Assays and methods relating to the treatment of melanoma

1. 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 the 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) (i) 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

(ii) surgically removing the melanoma and not administering adjuvant therapy if the value of NRP-2:MART is not increased at least 2 σ relative to a reference level.

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

3. The method of claim 2 , wherein the primers of SEQ ID NOs: 1-2 are present in a reaction mixture at about 0.3 FM, the primers of SEQ ID NO:s 7-8 are present in a reaction mixture at about 0.9 FM, or the primers of SEQ ID NOs: 10-11 are present in a reaction mixture at about 0.3 FM.

4. The method of claim 1 , 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, or 12.

5. 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) (i) 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

(ii) surgically removing the melanoma and not administering adjuvant therapy if the value of NRP-2:MART is not increased relative to a reference level.

6. The method of claim 5 , wherein the sample is an FFPE sample.

7. The method of claim 5 , wherein the levels of the mRNAs are measured using quantitative RT-PCR.

8. The method of claim 7 , wherein the amplicon products amplified during PCR are of less than 150 bp in length.

9. The method of claim 7 , wherein the amplicon products amplified during PCR are of less than 100 bp in length.

10. The method of claim 5 , 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.

11. The method of claim 5 , wherein steps (a) and (b) comprise performing duplex RT-PCR wherein the mRNA level of the internal control nucleic acid is measured simultaneously with the measurement of NRP-2 and Melan-A mRNAs.

12. The method of claim 11 , wherein the mRNA level of NRP-2 or Melan-A is normalized to the level of the internal control nucleic acid prior to performing step (c).

13. The method of claim 5 , wherein one or both of the measuring steps comprises performing PCR using one or more primers having the sequence of any of SEQ ID NOs: 1-2, 7-8, and 10-11.

14. The method of claim 13 , 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.

15. The method of claim 13 , wherein the primers of SEQ ID NOs: 1-2 are present in a reaction mixture at about 0.3 FM, the primers of SEQ ID NO:s 7-8 are present in a reaction mixture at about 0.9 FM, or the primers of SEQ ID NOs: 10-11 are present in a reaction mixture at about 0.3 FM.

16. The method of claim 5 , further comprising measuring the mRNA 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.

17. 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 by performing PCR;

(b) measuring the level of melan-A (MART) mRNA in the sample obtained from the subject by performing PCR;

(c) calculating the value of NRP-2:MART from the levels obtained in steps (a) and (b);

(d) performing PCR using known quantities of NRP-2 and melan-A nucleic acids to generate a standard curve;

(e) calculating copy numbers of NRP-2 and melan-A in the sample using the standard curve generated in step (d) and the value calculated in step (c);

(f) (i) surgically removing the melanoma and administering adjuvant therapy and follow-up monitoring if the copy number of NRP-2:MART is increased relative to a reference level; and

(ii) surgically removing the melanoma and not administering adjuvant therapy if the copy number of NRP-2:MART is not increased relative to a reference level.