ID NO: 3 and 4 and / or a probe having the sequence of SEQ ID NO: 10 for assaying the methylation status of an epigenetic biomarker in the target gene FBXL7 or a fragment thereof.

[0040] In some embodiments, the kit further comprises a primer pair having the sequences of SEQ ID NOs: 5 and 6 and / or a probe having the sequence of SEQ ID NO: 11 for assaying the methylation status of an epigenetic biomarker in the target gene CNTN4 or a fragment thereof, and / or a primer pair having the sequences of SEQ ID NOs: 7 and 8 and / or a probe having the sequence of SEQ ID NO: 12 for assaying the methylation status of an epigenetic biomarker in the target gene KLRG2 or a fragment thereof.

[0041] In some embodiments, the kit further comprises sodium bisulfite and adaptors for amplification of the entire target gene, and a polynucleotide (e.g., a detectably labeled polynucleotide) for quantifying the presence of converted methylated and / or converted unmethylated sequences of at least one cytosine from the DNA region of the epigenetic biomarker in the target gene. In some embodiments, the kit further comprises a methylation-sensitive restriction enzyme for amplification of the entire target sequence or gene.

[0042] In one embodiment, the disclosure provides a method for measuring the methylation level of one or more CpG sites in ZFP30 or a fragment thereof, the method comprising: Extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer; treating the obtained extract with hydrogen sulfate; amplifying the bisulfite-treated genomic DNA with a primer pair specific for ZFP30; measuring the methylation level of one or more CpG sites in ZFP30 by polymerase chain reaction (PCR).

[0043] In some further embodiments, the method further comprises measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, i.e., extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to CNTN4 or a fragment thereof and / or a primer pair specific to KLRG2 or a fragment thereof, and measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, by polymerase chain reaction (PCR).

[0044] In one embodiment, the present disclosure provides a method for measuring the methylation level of one or more (e.g., two, three, four, five, six, seven, or more) CpG sites in FBXL7 or a fragment thereof, comprising the steps of extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the resulting extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to FBXL7, and measuring the methylation level of the one or more CpG sites in FBXL7 by polymerase chain reaction (PCR).

[0045] In some further embodiments, the method further comprises measuring the methylation level of one or more (e.g., two, three, four, five, six, seven or more) CpG sites in CNTN4 and / or KLRG2, or fragments thereof, i.e., comprising extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to CNTN4 or a fragment thereof and / or a primer pair specific to KLRG2 or a fragment thereof, and measuring the methylation level of one or more (e.g., two, three, four, five, six, seven or more) CpG sites in CNTN4 and / or KLRG2, or fragments thereof, by polymerase chain reaction (PCR).

[0046] In one embodiment, the present disclosure provides a method for measuring the methylation level of one or more CpG sites in ZFP30 or a fragment thereof and FBXL7 or a fragment thereof, the method comprising the steps of extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the resulting extract with bisulfite, amplifying the bisulfite-treated genomic DNA with primers consisting of a ZFP30-specific primer pair and an FBXL7-specific primer pair, and measuring the methylation level of one or more CpG sites in ZFP30 and FBXL7 by polymerase chain reaction (PCR).

[0047] In some further embodiments, the method further comprises measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, i.e., extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to CNTN4 or a fragment thereof and / or a primer pair specific to KLRG2 or a fragment thereof, and measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, by polymerase chain reaction (PCR).

[0048] In some embodiments, the PCR described herein is methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR.

[0049] In one embodiment, the present disclosure provides a method comprising the steps of extracting genomic DNA from a biological sample obtained from a subject, treating the extracted genomic DNA with bisulfite, amplifying the bisulfite-treated genomic DNA using a primer specific to a CpG site of ZFP30, a primer specific to a CpG site of FBXL7, or a primer specific to a CpG site of ZFP30 and a primer specific to a CpG site of FBXL7, and measuring the methylation level of the CpG site of ZFP30, or the methylation level of the CpG site of FBXL7, or the methylation level of the CpG site of ZFP30 and the CpG site of FBXL7.

[0050] In some further embodiments, the method further comprises extracting genomic DNA from a biological sample obtained from the subject; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA using primers specific for CpG sites in CNTN4, primers specific for CpG sites in KLRG2, or primers specific for CpG sites in CNTN4 and CpG sites in KLRG2; and measuring the methylation level of the CpG sites in CNTN4, or the methylation level of the CpG sites in KLRG2, or the methylation levels of the CpG sites in CNTN4 and the CpG sites in KLRG2.

[0051] Certain embodiments of the primers described herein have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In some embodiments, the primers have the sequences of SEQ ID NOs: 1-8. In some embodiments, probes having the sequences of SEQ ID NOs: 9-12 are used to detect or measure the methylation status of one or more of the target genes ZFP30, FBXL7, CNTN4, and KLRG2 or fragments thereof.

[0052] In further embodiments, measuring the methylation level of the CpG sites is performed by PCR. In some embodiments, the PCR described herein is methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR. [Brief explanation of the drawings]

[0053] [Figures 1A-1D] Heatmaps of the differences in methylation status of several target genes between tumor tissues and adjacent normal tissues are shown (Figure 1A: ZFP30; Figure 1B: FBXL7; Figure 1C: CNTN4; and Figure 1D: KLRG2). Dark bands represent methylation sites on the genes. [Figure 2A] 1 shows the difference in early detection of the methylation status of the target gene ZFP30 in plasma samples from pancreatic cancer patients (n=31) and healthy individuals (n=12). [Figure 2B] 1 shows the difference in early detection of the methylation status of the target gene FBXL7 in plasma samples from pancreatic cancer patients (n=31) and healthy individuals (n=12). [Figure 3] 1 shows ROC curve analysis based on early detection of the methylation status of target genes in pancreatic cancer patients and healthy individuals. DETAILED DESCRIPTION OF THE INVENTION

[0054] It is understood that this invention is not limited to the particular materials and methods described herein, and that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

[0055] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "biomarker" includes a mixture of two or more biomarkers, and the like.

[0056] The term "AUC" as used herein is an abbreviation for area under a curve. In particular, it refers to the area under the receiver operating characteristic (ROC) curve. An ROC curve is a plot of the true positive rate against the false positive rate for different possible cut points of a diagnostic test. It shows a trade-off between sensitivity and specificity (increased sensitivity is accompanied by decreased specificity) depending on the cut point selected. The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better, with 1 being optimal. A random test would have an ROC curve lying on the diagonal with an area of ​​0.5. See: J.P. Egan. Signal Detection Theory and ROC Analysis, Academic Press, New York, 1975).

[0057] The term "biological sample" refers to a sample of tissue, cell, or fluid isolated from a subject, including, but not limited to, blood, buffy coat, plasma, serum, blood cells (e.g., peripheral blood mononuclear cells (PBMCS), band cells, neutrophils, metamyelocytes, monocytes, or T cells), excrement (such as stool), urine, bone marrow, bile, spinal fluid, lymphatic fluid, samples of skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, organs, biopsies, and samples of in vitro cell culture components, including, but not limited to, conditioned medium resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells and cellular components.

[0058] The term "biomarker" refers to a nucleic acid molecule present in a sample taken from a patient with human cancer compared to an equivalent sample taken from a control subject (e.g., one who has a negative diagnosis, one who has undetectable cancer, or one who is normal or healthy). A biomarker can be a nucleic acid, a fragment of a nucleic acid, a polynucleotide, or an oligonucleotide that can be detected and / or quantified. Biomarkers include polynucleotides that include a nucleotide sequence from a gene.

[0059] As used herein, the term "CpG site" refers to a stretch of DNA in the genome that is GC-rich relative to the rest of the genome. Typically, the GC content is 50% or more in these regions, spanning hundreds, and sometimes thousands, of base pairs. Often, these regions mark the 5' ends of genes.

[0060] A "control amount" of a biomarker can be any amount or range of amounts compared to a test amount of the biomarker.

[0061] As used herein, the term "early detection" refers to detecting possible cancer before metastasis. Preferably, it refers to detecting possible cancer before morphological changes in sample tissue or cells are observed.

[0062] As used herein, the terms "detect," "detecting," or "detection" can refer to either the general act of finding or identifying or specifically observing a detectably labeled composition.

[0063] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., non-coding RNA such as ribosomal RNA, transfer RNA, spliceosomal RNA, or microRNA). A polypeptide or non-coding RNA can be encoded by a full-length coding sequence or by any portion of a coding sequence, so long as the desired activity or functional properties of the full-length or fragment polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) are retained. Thus, a gene can include or exclude promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions. The term also includes the coding region of a structural gene and sequences flanking the coding region at both the 5' and 3' ends by at least about 1 kb, with the length of the gene corresponding to the full-length mRNA. The term "gene" further encompasses both cDNA and genomic forms of a gene.

[0064] As used herein, the term "identity" refers to a first sequence that shares a degree of sequence identity with a second sequence, but the sequence is not identical to the sequence of the second sequence. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous to but not identical to the sequence of the mutant gene. In some embodiments, the degree of identity between the two sequences is sufficient to allow homologous recombination between them under appropriate stringent conditions.

[0065] Techniques for determining the identity of nucleic acid and amino acid sequences include determining the nucleotide sequence of the mRNA for a gene and / or determining the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences (nucleic acid or amino acid sequences) is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100.

[0066] In some embodiments, the degree of sequence similarity between polynucleotides can be determined by hybridizing the polynucleotides under conditions that allow the formation of stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease and sizing the digested fragments. Two nucleic acid or two polypeptide sequences are substantially homologous to each other if the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, even more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecule, as determined using the methods described above. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a particular DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified, for example, in a Southern hybridization experiment under stringent conditions defined for that particular system. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors BD Hames and SJ Higgins, (1985) Oxford; Washington, DC; IRL Press.

[0067] As used herein, the term "prediction" refers to the likelihood that a patient will respond favorably or unfavorably to a drug or set of drugs, as well as the degree of such response. Thus, a treatment predictor is a variable that is related to an individual patient's response to a particular treatment, independent of prognosis.

[0068] As used herein, the term "methylation" refers to the presence of a methyl group added to a cytosine base or base in a region of a nucleic acid, e.g., a region of genomic DNA, by the action of a DNA methyltransferase enzyme.

[0069] The terms "methylation state," "methylation profile," or "methylation status" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (i.e., the methylation status of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated.

[0070] The term "hypermethylation" refers to an average methylation state corresponding to an increased presence of methylated nucleotide bases in nucleic acid molecules at one or more CpG dinucleotides within a DNA sequence of a test DNA sample compared to the amount of methylated nucleotide bases in nucleic acid molecules found at corresponding CpG dinucleotides in a normal test DNA sample.

[0071] The term "hypomethylation" refers to an average methylation state corresponding to a decreased presence of methylated nucleotide bases in nucleic acid molecules at one or more CpG dinucleotides within a DNA sequence of a test DNA sample compared to the amount of methylated nucleotide bases in nucleic acid molecules found at corresponding CpG dinucleotides in a normal test DNA sample.

[0072] The term "subject" refers to a human being.

[0073] The term "susceptibility" refers to a physical constitution or condition in which tissues react in a particular way to particular external stimuli, thus predisposing an individual to be more susceptible than normal to a particular disease.

[0074] "Target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0075] The term "risk" refers to the estimated chance of acquiring a disease within a particular period of time, such as within the next 10 years, or during a subject's lifetime.

[0076] The term "prognosis" as used herein generally refers to the prediction of the likely course and outcome of a clinical condition or disease. A patient's prognosis is usually made by assessing disease factors or symptoms that indicate a favorable or unfavorable course or outcome of the disease.

[0077] The term "weight sum score" refers to all possible alternatives evaluated by a score that includes all objectives individually weighted to emphasize the importance of different objectives.

[0078] Cancer is characterized by abnormal cell growth caused by one or more mutations or modifications in genes, leading to a dysregulated balance between cell proliferation and cell death. In many disease processes, such as cancer, gene promoter CpG islands acquire abnormal hypermethylation, resulting in transcriptional silencing that may be inherited by daughter cells after cell division. DNA methylation, which causes silencing in cancer, typically occurs at multiple CpG sites in CpG islands present in the promoters of protein-coding genes. Changes in DNA methylation have been recognized as a key factor in cancer development. DNA methylation profiling offers higher clinical sensitivity and dynamic range compared to other cancer detection methods. Therefore, the present disclosure provides methods and kits for early prediction, treatment response, and prognosis or recurrence monitoring of pancreatic cancer.

[0079] In some embodiments, the methylation status of one or more of ZFP30, FBXL7, CNTN4, and KLRG2 target DNA sequences or fragments thereof in a biological sample is measured to detect pancreatic cancer, or to detect a predisposition to or incidence of pancreatic cancer, or to predict treatment response, prognosis, or recurrence of pancreatic cancer in a human subject.

[0080] The ZFP30 gene encodes a ZFP30 zinc finger protein that is predicted to have DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity. The ZFP30 sequence and its function are known in the art, as described at the following website: https: / / www.genecards.org / cgi-bin / carddisp.pl?gene=ZFP30.

[0081] The FBXL7 gene encodes F-box and leucine-rich repeat protein 7, a member of the F-box protein family characterized by a 42-48 amino acid motif. The F-box protein binds to S-phase kinase-associated protein 1 (Skp1). The F-box protein constitutes one of the four subunits of the E3 ubiquitin protein ligase called SCF (SKP1-Cul1-F-box), which plays a role in the phosphorylation-dependent ubiquitination of proteins. The FBXL7 sequence and its function are known in the art, as described at the following website: https: / / www.genecards.org / cgi-bin / carddisp.pl?gene=FBXL7.

[0082] The CNTN4 gene encodes contactin 4, a member of the contactin family of immunoglobulins. Contactin is an axonal cell adhesion molecule that functions in the formation and plasticity of neural networks. The encoded protein is a glycosylphosphatidylinositol-anchored neuronal membrane protein that may play a role in the formation of axonal connections in the developing nervous system. The CNTN4 sequence and its function are known in the art, as described at the following website: https: / / www.genecards.org / cgi-bin / carddisp.pl?gene=CNTN4.

[0083] The KLRG2 gene encodes the killer cell lectin-like receptor G2, which confers carbohydrate-binding activity and is predicted to be an integral component of the membrane. The KLRG2 sequence and its function are known in the art, as described at the following website: https: / / www.genecards.org / cgi-bin / carddisp.pl?gene=KLRG2.

[0084] In some embodiments, the methylation comprises cytosine methylation sites. Optionally, the cytosine methylation comprises 5-methylcytosine (5-mCyt) and 5-hydroxymethylcytosine. Optionally, the cytosine methylation site occurs within a CpG dinucleotide motif. In other cases, the cytosine methylation site occurs in a CHG or CHH motif, which is adenine, cytosine, or thymine. Optionally, one or more CpG dinucleotide motifs or CpG sites form a CpG island, which is a short DNA sequence rich in CpG dinucleotides. Optionally, a CpG island is typically, but not always, about 0.2 to about 1 kb in length. Optionally, the methylation comprises CpG island methylation.

[0085] In some embodiments, the methylation status is analyzed by methylation-specific enzymatic digestion; an analysis selected from bisulfite sequencing, promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, and Ms-SNuPE; and other methods based on detection of amplified DNA. TM The term "refers to a fluorescence-based real-time PCR technique. MethylLight is described by Eads et al., Cancer Res. 59:2302-2306, 1999, which is incorporated herein by reference.

[0086] The term "HeavyMethyl" assay refers to an assay in which a methylation-specific blocking probe (also referred to herein as a blocker) covering the CpG positions between or covered by the amplification primers allows for methylation-specific selective amplification of a nucleic acid sample.

[0087] The term "Ms-SnuPE" refers to methylation-sensitive single-stranded nucleotide primer extension. MsSNuPE is described by Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997, which is incorporated herein by reference.

[0088] The term "MSP" refers to methylation-specific PCR. MSP is described by Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, and by U.S. Pat. No. 5,786,146, each of which is incorporated herein by reference.

[0089] Bisulfite modification of DNA is a method for assessing CpG methylation status. 5-methylcytosine is the most frequent covalent base modification in eukaryotic DNA. However, because 5-methylcytosine exhibits the same base-pairing behavior as cytosine, its location cannot be directly identified by sequencing or hybridization methods. Furthermore, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification, for example. Bisulfite sequencing is a method for analyzing DNA for the presence of 5-methylcytosine and is based on a specific reaction between bisulfite and cytosine. In this reaction, subsequent alkaline hydrolysis converts cytosine to uracil, which corresponds to thymine in its base-pairing behavior. However, 5-methylcytosine remains unmodified under the aforementioned conditions. Thus, the original DNA is converted so that methylcytosines, which were originally indistinguishable from cytosines in their hybridization behavior, can now be detected as the only remaining cytosines using molecular biology techniques, for example by amplification and hybridization or by sequencing.

[0090] In one embodiment, the methylation status is detected by polymerase chain reaction, nucleic acid sequencing (such as bisulfite sequencing or pyrosequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, or microarray. In one embodiment, the methylation status is detected by amplifying methylated CpGs of target genes using primers. In further embodiments, the detection of methylation is performed by PCR, methylation-specific PCR (MSP), real-time methylation-specific PCR, quantitative methylation-specific PCR (QMSP), PCR using methylated DNA-specific binding proteins, or quantitative PCR.

[0091] In one embodiment of the present disclosure, primers capable of amplifying methylated CpGs of the genes described herein can be used. The primers contain at least one CpG dinucleotide in the region that hybridizes to the methylated CpGs of the genes. Specifically, the primers for amplifying methylated CpGs of the genes include a sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence selected from the group consisting of the following sequences: JPEG2026505663000002.jpg49170

[0092] Probes hybridizable to the methylated CpGs of the genes described herein can be used. The probes hybridizable to the methylated CpGs of the genes contain at least one CpG dinucleotide in the region hybridizing to the methylated CpGs of the genes. Specifically, the probes can include sequences having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence selected from the group consisting of the following sequences: JPEG2026505663000003.jpg98170

[0093] In one embodiment, detecting the methylation status of the target gene includes the presence of hypermethylation in the gene relative to the normal state of the target gene.

[0094] In some embodiments, the biological sample is tissue, cells, blood, urine, serum, or plasma from a patient or test subject suspected of having breast cancer.

[0095] As used herein, the term "suspected cancer patient" refers to an individual who has received an initial diagnosis (e.g., a CT scan showing elevated or increased biomarker levels) but whose stage of cancer or the presence or absence of methylated genes indicative of cancer is unknown. The term further includes individuals who have once had cancer (e.g., individuals who are in remission).

[0096] In some embodiments, a detection test's ability to correctly predict a condition is measured as the sensitivity of the assay, the specificity of the assay, or the area under the receiver operating characteristic (ROC) curve (AUC). The greater the area under the ROC curve, the more accurate or powerful the predictive value of the test.

[0097] In one embodiment, a weighted sum score is measured to determine the methylation status of nucleic acid sequences and genes as indicators. The weighted sum model (WSM) is the most well-known and simplest multi-criteria decision analysis (MCDA) / multi-criteria decision making (MCDM) method for evaluating multiple alternatives in terms of multiple decision criteria.

[0098] In some embodiments, one or more of the biomarkers disclosed herein show a statistical difference in different samples of at least p<0.05, and detection tests using these biomarkers show an AUC of at least 0.9.

[0099] In some embodiments, the hypermethylation status of the epigenetic biomarkers in the DNA sequences described herein correlates with a "poor" prognosis, or the likelihood that a subject will respond poorly to a drug or set of drugs, leading to cancer progression and / or refractory to one or more therapeutic agents. In some cases, a "poor" prognosis refers to the likelihood that a subject will not respond to a drug or set of drugs, leading to cancer progression. In some cases, a "poor" prognosis refers to a subject survival of less than 5 years to less than 1 month. In some cases, a "poor" prognosis refers to a subject survival of less than 5 years to less than 1 month upon treatment. In some cases, a "poor" prognosis further refers to the likelihood that a subject will develop a cancer that is refractory to one or more drugs.

[0100] In some embodiments, the disclosure provides a probe having a sequence selected from the group consisting of SEQ ID NOs: 9 to 12. In some other embodiments, the disclosure provides a primer having a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

[0101] In some embodiments, the present disclosure provides kits for detecting and / or characterizing the methylation profile of a target DNA sequence described herein. In some embodiments, the target DNA sequence comprises a combination selected from the group consisting of: (1) ZFP30 and FBXL7, (2) ZFP30 and CNTN4, (3) FBXL7 and CNTN4, (4) ZFP30, FBXL7, and CNTN4, and (5) ZFP30, FBXL7, CNTN4, and KLRG2.

[0102] In some cases, the kit includes multiple primers or probes for detecting or measuring the methylation status / level of one or more target genes. Such kits optionally include at least one polynucleotide that hybridizes to at least one of the methylation biomarker sequences described herein and at least one reagent for detecting gene methylation. Reagents for detecting methylation include, for example, sodium bisulfate, a polynucleotide designed to hybridize to a sequence that is a product of the marker sequence when the marker sequence is unmethylated (e.g., contains at least one CU conversion), and / or a methylation-sensitive or methylation-dependent restriction enzyme. In some cases, the kit provides a solid support in the form of an assay device suitable for use in the assay. In some cases, the kit further includes a detectable label optionally linked to a polynucleotide (e.g., a probe) in the kit. In some embodiments, the kit further includes a process unit for obtaining a weighted sum score as described herein.

[0103] Optionally, the kit also includes one or more detectably labeled polypeptides capable of hybridizing to the amplified portions. In some embodiments, the kit includes sufficient primers to amplify the target DNA sequences described herein, and optionally includes a detectably labeled polynucleotide capable of hybridizing to each amplified DNA region or portion. The kit can further include a methylation-dependent or methylation-sensitive restriction enzyme and / or sodium bisulfite.

[0104] In some embodiments, the kits include sodium bisulfite, primers, and adapters for amplification of the entire target gene, and a polynucleotide (e.g., a detectably labeled polynucleotide) for quantifying the presence of converted methylated and / or converted unmethylated sequences of at least one cytosine from the DNA region of an epigenetic biomarker described herein.

[0105] In some embodiments, the kits include methylation-sensitive restriction enzymes, primers and adapters for amplification of the entire target gene, and polynucleotides for quantifying the copy number of at least a portion of a DNA region of an epigenetic marker described herein. In some embodiments, the kits include a methylation-binding moiety and one or more polynucleotides for quantifying the copy number of at least a portion of a DNA region of a marker described herein.

[0106] The invention described and claimed herein has many attributes and embodiments, including, but not limited to, those set forth, described, or referenced in this detailed disclosure. It is not intended to be all-inclusive, and the invention described and claimed herein is not limited to or constrained by the features or embodiments identified in this detailed disclosure, which are included for purposes of illustration only and not limitation. Those skilled in the art will readily recognize that many components and parameters may be changed or modified to a certain extent or substituted with known equivalents without departing from the scope of the invention. It is to be understood that such modifications and equivalents are incorporated herein as if individually set forth. The invention also includes all steps, features, compositions, and compounds referenced or shown herein, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0107] All patents, publications, scientific papers, websites, and other documents and materials referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is incorporated by reference to the same extent as if it were individually incorporated by reference in its entirety or as if expressly set forth herein in its entirety. Applicant reserves the right to physically incorporate into this specification any and all materials and information from such patents, publications, scientific papers, websites, electronically available information, and other referenced materials or documents. The reference herein to any applications, patents, and publications is not, and should not be construed as, an admission or suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0108] The specific methods and compositions described herein are representative of preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention. Those skilled in the art will recognize other objects, aspects, and embodiments upon review of this specification, which are encompassed within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein can suitably be practiced in the absence of any element or elements or limitation not specifically disclosed herein as essential. Thus, for example, in each instance herein in an embodiment or example of the invention, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms in the specification. Furthermore, terms such as "comprising," "including," and "containing" are to be interpreted expansively, rather than limiting. Illustratively, the methods and processes described herein may suitably be practiced with steps in a different order and are not necessarily limited to the order of steps set forth herein or in the claims. Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Under no circumstances shall this patent be construed as limited to the particular examples or embodiments or methods specifically disclosed herein. Under no circumstances shall this patent be construed as limited by any statements made by examiners or other officers or employees of the Patent and Trademark Office, unless such statements have been specifically and without limitation or reservation expressly adopted in applicant's reply brief.Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, but should not be construed as limiting the scope of the invention. Any examples of aspects, embodiments, or components of the invention referenced herein should be considered non-limiting.

[0109] The terms and expressions used are terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it will be understood that those skilled in the art may make modifications and variations of the concepts disclosed herein, and that such modifications and variations are deemed to be within the scope of the invention as defined by the appended claims.

[0110] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes any generic description of the invention with a proviso or negative limitation excluding any subject of that genus, regardless of whether the excluded item is specifically recited herein.

[0111] Other embodiments are within the scope of the following claims. Furthermore, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is thereby also described in terms of any individual member of the Markush group or subgroup of members thereof.

[0112] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the preceding description. Therefore, the following examples are merely illustrative and should not be construed as limiting the scope of the present invention in any way.

[0113] Although the present disclosure has been described with exemplary embodiments, various changes and modifications may be suggested by those skilled in the art, and the present disclosure is intended to cover such changes and modifications that fall within the scope of the appended claims. <Example>

[0114] Example 1: Methylation status of target nucleic acids and genes in pancreatic cancer tissues

[0115] The beta values ​​for the Illumina Methylation 450K array-based data were obtained from The Cancer Genome Atlas (TCGA) Research Network. The target nucleic acids and genes shown in Table 1 were selected if their Δβ values ​​(tumor tissue values ​​minus normal tissue values) exceeded 0.3.

[0116] JPEG2026505663000004.jpg45170

[0117] Figures 1A-1D show the differences in methylation status (β values) of target nucleic acids and genes in tumor tissues and adjacent normal tissues (n=185). Darker colors indicate tissues with higher methylation status according to Illumina Methylation 450K array-based data.

[0118] Example 2: Early detection of methylation status of target genes in plasma samples from pancreatic cancer patients and healthy individuals

[0119] cfDNA extracted from plasma of pancreatic cancer patients and healthy controls was analyzed by probe-based methylation-specific real-time PCR (qMSP). Representative figures, Figures 2A and 2B, show the methylation status of the target genes ZFP30 (Figure 2A) and FBXL7 (Figure 2B) in plasma samples from pancreatic cancer patients (n = 31) and healthy controls (n = 12), revealing a distinction between the two groups. Pancreatic cancer patients exhibit higher methylation levels for both target genes compared to healthy controls.

[0120] Example 3: Performance of early detection of methylation status of target genes in plasma samples from pancreatic cancer patients and healthy individuals

[0121] The methylation status results of the target genes ZFP30 and FBXL7 in the plasma samples of the two groups were combined and analyzed. The table below shows the sensitivity, specificity, and accuracy of early detection of the methylation status of the two target genes in the Taiwanese cohort and the translational cohort. The sensitivity, specificity, and accuracy were 100.0%, 87.6%, and 89.1%, respectively, in the Taiwanese cohort, and 87.5%, 87.6%, and 87.6%, respectively, in the Taiwanese cohort-American cohort comparison.

[0122] JPEG2026505663000005.jpg44170

[0123] JPEG2026505663000006.jpg42170

[0124] Example 4: Receiver operating characteristic curve analysis of early detection of methylation status of target genes in plasma samples from pancreatic cancer patients and healthy individuals

[0125] Based on the early detection results of the target genes ZFP30 and FBXL7 in the plasma samples of the two groups, receiver operating characteristic (ROC) curves were constructed for the two target genes ZFP30 and FBXL7, including both individual and combined analyses. The analysis results showed that the optimal diagnostic model was achieved when using the ZFP30 gene, with an area under the curve (AUC) value of 0.911, while the area under the curve (AUC) value of 0.816 was obtained when using the FBXL7 gene.

Claims

1. 1. A method for detecting a methylation status in a subject in need thereof, detecting a predisposition to or incidence of pancreatic cancer, or predicting a treatment response, prognosis, or recurrence of pancreatic cancer in a subject, comprising: (a) providing a biological sample containing DNA from the subject, the DNA comprising the target gene ZFP30 or a fragment thereof; (b) assaying the methylation status of one or more epigenetic biomarkers having CpG sites in the target gene ZFP30 or a fragment thereof; (c) identifying the target gene ZFP30 or a fragment thereof in a biological sample having a methylation level higher than that of a healthy individual as hypermethylated, wherein hypermethylation of the target gene ZFP30 or a fragment thereof indicates pancreatic cancer, or a predisposition to pancreatic cancer, or the incidence, poor therapeutic response, poor prognosis or recurrence of pancreatic cancer.

2. The method of claim 1, wherein the step of assaying the methylation status in (b) comprises extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, amplifying the genomic DNA with a primer pair specific to ZFP30, and measuring the methylation level of one or more CpG sites in ZFP30 by polymerase chain reaction (PCR).

3. 3. The method of claim 1, wherein the step of assaying the methylation status in (b) further comprises extracting genomic DNA from the biological sample followed by treating the resulting extract with bisulfite and amplifying the bisulfite-treated genomic DNA.

4. The method of claim 2 or 3, wherein the pair of primers specific to ZFP30 used to amplify the genomic DNA or bisulfite-treated genomic DNA has at least about 85% sequence identity to the sequences of SEQ ID NOs: 1 and 2.

5. 4. The method according to claim 2 or 3, wherein the methylation level of the target gene ZFP30 or a fragment thereof is assayed using a ZFP30 methylation-specific probe.

6. 6. The method of claim 5, wherein the ZFP30 methylation-specific probe has a sequence having at least 85% identity to SEQ ID NO:

9.

7. 10. The method according to any one of the preceding claims, wherein the one or more epigenetic biomarkers having a CpG site are located at Chr19 37692300-37692450 of the target gene ZFP30.

8. 1. A method for detecting a methylation status in a subject in need thereof, detecting a predisposition to or incidence of pancreatic cancer, or predicting a treatment response, prognosis, or recurrence of pancreatic cancer in a subject, comprising: (aii) providing a biological sample containing DNA from the subject, wherein the DNA comprises a target gene ZFP30 or a fragment thereof, and a target gene FBXL7 or a fragment thereof; (Bii) assaying the methylation status of one or more epigenetic biomarkers having a CpG site in the target gene ZFP30 or a fragment thereof, and the methylation status of one or more epigenetic biomarkers having a CpG site in the target gene FBXL7 or a fragment thereof; (cii) identifying the target genes ZFP30 and FBXL7 or fragments thereof in a biological sample having a methylation level higher than that of a healthy individual as hypermethylated, wherein the hypermethylation of the target genes ZFP30 and FBXL7 or fragments thereof indicates pancreatic cancer, or a predisposition to pancreatic cancer, or incidence, poor treatment response, poor prognosis, or recurrence of pancreatic cancer.

9. 9. The method of claim 8, wherein the step of assaying the methylation status in (bii) comprises extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, amplifying the genomic DNA with primers consisting of a pair of primers specific to ZFP30 and a pair of primers specific to FBXL7, and measuring the methylation level of one or more CpG sites in ZFP30 and the methylation level of one or more CpG sites in FBXL7 by polymerase chain reaction (PCR).

10. 10. The method of claim 9, wherein the step of assaying the methylation status of (bii) further comprises extracting genomic DNA from the biological sample followed by treating the resulting extract with bisulfite and amplifying the bisulfite-treated genomic DNA.

11. The method of claim 9 or 10, wherein the pair of primers specific to ZFP30 used to amplify the genomic DNA or bisulfite-treated genomic DNA has at least about 85% sequence identity to the sequences of SEQ ID NOs: 1 and 2, and the pair of primers specific to FBXL7 used to amplify the genomic DNA or bisulfite-treated genomic DNA has at least about 85% sequence identity to the sequences of SEQ ID NOs: 3 and 4.

12. The method of claim 9 or 10, characterized in that the methylation levels of the target gene ZFP30 or a fragment thereof and the target gene FBXL7 or a fragment thereof are assayed using a ZFP30 methylation-specific probe and an FBXL7 methylation-specific probe.

13. The method of claim 12, wherein the ZFP30 methylation-specific probe has a sequence having at least about 85% identity to SEQ ID NO: 9, and the FBXL7 methylation-specific probe has a sequence having at least 85% identity to SEQ ID NO:

10.

14. 10. The method of claim 9, wherein the one or more epigenetic biomarkers having a CpG site are located at Chr19 37692300-37692450 of the target gene ZFP30, and the one or more epigenetic biomarkers having a CpG site are located at Chr5 15500544-15500694 of the target gene FBXL7.

15. In the method (a) or (aii), the DNA further comprises one or more of the target genes CNTN4 and KLRG2 or fragments thereof; (biii) further comprises assaying the methylation status of one or more epigenetic biomarkers of said target genes CNTN4 and KLRG2 or fragments thereof; 10. The method of claim 9, further comprising identifying one or more of the target genes CNTN4 and KLRG2 or fragments thereof as hypermethylated in a biological sample having a methylation level higher than the methylation level of a control gene compared to a healthy individual, wherein the hypermethylation of the target genes ZFP30, FBXL7, and one or more of CNTN4 and KLRG2 indicates pancreatic cancer, or a predisposition to pancreatic cancer, or incidence, poor treatment response, poor prognosis, or recurrence of pancreatic cancer.

16. 16. The method of claim 15, wherein the step of assaying the methylation status in (biii) comprises extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, amplifying the genomic DNA with primers consisting of a pair of primers specific to CNTN4 and / or a pair of primers specific to KLRG2, and measuring the methylation level of one or more CpG sites in CNTN4 and / or the methylation level of one or more CpG sites in KLRG2 by polymerase chain reaction (PCR).

17. 17. The method of claim 16, wherein the step of assaying the methylation status of (biii) further comprises extracting genomic DNA from the biological sample followed by treating the resulting extract with bisulfite and amplifying the bisulfite-treated genomic DNA.

18. 18. The method of claim 16 or 17, wherein the pair of CNTN4-specific primers used to amplify the genomic DNA or bisulfite-treated genomic DNA has at least about 85% sequence identity to the sequences of SEQ ID NOs: 5 and 6, and the pair of KLRG2-specific primers used to amplify the genomic DNA or bisulfite-treated genomic DNA has at least about 85% sequence identity to the sequences of SEQ ID NOs: 7 and 8.

19. 18. The method of claim 16 or 17, wherein a CNTN4 methylation-specific probe, a KLRG2 methylation-specific probe, or a combination thereof is used to assay the methylation levels of one or more of the target gene CNTN4 and KLRG2 DNA or fragments thereof and a control DNA sequence in the biological sample.

20. 20. The method of claim 19, wherein the CNTN4 methylation-specific probe has a sequence having at least 85% identity to SEQ ID NO: 11, and the KLRG2 methylation-specific probe has a sequence having at least 85% identity to SEQ ID NO:

12.

21. 10. The method according to any one of the preceding claims, characterized in that the biological sample is a tissue sample, a pancreatic juice sample, a pancreatic cyst fluid sample, a stool sample, a cell sample, a blood sample, a urine sample, a serum sample or a plasma sample.

22. 10. The method of any one of the preceding claims, wherein the methylation status is detected by methylation specific PCR (MSP), real-time methylation specific PCR, quantitative methylation specific PCR (QMSP), methylation sensitive DNA restriction enzyme analysis, bisulfite genomic sequencing PCR, or PCR using a methylated DNA specific binding protein.

23. 1. A method for measuring the methylation level of one or more CpG sites in ZFP30 or a fragment thereof, comprising: Extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer; treating the obtained extract with hydrogen sulfate; amplifying the bisulfite-treated genomic DNA with a primer pair specific for ZFP30; measuring the methylation level of one or more CpG sites in ZFP30 by polymerase chain reaction (PCR).

24. The method of claim 23, characterized in that the method further comprises measuring the methylation level of one or more CpG sites in FBXL7 or a fragment thereof, i.e., extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to FBXL7, and measuring the methylation level of one or more CpG sites in FBXL7 by polymerase chain reaction (PCR).

25. The method of claim 24, further comprising measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, i.e., comprising the steps of extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to CNTN4 or a fragment thereof and / or a primer pair specific to KLRG2 or a fragment thereof, and measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, by polymerase chain reaction (PCR).

26. A method for measuring the methylation level of one or more CpG sites in ZFP30 or a fragment thereof and FBXL7 or a fragment thereof, comprising the steps of extracting genomic DNA from a biological sample of a subject suspected of having or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with primers consisting of a pair of primers specific to ZFP30 and a pair of primers specific to FBXL7, and measuring the methylation level of one or more CpG sites in ZFP30 and FBXL7 by polymerase chain reaction (PCR).

27. The method of claim 26, further comprising measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, i.e., comprising the steps of extracting genomic DNA from a biological sample of a subject suspected of or suffering from pancreatic cancer, treating the obtained extract with bisulfite, amplifying the bisulfite-treated genomic DNA with a primer pair specific to CNTN4 or a fragment thereof and / or a primer pair specific to KLRG2 or a fragment thereof, and measuring the methylation level of one or more CpG sites in CNTN4 and / or KLRG2, or fragments thereof, by polymerase chain reaction (PCR).

28. A method comprising the steps of extracting genomic DNA from a biological sample obtained from a subject, treating the extracted genomic DNA with bisulfite, amplifying the bisulfite-treated genomic DNA using a primer specific to a CpG site of ZFP30, a primer specific to a CpG site of FBXL7, or a primer specific to a CpG site of ZFP30 and a primer specific to a CpG site of FBXL7, and measuring the methylation level of the CpG site of ZFP30, or the methylation level of the CpG site of FBXL7, or the methylation levels of the CpG site of ZFP30 and the CpG site of FBXL7.

29. 29. The method of claim 28, further comprising the steps of: extracting genomic DNA from a biological sample obtained from the subject; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA using a primer specific to a CpG site of CNTN4, a primer specific to a CpG site of KLRG2, or a primer specific to a CpG site of CNTN4 and a primer specific to a CpG site of KLRG2; and measuring the methylation level of the CpG site of CNTN4, or the methylation level of the CpG site of KLRG2, or the methylation levels of the CpG site of CNTN4 and the CpG site of KLRG2.

30. 29. The method of any one of claims 13 to 28, wherein the primer has at least about 85% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

31. 29. The method of any one of claims 13 to 28, wherein the probe has a sequence of SEQ ID NO: 9 to 12 and is used to detect or measure the methylation status of one or more of the target genes ZFP30, FBXL7, CNTN4, and KLRG2 or fragments thereof.

32. 29. The method according to any one of claims 13 to 28, wherein the PCR is methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis or bisulfite genomic sequencing PCR.

33. A kit for detecting the methylation status of a subject in need of pancreatic cancer detection, or for detecting a predisposition to pancreatic cancer or the incidence of pancreatic cancer, or for predicting the therapeutic response, prognosis or recurrence of pancreatic cancer in a subject, the kit comprising a primer pair having the sequences of SEQ ID NO: 1 and 2 and / or a probe having the sequence of SEQ ID NO: 9 for assaying the methylation status of an epigenetic biomarker in the target gene ZFP30 or a fragment thereof.

34. The kit of claim 33, further comprising a primer pair having the sequences of SEQ ID NO: 3 and 4 and / or a probe having the sequence of SEQ ID NO: 10 for assaying the methylation status of an epigenetic biomarker in the target gene FBXL7 or a fragment thereof.

35. The kit of claim 34, further comprising a primer pair having sequences of SEQ ID NOs: 5 and 6 and / or a probe having the sequence of SEQ ID NO: 11 for assaying the methylation status of an epigenetic biomarker in the target gene CNTN4 or a fragment thereof, and / or a primer pair having sequences of SEQ ID NOs: 7 and 8 and / or a probe having the sequence of SEQ ID NO: 12 for assaying the methylation status of an epigenetic biomarker in the target gene KLRG2 or a fragment thereof.

36. 35. The kit of claim 33 or 34, further comprising sodium bisulfite and adaptors for amplification of the entire target gene, and a polynucleotide (e.g., a detectably labeled polynucleotide) for quantifying the presence of converted methylated and / or converted unmethylated sequences of at least one cytosine from the DNA region of the epigenetic biomarker in the target gene.

37. A primer pair having the sequences of SEQ ID NOs: 3 and 4, the sequences of SEQ ID NOs: 5 and 6, or the sequences of SEQ ID NOs: 7 and 8.

38. A probe having the sequence of SEQ ID NO: 9, 10, 11 or 12.

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