WO2016115354A1

WO2016115354A1 - Methods for cancer diagnosis and prognosis

Info

Publication number: WO2016115354A1
Application number: PCT/US2016/013421
Authority: WO
Inventors: Hung-Cheng Lai; Rui-lan HUANG
Original assignee: Taipei Medical University; Yen, Yun
Priority date: 2015-01-14
Filing date: 2016-01-14
Publication date: 2016-07-21
Also published as: TW201632629A

Abstract

Disclosed herein are various methods for assessing the presence of ovarian tumor or malignant ovarian tumor, or the prognosis of the subject, based on the hypermethylation of one or more markers. Also disclosed herein are methods for determining whether a tissue sample derived from a subject is neoplastic based on the hypermethylation of one or more markers; said tissue sample is a bladder sample, breast sample, cervical scraping sample, colon sample, endometrium sample, liver sample, oral scraping sample, or osteosarcoma sample.

Description

METHODS FOR CANCER DIAGNOSIS AND PROGNOSIS

BACKGROUND OF THE INVENTION

[0001 ] 1 . FIELD OF THE INVENTION

[0002] The present disclosure relates to cancer diagnosis and prognosis. More particularly, the disclosed invention relates to method for cancer diagnosis and prognosis based on the methylation state of selected markers.

[0003] 2. DESCRIPTION OF RELATED ART

[0004] Ovarian cancer is the fifth most common cancer for women. The mortality rate of ovarian cancer is the highest among female reproductive cancers. Ovarian cancer symptoms are often vague during the early stage, and hence ovarian cancer is rarely diagnosed in its early stages. By the time the diagnosis is made, it is usually quite advanced and the tumor has often spread beyond the ovaries. To be specific, about 90 percent of ovarian cancer cases develop from epithelial cells, and up to 70 percent of epithelial ovarian cancer cases are diagnosed at stage III or IV.

[0005] If treated early, ovarian cancer may be curable; for women diagnosed with stage I ovarian cancer, the 10-year survival rate is close to 90%. In contrast, the five-year survival rate for women in stage III or IV is only about 15%-20%. Accordingly, a suitable method for screening women for early stage ovarian cancer would be of great benefit.

[0006] Women with BRCA1 and BRCA2 genetic mutations, the Lynch syndrome, or a family history of ovarian cancer are at increased risk for ovarian cancer. These women are recommended for genetic counseling to evaluate their potential risks. Women whose family history is associated with an increased risk of BRCA1 or BRCA2 mutations are often subject to BRCA testing.

[0007] For asymptomatic women without known genetic mutations that increase risk for ovarian cancer, transvaginal ultrasonography and serum cancer antigen (CA)-125 testing are the most commonly suggested screening tests. However, researchers have reported that such screenings do not decrease ovarian cancer mortality. Moreover, the high false-positive rate (about 10%) of these screenings leads to unneeded oophorectomy that may cause major complications. Since the harms caused by current screening procedures for ovarian cancer outweigh the benefits, in 2012, the U.S. Preventive Service Task Force recommended against screening for ovarian cancer in asymptomatic, average-risk women using serum CA-125 testing and transvaginal ultrasonography.

[0008] In view of the foregoing, the diagnostic tests presently available for ovarian cancer are not well suited to patient screening. Accordingly, there exists a need in the art for providing methods for use in the diagnosis and prognosis of ovarian cancer.

SUMMARY

[0009] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0010] In the first aspect, the present disclosure is directed to a method for assessing the tumor malignancy or the prognosis of a subject diagnosed with ovarian neoplasm.

[001 1 ] According to one embodiment of the present disclosure, the method comprises the following steps:

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, TMEM 132D (SEQ ID NO. 75), I RX1 (SEQ ID NO. 76), MIR124-2 (SEQ I D NO. 77), TRH (SEQ ID NO. 78), PCDHB4 (SEQ I D NO.

79), ZNF132 (SEQ ID NO. 80), KLHL33 (SEQ ID NO. 81 ), DPP6 (SEQ ID NO. 82), ADCY8 (SEQ ID NO. 83), C20orf85 (SEQ ID NO. 84), C17orf46 (SEQ ID NO. 85), FOXI2 (SEQ ID NO. 86), HIST1 H4I (SEQ I D NO. 87), HIST1 H2BB (SEQ ID NO. 88), SLC6A3 (SEQ ID NO. 89), HTR1 A (SEQ ID NO. 90), CD01 (SEQ ID N0.91 ), CDH8 (SEQ ID NO. 92), PCDHGA5 (SEQ

ID NO. 93), COL14A1 (SEQ ID NO. 94), SPAG6 (SEQ ID NO. 95), RAX (SEQ ID NO. 96), PRDM14 (SEQ ID NO. 97), HIST1 H3E (SEQ ID NO. 98), ZIC1 (SEQ ID NO. 99), COX7A1 (SEQ ID NO. 100), and ECEL1 P2 (SEQ ID NO. 101 );

(c) determining whether the at least one target gene is hypermethylated; and

(d) assessing the tumor malignancy or prognosis of the subject based on the result of step (c); wherein the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian neoplasm or a poor prognosis.

[0012] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(e) determining the methylation state of GHSR gene (SEQ ID NO. 102); and

(f) determining whether the GHSR gene is hypermethylated; wherein in the step (d), the tumor malignancy or prognosis of the subject is determined based on the results of both step (c) and step (f), and the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has malignant ovarian neoplasm or a poor prognosis.

[0013] Still optionally, in some embodiments, the method further comprises the following steps:

(e) determining the methylation state of HIST1 H3C gene; and

(f) determining whether the HIST1 H3C gene is hypermethylated; wherein in the step (d), the prognosis of the subject is determined based on the results of both step (c) and step (f), and the hypermethylation of the HIST1 H3C gene and the hypermethylation of the at least one HIST1 H2BB, HIST1 H3E, and HIST1 H4I indicate that the subject has a poor progression-free survival.

[0014] According to various embodiments of the present disclosure, the hypermethylation of the at least one target gene may indicate that the subject has (1 ) a poor progression-free survival or (2) a poor overall survival.

[0015] In certain embodiments, the at least one gene is TRH, the prognosis is associated with progression-free survival, and the method further comprising the steps of,

(e) determining the methylation state of GHSR gene; and (f) determining whether the GHSR gene is hypermethylated, wherein in the step (d), the prognosis of the subject is determined based on the results of step (c) and step (f), and the hypermethylation of the GHSR gene and the hypermethylation of the TRH indicate that the subject has a poor progression-free survival.

[0016] According to various embodiments of the present disclosure, the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage), tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma), serum, lymph, spinal fluid). According to certain working examples of the present disclosure, the sample is obtained from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.

[0017] In the second aspect, the present disclosure is directed to a method for assessing whether a subject has ovarian neoplasm.

[0018] According to one embodiment of the present disclosure, the method comprises the following steps:

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, TMEM132D, I RX1 , MI R124-2, TRH, PCDHB4, ZNF132, KLHL33, DPP6, ADCY8, C20orf85, C17orf46, FOXI2, HIST1 H4I,

HIST1 H2BB, SLC6A3, HTR1A, CD01 , CDH8, PCDHGA5, COL14A1 , SPAG6, RAX, PRDM 14, HIST1 H3E, ZIC1 , COX7A1 , and ECEL1 P2;

(c) determining whether the at least one target gene is hypermethylated; and

(d) assessing whether the subject has ovarian neoplasm based on the result of step (c), wherein the hypermethylation of the at least one target gene indicates the presence of ovarian neoplasm in the subject.

[0019] According to optional embodiments of the present disclosure, the method further comprises the following steps: (e) determining the methylation state of GHSR gene; and

(f) determining whether the GHSR gene is hypermethylated; wherein in the step (d), the presence of ovarian neoplasm in the subject is determined based on the results of step (c) and step (f), and the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate the presence of ovarian neoplasm in the subject.

[0020] In certain optional embodiments, the sample is a serum or plasma sample, and when the results of the step (d) indicates that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of FOXI2, MIR-124, or both.

[0021 ] In other optional embodiments, the sample is obtained from cervical scraping cells, and the at least one target gene is selected from the group consisting of, MIR124-2, TRH, and FOXI2. In this case, when in the step (d), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

[0022] According to various embodiments of the present disclosure, the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage), tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma), serum, lymph, spinal fluid). According to certain working examples of the present disclosure, the sample is obtained from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.

[0023] In the third aspect, the present disclosure is directed to a method for assessing whether a subject has malignant ovarian cancer.

[0024] According to one embodiment of the present disclosure, the method comprises the following steps: (a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, and MIR124-2; and

(c) determining whether the at least one target gene is hypermethylated; wherein the subject has malignant ovarian cancer if the at least one target gene is hypermethylated.

[0025] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(d) determining the methylation state of GHSR gene; and

(e) determining whether the GHSR gene is hypermethylated; wherein the subject has malignant ovarian cancer if the GHSR gene and the at least one target gene are hypermethylated.

[0026] As an alternative to, or in addition to, steps (d) and (e) described in the previous embodiments, the method according to certain embodiments of the present disclosure further comprises the following steps:

(f) determining the methylation state of HIST1 H3C gene; and

(g) determining whether the HIST1 H3C gene is hypermethylated; wherein the subject has malignant ovarian cancer if the at least one target gene and at least one of the HIST1 H3C gene and the GHSR gene are hypermethylated.

[0027] According to various embodiments of the present disclosure, the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage), tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma), serum, lymph, spinal fluid). According to certain working examples of the present disclosure, the sample is obtained from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject. [0028] In the fourth aspect, the present invention is directed to a method for assessing whether a subject has ovarian tumor.

[0029] According to one embodiment of the present invention, the method comprises the following steps:

(a) obtaining a sample from the subject;

(c) determining whether the at least one target gene is hypermethylated; wherein the subject has ovarian tumor if the at least one target gene is hypermethylated.

[0030] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(d) determining the methylation state of GHSR gene; and

(e) determining whether the GHSR gene is hypermethylated; wherein the subject has ovarian tumor if the GHSR gene and the at least one target gene are hypermethylated.

[0031 ] In certain embodiments, when in the step (c), it is determined that he subject has ovarian tumor, the method further comprises the step of determining whether the ovarian tumor is malignant depending on the methylation state of the at least one target gene.

[0032] According to various embodiments of the present disclosure, the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage), tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma), serum, lymph, spinal fluid). According to certain working examples of the present disclosure, the sample is obtained from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject. [0033] In certain optional embodiments, the sample is a serum or plasma sample, and when the results of the step (c) indicates that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of FOXI2, MIR-124, or both.

[0034] In other optional embodiments, the sample is obtained from cervical scraping cells, and the at least one target gene is selected from the group consisting of, MIR124-2, TRH, and FOXI2. In this case, when in the step (d), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

[0035] In the fifth aspect, the present invention is directed to a method for assessing whether a subject has malignant ovarian cancer.

[0036] According to one embodiment of the present invention, the method comprises the following steps:

(a) obtaining a sample from the subject, wherein the sample is derived from scraping cells;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, TRH, FOXI2, and Ml R124-2; and

[0037] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(d) determining the methylation state of GHSR gene using the method of the step (b); and

(e) determining whether the GHSR gene is hypermethylated using the method of the step (c); wherein the subject has malignant ovarian cancer if the GHSR gene and the at least one target gene are hypermethylated.

[0038] In the sixth aspect, the present invention is directed to a method for assessing whether a subject has ovarian tumor. [0039] According to one embodiment of the present invention, the method comprises the following steps:

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is TRH or MIR124-2; and

[0040] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(e) determining whether the GHSR gene is hypermethylated using the method of the step (c); wherein the subject has ovarian tumor if the GHSR gene and the at least one target gene are hypermethylated.

[0041 ] In the seventh aspect, the present invention is directed to a method for assessing whether a subject has malignant ovarian cancer.

[0042] According to one embodiment of the present invention, the method comprises the following steps:

(a) obtaining a body fluid sample from the subject;

(b) determining the methylation state of at least one target gene in the body fluid sample, wherein the at least one target gene is FOXI2 or Ml R124-2; and

[0043] According to optional embodiments of the present disclosure, the body fluid sample is a serum or plasma sample.

[0044] In the eighth aspect, the present invention is directed to a method for assessing whether a subject has bladder neoplasm using a bladder sample derived from the subject.

[0045] According to one embodiment of the present invention, the method comprises the following steps: (a) determining the methylation state of at least one target gene in the bladder sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 ; and

(b) determining whether the at least one target gene is hypermethylated; wherein the bladder sample is neoplastic when the at least one target gene is hypermethylated.

[0046] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the bladder sample using the method of the step (a); and

(d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the bladder sample is neoplastic when the GHSR gene and the at least one target gene are hypermethylated.

[0047] In the ninth aspect, the present invention is directed to a method for assessing whether a subject has breast neoplasm using a breast sample derived from the subject.

[0048] According to one embodiment of the present invention, the method comprises the following steps:

(a) determining the methylation state of at least one target gene in the breast sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM 14, RAX, SPAG6, TRH, and Z1 C1 ; and

(b) determining whether the at least one target gene is hypermethylated; wherein the subject has breast neoplasm when the at least one target gene is hypermethylated.

[0049] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the breast sample using the method of the step (a); and (d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the subject has breast neoplasm when the GHSR gene and the at least one target gene are hypermethylated.

[0050] In the tenth aspect, the present invention is directed to a method for assessing whether a subject has cervical neoplasm using a cervical scraping sample derived from the subject.

[0051 ] According to one embodiment of the present invention, the method comprises the following steps:

(a) determining the methylation state of at least one target gene in the cervical scraping sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM 14, RAX, SPAG6, TRH, and Z1 C1 ; and

(b) determining whether the at least one target gene is hypermethylated; wherein the subject has cervical neoplasm when the at least one target gene is hypermethylated.

[0052] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the cervical scraping sample using the method of the step (a); and

(d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the subject has cervical neoplasm when the GHSR gene and the at least one target gene are hypermethylated.

[0053] In the eleventh aspect, the present invention is directed to a method for assessing whether a subject has colon neoplasm using a colon sample derived from the subject.

[0054] According to one embodiment of the present invention, the method comprises the following steps:

(a) determining the methylation state of at least one target gene in the colon sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM 14, RAX, SPAG6, TRH, and Z1 C1 ; and (b) determining whether the at least one target gene is hypermethylated; wherein the subject has colon neoplasm when the at least one target gene is hypermethylated.

[0055] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the colon sample using the method of the step (a); and

(d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the subject has colon neoplasm when the GHSR gene and the at least one target gene are hypermethylated.

[0056] In the twelfth aspect, the present invention is directed to a method for assessing whether a subject has endometrium neoplasm.

[0057] According to one embodiment of the present invention, the method comprises the following steps:

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the endometrium sample, wherein the at least one target gene is selected from the group consisting of, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, SPAG6, TRH, PRDM14, TRH, and Z1 C1 ; and

(c) determining whether the at least one target gene is hypermethylated; wherein the subject has endometrium neoplasm when the at least one target gene is hypermethylated.

[0058] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(d) determining the methylation state of GHSR gene in the sample using the method of the step (b); and

(e) determining whether the GHSR gene is hypermethylated using the method of the step (c); wherein the subject has endometrium neoplasm when the GHSR gene and the at least one target gene are hypermethylated.

[0059] In certain embodiments, the sample is obtained from endometrium cells, and the at least one target genes is selected from the group consisting of, PRDM14, TRH, MIR124-2, Z1 C1 , and FOXI2. Alternatively, the sample is obtained from cervical scraping cells, and the at least one target gene is selected from the group consisting of, PRDM14, TRH, MIR124-2, Z1 C1 , and FOXI2.

[0060] In the thirteenth aspect, the present invention is directed to a method for assessing whether a subject has liver neoplasm using a liver sample derived from the subject.

[0061 ] According to one embodiment of the present invention, the method comprises the following steps:

(a) determining the methylation state of at least one target gene in the liver sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2,

FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM 14, RAX, SPAG6, TRH, and Z1 C1 ; and

(b) determining whether the at least one target gene is hypermethylated; wherein the subject has liver neoplasm when the at least one target gene is hypermethylated.

[0062] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the liver sample using the method of the step (a); and

(d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the subject has liver neoplasm when the GHSR gene and the at least one target gene are hypermethylated.

[0063] In the fourteenth aspect, the present invention is directed to a method for assessing whether a subject has oral neoplasm using an oral scraping sample derived from the subject.

[0064] According to one embodiment of the present invention, the method comprises the following steps:

(a) determining the methylation state of at least one target gene in the oral scraping sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 ; and (b) determining whether the at least one target gene is hypermethylated; wherein the oral scraping sample is neoplastic subject has oral neoplasm when the at least one target gene is hypermethylated.

[0065] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the oral scraping sample using the method of the step (a); and

(d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the oral scraping sample is neoplastic subject has oral neoplasm when the GHSR gene and the at least one target gene are hypermethylated.

[0066] In the fifteenth aspect, the present invention is directed to a method for assessing whether a subject has osteosarcoma using an osteosarcoma sample derived from the subject.

[0067] According to one embodiment of the present invention, the method comprises the following steps:

(a) determining the methylation state of at least one target gene in the osteosarcoma sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2,

PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 ; and

(b) determining whether the at least one target gene is hypermethylated;

[0068] According to optional embodiments of the present disclosure, the method further comprises the following steps:

(c) determining the methylation state of GHSR gene in the osteosarcoma sample using the method of the step (a); and

(d) determining whether the GHSR gene is hypermethylated using the method of the step (b); wherein the subject has osteosarcoma when the GHSR gene and the at least one target gene are hypermethylated.

[0069] Optionally, the step of determining the methylation state of a gene, as described in the above-mentioned aspects/embodiments of the present disclosure, can be achieved by performing methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq), or nanopore sequencing.

[0070] Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071 ] The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:

[0072] Figures 1A and 1 B summarize the methylation levels of 21 candidates genes and 1 control genes in the ovarian tissue specimens according to one embodiment of the present disclosure;

[0073] Figure 2 provides the methylation profiles of 1 1 candidates genes in the ovarian tissue specimens according to one embodiment of the present disclosure;

[0074] Figure 3 summarizes the methylation indexes of 2 candidates genes in the pooled cell-free DNA sample according to one embodiment of the present disclosure;

[0075] Figures 4A and 4B provide line graphs illustrating the prognostic power of four histone cluster genes in the ovarian tissue specimens according to one embodiment of the present disclosure;

[0076] Figures 5A and 5B provide line graphs illustrating the prognostic power of TRH gene, GHSR gene and both in the ovarian tissue specimens according to one embodiment of the present disclosure;

[0077] Figures 6A, 6B, and 6C summarize the methylation indexes of 21 candidate genes and one control genes in the various tissue specimens according to one embodiment of the present disclosure; [0078] Figures 7A and 7B provides the methylation profiles and sensitivity of CD01 in tissue and cervical scraping specimens according to one embodiment of the present disclosure;. DESCRIPTION

[0079] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0080] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention belongs.

[0081 ] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1 %, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0082] Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms "a" and "an" include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0083] As used herein, the term "diagnosis" refers to the identification of a pathological state, disease, or condition, such as neoplasms of various tissue origins, including ovary, bladder, breast, cervix, colon, endometrium, liver, oral tissue, osteosarcoma, and soft tissue. In some cases, the term diagnosis also refers to distinguishing between the malignant and benign neoplasms.

[0084] The term "prognosis" as used herein refers to the prediction of the likelihood of cancer-attributable death or progression, including any of, the recurrence rate and metastatic spread of a neoplastic disease, and the recurrence-free survival time, progression-free time, or the overall survival time of a subject diagnosed with a neoplastic disease. According to certain embodiments of the present application, the term "prognosis" concerns an estimation of the recurrence-free survival (RFS), progression-free survival (PFS), or overall survival (OS). Further, the term "poor prognosis" as used herein means that a subject's chance of having a given remaining expectancy of life is substantially decreased, as compared with another subject who has a normal methylation profile of one or more markers identified herein. In some instance, a subject with a poor RFS suggests that the subject's RFS time may be less than 12 or 24 months. Similarly, a poor overall or progression-free survival indicates that the subject's OS or PFS time may be less than 12 or 24 months. Alternatively, a poor RFS or PFS may suggest that the subject has a higher probability of experiencing tumor recurrence or progression during a given time period, as compared with a reference subject group. [0085] Throughout the present disclosure, the term "neoplasm" refers to a new and abnormal growth of cells or a growth of abnormal cells that reproduce faster than normal. A neoplasm creates an unstructured mass (a tumor), which can be either benign or malignant. The term "benign" refers to a neoplasm or tumor that is noncancerous, e.g. its cells do not invade surrounding tissues or metastasize to distant sites; whereas the term "malignant" refers to a neoplasm or tumor that is metastatic, invades contiguous tissue or no longer under normal cellular growth control.

[0086] As used herein, the term "cancer" refers to all types of cancer or malignant neoplasm or tumors found in animals, including leukemia, carcinoma, melanoma, and sarcoma. The methods according to various embodiments of the present disclosure are directed to the diagnosis and/or prognosis of one or more carcinomas and sarcomas. The term "carcinoma" refers to a malignant tumor originating from epithelial cells. Exemplary carcinomas of embodiments of the present disclosure include, but are not limited to, ovarian cancer, cervical cancer, endometrial cancer, breast cancer, hepatocellular carcinoma, bladder cancer, colon cancer, and oral cancer. The term "sarcoma" generally refers to a malignant tumor that develops from osteosarcoma, cartilage (e.g., chondrosarcoma) or soft tissues like fat, muscle, nerves, fibrous tissues, blood vessels, or deep skin tissues.

[0087] The term "methylation" as used herein, refers to the covalent attachment of a methyl group at the C5-position of cytosine within the CpG dinucleotides of the core promoter region of a gene. The term "methylation state" refers to the presence or absence of 5-methyl- cytosine (5-mCyt) at one or a plurality of CpG dinucleotides within a gene or nucleic acid sequence of interest. As used herein, the term "methylation level" refers to the amount of methylation in one or more copies of a gene or nucleic acid sequence of interest. The methylation level may be calculated as an absolute measure of methylation within the gene or nucleic acid sequence of interest. Also a "relative methylation level" may be determined as the amount of methylated DNA, relative to the total amount DNA present or as the number of methylated copies of a gene or nucleic acid sequence of interest, relative to the total number of copies of the gene or nucleic acid sequence. Additionally, the "methylation level" can be determined as the percentage of methylated CpG sites within the DNA stretch of interest.

[0088] As used herein, the term "methylation profile" refers to a set of data representing the methylation level of one or more target genes in a sample of interest. In some embodiments, the methylation profile is compared to a reference methylation profile derived from a known type of sample (e.g. , cancerous or noncancerous samples or samples from different stages of cancer).

[0089] As used herein, the term "differential methylation" refers to a difference in the methylation level of one or more target genes in one sample or group, as compared with the methylation level of said one or more target genes in another sample or group. The differential methylation can be classified as an increased methylation ("hypermethylation") or a decreased methylation ("hypomethylation"). As used herein, the term "hypermethylation" of a target gene in a test sample refers to an increased methylation level of at least 10%, relative to the average methylation level of the target gene in a reference sample. According to various embodiments of the present disclosure, the increased methylation level may be at least 15, 20, 25, 30, 35, 40, 45, or 50%.

[0090] "Percentage (%) nucleotide sequence identity" with respect to a gene or nucleotide sequence identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the referenced polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percentage nucleotide sequence identity of a given polynucleotide sequence A to a referenced polynucleotide sequence B (which can alternatively be phrased as a given polynucleotide sequence A that has a certain % nucleotide sequence identity to a referenced polynucleotide sequence B) is calculated by the formula as follows:

where X is the number of nucleotide residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of nucleotide residues in A or B, whichever is shorter.

[0091 ] As could be appreciated, all the genes or polynucleotide sequences described herein respectively comprise their variants that have at least 75% nucleotide sequence identity to the named genes or polynucleotide sequences. Accordingly, unless otherwise expressly specified, all of the genes or polynucleotide sequences described herein should be understood as modified in all instances by the phrase "and a polynucleotide sequence having at least 75% nucleotide sequence identity thereto."

[0092] The term "subject" refers to a mammal including the human species that can be subjected to the diagnosis and/or prognosis methods of the present invention. The term "subject" is intended to refer to both the male and female gender unless one gender is specifically indicated.

[0093] The term "sample" used herein comprises any samples obtained from a patient. According to embodiments of the present disclosure, the sample contains DNA molecules and the methylation level thereof can be determined. Examples of such body samples include, but are not limited to, blood, smears, sputum, urine, stool, liquor, bile, gastrointestinal secretions, lymph fluid, osteosarcoma marrow, organ aspirates and organ or tissue biopsies. These body samples can be obtained from the patient by routine measures known to persons having ordinary skill in the art. Further, persons having ordinary skill in the art are also familiar with methods and reagents for the DNA isolation from the sample, e.g. extraction with phenol/chloroform or by means of commercial kits.

[0094] The present disclosure is based, at least in part, on the finding that differential methylation (in particular, hypermethylation) of one or more target genes, as identified hereinbelow, relates to the tumor progression, or the absence thereof, in a subject. Accordingly, these target genes, alone or in combination, can be used as biomarkers for the prediction of risk or susceptibility of a subject developing a neoplasm, the determination of the malignancy of the neoplasm, and/or making prognosis of the patient being diagnosed with said neoplasm. Further, the methylation profile of relevant genes of the patient can be used as a guide for tailoring suitable therapy regime individually. For example, for patients with one or more hypermethylated target genes listed herein, de-methylation agents or other epigenetic drugs can be administered to the patients to treat the neoplasm.

[0095] In view of the foregoing, the present disclosure provides various diagnostic and/or prognostic methods, which will be separately addressed below. For example, all methods involve the determination of the methylation state or methylation level of at least one target gene. Hence, the steps common to most, if not all, claimed methods are first described in the following paragraph.

[0096] According to various aspects and/or embodiments of the present disclosure, for methods that are practiced on a live subject, common steps include, at least, a sampling step (a), in which a biological sample is obtained from the subject; at least one methylation determination step (b), in which the methylation state of at least one target gene in the sample is determined; and at least one determining step (c), in which the presence or absence of hypermethylation of said at least one target gene is determined. As to methods that are directly practiced on biological samples, such as tissue biopsy samples, cervical scraping cells, and body fluid samples (e.g., blood or urine), the afore-mentioned sampling step (a) is omitted. The methylation profile thus obtained is then used in making the diagnostic and/or prognostic assessments, as respectively described below in connection with each aspect and representative embodiments of the present disclosure.

[0097] In the first aspect, the present disclosure provides a method for assessing the tumor malignancy or the prognosis of a subject diagnosed with ovarian neoplasm, which comprises the common steps (a), (b) and (c) as described above, and an assessement step (d), in which the tumor malignancy or prognosis of the subject is determined based on the result of step (c). Specifically, the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian neoplasm or a poor prognosis.

[0098] According to various embodiments of the present disclosure, the at least one target gene is selected from the group consisting of, TMEM132D, IRX1 , MIR124-2, TRH, PCDHB4, ZNF132, KLHL33, DPP6, ADCY8, C20orf85, C17orf46, FOXI2, HIST1 H4I, HIST1 H2BB, SLC6A3, HTR1 A, CD01 , CDH8, PCDHGA5, COL14A1 , SPAG6, RAX, PRDM14, HIST1 H3E, ZIC1 , COX7A1 , and ECEL1 P2.

[0099] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (e), in which the methylation state of GHSR gene is determined; and a second determining step (f), in which the presence or absence of hypermethylation of the GHSR gene is determined.

[0100] In this optional embodiment, in the assessment step (d), the tumor malignancy or prognosis of the subject is determined based on the results of both step (c) and step (f). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has malignant ovarian neoplasm or a poor prognosis. For example, the at least one target gene to be used together with the GHSR gene may be the TRH gene, and the combination of GHSR and TRH is effective in assessing the progression-free survival of the subject. As is evident from results of the working examples provided below, taking the methylation profile of the GHSR gene into account while assessing the tumor malignancy or prognosis of the subject provides a more accurate assessment. Accordingly, this embodiment can be used in combination with any other embodiments provided herein.

[0101 ] It should be noted that the first and second methylation determination steps could be performed simultaneously or sequentially, in a single procedure or in separates procedures. Similarly, the first and second determining steps can be performed at the same time or in sequence. As could be appreciated, the above-mentioned description regarding the sequence in which these steps are performed or the procedures used for carried out these steps is also applicable in other embodiments described herein. [0102] According to various embodiments of the present disclosure, the hypermethylation of the at least one target gene may indicate (1 ) a poor progression-free survival or (2) a poor overall survival.

[0103] In optional embodiments, the at least one target gene is selected from the group consisting of, ADCY8, CD01 , HIST1 H4I, ECEL1 P2, HTR1 A, SPAG6, CDH8, DPP6, HIST1 H3E, PCDHGA5, RAX, C17orf46, COL14A1 , FOXI2, PRDM14, ZIC1 , HIST1 H2BB, MIR124-2, and TRH, and the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian neoplasm.

[0104] Still optionally, the at least one target gene is selected from the group consisting of, MIR124-2, TRH, PCDHB4, ZNF132, KLHL33, C20orf85, C17orf46, HIST1 H4I, HIST1 H2BB, CD01 , CDH8, PCDHGA5, COL14A1 , SPAG6, HIST1 H3E, ZIC1 , COX7A1 , and ECEL1 P2, and the hypermethylation of the at least one target gene indicates that the subject has a poor progression-free survival.

[0105] Alternatively, the at least one target gene is selected from the group consisting of, TMEM132D, IRX1 , KLHL33, DPP6, ADCY8, FOXI2, SLC6A3, HTR1A, CD01 , CDH8, RAX, and PRDM14, and the hypermethylation of the at least one target gene indicates that the subject has a poor overall survival.

[0106] In certain optional embodiments, the at least one target gene is selected from a specific set of markers that consists of, HIST1 H2BB, HIST1 H3E, and HIST1 H4I. This set of markers is particularly useful for assessing the progression-free survival of the subject. According to further embodiments, the hypermethylation of two or three of the HIST1 H2BB, HIST1 H3E, and HIST1 H4I genes indicates that the subject has a poor progression-free survival.

[0107] Moreover, this set of markers can be used in connection with an additional marker, HIST1 H3C gene. According to optional embodiments of the present disclosure, the method further comprises a second methylation determination step (e) in which the methylation state of HIST1 H3C gene is determined; and a second determining step (f), in which the presence or absence of hypermethylation of the HIST1 H3C gene is determined. In this embodiment, in the assessment step (d), the tumor malignancy or prognosis of the subject is determined based on the results of both step (c) and step (f). Specifically, the hypermethylation of the HIST1 H3C gene and the hypermethylation of the above-mentioned set of markers indicate that the subject has a poor progression-free survival.

[0108] In some embodiments, the sample is obtained from the subject's body fluid (such as, serum or plasma). In this case, the target gene can be FOXI2, MIR124-2, or both.

[0109] In some other embodiments, the sample is obtained from the ovarian tissue (e.g. , from cervical scraping cells) of the subject, and the target gene is MIR124-2, TRH, or FOXI2, or a combination thereof. Further, the assessment step (d) can be performed based on the methylation profiles of GHSR and at least one target gene selected from the group consisting of MIR124-2, TRH, and FOXI2.

[01 10] According to various embodiments of the present disclosure, the methylation state of a gene can be determined by procedures, which include but are not limited to, methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing.

[01 1 1 ] In the working examples provided below, the methylation state is determined using qMSP or bisulfite pyrosequencing. As could be appreciated, the present method is not limited to the methods described above; rather, the scope of the claimed invention encompasses the use of other equivalent methods for quantitatively determining the methylation state or level of a particular gene. Further, the above-mentioned methods and equivalents thereof are also applicable to the embodiments described hereinbelow, hence, the method suitable for determining the methylation state or methylation level of the gene is not repeated in the following aspects/embodiments, for the sake of brevity.

[01 12] In the second aspect, the present disclosure provides a method for assessing whether the subject has ovarian neoplasm, comprising the steps of. The method also comprises the common steps (a), (b) and (c) as described above, and an assessement step (d) in which the presence of ovarian neoplasm in the subject is determined based on the result of step (c). In particular, the hypermethylation of the at least one target gene indicates that the subject has ovarian neoplasm.

[01 13] According to various embodiments of the present disclosure, the at least one target gene is selected from the group consisting of, TMEM132D, IRX1 , MIR124-2, TRH, PCDHB4, ZNF132, KLHL33, DPP6, ADCY8, C20orf85, C17orf46, FOXI2, HIST1 H4I, HIST1 H2BB, SLC6A3, HTR1 A, CD01 , CDH8, PCDHGA5, COL14A1 , SPAG6, RAX, PRDM14, HIST1 H3E, ZIC1 , COX7A1 , and ECEL1 P2. In some optional embodiments, the at least one target gene is selected from the group consisting of, MIR124-2, CD01 , HTR1A, and TRH.

[01 14] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (e) and a second determining step (f), as described above in connection with the methods of the first aspect of the present disclosure.

[01 15] In this optional embodiment, in the assessment step (d), the presence of the ovarian neoplasm is determined based on the results of both step (c) and step (f). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has ovarian neoplasm. For example, the GHSR gene can be used together with the MIR124-2 gene in assessing the presence of the ovarian neoplasm in the subject to attain a more accurate diagnosis. As could be appreciated, this embodiment can be used in combination with any other embodiments provided herein.

[01 16] In some embodiments, the sample is obtained from the subject's body fluid (such as, serum or plasma). In this case, the target gene can be FOXI2, MIR124-2, or both. Still optionally, when the results of the step (d) indicates that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of FOXI2, MIR-124, or both.

[01 17] In some other embodiments, the sample is obtained from the ovarian tissue (e.g. , from cervical scraping cells) of the subject, and the target gene is MIR124-2, TRH, or FOXI2, or a combination thereof. Further, the assessment step (d) can be performed based on the methylation profiles of GHSR and at least one target gene selected from the group consisting of MIR124-2, TRH, and FOXI2. Optionally, when in the step (d), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

[01 18] In a third aspect, the present disclosure directs to a method for providing prognosis to a subject on whether she has malignant ovarian cancer, According to various embodiments of the present disclosure, the malignancy of the ovarian cancer is determined based on the result of step (c). In particular, the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian cancer.

[01 19] According to various embodiment, the at least one target gene is selected from the group consisting of, ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, TRH, FOXI2, And MIR124-2.

[0120] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the tumor malignancy of the subject is determined based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has malignant ovarian cancer. As is evident from results of the working examples provided below, taking the methylation profile of the GHSR gene into account while assessing the malignancy of the ovarian cancer provides a more accurate assessment. Accordingly, this embodiment can be used in combination with any other embodiments provided herein.

[0121 ] Still optionally, the method according to certain embodiments of the present disclosure further comprises a second methylation determination step (f), in which the methylation state of HIST1 H3C gene is determined; and a second determining step (g), in which the presence or absence of hypermethylation of the HIST1 H3C gene is determined. In this case, the tumor malignancy of the subject is determined based on the results of both step (c) and step (g). in particular, the hypermethylation of the HIST1 H3C gene and the hypermethylation of the at least one target gene indicate that the subject has malignant ovarian cancer. Like the GHSR gene, incorporating the methylation profile of the HIST1 H3C provides a more accurate result while assessing the malignancy of the ovarian cancer. Accordingly, this embodiment can be used in combination with any other embodiments provided herein.

[0122] For example, in another optional embodiment, the steps (a), (b), (c), (d), (e), and (f) are performed, and the prognosis on the malignancy of the ovarian cancer is determined based on the results of steps (c), (e), and (g). In other words, the subject has malignant ovarian cancer if the at least one target gene, and at least one of the HIST1 H3C gene and the GHSR gene are hypermethylated.

[0123] In certain embodiments, the methylation states of CD01 , HIST1 H3E, HIST1 H4I, HTR1 A, TRH, FOXI2, And MIR124-2 are determined in the step (b), and the methylation profile of these markers, alone or together with the methylation profile of the GHSR gene and/or the HIST1 H3C gene, is used to making prognosis on the malignancy of the subject's ovarian cancer.

[0124] In some embodiments, the methylation states of CD01 , FOXI2, TRH, And MIR124-2 are determined in the step (b), and the methylation profile of these markers, alone or together with the methylation profile of the GHSR gene, is used to making prognosis on the malignancy of the subject's ovarian cancer.

[0125] In some embodiments, the sample is obtained from the subject's body fluid (such as, serum or plasma). In this case, the target gene can be FOXI2, MIR124-2, or both.

[0126] In some other embodiments, the sample is obtained from the ovarian tissue (e.g., from cervical scraping cells) of the subject, and the target gene is MIR124-2, TRH, FOXI2, or a combination thereof. Further, the making prognosis on the malignancy of the subject's ovarian cancer can be determined based on the methylation profiles of GHSR and at least one target gene selected from the group consisting of MIR124-2, TRH, and FOXI2. [0127] As could be appreciated, if it is determined that the subject does not have malignant ovarian cancer, the method (or relevant steps thereof) described in the fourth aspect of the present disclosure could be carried out to determine whether the subject has the ovarian tumor or not.

[0128] In the fourth aspect, the present invention is directed to a method for assessing whether a subject has ovarian tumor (whether benign or malignant), in which the hypermethylation of the at least one target gene as determined in the step (c) indicates that the subject has the ovarian tumor.

[0129] According to various embodiment, the at least one target gene is selected from the group consisting of, ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, TRH, FOXI2, And MIR124-2.

[0130] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined.

[0131 ] In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has ovarian tumor (benign or malignant). As is evident from results of the working examples provided below, taking the methylation profile of the GHSR gene into account while assessing the presence of the ovarian tumor provides a more accurate assessment. Accordingly, this embodiment can be used in combination with any other embodiments provided herein.

[0132] In certain embodiments, the methylation states of ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HTR1A, TRH, FOXI2, And MIR124-2 are determined in the step (b), and the methylation profile of these markers, alone or together with the methylation profile of the GHSR gene, is used to making diagnosis on whether the subject has the ovarian tumor.

[0133] In some embodiments, the methylation states of CD01 , HTR1A, TRH, And MIR124-2 are determined in the step (b), and the methylation profile of these markers, alone or together with the methylation profile of the GHSR gene, is used to making diagnosis on whether the subject has the ovarian tumor. [0134] In some embodiments, the sample is obtained from the subject's body fluid (such as, serum or plasma). In this case, the target gene can be FOXI2, MIR124-2, or both. Still optionally, when the results of the step (c) indicates that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of FOXI2, MIR-124, or both.

[0135] In some other embodiments, the sample is obtained from the ovarian tissue (e.g., from cervical scraping cells) of the subject, and the target gene is selected from the group consisting of MIR124-2, TRH, and FOXI2. Further, the prognosis of the malignancy of the subject's ovarian cancer can be determined based on the methylation profiles of GHSR and at least one target gene selected from the group consisting of MIR124-2, TRH, and FOXI2. In this case, when in the step (c), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

[0136] Still optionally, if it is determined that the subject has ovarian tumor, the present method further comprises the step of determining whether the ovarian tumor is malignant depending on the methylation state of the at least one target gene. For example, the method (or relevant steps thereof) described in the third aspect of the present disclosure could be performed to determine whether the ovarian tumor is malignant.

[0137] In the fifth aspect, the present disclosure provides a method for making diagnosis on whether a subject has malignant ovarian cancer. According to various embodiments of the present disclosure, the sample used in the step (a) is obtained from cervical scraping cells, the at least one target gene is selected from the group consisting of, TRH, FOXI2, And MIR124-2, and the malignancy of the ovarian cancer is determined based on the result of step (c). In particular, the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian cancer.

[0138] Like the embodiments related to the second aspect of the present disclosure, the malignancy of the ovarian cancer can be determined by taking into further consideration of the expression profile of the GHSR gene. Specifically, the subject has malignant ovarian cancer if the GHSR gene and the at least one target gene selected from the group consisting of TRH, FOXI2, And MIR124-2 are hypermethylated.

[0139] In the sixth aspect, the present invention is directed to a method for assessing whether a subject has ovarian cancer. According to various embodiments of the present disclosure, the sample used in the step (a) is obtained from cervical scraping cells, the at least one target gene is TRH or MIR124-2, and the hypermethylation of the at least one target gene as determined in the step (c) indicates that the subject has the ovarian tumor.

[0140] Like the embodiments related to the third aspect of the present disclosure, the assessment of the presence of ovarian tumor (whether benign or malignant) can be determined further in consideration of the expression profile of the GHSR gene. Specifically, the hypermethylation e subject has malignant ovarian cancer if the GHSR gene and the at least one target gene of TRH or MIR124-2 are hypermethylated.

[0141 ] In the seventh aspect, the present invention is directed to a method for assessing whether a subject has malignant ovarian cancer. According to various embodiments of the present disclosure, the sample used in the step (a) is a body fluid sample, the at least one target gene is FOXI2, MIR124-2, or both, and the malignancy of the ovarian cancer is determined based on the result of step (c). In particular, the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian cancer.

[0142] According to optional embodiments of the present disclosure, the body fluid sample is a serum or plasma sample.

[0143] In the eighth aspect, the present invention is directed to a method for assessing whether a subject has bladder neoplasm using a bladder sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is a bladder sample, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has bladder neoplasm. [0144] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has bladder neoplasm (meaning, the subject may have benign or malignant bladder tumor).

[0145] In the ninth aspect, the present invention is directed to a method for assessing whether a subject has breast neoplasm using a breast sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is a breast sample, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has breast neoplasm.

[0146] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has breast neoplasm (meaning, the subject may have benign or malignant breast tumor).

[0147] In the tenth aspect, the present invention is directed to a method for assessing whether a subject has cervical neoplasm using a cervical scraping sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is a sample obtained from cervical scraping cells, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has cervical neoplasm.

[0148] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has cervical neoplasm (meaning, the subject may have benign or malignant cervix tumor).

[0149] In the eleventh aspect, the present invention is directed to a method for assessing whether a subject has colon neoplasm using a colon sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is a colon sample, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has colon neoplasm.

[0150] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has colon neoplasm (meaning, the subject may have benign or malignant colon tumor).

[0151 ] In the twelfth aspect, the present invention is directed to a method for assessing whether a subject has endometrium neoplasm. According to various embodiments of the present disclosure, the sample used in the step (a) can be an endometrium sample, the at least one target gene is CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, SPAG6, TRH, PRDM14, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has endometrium neoplasm. Preferably, the at least one target gene can be any of PRDM14, TRH, MIR124-2, Z1 C1 , and FOXI2.

[0152] Alternatively, the sample may be obtained from cervical scraping cells, the at least one target gene is selected from the group consisting of PRDM14, TRH, MIR124-2, Z1 C1 , and FOXI2, and when the at least one target gene is hypermethylated, it is determined that the subject has endometrium neoplasm.

[0153] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has endometrium neoplasm (meaning, the subject may have benign or malignant endometrial tumor).

[0154] In the thirteenth aspect, the present invention is directed to a method for determining whether a liver sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is a liver sample, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has liver neoplasm.

[0155] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has liver neoplasm (meaning, the subject may have benign or malignant liver tumor).

[0156] In the fourteenth aspect, the present invention is directed to a method for determining whether an oral scraping sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is an oral scraping sample, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the oral scraping sample is neoplastic subject has oral neoplasm.

[0157] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of bot step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the oral scraping sample is neoplastic subject has oral neoplasm (meaning, the subject may have benign or malignant oral tumor).

[0158] In the fifteenth aspect, the present invention is directed to a method for assessing whether an osteosarcoma sample derived from the subject. According to various embodiments of the present disclosure, the sample used in the step (a) is an osteosarcoma sample, the at least one target gene is ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, and Z1 C1 , and when the at least one target gene is hypermethylated, it is determined that the subject has osteosarcoma.

[0159] According to an optional embodiment of the present disclosure, the method further comprises a second methylation determination step (d), in which the methylation state of GHSR gene is determined; and a second determining step (e), in which the presence or absence of hypermethylation of the GHSR gene is determined. In this case, the diagnosis is made based on the results of both step (c) and step (e). Specifically, the hypermethylation of the GHSR gene and the hypermethylation of the at least one target gene indicate that the subject has osteosarcoma (meaning, the subject may have benign or malignant sarcoma).

[0160] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

[0161 ] Materials and Methods

[0162] 1 . Tissue Specimens

[0163] All tissue specimens were collected from volunteers recruited at Tri-Service Hospital (Taipei, Taiwan, R.O.C.). From 1994 to 2013, a total of 288 adult subjects were enrolled under the approval of the Institutional Review Board of Tri-Service Hospital with written informed consent of the patients.

[0164] The ovarian tumor specimens were obtained during surgery and were frozen immediately in liquid nitrogen and stored at -80°C until analysis. All clinical information was obtained from the patient's medical record at the Tri-Service Hospital. The pathological diagnosis of each sample was confirmed with histological examination by gynecologic pathologists. Normal epithelial ovarian tissue was collected by scraping the ovarian epithelium from patients diagnosed with uterine leiomyoma.

[0165] In the discovery cohort, 75 malignant ovarian tumor specimens, 20 benign ovarian tumor specimens, and 6 normal ovarian tissue specimens, all from independent patients, were used for investigating the genome-wide methylation profiles. In the validation cohort, 1 18 malignant ovarian tumor specimens, 43 benign ovarian tumor specimens, and 8 normal ovarian tissue specimens, all from independent patients, were used to validate the differential methylation profiles.

[0166] Serum and plasma specimens were collected from patients diagnosed with benign ovarian tumors (N=26) and malignant tumors (N=33), as well as from 18 female patients who were free of ovarian tumors at the sampling time. The collected blood samples were stored at -4°C, and within three days, the serum and plasma in the blood sample were separated and respectively stored in microcentrifuge tubes at -80°C until analysis.

[0167] Cervical scraping cells were collected from patients diagnosed with benign ovarian tumors (N=6) or malignant tumors (N=13), and 25 female patients were free of ovarian and cervical diseases at the sampling time. The endocervical scraping cells were collected as follows. Inserting a new endocervical brush into the endocervical canal and gently rotating the brush three to five times to ensure appropriate sampling. The brush was then plunged into a centrifuge tube (15 ml) containing 2 ml RHAIater® (Ambion, Life technologies, USA), and closed the cap of the centrifuge tube. The endocervical scraping cells specimens were stored at 4°C. After vortexing for 10 seconds, the swab cells-containing sample was aliquoted into three microcentrifuge tubes (1 ml per tube), which were stored at -80°C until further analysis.

[0168] Specimens from patients diagnosed with cervical cancer (N=20), bladder cancer (N =20), hepatocellular carcinoma (N=24), breast cancer (N=15), colon cancer (N=15), endometrial cancer (N=15), oral cancer (N=15), and osteosarcoma (N=15) were residual tissues removed during surgery or pathological examination.

[0169] Specimens from normal (i.e., non-cancerous) cervix epithelial scraping cells (N=24), normal bladder epithelial tissue adjacent to the benign tumor (N =20), hepatitis tissue (N =24), normal breast tissue adjacent to the benign tumor (N=15), normal colon tissue adjacent to the benign tumor (N=15), hyperplasia endometrial tissue (N=15), normal oral scraping cells (N=15), and osteoarthritis tissue (N=15) were respectively obtained from patients who were not diagnosed with malignant carcinoma.

[0170] 2. Preparation of Genomic DNA and Bisulfite Conversion

[0171 ] Genomic DNA was extracted from specimens using QIAGENE^®DNA Extraction Kits (purchased from Taigen Bioscience Corp, Taipei, Taiwan, R.O.C.), and the DNA concentration was determined using the NanoDrop 1000 spectrophotometer. For validation, Pooled DNA sample was derived from 5 specimens of the same type of disease and tissues, in which the DNA was present in equal amount.

[0172] The genomic DNA (1 μg) was bisulfite modified using the CpGenome Fast DNA Modification Kit (Chemicon-Millipore, MA, USA) according to the manufacturer's recommendations, and dissolved in 70 μΙ_ of nuclease-free water. The bisulfite DNA was used as the temple for the DNA methylation analysis.

[0173] 3. MethylCap Sequencing

[0174] Methylated DNA was enriched from 2 μg genomic DNA using the MethylMiner methylated DNA Enrichment Kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Briefly, genomic DNA was sonicated to about 200-bp, captured by MBD proteins and precipitated using 1 M salt buffer. Enriched methylated DNA was used to generate libraries for sequencing following the standard protocols from lllumina (San Diego, CA). MethylCap-seq libraries were sequenced using the lllumina Genome Analyzer l lx System. Image analysis and base calling were performed using the standard lllumina pipeline. Unique reads (up to 36-bp reads) were mapped to the human reference genome (hg18) using the ELAND algorithm, with up to two base-pair mismatches.

[0175] 4. Bisulfite Pyrosequencing

[0176] Bisulfite pyrosequencing was used to quantitate the DNA methylation level, in which the methylation level is expressed as the percentage of methylation (e.g., 0% to 100%) for each CpG site of the pyrosequencing amplicons.

[0177] PCR products were amplified using the PyroMark PCR Kit (QIAGEN). Sample preparation, pyrosequencing and analysis of the results were performed using the PyroMark Q24 System (QIAGEN) per the manufacturer's instructions.

[0178] Primers for use in the bisulfite pyrosequencing are listed in Table 1 below.

[0179] 5. Quantitative Methylation-Specific Polymerase Chain Reaction (qMSP)

[0180] Quantitative methylation-specific PCR (qMSP) using specific probes and primers (Table 2) was performed to identify the relative DNA methylation level of 3-8 CpG sites by making reference to the un-methylated (COL2A1 ) total input DNA. If the Cp value of COL2A1 was greater than 36, the specimen DNA was identified as an unqualified sample.

[0181 ] For qMSP, fluorescence-based real-time PCR was carried out using the Roche 480 PCR system. The reaction mixture (20μΙ_) contained 2μΙ_ of bisulfited genomic DNA, 250 nM of each primer (HPLC-grade), 225 nM TaqMan probe, and 10μΙ_ of FastStart Universal Probe Master (Rox) (Roche). The 5' -end of probes was labeled with 6-carboxy-fluorescein (6-FAM), and the 3'-end was labeled with a quencher dye (MGB by Applied Biosystems or BHQ1 by TIB). The reactions were performed using a thermal cycler protocol in the standard mode with an initial incubation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and annealing and extension for 1 minute at 60°C.

[0182] 6. Statistics

[0183] In an independence cohort, the true positive rate (i.e. , the sensitivity) was plotted against the false positive rate (that is, 1 -specificity) to obtain a receiver operating characteristic (ROC) curve. To assess the best accuracy, the area under the curve (AUC) of the ROC was used to identify the optimal cut-off value for distinguishing two groups of samples. The best accuracy and cut-off value were evaluated using 100 times of bootstrapping and Youden methods (the maximal sum of sensitivity and specificity).

[0184] To assess the best combination of candidate genes, the multiple regression model was used to analyze the relationship between the presence (assigned = 1 ) and absence (assigned = 0) of disease and the methylation levels of multiple genes. The correlation coefficient in our analysis model is a measure of how tightly the data of methylation levels around the patients diagnosed with ovarian tumor. The correlation coefficient was calculated by taking the square root of the coefficient of determination. A higher correlation coefficient indicates that the gene combination is more correlated to the presence of ovarian tumor. The correlation coefficients were also referenced to the weighting value to find the optimal cutoff values of the gene combination.

[0185] Both the Enter mode and the Stepwise mode were used to introduce variables into the regression model. In the Enter method, all variables were entered into the model in one single step. In the Stepwise method, significant variables were entered sequentially; and after the entry of one variable, the p-value of the variable was check to determine whether the variable should be included in or removed from the model.

[0186] The Kruskal-Wallis test was used to assess the effect of the methylation levels by the ranks in numerous pathological types of ovarian tissue.

[0187] P-values less than 0.05 were considered statistically significant.

[0188] Example 1

[0189] Identification of Hypermethylated Genes Associated with Diagnosis and Prognosis of Ovarian Cancer [0190] In the discovery cohort, specimens are subject to MethylCap-sequencing, and 29 candidate genes highly related to the progression-free survival (PFS) and overall survival (OS) of the subject were identified (see, Table 3).

[0191 ] The 29 candidate genes identified in the discovery cohort were subject to further verification in which pooled DNA from normal ovarian tissues (N) and malignant ovarian cancer tissues (T) were subject to bisulfite pyrosequencing. The results (Figures 1 A and 1 B) indicated that 21 of the candidate genes exhibit significant hypermethylation in malignant ovarian cancer tissues.

[0192] The methylation profiles of 1 1 of the candidate genes were further investigated. Results provided in Figure 2 indicated that for each of these 1 1 genes, the methylation profiles thereof differ among normal ovarian tissues (N, n = 8), benign ovarian tumor tissues (B, n = 43) and malignant ovarian cancer tissues (M, n = 1 18), with this difference being statistically significant (Kruskal-Wallis test; p < 0.001 ).

[0193] These 1 1 genes, namely, ADCY8, CD01 , GHSR, HIST1 H2BB, HIST1 H3C, HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, and MIR124-2 were used in the subsequent independent cohort to assess the best accuracy for the diagnostic and/or prognostic stratification power.

[0194] Example 2

[0195] Assessment of Best Accuracy for Distinguishing among Subjects with Normal Ovarian Tissue, Benign Ovarian Neoplasm and Malignant Ovarian Neoplasm by Bisulfite Pyrosequencing

[0196] In the independent cohort, the ROC graph was plotted; also, the cut-off point, AUC, and 95% confidence interval (CI) of the nine genes identified in Example 1 , above, were determined, and the results are summarized in Table 4.

[0197] The data in Table 4 indicates that nine (ADCY8, CD01 , GHSR, HIST1 H2BB, HIST1 H3E, HTR1A, TRH, FOXI2, And MIR124-2) out of the 1 1 genes exhibited significant accuracies (0.759 to 0.958) in distinguishing subjects with the normal ovarian tissues (N) from those with wither the benign or malignant ovarian tumor tissues (B+M).

[0198] With respect to the stratification between the subjects with the benign ovarian tumor tissues (B) and those with the malignant ovarian cancer tissues (M), nine (CD01 , GHSR, HIST1 H3C, HIST1 H3E, HIST1 H4I , HTR1 A, TRH, FOXI2, And MIR124-2) of the 1 1 genes manifested significant accuracies (0.749 to 0.956), while a greater cut-off point was used.

[0199] Example 3

[0200] Assessment of Best Coefficients for Distinguishing among Subjects with Normal Ovarian Tissue, Benign Ovarian Neoplasm and Malignant Ovarian Neoplasm by Bisulfite Pyrosequencing

[0201 ] The multivariate regression analysis was first carried out using the Enter mode to evaluate the top five coefficients; see, Table 5.

[0202] Based on the findings summarized in Table 5, it was noted that MIR124-2, CD01 , HTR1A, GHSR, and TRH genes exhibited relatively higher correlation coefficients that may be used to discriminate subjects with normal ovarian tissues (N) from those with the benign or malignant tumor tissues (B+M).

[0203] On the other hand, MI R124-2, CD01 , TRH, GHSR, and FOXI2 genes exhibited better correlation in terms of stratifying subjects with benign ovarian tumor tissues (B) and those with malignant ovarian cancer tissues (M).

[0204] In view of the foregoing, CD01 , F0XI2, GHSR, HTR1A, MIR124-2 and TRH genes were used in the subsequent qMSP platform to investigate the stratification power of these genes.

[0205] Example 4

[0206] Assessment of Best Gene Combination for Distinguishing Subjects with Normal Ovarian Tissue from Subjects with Benign or Malignant Ovarian Neoplasm Using Ovarian Tissues Derived from Cervical scraping Cells by qMSP

[0207] In this example, the ovarian tissues specimens were obtained using cervical scraping cells. The methylation profiles determined by qMSP were subject to stepwise multivariate regression so as to evaluate the best combination of genes for distinguishing the subjects with normal ovarian tissues (N) from those with benign or malignant neoplastic ovarian tissues (B + M).

[0208] The results, as summarized below in Table 6, indicated that the best combination of genes for distinguishing subjects with N from those with B+M ovarian tissues was the combination of GHSR and MIR124-2. The same combination was also most effective in distinguishing B from M ovarian tissues.

[0209] Example 5

[0210] Cell-Free DNA Genes

[02] 1 ] In this example, the pooled cell-free DNA sample derived from the serum was subject to qMSP to distinguish benign (B) and malignant (M) ovarian cancer. The preliminary data provided in Figure 3 indicated that in the blood sample of patients with malignant ovarian cancer, the candidate genes (FOXI2 and MIR24-2) were highly methylated.

[0212] Example 6

[0213] Determination of Prognostic Power of Histone Cluster Genes

[0214] In the validation cohort, the prognostic power of the histone cluster genes (including HIST1 H2BB, HIST1 H3C, HIST1 H3E, and HIST1 H4I) were investigated using ovarian tissues specimens and bisulfite pyrosequencing. These histone cluster genes exhibited similar biological function in maintaining the chromatin structure stability and participating the DNA replication process. Results are summarized in Figure 4.

[0215] Figure 4A, the hypermethylation of each of the four histone cluster genes identified above was found to be significantly related to the poor survival outcome (the shorter recurrence interval and the lower percentage of PFS at the same interval, solid line). In the following description, the subjects were categorized into three groups based on the number of the hypermethylated histone cluster genes in the samples: low HS-HM group (samples with no or one hypermethylated histone cluster gene); medium HS-HM group (samples with two or three hypermethylated histone cluster genes); and high HS-HM group (samples with four hypermethylated histone cluster genes).

[0216] The data illustrated in Figure 4B indicated that the subjects in the medium and high HS-HM groups were significantly related to the poor survival outcome (solid and broken lines). Moreover, subjects in the high HS-HM group exhibited the worst survival profile (solid line).

[0217] Example 7

[0218] Cut-off Point and Univariate COX Regression Analysis of Various Genes on Progression-Free Survival in Ovarian Cancer

[0219] Regarding the efficacy of histone cluster genes (including HIST1 H2BB, HIST1 H3C, HIST1 H3E and HIST1 H4I) in predicting the progression-free survival (PFS) of patients diagnosed with ovarian cancer, the cut-off point, hazard ratio (HR), and 95% confidence interval (95% CI) of histone cluster genes (including HIST1 H2BB, HIST1 H3C, HIST1 H3E and HIST1 H4I) were determined from the ROC graph produced using the bisulfite pyrosequencing data; the results are summarized in Table 7.

[0220] The data in Table 7 indicated that HIST1 H3E is most effective in predicting the PFS of the ovarian cancer patient (p < 0.001 ).

[0221 ] COX regression analysis was used to investigate the effects of the combination of histone cluster genes on progression-free survival in ovarian cancer, and the results are provided in Table 8.

[0222] As is evident from the data in Table 8, the hypermethylation of the four above-mentioned histone cluster genes is an important factor associated with the outcome of ovarian cancer (the high HS-HM group; adjusted HR = 1 0.45, p < 0.001 ). Further, subjects in the medium and high HS-HM groups were associated with the worse recurrence outcome. Additionally, subjects in the high HS-HM group were associated with the worst risk (1 0.45 times), as compared with subjects in the low HS-HM group.

[0223] COX regression analysis was also used to investigate the effects of the combination of two hormone-associated genes (GSHR and TRH) on progression-free survival (PFS) in ovarian cancer, and the results are provided in Table 9 and Figures 5A and 5B.

[0224] As can be seen in Table 9, the hypermethylation of both of the GSHR and TRH genes is also an important factor related to the outcome of ovarian cancer (2-HM group; adjusted HR = 7.13, p = 0.012). Also, the hypermethylation of one (1 -HM group) or both (2-HM group) of the two hormone-associated genes were associated with subjects with the worse recurrence outcome (see, Figures 5A and 5B). Additionally, the subjects in the 2-HM group had higher risk (7.13 times) for recurrence, as compared with subjects with no hypermethylation of either gene (0-HM group).

[0225] Summarized in Table 10 is the PFS probability of patients (meaning the percentage of recurrence-free patients) with different hypermethylation profiles during the first year and the second years from the treatment.

[0226] The data in Table 10 indicated that subjects in the high HS-HM and 2-HM groups exhibited the lowest percentage of recurrence-free patients (28.6% and 56.7% during the first-year interval; 14.4% and 30.9% during second-year interval) after the treatment.

[0227] Example 8

[0228] Methylation Profiles of Candidate Genes in Various Tissues

[0229] The methylation profile of the 21 candidates genes identified in Example 1 , above, in various tissues (including, bladder (Bl), breast (Br), colon (CI), cervix (Cx), endometrium (Em), liver (Hepa), oral (Oral), and sarcoma (SA) tissues) were determined. The results are provided in Figures 6A, 6B, and 6C.

[0230] Example 9

[0231 ] Assessment of Best Accuracy for Distinguishing Subjects with Normal Bladder Tissue from Subjects with Benign or Malignant Bladder Neoplasm

[0232] In this and the following examples, the ROC graph was plotted based on the methylation profile obtained from the Example 8, above. Then, the cut-off point, AUC and 95% confidence interval (CI) of the 21 candidate genes were determined, and the results are summarized in Table 1 1 .

[0233] The results in Table 1 1 indicated that the ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, GHSR, HIST1 H3E, HIST1 H4I, HTR1A, MI R124-2, PRDM14, RAX, SPAG6 and TRH genes are effective markers for distinguishing subjects with normal bladder tissues (N) from those with benign or malignant bladder tumor tissues (B+M).

[0234] Example 10

[0235] Assessment of Best Accuracy for Distinguishing Subjects with Normal Breast Tissue from Subjects with Benign or Malignant Breast Neoplasm

[0236] In this example, the optimal cutoff value for each candidate gene in discriminating subjects with normal breast tissues (N) from those with benign or malignant breast tumor tissues (B+M), as well as the sensitivity and specificity of each marker regarding the optimal cutoff value, was determined. The results are summarized in Table 12.

[0237] The results in Table 12 indicated that the ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H3C, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6 and TRH genes are effective markers for distinguishing subjects with normal breast tissues (N) from those with benign or malignant breast tumor tissues (B+M).

[0238] Example 11

[0239] Assessment of Best Accuracy for Distinguishing Subjects with Normal Ovarian Tissue from Subjects with Benign or Malignant Cervical Neoplasm Using Cervical Scraping

[0240] Cervical scraping samples were used to determine effective markers for discriminating subjects with/without cervical neoplasm, and the data are summarized in Table 13.

[0241 ] The results in Table 13 indicated that the ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, GHSR, HIST12BB, HIST1 H3C, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH and ZIC1 genes are effective markers for distinguishing the subjects with normal cervical tissues (N) from the subjects with benign or malignant cervical tumor tissues (B+M).

[0242] Example 12

[0243] Assessment of Best Accuracy for Distinguishing Subjects with Normal Colon Tissue from Subjects with Benign or Malignant Colon Neoplasm

[0244] Colon tissue samples were used to determine effective markers for discriminating subjects with/without colon neoplasm, and the data are summarized in Table 14.

[0245] The results in Table 14 indicated that the ADCY8, C17orf46, CDH8, CD01 , C0L14A1 , DPP6, ECEL1 P2, F0XI2, HIST1 H3C, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PRDM14, RAX, SPAG6 and TRH genes are effective markers for distinguishing subjects with normal colon tissues (N) from those with benign or malignant colon tumor tissues (B+M).

[0246] Example 13

[0247] Assessment of Best Accuracy for Distinguishing Subjects with Normal Endometrium Tissue from Subjects with Benign or Malignant Endometrium Neoplasm

[0248] Samples obtained from endometrium cells were used to determine effective markers for discriminating subjects with/without endometrium neoplasm , and the data are summarized in Table 15.

[0249] The results in Table 15 indicated that, when using samples obtained from endometrium cells, the CDH8, ECEL1 P2, FOXI2, GHSR, HIST1 H3C, HIST1 H4I, HTR1A and TRH genes are effective markers for distinguishing subjects with normal endometrium tissues (N) from those with benign or malignant endometrial tumor tissues (B+M). [0250] Samples obtained from cervical scraping cells were also used to determine effective markers for discriminating subjects with/without endometrium neoplasm, and the data are summarized in Table 16.

[0251 ] The results in Table 16 indicated that, when using the samples obtained from cervical scraping cells, the PRDM14, GHSR, TRH, FOXI2, Z1 C1 and MIR124-2 genes are effective markers for distinguishing subjects with normal endometrium tissues (N) from those with benign or malignant endometrial tumor tissues (B+M).

[0252] The methylation levels of CD01 in tissues and cervical scrapings from subjects with endometrial cancer (EC) or healthy subjects were further validated. The data, as summarized in Figure 7A indicated that the DNA methylation status of CD01 in endometrial cancer is higher than in normal endometrium and myoma (n = total case number). The diagram in Figure 7B indicated that the methylation of CD01 has 0.94 area under an receiver operating curve (AUC) with 77.4% sensitivity (Sn.) and 96.9% specificity (Sp.) for distinguishing EC and none carcinoma .

[0253] Example 14

[0254] Assessment of Best Accuracy for Distinguishing Subjects with Normal Liver Tissue from Subjects with Benign or Malignant Liver Neoplasm Using Liver Biopsy

[0255] Samples obtained from liver tissues were used to determine effective markers for discriminating subjects with/without liver neoplasm , and the data are summarized in Table 17. [0256] The results in Table 17 indicated that the ADCY8, C17orf46, CDH8, C0L14A1 , DPP6, ECEL1 P2, F0XI2, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH and ZIC1 genes are effective markers for distinguishing subjects with normal liver tissues (N) from those with benign or malignant hepatocellular tumor tissues (B+M).

[0257] Example 15

[0258] Assessment of Best Accuracy for Distinguishing Assessment of Best Accuracy for Distinguishing Subjects with Normal Oral Tissue from Subjects with Benign or Malignant Oral Neoplasm Using Oral Scraping Cells

[0259] Samples obtained from oral scraping cells were used to determine effective markers for discriminating subjects with/without oral neoplasm , and the data are summarized in Table 18. [0260] The results in Table 18 indicated that the CDH8, C0L14A1 , DPP6, ECEL1 P2, F0XI2 and HIST1 H3E genes are effective markers for distinguishing the subject with normal oral tissues (N) from those with benign or malignant oral tumor tissues (B+M) by oral scraping cells.

[0261 ] Example 16

[0262] Assessment of Best Accuracy for Distinguishing Subjects with Normal Osteosarcoma Tissue from Subjects with Benign or Malignant Osteosarcoma Neoplasm

[0263] Samples obtained from osteosarcoma cells were used to determine effective markers for discriminating subjects with/without osteosarcoma, and the data are summarized in Table 19.

[0264] The results in Table 19 indicated that the ADCY8, C17orf46, CD01 , C0L14A1 , ECEL1 P2, F0XI2, HIST1 H3C, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH and ZIC1 genes are effective markers for distinguishing subjects with normal chondrocyte (N) from those with benign or malignant osteosarcoma tissues (B+M).

[0265] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

Claims

WHAT IS CLAIMED IS:

1 . A method for assessing the tumor malignancy or the prognosis of a subject diagnosed with ovarian neoplasm, comprising the steps of,

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, TMEM132D, IRX1 , MIR124-2, TRH, PCDHB4, ZNF132, KLHL33, DPP6, ADCY8, C20orf85, C17orf46, FOXI2, HIST1 H4I, HIST1 H2BB, SLC6A3, HTR1A, CD01 , CDH8, PCDHGA5, COL14A1 , SPAG6, RAX, PRDM14, HIST1 H3E, ZIC1 , COX7A1 , ECEL1 P2, and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes;

(c) determining whether the at least one target gene is hypermethylated; and

(d) assessing the tumor malignancy or prognosis of the subject based on the result of step (c), wherein the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian neoplasm or a poor prognosis.

2. The method of claim 1 , further comprising the steps of,

(e) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto; and

(f) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated,

wherein in the step (d), the tumor malignancy or prognosis of the subject is determined based on the results of step (c) and step (f), and the hypermethylation of the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the hypermethylation of the at least one target gene indicate that the subject has malignant ovarian neoplasm or a poor prognosis.

3. The method of claim 1 , wherein,

the at least one target gene is selected from the group consisting of,

ADCY8, CD01 , HIST1 H4I, ECEL1 P2, HTR1 A, SPAG6, CDH8, DPP6, HIST1 H3E, PCDHGA5, RAX, C17orf46, COL14A1 , FOXI2, PRDM14, ZIC1 , HIST1 H2BB, MIR124-2, and TRH, and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

the hypermethylation of the at least one target gene indicates that the subject has malignant ovarian neoplasm.

4. The method of claim 1 , wherein,

the at least one target gene is selected from the group consisting of, MIR124-2, TRH, PCDHB4, ZNF132, KLHL33, C20orf85, C17orf46, HIST1 H4I, HIST1 H2BB, CD01 , CDH8, PCDHGA5, COL14A1 , SPAG6, HIST1 H3E, ZIC1 , COX7A1 , and ECEL1 P2, and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes;

the prognosis is associated with progression-free survival; and

the hypermethylation of the at least one target gene indicates that the subject has a poor progression-free survival.

5. The method of claim 1 , wherein,

the at least one target gene is selected from the group consisting of,

TMEM 132D, IRX1 , KLHL33, DPP6, ADCY8, FOXI2, SLC6A3, HTR1A, CD01 , CDH8, RAX, and PRDM14, and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes;

the prognosis is associated with overall survival; and

the hypermethylation of the at least one target gene indicates a poor overall survival.

6. The method of claim 1 , wherein,

the at least one target gene is selected from the group consisting of, HIST1 H2BB, HIST1 H3E, and HIST1 H4I, and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

the prognosis is associated with progression-free survival.

7. The method of claim 6, further comprising the steps of,

(e) determining the methylation state of HIST1 H3C gene or a polynucleotide sequence having at least 75% sequence identity thereto; and

(f) determining whether the HIST1 H3C gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated, wherein in the step (d), the prognosis of the subject is determined based on the results of step (c) and step (f), and the hypermethylation of the HIST1 H3C gene or the polynucleotide sequence having at least 75% sequence identity thereto and the hypermethylation of the at least one of HIST1 H2BB, HIST1 H3E, and HIST1 H4I, and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes indicate that the subject has a poor progression-free survival.

8. The method of claim 6, wherein the hypermethylation of at least two of the target genes indicates that the subject has a poor progression-free survival.

9. The method of claim 1 , wherein, the at least one target gene is TRH, the prognosis is associated with progression-free survival, and the method further comprising the steps of,

wherein in the step (d), the prognosis of the subject is determined based on the results of step (c) and step (f), and the hypermethylation of the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the hypermethylation of the TRH or the polynucleotide sequence having at least 75% sequence identity thereto indicate that the subject has a poor progression-free survival.

10. The method of claim 1 , wherein the sample is obtained from a tissue, cell, tumor, or biological fluid of the subject.

1 1 . The method of claim 10, wherein the sample is a serum or plasma sample.

12. The method of claim 1 1 , wherein the at least one target gene is FOXI2 or MIR124-2.

13. The method of claim 10, wherein the sample is obtained from cervical scraping cells.

14. The method of claim 13, wherein the at least one target gene is selected from the group consisting of, MIR124-2, TRH, and FOXI2.

15. The method of claim 2, wherein the sample is obtained from cervical scraping cells.

16. The method of claim 15, wherein the at least one target gene is selected from the group consisting of, MIR124-2, TRH, and FOXI2.

17. The method of claim 1 , wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

18. A method for assessing whether the subject has ovarian neoplasm, comprising the steps of,

(a) obtaining a sample from the subject;

(c) determining whether the at least one target gene is hypermethylated; and

19. The method of claim 18, further comprising the steps of, (e) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto; and

wherein in the step (d), the presence of ovarian neoplasm in the subject is determined based on the results of step (c) and step (f), and the hypermethylation of the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the hypermethylation of the at least one target gene indicate the presence of ovarian neoplasm in the subject.

20. The method of claim 18, wherein the at least one target gene is selected from the group consisting of, MIR124-2, CD01 , HTR1A, and TRH.

21 . The method of claim 18, wherein the sample is obtained from a tissue, cell, tumor, or biological fluid of the subject.

22. The method of claim 21 , wherein the sample is a serum or plasma sample.

23. The method of claim 22, wherein when in the step (d), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of FOXI2, MIR-124, or a combination of both.

24. The method of claim 21 , wherein the sample is obtained from cervical scraping cells.

25. The method of claim 24, wherein the at least one target gene is selected from the group consisting of, MIR124-2, TRH, and FOXI2.

26. The method of claim 25, t wherein when in the step (d), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

27. The method of claim 19, wherein the sample is obtained from cervical scraping cells.

28. The method of claim 27, wherein the at least one target gene is selected from the group consisting of, MIR124-2, TRH and FOXI2.

29. The method of claim 18, wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

30. A method for assessing whether a subject has malignant ovarian cancer, comprising the steps of,

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, MIR124-2, and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(c) determining whether the at least one target gene is hypermethylated, wherein the subject has malignant ovarian cancer if the at least one target gene is hypermethylated.

31 . The method of claim 30, further comprising the steps of,

(d) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto; and

(e) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated, wherein the subject has malignant ovarian cancer if the GHSR gene and the at least one target gene are hypermethylated.

32. The method of claim 31 , further comprising the steps of,

(f) determining the methylation state of HIST1 H3C gene or a polynucleotide sequence having at least 75% sequence identity thereto; and

(g) determining whether the HIST1 H3C gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated, wherein the subject has malignant ovarian cancer if the HIST1 H3C gene or the polynucleotide sequence having at least 75% sequence identity thereto, the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto, and the at least one target gene are hypermethylated.

33. The method of claim 30, wherein the at least one target gene includes, CD01 , HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, and MIR124-2.

34. The method of claim 31 , wherein the at least one target gene includes, CD01 , HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, and MIR124-2.

35. The method of claim 32, wherein the at least one target gene includes, CD01 , HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, and MIR124-2.

36. The method of claim 30, wherein the at least one target gene includes CD01 , FOXI2, TRH, and MIR124-2.

37. The method of claim 31 , wherein the at least one target gene includes CD01 , FOXI2, TRH, and MIR124-2.

38. The method of claim 30, wherein the sample is obtained from a tissue, cell, tumor, or biological fluid of the subject.

39. The method of claim 38, wherein the sample is a serum or plasma sample.

40. The method of claim 39, wherein the at least one target gene is FOXI2 or MIR124-2.

41 . The method of claim 38, wherein the sample is obtained from cervical scraping cells.

42. The method of claim 41 , wherein the at least one target gene is selected from the group consisting of, FOXI2, MIR124-2, and TRH.

43. The method of claim 31 , wherein the sample is obtained from cervical scraping cells.

44. The method of claim 43, wherein the at least one target gene is selected from the group consisting of, FOXI2, MIR124-2, and TRH.

45. The method of claim 30, wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

46. A method for assessing whether a subject has ovarian tumor, comprising the steps of,

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, TRH, FOXI2, Ml R124-2, and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(c) determining whether the at least one target gene is hypermethylated, wherein the subject has ovarian tumor if the at least one target gene is hypermethylated.

47. The method of claim 46, further comprising the steps of,

(e) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated, wherein the subject has ovarian tumor if the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

48. The method of claim 46, wherein the at least one target gene includes ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HTR1A, TRH, FOXI2, and MIR124-2.

49. The method of claim 47, wherein the at least one target gene includes ADCY8, CD01 , HIST1 H2BB, HIST1 H3E, HTR1A, TRH, FOXI2, and MIR124-2.

50. The method of claim 46, wherein the at least one target gene includes CD01 , HTR1 A, TRH, and MIR124-2.

51 . The method of claim 47, wherein the at least one target gene includes FOXI2, CD01 , HTR1 A, TRH, and MIR124-2.

52. The method of claim 46, wherein the sample is obtained from a tissue, cell, tumor, or biological fluid of the subject.

53. The method of claim 52, wherein the sample is a serum or plasma sample.

54. The method of claim 53, wherein when in the step (c), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of FOXI2, MIR124-2, or both.

55. The method of claim 52, wherein the sample is obtained from cervical scraping cells.

56. The method of claim 55, wherein the at least one target gene is selected from the group consisting of MIR124-2, TRH and FOXI2.

57. The method of claim 56, wherein when in the step (c), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

58. The method of claim 47, wherein the sample is obtained from cervical scraping cells.

59. The method of claim 58, wherein the at least one target gene is selected from the group consisting of MIR124-2, TRH and FOXI2.

60. The method of claim 59, wherein when in the step (c), it is determined that the subject has ovarian neoplasm, the method further comprises the step of determining whether the ovarian neoplasm is malignant depending on the methylation state of MIR124-2, TRH, FOXI2, or any combination thereof.

61 . The method of claim 46, wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

62. A method for assessing whether a subject has malignant ovarian cancer, comprising the steps of,

(a) obtaining a sample from the subject, wherein the sample is obtained from cervical scraping cells;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, TRH, FOXI2, MIR124-2, and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(c) determining whether the at least one target gene is hypermethylated, wherein the subject has malignant ovarian cancer if the at least one target gene is hypermethylated .

63. The method of claim 62, further comprising the steps of,

(d) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto using the method of the step (b); and

(e) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (c), wherein the subject has malignant ovarian cancer if the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated .

64. The method of claim 62, wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

65. A method for assessing whether a subject has ovarian tumor, comprising the steps of,

(a) obtaining a sample from the subject, wherein the sample is obtained from;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of TRH, MIR124-2 and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

66. The method of claim 65, further comprising the steps of,

(e) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (c), wherein the subject has ovarian tumor if the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

67. The method of claim 65, wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

68. A method for assessing whether a subject has malignant ovarian cancer, comprising the steps of,

(a) obtaining a body fluid sample from the subject; (b) determining the methylation state of at least one target gene in the body fluid sample, wherein the at least one target gene is selected from the group consisting of FOXI2, MIR124-2, and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

69. The method of claim 68, wherein the body fluid sample is a serum or plasma sample.

70. The method of claim 68, wherein the methylation state is measured by methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (qMSP), bisulfite sequencing (BS), bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) or nanopore sequencing..

71 . A method for assessing whether a subject has bladder neoplasm using a bladder sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the bladder sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and the polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the subject has bladder neoplasm when the at least one target gene is hypermethylated.

72. The method of claim 71 , further comprising the steps of,

(c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the bladder sample using the method of the step (a); and (d) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the subject has bladder neoplasm when the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

73. A method for assessing whether a subject has breast neoplasm using a breast sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the breast sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the subject has breast neoplasm when the at least one target gene is hypermethylated.

74. The method of claim 73, further comprising the steps of,

(c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the breast sample using the method of the step (a); and

(d) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the subject has breast neoplasm when the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

75. A method for determining whether a subject has cervical neoplasm using a cervical scraping sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the cervical scraping sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the subject has cervical neoplasm when the at least one target gene is hypermethylated.

76. The method of claim 75, further comprising the steps of,

(c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the cervical scraping sample using the method of the step (a); and

(d) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the subject has cervical neoplasm when the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

77. A method for determining whether a subject has colon neoplasm using a colon sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the colon sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the subject has colon neoplasm when the at least one target gene is hypermethylated.

78. The method of claim 77, further comprising the steps of,

(c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the colon sample using the method of the step (a); and

(d) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the subject has colon neoplasm when the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

79. A method for assessing whether a subject has endometrium neoplasm, comprising the steps of,

(a) obtaining a sample from the subject;

(b) determining the methylation state of at least one target gene in the sample, wherein the at least one target gene is selected from the group consisting of, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, SPAG6, TRH, PRDM 14, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(c) determining whether the at least one target gene is hypermethylated, wherein the subject has endometrium neoplasm when the at least one target gene is hypermethylated.

80. The method of claim 79, further comprising the steps of,

(d) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the sample using the method of the step (b); and

(e) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (c), wherein the subject has endometrium neoplasm when the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

81 . The method of claim 79, wherein the sample is obtained from endometrium cells.

82. The method of claim 81 , wherein the at least one target gene is selected from the group consisting of, PRDM14, TRH, MIR124-2, Z1 C1 , and FOXI2.

83. The method of claim 79, wherein the sample is obtained from cervical scraping cells.

84. The method of claim 83, wherein the at least one target gene is selected from the group consisting of, PRDM14, TRH, MIR124-2, Z1 C1 , and FOXI2.

85. A method for determining whether a subject has liver neoplasm using a liver sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the liver sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the subject has liver neoplasm when the at least one target gene is hypermethylated.

86. The method of claim 85, further comprising the steps of,

(c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the liver sample using the method of the step (a); and

(d) determining whether the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the subject has liver neoplasm when the GHSR gene or the polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

87. A method for determining whether a subject has oral neoplasm using an oral scraping sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the oral scraping sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the oral scraping sample is neoplastic subject has oral neoplasm when the at least one target gene is hypermethylated.

88. The method of claim 87, further comprising the steps of, (c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the oral scraping sample using the method of the step (a); and

(d) determining whether the GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the oral scraping sample is neoplastic subject has oral neoplasm when the GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.

89. A method for determining whether a subject has osteosarcoma using an osteosarcoma sample derived from the subject, comprising the steps of,

(a) determining the methylation state of at least one target gene in the osteosarcoma sample, wherein the at least one target gene is selected from the group consisting of, ADCY8, C17orf46, CDH8, CD01 , COL14A1 , DPP6, ECEL1 P2, FOXI2, HIST1 H2BB, HIST1 H3E, HIST1 H4I, HTR1 A, MIR124-2, PCDHGA5, PRDM14, RAX, SPAG6, TRH, Z1 C1 , and a polynucleotide sequence having at least 75% sequence identity to any of the above-mentioned genes; and

(b) determining whether the at least one target gene is hypermethylated, wherein the subject has osteosarcoma when the at least one target gene is hypermethylated.

90. The method of claim 89, further comprising the steps of,

(c) determining the methylation state of GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto in the osteosarcoma sample using the method of the step (a); and

(d) determining whether the GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto is hypermethylated using the method of the step (b), wherein the subject has osteosarcoma when the GHSR gene or a polynucleotide sequence having at least 75% sequence identity thereto and the at least one target gene are hypermethylated.