Assays and methods relating to the treatment of melanoma

Patent metadata

Field	Value
Publication number	US-2016312300-A1
Application number	US-201415105192-A
Country	US
Kind code	A1
Filing date	Dec 19, 2014
Priority date	Dec 20, 2013
Publication date	Oct 27, 2016
Grant date	—

Abstract

The technology described herein relates to assays and methods for the diagnosis, prognosis, and/or treatment of melanoma, e.g. relating to measuring the level of neurophilin-2 (NRP-2) mRNA expressed in melanoma cells. In some embodiments, the level of NRP-2 can be normalized to the level of Melan-A (MART) mRNA.

First claim

1 . An assay for detecting a malignant melanoma, the assay comprising:
(a) performing quantitative duplex RT-PCR on a sample obtained from a subject to measure:
1) the level of neurophilin-2 (NRP-2) in the sample; and
2) a known quantity of an internal control nucleic acid added to the sample; and
normalizing the level of NRP-2 to the level of the internal control nucleic acid; (b) performing quantitative duplex RT-PCR on a sample obtained from a subject to measure:
1) the level of melan-A (MART) in the sample; and
2) a known quantity of an internal control nucleic acid added to the sample; and
normalizing the level of MART to the level of the internal control nucleic acid; (c) calculating the value of NRP-2:MART from the levels obtained in steps (a) and (b); wherein malignant melanoma is detected in the sample if the value of NRP-2:MART is increased by at least 2 σ relative to a reference level.

2 . A method of treatment for melanoma, the method comprising:
(a) performing quantitative duplex RT-PCR on a sample obtained from a subject to measure:
1) the level of neurophilin-2 (NRP-2) in the sample; and
2) a known quantity of an internal control nucleic acid added to the sample; and
normalizing the level of NRP-2 to the level of the internal control nucleic acid; (b) performing quantitative duplex RT-PCR on a sample obtained from a subject to measure:
1) the level of melan-A (MART) in the sample; and
2) a known quantity of an internal control nucleic acid added to the sample; and
normalizing the level of MART to the level of the internal control nucleic acid; (c) calculating the value of NRP-2:MART from the levels obtained in steps (a) and (b); and (d) surgically removing the melanoma and administering adjuvant therapy and follow-up monitoring if the value of NRP-2:MART is increased at least 2 σ relative to a reference level; and not administering adjuvant therapy if the value of NRP-2:MART is not increased at least 2 σ relative to a reference level.

3 . The method of claim 2 , wherein PCR is performed using one or more primers having the sequence of any of SEQ ID NOs: 1-2, 7-8, and 10-11.

4 . The method of claim 2 , wherein the level of amplicons resulting from PCR is detected using one or more probes having the sequence of any of SEQ ID NOs: 3, 9, and 12.

5 . The method of claim 2 , wherein the primers or probes are present in a reaction mixture at about the concentrations shown in Table 1.

6 . An assay for detecting a malignant melanoma, the assay comprising:
(a) measuring the level of neurophilin-2 (NRP-2) mRNA in a sample obtained from a subject with melanoma; (b) measuring the level of Melan-A (MART) mRNA in the sample obtained from the subject; and (c) calculating the value of NRP-2:MART from the levels obtained in steps (a) and (b); wherein malignant melanoma is detected in the sample if the value of NRP-2:MART is increased relative to a reference level.

7 .- 17 . (canceled)

18 . A method of treatment for melanoma, the method comprising:
(a) measuring the level of neurophilin-2 (NRP-2) mRNA in a sample obtained from a subject with melanoma; (b) measuring the level of Melan-A (MART) mRNA in the sample obtained from the subject; (c) calculating the value of NRP-2:MART from the levels obtained in steps (a) and (b); and (d) surgically removing the melanoma and administering adjuvant therapy and follow-up monitoring if the value of NRP-2:MART is increased relative to a reference level; and not administering adjuvant therapy if the value of NRP-2:MART is not increased relative to a reference level.

19 . The method of claim 18 , wherein the sample is an FFPE sample.

20 . The method of claim 18 , wherein the levels of the mRNAs are measured using quantitative RT-PCR.

21 . The method of claim 20 , wherein amplicons of less than 150 bp are amplified during PCR.

22 . The method of claim 20 , wherein amplicons of less than 100 bp are amplified during PCR.

23 . The method of claim 18 , wherein a known quantity of an internal control nucleic acid is added to the sample prior to measuring the level of NRP-2 and Melan-A mRNAs.

24 . The method of claim 18 , wherein steps (a) and (b) comprise performing duplex RT-PCR wherein the level of the internal control nucleic acid is measured simultaneously with the measurement of NRP-2 and Melan-A mRNAs.

25 . The method of claim 24 , wherein the level of NRP-2 or Melan-A is normalized to the level of the internal control nucleic acid prior to performing step (c).

26 . The method of claim 18 , wherein PCR is performed using one or more primers having the sequence of any of SEQ ID NOs: 1-2, 7-8, and 10-11.

27 . The method of claim 18 , wherein the level of amplicons resulting from PCR is detected using one or more probes having the sequence of any of SEQ ID NOs: 3, 9, and 12.

28 . The method of claim 18 , wherein the primers or probes are present in a reaction mixture at about the concentrations shown in Table 1.

29 . The method of claim 18 , further comprising
performing PCR using known quantities of NRP-2 and Melan-A nucleic acids to generate a standard curve; and calculating copy numbers of NRP-2 and Melan-A in the sample using the standard curve.

30 .- 41 . (canceled)

42 . The method of claim 18 , wherein the assay or method further comprises measuring the level of one or more marker genes selected from the group consisting of:
IL8; AREG; MMP1; CSPG2; SerpinB2; RAP1A; FLRT3; CSPG2; COL4A1; TK1; DHFR; CDH3; HELLS; KIT; CXCL1; Ki67; MITF; p53; and p21;
wherein an increase in the marker gene relative to a reference level indicates malignant melanoma is detected in the sample or that the melanoma has a predisposition to become malignant.

43 .- 46 . (canceled)

Assignees

• Univ Boston

Inventors

• Alani Rhoda M
• Ryu Byungwoo

Classifications

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q2600/112

  Disease subtyping, staging or classification · CPC title