Detecting neoplasm

• US 9,121,070 B2
• Filed: 01/30/2014
• Issued: 09/01/2015
• Est. Priority Date: 02/15/2008
• Status: Active Grant

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Taylor, William R., Harrington, Jonathan J., Quint, Patrick S., Zou, Hongzhi, Bergen, Harold R. III, Smith, David I., Ahlquist, David A.
• Primary Examiner(s): AEDER, SEAN E
• Application Number:
  US14/168,552
• Publication Number:
  US 20140221252A1
• Time in Patent Office: 579 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/40 involving amylase
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)
• CPC Class Codes: C12Y 304/2107 Pancreatic endopeptidase E (3.4.21.70)
• CPC Class Codes: G01N 2333/928 acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
• CPC Class Codes: G01N 2333/966 Elastase
• CPC Class Codes: G01N 2560/00 Chemical aspects of mass spectrometric analysis of biological material
• CPC Class Codes: G01N 33/57407 Specifically defined cancers
• CPC Class Codes: G01N 33/57419 of colon
• CPC Class Codes: G01N 33/57438 of liver, pancreas or kidney

Abstract

This document relates to methods and materials for detecting premalignant and malignant neoplasms. For example, methods and materials for determining whether or not a stool sample from a mammal contains nucleic acid markers or polypeptide markers of a neoplasm are provided.

Claims

1. A system for detecting colorectal cancer or pre-cancer in a mammal, said system comprising:

a) a stool sample,

b) reagents sufficient for measuring a K-ras mutation score within said stool sample, wherein said reagents comprise K-ras specific primers,

c) reagents sufficient for converting unmethylated cytosine residues to uracil residues within said stool sample while leaving 5-methylcytosine residues unchanged, and reagents sufficients for measuring BMP3 methylation within said stool sample, wherein said reagents sufficient for measuring BMP3 methylation comprise BMP3 specific primers, and

d) reagents sufficient for measuring elevated human DNA level within said stool sample.

2. The system of claim 1, further comprising a DNA stabilization buffer.

3. The system of claim 1, further comprising DNA extraction reagents.

4. The system of claim 1, wherein said reagents for measuring a K-ras mutation score within said stool sample comprise reagents for conducting a digital melt curve analysis.

5. The system of claim 1, wherein said reagents for measuring a K-ras mutation score within said stool sample comprise reagents for conducting quantitative allele-specific PCR.

6. The system of claim 1, wherein said reagents for measuring BMP3 methylation within said stool sample comprise reagents for conducing real-time methylation-specific PCR.

7. The system of claim 1, wherein said reagents for measuring elevated human DNA level comprise reagents for conducting real-time Alu PCR.

8. The system of claim 1, wherein said K-ras specific primers comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO:

2, 3, 4, 5, 6 and 7.

9. The system of claim 1, wherein said reagents sufficient for measuring a K-ras mutation score within said stool sample further comprise one or more K-ras specific probes.

10. The system of claim 9, wherein said one or more K-ras specific probes comprise SEQ ID NO:

1.

11. The system of claim 1, wherein said reagents sufficient for measuring elevated human DNA level within said stool sample comprise one or more primers specific for Alu sequences.

12. The system of claim 11, wherein said primers specific for Alu sequences comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

44, 45, 46, 47 and 48.

13. The system of claim 1, further comprising a stool stability buffer comprising Tris PH 9.0, EDTA, and NaCl.