Detecting neoplasm

• US 9,399,800 B2
• Filed: 08/14/2015
• Issued: 07/26/2016
• 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:
  US14/827,013
• Publication Number:
  US 20150354013A1
• Time in Patent Office: 347 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, and

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

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 K-ras specific primers comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

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

8. 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.

9. The system of claim 8, wherein said one or more K-ras specific probes comprise SEQ ID NO:

1.

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

11. The system of claim 1, wherein the bisulfite reagent is sodium bisulfite.