Molecular prognostic signature for predicting breast cancer metastasis, and uses thereof
View Patent ↗The present invention is based on the discovery of a unique 14-gene molecular prognostic signature that is useful for predicting breast cancer metastasis. In particular, the present invention relates to methods and reagents for detecting and profiling the expression levels of these genes, and methods of using the expression level information in predicting risk of breast cancer metastasis.
1. A method of determining risk of tumor metastasis in a human having breast cancer, the method comprising detecting the expression level of at least CENPA, PKMYT1, MELK, MYBL2, BUB1, RACGAP1, TK1, UBE2S, DC13, RFC4, PRR11, DIAPH3, ORC6L and CCNB1 in breast tumor cells of said human, and correlating increased expression levels of said genes with an increased risk of tumor metastasis in said human.
2. The method of claim 1 , further comprising combining the expression level of each of said genes, thereby obtaining a metastasis score (MS) which indicates the risk of tumor metastasis in said human.
3. The method of claim 2 , wherein the method comprises either equally weighing or weighing by a particular constant value (ai) the expression level of each of said genes, and adding together the expression level of each of said genes to obtain said metastasis score (MS).
4. The method of claim 2 , wherein the method comprises identifying said human as having an increased risk of tumor metastasis based on said metastasis score (MS).
5. The method of claim 2 , wherein the method comprises calculating said expression level as a Δ(ΔCt) value as follows for each of said genes:
Δ(Δ Ct ) =( Ct GOI −Ct EC ) test RNA −( Ct GOI −Ct EC ) refRNA
wherein Ct is the PCR threshold cycle of exponential target amplification, GOI =gene of interest, test RNA=patient sample RNA, ref RNA=reference RNA, and EC=endogenous control.
6. The method of claim 5 , wherein the method comprises calculating the metastasis score (MS) by the following:
MS
=
a
0
+
∑
i
=
1
M
ai
*
Gi
wherein M=14, α0=0.022 or 0, αi corresponds to the value presented in Table 2 for each of said genes, and Gi=the expression level as indicated by said Δ(ΔCt) value for each said gene (i).
7. The method of claim 5 , wherein the method comprises calculating the metastasis score (MS) by the following:
MS
=
a
0
+
∑
i
=
1
M
ai
*
Gi
wherein M=14, α0=0 or 0.022, αi=1, and Gi= the expression level as indicated by said Δ(ΔCt) value for each said gene (i).
8. The method of claim 5 , wherein the method comprises calculating the metastasis score (MS) by the following:
MS
=
a
0
+
b
*
[
∑
i
=
1
M
ai
*
Gi
]
wherein M=14, α0=0.022 or 0, b=−0.251 or −1, αi corresponds to the value presented in Table 2 for each of said genes, and Gi= the expression level as indicated by said Δ(ΔCt) value for each said gene (i).
9. The method of claim 5 , wherein the method comprises calculating the metastasis score (MS) by the following:
MS
=
a
0
+
b
*
[
∑
i
=
1
M
ai
*
Gi
]
wherein M=14, α0=0.8657 or 0, b=−0.04778 or −1, αi=1 for each of said genes, and Gi =the expression level as indicated by said Δ(ΔCt) value for each said gene (i).
10. The method of claim 5 , wherein the method comprises calculating the metastasis score (MS) by the following:
MS
(
new
)
=
-
(
1
/
14
)
*
[
∑
i
=
1
14
Gi
]
wherein Gi =the expression level as indicated by said Δ(ΔCt) value for each said gene (i).
11. The method of claim 2 , further comprising comparing said metastasis score (MS) to at least one predefined metastasis score cutoff threshold (MS threshold).
12. The method of claim 11 , wherein the method comprises:
(a) identifying said human as having an increased risk of tumor metastasis if their MS is higher than the predefined MS threshold, or
(b) identifying said human as having a decreased risk of tumor metastasis if their MS is lower than the predefined MS threshold, or
(c) identifying said human as having an intermediate risk if their MS is between two or more MS thresholds.
13. The method of claim 11 , wherein the method comprises comparing said metastasis score (MS) against a lower MS threshold value of −1.47 and an upper MS threshold value of −0.843, wherein the method comprises:
(a) identifying said human as having a decreased risk of tumor metastasis if their MS is lower than −1.47, or
(b) identifying said human as having an increased risk of tumor metastasis if their MS is greater than −0.843, or
(c) identifying said human as having an intermediate risk of tumor metastasis if their MS is between −1.47 and −0.843.
14. The method of claim 1 , wherein said breast tumor cells are estrogen receptor (ER)-positive.
15. The method of claim 1 , wherein said human has no detectable tumor cells in their lymph nodes (node-negative).
16. The method claim 1 , wherein the method comprises obtaining the breast tumor cells from a formalin-fixed paraffin-embedded (FFPE) tissue section, a tumor biopsy, or a frozen tumor tissue sample.
17. The method of claim 1 , wherein the method comprises reverse transcribing and amplifying mRNA of each of said genes using the two primers associated with the corresponding gene as set forth in Table 3, SEQ ID NOS:1-28.
18. The method of claim 1 , wherein the method comprises normalizing the expression levels of said genes against the expression level of at least one control gene, or an average of two or more control genes.
19. The method of claim 18 , wherein said control gene is selected from the group consisting of NUP214, PPIG and SLU7.
20. The method of claim 1 , wherein the method comprises detecting the expression level of each of said genes using real-time PCR or a microarray.