Methods and kits for the molecular subtyping of tumors

Abstract

The present invention relates to an in vitro method of identifying a molecular subtype of a tumor in a cancer patient and to a method of stratifying a cancer patient for tumor treatment. The present invention further relates to kits that are useful for identifying a molecular subtype of a tumor in a cancer patient.

1 .- 8 . (canceled) 
     
     
         39 . A kit for identifying a molecular subtype of a tumor in a cancer patient by means of reverse transcription (RT) quantitative PCR, said kit comprising:
 at least one pair of HER2-specific primers and at least one HER2-specific probe;   at least one pair of ESR1-specific primers and at least one ESR1-specific probe;   at least one pair of PGR-specific primers and at least one PGR-specific probe; and   at least one pair of Ki67-specific primers and at least one Ki67-specific probe.   
     
     
         40 . The kit of  claim 39 , wherein the quantitative PCR is fluorescence-based quantitative real-time PCR. 
     
     
         41 . The kit of  claim 39 , wherein detection of the probe is based on amplification-mediated probe displacement. 
     
     
         42 . The kit of  claim 41 , wherein the probe is a dual-label probe comprising a fluorescence reporter moiety and a fluorescence quencher moiety. 
     
     
         43 . The kit of  claim 39 , further comprising a reverse transcriptase and a DNA polymerase. 
     
     
         44 . The kit of  claim 43 , wherein the reverse transcriptase and the polymerase are provided in the form of an enzyme-mix which allows a one-step reverse transcription (RT) quantitative PCR. 
     
     
         45 . The kit of  claim 39 , further comprising at least one pair of reference gene-specific primers and at least one reference gene-specific probe. 
     
     
         46 . The kit of  claim 45 , wherein the reference gene is one or more selected from the group comprising CALM2, B2M, RPL37A, GUSB, HPRT1 and GAPDH. 
     
     
         47 . The kit of  claim 39 , further comprising at least one control RNA sample. 
     
     
         48 . The kit of  claim 39 , wherein the primers provide an amplicon size of less than 120 bp. 
     
     
         49 . The kit of  claim 39 , wherein the ESR1-specific primers have a length of 15 to 30 nucleotides and comprise at least 10 contiguous nucleotides of the sequences of SEQ ID NOs: 1 and 2, and/or wherein the HER2-specific primers have a length of 15 to 30 nucleotides and comprise at least 10 contiguous nucleotides of the sequences of SEQ ID NOs: 4 and 5, and/or wherein the Ki67-specific primers have a length of 15 to 30 nucleotides and comprise at least 10 contiguous nucleotides of the sequences of SEQ ID NOs: 7 and 8, and/or wherein the PGR-specific primers have a length of 15 to 30 nucleotides and comprise at least 10 contiguous nucleotides of the sequences of SEQ ID NOs: 10 and 11. 
     
     
         50 . The kit of  claim 39 , wherein the ESR1-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 3, and/or wherein the HER2-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 6, and/or wherein the Ki67-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 9, and/or wherein the PGR-specific probe has a length of 20 to 35 nucleotides and comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 12. 
     
     
         51 . The kit of  claim 39 , wherein the tumor is a solid tumor. 
     
     
         52 . The kit of  claim 39 , wherein the tumor is a breast tumor or is derived from a breast tumor. 
     
     
         53 . The kit of  claim 39 , wherein the cancer is breast cancer. 
     
     
         54 . The kit of  claim 39 , further comprising at least one pair of RACGAP1-specific primers and at least one RACGAP1-specific probe 
     
     
         55 . The kit of  claim 39 , wherein the kit comprises:
 a pair of HER2-specific primers and an HER2-specific probe;   a pair of ESR1-specific primers and an ESR1-specific probe;   a pair of Ki67-specific primers and a Ki67-specific probe; and   a pair of PGR-specific primers and a PGR-specific probe,   
       wherein the ESR1-specific primers have the sequences of SEQ ID NOs: 1 and 2, wherein the HER2-specific primers have the sequences of SEQ ID NOs: 4 and 5, wherein the Ki67-specific primers have the sequences of SEQ ID NOs: 7 and 8, and wherein the PGR-specific primers have the sequences of SEQ ID NOs: 10 and 11. 
     
     
         56 . The kit of  claim 54 , wherein the RACGAP1-specific primers provide an amplicon size of less than 120 bp. 
     
     
         57 . The kit of  claim 55 , wherein the ESR1-specific probe has the sequence of SEQ ID NO: 3, the HER2-specific probe has the sequence of SEQ ID NO: 6, the Ki67-specific probe has the sequence of SEQ ID NO: 9, or the PGR-specific probe has the sequence of SEQ ID NO: 12. 
     
     
         58 . The kit of  claim 55 , wherein the ESR1-specific probe has the sequence of SEQ ID NO: 3, the HER2-specific probe has the sequence of SEQ ID NO: 6, the Ki67-specific probe has the sequence of SEQ ID NO: 9, and the PGR-specific probe has the sequence of SEQ ID NO: 12.