Methods and signatures for oropharyngeal cancer prognosis

1. A method of identifying a subject with oropharyngeal cancer as having a poor or a favorable prognosis, the method comprising:

(a) obtaining a tissue sample yielding low quality RNA from a subject;

(b) using a PCR-based profiling method for miRNA expression, to determine in the sample, the levels of six prognostic miRNAs selected from the group consisting of miR-142-3p, miR-31, miR-146a, miR-26b, miR-24, and miR-193b;

(c) calculating a risk score using the levels of miRNAs determined, and

(d) comparing the risk score to a median risk score,

wherein, if (i) the risk score is equal to or higher than the median risk score, the subject has a poor prognosis, and (ii) the risk score is lower than the median risk score, the subject has a favorable prognosis, wherein poor prognosis is indicative of a significantly greater risk of death.

2. The method of claim 1 , wherein the sample is processed by real time PCR.

3. The method of claim 2 , wherein the real time PCR is preceded by reverse transcription of each miRNA into a cDNA using reverse transcription primers specific for each miRNA.

4. The method of claim 3 , wherein each reverse transcription primer specific for each miRNA comprises bases that anneal to the 3′ end of the mature miRNA sequence and a unique tag sequence.

5. The method of claim 4 , wherein the reverse transcription of the prognostic miRNAs is performed in a multiplexed reaction.

6. The method of claim 2 , wherein the real time PCR for each miRNA is performed individually.

7. The method of claim 6 , wherein each real time PCR is performed using two PCR primers, wherein the first PCR primer anneals to the unique tag sequence of the reverse transcription primer, and the second PCR primer anneals to a unique sequence in the reverse transcribed sequence of the miRNA.

8. The method of claim 1 , wherein the median risk score is calculated from the risk score of a population of subjects with oropharyngeal squamous cell carcinoma.

9. A method for determining the prognosis of a subject with oropharyngeal carcinoma, the method comprising:

(a) obtaining a tissue sample yielding low quality RNA from a subject;

(b) using a PCR-based profiling method for miRNA expression, to determine in the sample, the levels of six prognostic miRNAs selected from the group consisting of miR-142-3p, miR-31, miR-146a, miR-26b, miR-24, and miR-193b;

(c) calculating a risk score (S) according to the following formula:

S=C 1 E 1 +C 2 E 2 +C 3 E 3 −C 4 E 4 −C 5 E 5 −C 6 E 6 , wherein:

C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 are coefficients;

E 1 is the expression level of miR-24;

E 2 is the expression level of miR-31;

E 3 is the expression level of miR-193b;

E 4 is the expression level of miR-26b;

E 5 is the expression level of miR-142-3p;

E 6 is the expression level of miR-146a; and

assessing prognosis by comparing the risk score to a median risk score, wherein, if (i) the risk score is equal to or higher than the median risk score, the subject has a poor prognosis, and (ii) the risk score is lower than the median risk score, the subject has a favorable prognosis, wherein poor prognosis is indicative of a significantly greater risk of death.

10. The method of claim 9 , wherein the method further comprises measuring expression level of at least one miRNA selected from the group consisting of miR-29b, miR-203, miR-155, miR-26a, miR-215, miR-101, and let-7i.

11. The method of claim 9 , wherein the median risk score is calculated from a population of subjects having oropharyngeal carcinoma.

12. The method of claim 9 , wherein each of C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 is a Cox regression analysis Z score.

13. The method of claim 12 , wherein the Z score is equal to Cox regression coefficient/standard error of the coefficient.

14. The method of claim 9 , wherein the expression level of each miRNA is a normalized level of expression.

15. The method of claim 9 , wherein the oropharyngeal carcinoma is a squamous cell carcinoma.

16. The method of claim 1 , wherein the method further comprises determining in the sample the levels of at least one prognostic miRNA selected from the group consisting of miR-29b, miR-203, miR-155, miR-26a, miR-215, miR-101, and let-7i.

17. The method of claim 1 , wherein the tissue sample yielding low-quality RNA is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

18. The method of claim 9 , wherein the tissue sample yielding low-quality RNA is a formalin-fixed paraffin-embedded (FFPE) tissue sample.