Method of analysing a blood sample of a subject for the presence of a disease marker

Abstract

The present invention relates to a method of analysing a blood sample of a subject for the presence of a disease marker, said method comprising the steps of a) extracting nucleic acid from anucleated blood cells in said blood sample to provide an anucleated blood cells-extracted nucleic acid fraction, and b) analysing said anucleated blood cells-extracted nucleic acid fraction for the presence of a disease marker, wherein said disease marker is a disease-specific mutation in a gene of a cell of said subject, or wherein said disease marker is a disease-specific expression profile of genes of a cell of said subject.

1 . A method of analysing a blood sample of a subject for the presence of a disease cancer marker, said method comprising the steps of
 a) extracting nucleic acid from anucleated blood cells, in said blood sample to provide an anucleated blood cell-extracted nucleic acid fraction, and   b) analysing said anucleated blood cell-extracted nucleic acid fraction for the presence of a cancer marker,   wherein said cancer marker is a cancer-specific mutation in a gene of a nucleated cell of said subject, or   wherein said cancer marker is a cancer-specific expression profile of genes of a nucleated cell of said subject,   wherein said cancer marker is not a megakaryocyte derived nucleic acid, and wherein said nucleated cell is not a megakaryocyte.   
     
     
         2 . The method of  claim 1 , wherein said anucleated blood cells are thrombocytes. 
     
     
         3 - 8 . (canceled) 
     
     
         9 . The method of any  claim 1 , wherein said cancer-specific mutation is in a chromosomal gene, or wherein said cancer-specific expression profile is of chromosomal genes. 
     
     
         10 . The method of  claim 1 , wherein said nucleic acid is ribonucleic acid (RNA). 
     
     
         11 . The method of  claim 1 , wherein said step b) of analysing said anucleated blood cell-extracted nucleic acid fraction for the presence of a cancer marker comprises the selective amplification of
 i) said mutation by reverse transcriptase polymerase chain reaction amplification using at least one nucleic acid mutation-specific amplification primer or probe, or   ii) a plurality of mRNAs by reverse transcriptase polymerase chain reaction amplification to determine the expression level of the chromosomal genes encoding said mRNAs to thereby provide an expression profile for said genes and comparing said expression profile to a reference profile.   
     
     
         12 . The method of  claim 1 , wherein said method is part of a method of diagnosing said cancer in a subject, and wherein the presence of said cancer marker in said anucleated blood cell-extracted nucleic acid fraction is indicative of said subject suffering from said cancer. 
     
     
         13 . A method for determining the stage of disease or the efficacy of a disease treatment in a subject, comprising the steps of:
 analysing a blood sample of a subject for the presence of a cancer marker using the method according to  claim 1  at a first time point to thereby provide a first value for the level of said marker in said subject,   analysing a blood sample of said subject for the presence of a cancer marker using the method according to  claim 1  at a second time point to thereby provide a second value for the level of said disease marker in said subject, wherein said subject has been subjected to a treatment between said first and second time point, and   comparing said first and second value to determine the efficacy of said treatment in said subject.   
     
     
         14 . A method for determining the stage of a disease in a subject, comprising the steps of:
 analysing a blood sample of a subject for the presence of a cancer marker using the method according to  claim 1  to thereby provide a test value for the level of said marker in said subject,   providing a reference value for the level of said cancer marker wherein said reference value is correlated to a particular stage of disease, and   comparing said test and reference value to determine the cancer stage in said subject.   
     
     
         15 . A kit of parts adapted for performing the method recited in  claim 1 , the kit comprising a packaging material which comprises at least one of:
 a container for holding anucleated blood cells separated from a blood sample of a subject;   an agent for extracting nucleic acids from said anucleated blood cells;   an agent for selectively amplifying from said nucleic acids extracted from said anucleated blood cells a disease-specific mutation in a gene of a nucleated cell of said subject by reverse transcriptase polymerase chain reaction amplification, and   a printed or electronic instruction for performing the method recited in  claim 1 ,   
       the kit further comprising:
 a reference for said cancer marker, wherein said reference is indicative for the presence or absence of said marker in said anucleated blood cells-extracted nucleic acid fraction. 
 
     
     
         16 . The kit of  claim 15 , wherein said reference is a reference value for the level of nucleic acids comprising said cancer-specific mutation in thrombocytes in a healthy control subject or in a control subject suffering from said cancer, or
 wherein said reference is a reference expression profile for said plurality of mRNAs in anucleated blood cells from a healthy control subject or from a control subject suffering from said.   
     
     
         17 . A kit of  claim 15 , wherein said agent is selected from a particle or fluorescent marker-labeled anti-anucleated blood cell antibody, or wherein said instruction is selected from an instruction for bead-based anucleated blood cells isolation, an instruction for FACS sorting of anucleated blood cells, an instruction for anucleated blood cell recovery by centrifugation, or negative selection of non-anucleated blood cell components. 
     
     
         18 . A device for diagnosing cancer, the device comprising a support and at least one agent for specifically determining a level and/or activity of at least one nucleic acid mutant in an anucleated blood cell sample of the subject attached to said support, and
 a computer-readable medium having computer-executable instructions for performing the method recited in  claim 1 .   
     
     
         19 . The device of  claim 18 , wherein said at least one agent is an oligonucleotide probe or sequencing primer. 
     
     
         20 . The device of  claim 18 , comprising a lateral flow device, a dipstick or a cartridge for performing a nucleic acid hybridization reaction between:
 an anucleated blood cells-extracted nucleic acid and at least one nucleic acid mutation-specific amplification primer or oligonucleotide probe, wherein said nucleic acid mutation-specific amplification primer or oligonucleotide probe is specific for a cancer-specific mutation, or   an anucleated blood cells-extracted nucleic acid and a plurality of gene-specific amplification primers or oligonucleotide probes for providing a cancer-specific gene expression profile.   
     
     
         21 . The method of  claim 10 , wherein said nucleic acid is messenger ribonucleic acid (mRNA).