SYSTEM AND METHOD FOR DETECTING CANCER

• US 20150285807A1
• Filed: 06/11/2013
• Published: 10/08/2015
• Est. Priority Date: 06/11/2012
• Status: Abandoned Application

• Current Assignee: Brigham and Women's Hospital Incorporated
• Original Assignee: Brigham and Women's Hospital Incorporated
• Inventors: Lian, Christine Guo, Shi, Yujiang Geno
• Application Number:
  US14/405,673
• Publication Number:
  US 20150285807A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 38/443 acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Y 101/01042 Isocitrate dehydrogenase (NADP+) (1.1.1.42)
• CPC Class Codes: C12Y 114/11 with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/5743 of skin, e.g. melanoma
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

Disclosed herein are methods of detecting malignancy in a tumor of a subject comprising measuring the amount of 5-hmC in a tumor sample and comparing to a control to thereby detect a level of reduction of 5-hmC in the tumor, wherein the tumor is characterized as malignant if a threshold level of reduction is detected. The tumor may be a solid tumor such as a melanocytic lesion. Also disclosed are methods of determining the prognosis of a subject with a tumor by determination of the amount of 5-hmC in the tumor. Methods for treatment of a tumor with reduced 5-hmC are also disclosed.

Claims

1-30. -30. (canceled)

31. A method of diagnosing a subject with a melanocytic lesion comprising,

a) processing a tissue sample of the melanocytic lesion of the subject to thereby label 5-hmC present in the tissue sample;

b) measuring the 5-hmC in the sample by detection of the labeled 5-hmC in the tissue sample;

c) quantitating the 5-hmC in the tissue sample as compared to a healthy control to thereby detect a threshold level of reduction of 5-hmC in the sample; and

d) characterizing the melanocytic lesion by the detected threshold level of reduction of 5-hmC to thereby diagnose the subject.

32. The method of claim 31, wherein the processing step a) is by immunohistochemical staining or by immunofluorescence and/or wherein quantitating step c) is by assignment of a 5-hmC staining score or by assignment of a 5-hmC cell count score.

33. The method of claim 31, wherein the threshold level of reduction is equivalent to:

a) a 5-hmC staining score of ≦

2 which indicates the melanocytic lesion is a stage 2-3 melanoma, with a Breslow of >

1 mm, and >

1 mitosis;

b) a 5-hmC staining score of ≧

3 which indicates the melanocytic lesion is a stage 1 melanoma, with a Breslow of ≦

1 mm, and ≦

1 mitosis;

c) a 5-hmC staining score of ≧

1.5 which indicates the melanocytic lesion is a melanoma without ulceration;

or

d) a 5-hmC staining score of <

1.5 which indicates the melanocytic lesion is a melanoma with ulceration.

34. The method of claim 31 wherein the threshold level of reduction is equivalent to:

a) a 5-hmC cell count score of ≧

2 which indicates the melanocytic lesion is a benign melanocytic nevus;

b) a 5-hmC cell count score of ≧

3 which indicates the melanocytic lesion is a benign melanocytic nevus;

c) a 5-hmC cell count score of <

1 which indicates the melanocytic lesion is a primary cutaneous melanoma or a visceral metastatsic melanoma;

d) a 5-hmC cell count score of <

0.5 which indicates the melanocytic lesion is a primary cutaneous melanoma or a visceral metastatsic melanoma;

or

e) a 5-hmC cell count score of <

0.25 which indicates the melanocytic lesion is a lymphnode metastatic melanoma.

35. The method of claim 31, wherein processing step a) is by purifying genomic DNA from a tissue sample of the melanocytic lesion and measuring step b) is by sequencing genomic DNA identified as containing 5-hmC in the purified genomic DNA or by detection of the 5-hmC in specific genes of the genomic DNA, or by an anti-5-hmC antibody-based detection system.

36. The method of claim 35, wherein the threshold level of reduction is a ≧

5 fold reduction in the 5-hmC of the specific genes of the genomic DNA which indicates the melanocytic lesion is a melanoma.

37. The method of claim 35, wherein the anti-5-hmC antibody-based detection system is a dot blot assay and/or the 5-hmC levels are detected by a 5-hmC glucosylation assay.

38. The method of claim 37 wherein the 5-hmC glucosylation assay is a T4 phage β

-glucosyltransferase-mediated 5-hmC glucosylation assay.

39. A method of diagnosing a subject having a melanocytic lesion comprising,

a) processing a tissue sample of the melanocytic lesion of the subject to thereby label expression product of one or more of the genes IDH2, TET1, TET2, TET3;

b) measuring the expression product of the one or more genes in the sample by detection of the labeled expression product in the tissue sample;

c) quantitating the labeled expression product(s) in the sample as compared to a healthy control to thereby detect a threshold level of reduction of the expression product(s) in the sample; and

d) diagnosing the subject as having a malignancy if a level of reduction of TET3 and/or IDH2 of >

50% is detected, and/or if a level of reduction of TET1 and/or TET2 of >

75% is detected.

40. The method of claim 39 further comprising treating a subject diagnosed with a melanoma comprising contacting melanoma cells of the subject with an effective amount of an agent that increases expression of IDH2 and/or TET2 sufficient to increase 5-hmC in the genome of the cell.

41. The method of claim 40, wherein the agent is selected from an expression vector encoding IDH2 and/or TET2, a regulatory molecule which increases transcription or translation of the IDH2 and/or TET2 gene, and combinations thereof.

42. The method of claim 40, wherein contacting is by administering to the subject a therapeutic amount of a pharmaceutical composition comprising the agent.

43. The method of claim 42, wherein administering is intravenous (I.V.), intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (I.P.), intrathecal (I.T.), intrapleural, intrauterine, rectal, vaginal, topical, or intratumor.

44. A method of determining prognosis of a subject with a melanocytic lesion comprising,

a) processing a tissue sample of the melanocytic lesion of the subject to thereby label the 5-hmC present in the tissue sample;

b) measuring the 5-hmC in the sample by detection of the labeled 5-hmC in the tissue sample;

c) quantitating the 5-hmC in the tissue sample as compared to a healthy control to thereby detect a threshold level of reduction of 5-hmC in the sample;

d) correlating the threshold level of reduction detected in step c) with one or more melanoma staging parameters; and

e) determining the prognosis of the subject based on that of the staging parameter to which the amount of 5-hmC is correlated.

45. The method of claim 44, wherein the staging parameter is selected from the group consisting of Breslow depth, mitosis rate, presence or absence of ulceration, overall stage of melanoma, melanocytic lesion type, and combinations thereof.

46. The method of claim 44, wherein the processing step a) is by immunohistochemical staining or by immunofluorescence.

47. The method of claim 46, wherein quantitating step c) is by assignment of a 5-hmC staining score.

48. The method of claim 44, wherein the threshold level of reduction is equivalent to:

a) a 5-hmC staining score of ≦

2 which indicates the melanocytic lesion is a stage 2-3 melanoma, with a Breslow of >

1 mm, and >

1 mitosis;

b) a 5-hmC staining score of ≧

3 which indicates the melanocytic lesion is a stage 1 melanoma, with a Breslow of ≦

1 mm, and ≦

1 mitosis;

c) a 5-hmC staining score of ≧

1.5 which indicates the melanocytic lesion is a melanoma without ulceration;

or

d) a 5-hmC staining score of <

1.5 which indicates the melanocytic lesion is a melanoma with ulceration.

49. The method of claim 46, wherein quantitating step c) is by assignment of a 5-hmC cell count score.

50. The method of claim 44, wherein the threshold level of reduction is equivalent to:

a) a 5-hmC cell count score of ≧

2 which indicates the melanocytic lesion is a benign melanocytic nevus;

b) a 5-hmC cell count score of ≧

3 which indicates the melanocytic lesion is a benign melanocytic nevus;

c) a 5-hmC cell count score of <

1 which indicates the melanocytic lesion is a primary cutaneous melanoma or a visceral metastatsic melanoma;

d) a 5-hmC cell count score of <

0.5 which indicates the melanocytic lesion is a primary cutaneous melanoma or a visceral metastatsic melanoma;

or

e) a 5-hmC cell count score of <

0.25 which indicates the melanocytic lesion is a lymphnode metastatic melanoma.