Composition and method for determination of ck19 expression

Abstract

Disclosed is a method for quantitative determination of CK-19 mRNA positive cells in a biological sample. The method can be used, for instance, with peripheral blood to detect cancer in a patient. In one embodiment, the method can be used to detect the cancer before initiation of adjuvant treatment, thereby providing information about therapeutic efficacy. Practice of the invention method is sensitive, reliable, and easy to perform.

1 - 5 . (canceled) 
     
     
         6 . A primer pair consisting of one primer having the sequence of SEQ ID NO: 1 and one primer having the sequence of SEQ ID NO: 2. 
     
     
         7 . The primer pair according to  claim 6 , wherein the primers comprises nucleotides, analogues of nucleotides or labels. 
     
     
         8 . A method of quantitatively determining the presence in a test sample of a mRNA derived from a gene comprising at least one intron using a pair of primers, wherein at least one of said primers comprises at least one intron-spanning site comprising the steps of
 (i) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of  claim 6  and a test sample;   (ii) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; and   (iii) determining the amount of the mRNA in the sample using real-time monitoring during PCR.   (iv)   
     
     
         9 . The method according to  claim 8 , wherein the test sample is subjected to reverse transcription prior to forming the reaction mixture of step (i). 
     
     
         10 . The method according to  claim 8 , wherein the test sample is selected from a blood sample, a sample from the bone marrow and a sample derived from the lymph nodes. 
     
     
         11 . The method according to  claim 10 , wherein the sample is a blood sample. 
     
     
         12 . The method according to  claim 11 , wherein the blood sample is centrifuged before forming the reaction mixture. 
     
     
         13 . The method according to any of the  claim 8 , wherein the reaction mixture of step (i) further comprises a housekeeping primer pair. 
     
     
         14 . The method according to  claim 13 , wherein the housekeeping primer pair has the sequence according to SEQ ID NO:5 and SEQ ID NO: 6. 
     
     
         15 . A method of determining the prospects of adjuvant therapy in a patient suffering from cancer comprising the steps of
 (i) providing a biological sample from the patient;   (ii) isolating nucleic acids from the biological sample;   (iii) optionally reverse transcribing the isolated nucleic acids, when the origin of the nucleic acid is RNA;   (iv) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of  claim 6  and an aliquot of the nucleic acids isolated in step (ii) or the reverse transcribed nucleic acid of step (iii);   (v) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence;   (vi) quantitating the CK-19 mRNA positive cells in the sample using real-time monitoring during PCR; and   (vii) based on the amount of CK-19 mRNA positive cells in the sample determining the prospects of adjuvant therapy.   
     
     
         16 . The method according to  claim 15 , wherein the cancer is breast cancer, preferably operable breast cancer. 
     
     
         17 . The Diagnostic method according to  claim 15 , wherein the biological sample is a blood sample. 
     
     
         18 . The method according to any of the  claim 15 , wherein the reaction mixture of step (iii) further comprises a housekeeping primer pair. 
     
     
         19 . The method according to  claim 18 , wherein the housekeeping primer pair has the sequence according to SEQ ID NO:5 and SEQ ID NO: 6. 
     
     
         20 . A diagnostic kit for amplifying a CK19 target sequence comprising:
 (i) the primer pair of  claim 6 ;   (ii) optionally sequences hybridizing to additional markers on cancer cells;   (iii) amplification reagents.   
     
     
         21 . The diagnostic kit according to  claim 20 , wherein the amplification reagents comprise the hybridization probes of SEQ ID NO: 3 and SEQ ID NO: 4. 
     
     
         22 . The diagnostic kit according to  claim 20 , wherein the kit further comprises a housekeeping primer pair, preferably with the sequences according to SEQ ID NO: 5 and SEQ ID NO: 6. 
     
     
         23 . The diagnostic kit according to any of the  claim 20 , wherein the amplification reagents and the primer pair(s) are lyophilized. 
     
     
         24 . A primer pair comprising a first primer having at least about 8 nucleobases of the sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the first primer being from between about 10 to about 20 nucleobases in length and further being capable of specifically hybridizing with the CK-19 cDNA sequence shown in  FIG. 1 ,
 and further comprising a second primer having at least about 8 nucleobases of the sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the second primer being from between about 10 to about 20 nucleobases in length and further being capable of specifically hybridizing with the CK-19 cDNA sequence shown in  FIG. 1 .   
     
     
         25 . (canceled) 
     
     
         26 . The primer pair of  claim 24 , wherein at least one of the first and second primers comprises a modified internucleoside linkage, modified sugar moiety or modified nucleobase. 
     
     
         27 . The primer pair of  claim 26 , wherein at least one of the first and second primers includes at least one locked nucleic acid (LNA) unit. 
     
     
         28 . A method of determining the presence in a test sample of a mRNA derived from a gene comprising at least one intron using a pair of primers, wherein at least one of said primers comprises at least one intron-spanning site comprising the steps of
 (v) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of  claim 24  and a test sample;   (vi) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; and   (vii) determining the amount of the mRNA in the sample using real-time monitoring during PCR.   
     
     
         29 . The method of  claim 28  further comprising use of a housekeeping primer pair. 
     
     
         30 . A diagnostic kit comprising the primer or primer pair of  claim 24 . 
     
     
         31 . A method of determining presence of CK-19 mRNA in a biological sample, the method comprising the following steps:
 a) separating any mononuclear cells from the biological fluid,   B) contacting the separated mononuclear cells with an antibody that specifically binds antigen expressed by the mononuclear cells (or antigen binding fragment thereof), wherein the antibody (or fragment) is bound to a solid support, the contacting being sufficient to form a binding complex between the cells, the antibody (or fragment) and solid support,   c) separating the complex from any unbound material,   d) isolating nucleic acid from endothelial mononuclear cells bound to the complex,   e) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of  claim 24  and the nucleic acid,   f) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the CK-19 target sequence; and   g) detecting CK-19 mRNA in the biological sample using PCR.   
     
     
         32 . The method of  claim 31 , wherein the epithelial mononuclear cells are separated by filtration or centrifugation. 
     
     
         33 . The method of  claim 32 , wherein the centrifugation step comprises use of a Ficoll density gradient. 
     
     
         34 . The method of  claim 31 , wherein the antibody is a monoclonal that specifically binds a glycoprotein expressed by the endothelial mononuclear cells. 
     
     
         35 . The method of  claim 34 , wherein the monoclonal antibody is Ber-EP4. 
     
     
         36 . The method of  claim 31 , wherein the solid support is an immunomagnetic bead. 
     
     
         37 . The method of  claim 36 , wherein step c) of the method further comprises impressing a magnetic field on the binding complex to separate the complex from any unbound material. 
     
     
         38 . The method of  claim 31 , wherein the primer pair are the sequences represented by SEQ ID NO:1 and SEQ ID NO:2. 
     
     
         39 . The method of  claim 31 , wherein the biological fluid is peripheral blood.