Method of diagnosis/prognosis of human chronic lymphocytic leukemia comprising the profiling of lpl/adam genes

Abstract

The present invention provides methods of diagnosis and prognosis of human chronic lymphocytic leukemia (CLL) in a subject a patient in need thereof. The methods of the present invention involve measuring the expression profile of two known genes: LPL and ADAM29; and comparing the ratio of their expression to diagnose the presence of CLL or to prognose the likelihood of developing CLL or the symptoms consistent with CLL.

1 : A method of identifying a subject with a significant probability of having lymphocytic leukemia in a subject in need thereof, comprising:
 determining the gene expression levels of LPL and ADAM29 in a sample containing mRNA obtained from said subject; and   designating said subject as having a significant probability of having lymphocytic leukemia if said determining evidences expression of at least one of LPL and ADAM29 in said sample.   
     
     
         2 : The method according to  claim 1 , further comprising, prior to said determining, extracting a specimen from said subject, wherein said specimen contains mRNA. 
     
     
         3 : The method according to  claim 2 , wherein said specimen contains at least one biological material selected from the group consisting of peripheral mononuclear blood cells, a tissue containing B cells, and extracted B cells. 
     
     
         4 : The method according to  claim 3 , wherein said specimen is a peripheral blood sample. 
     
     
         5 : The method according to  claim 1 , wherein said subject is a human. 
     
     
         6 : The method according to  claim 1 , wherein said determining is by an amplification method. 
     
     
         7 : The method according to  claim 6 , wherein said amplification method is a PCR method and is selected from the group consisting of real-time PCR and multiplex PCR. 
     
     
         8 : The method according to  claim 1 , further comprising classifying said lymphocytic leukemia as chronic lymphocytic leukemia by classifying the IgVH mutational status of said subject based on the expression ratio of LPL and ADAM29. 
     
     
         9 : A method of classifying IgVH mutational status of a subject having chronic lymphocytic leukemia, comprising
 obtaining a peripheral blood sample from said subject;   determining the gene expression levels of LPL and ADAM29 in said peripheral blood sample;   evaluating the LPL/ADAM29 gene expression ratio; and   classifying the IgVH gene as:
 mutated if the LPL/ADAM29 ratio is less than one, or 
 unmutated if the LPL/ADAM29 ratio is greater than or equal to one. 
   
     
     
         10 : The method according to  claim 9 , wherein said determining is by an amplification method. 
     
     
         11 : The method according to  claim 10 , wherein said amplification method is a PCR method and is selected from the group consisting of real-time PCR and multiplex PCR. 
     
     
         12 : The method according to  claim 9 , further comprising validating the IgVH mutational status wherein said validating comprises
 determining the percentage of CD3+CD56+ cells present in a specimen obtained from said subject that are positive for ZAP-70 intracellular expression; and   classifying the IgVH gene sequence as:
 mutated if the percentage of CD19+CD3−CD56-cells present in said specimen that are positive for ZAP-70 intracellular expression is less than 20%, or 
 unmutated if the percentage of CD19+CD3−CD56-cells present in said specimen that are positive for ZAP-70 intracellular expression is greater than or equal to 20%. 
   
     
     
         13 : A method of classifying IgVH mutational status of a subject having chronic lymphocytic leukemia, comprising
 obtaining a sample containing mRNA from said subject;   performing a competitive multiplex PCR assay in the presence of PCR primers for LPL and ADAM29, wherein said PCR primers are SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6,   separating the PCR amplification product; and   classifying the IgVH mutational status by determining the relative intensities of the bands corresponding to 410 bp and 445 bp,
 wherein the 410 bp band corresponds to LPL and the 445 bp band corresponds to ADAM29; and 
 wherein
 when the intensity of the band at 410 bp is greater than the intensity of the band at 445 bp or when there is only a single band at 410 bp then the IgVH mutational status is classified as being unmutated; and 
 when the intensity of the band at 410 bp is less than the intensity of the band at 445 bp or when there is only a single band at 445 bp then the IgVH mutational status is classified as being mutated. 
 
   
     
     
         14 : The method according to  claim 13 , wherein said sample contains at least one biological material selected from the group consisting of peripheral mononuclear blood cells, a tissue containing B cells, extracted B cells, and pre-extracted mRNA. 
     
     
         15 : The method according to  claim 13 , wherein when the intensity of the band at 410 bp and the band at 445 bp are both present, said method further comprises validating the IgVH mutational status. 
     
     
         16 : The method according to  claim 15 , wherein said validating comprises
 determining the gene expression levels of LPL and ADAM29 in a sample containing mRNA from said subject;   evaluating the LPL/ADAM29 gene expression ratio; and   classifying the IgVH gene as:
 mutated if the LPL/ADAM29 ratio is less than one, or 
 unmutated if the LPL/ADAM29 ratio is greater than or equal to one 
   
     
     
         17 : The method according to  claim 16 , further comprising, prior to said determining, obtaining a specimen from said subject, wherein said specimen contains mRNA. 
     
     
         18 : The method according to  claim 17 , wherein said specimen contains at least one biological material selected from the group consisting of peripheral mononuclear blood cells, a tissue containing B cells, and extracted B cells. 
     
     
         19 : The method according to  claim 16 , wherein said classifying is confirmed by a method comprising
 determining the percentage of CD3+CD56+ cells present in a specimen obtained from said subject that are positive for ZAP-70 intracellular expression; and   classifying the IgVH gene sequence as:
 mutated if the percentage of CD19+CD3−CD56-cells present in said specimen that are positive for ZAP-70 intracellular expression is less than 20%, or 
 unmutated if the percentage of CD19+CD3−CD56-cells present in said specimen that are positive for ZAP-70 intracellular expression is greater than or equal to 20%. 
   
     
     
         20 : A method of classifying IgVH mutational status of a subject having chronic lymphocytic leukemia, comprising
 obtaining a sample containing mRNA from said subject;   performing a competitive multiplex PCR assay in the presence of PCR primers for LPL and ADAM29, wherein said PCR primers are selected such that the size differences between the bands corresponding to LPL and ADAM29 are resolvable by electrophoresis,   separating the PCR amplification product; and   classifying the IgVH mutational status by determining the relative intensities of the bands corresponding to LPL and ADAM29,
 wherein
 when the intensity of the band corresponding to LPL is greater than the intensity of the band corresponding to ADAM29 or when there is only a single band corresponding to LPL then the IgVH mutational status is classified as being unmutated; and 
 when the intensity of the band corresponding to LPL is less than the intensity of the band corresponding to ADAM29 or when there is only a single band corresponding to ADAM29 then the IgVH mutational status is classified as being mutated. 
 
   
     
     
         21 : The method according to  claim 20 , wherein said sample contains at least one biological material selected from the group consisting of peripheral mononuclear blood cells, a tissue containing B cells, extracted B cells, and pre-extracted mRNA. 
     
     
         22 : The method according to  claim 20 , wherein when the intensity of the band at 410 bp and the band at 445 bp are both present, said method further comprises validating the IgVH mutational status. 
     
     
         23 : The method according to  claim 22 , wherein said validating comprises
 determining the gene expression levels of LPL and ADAM29 in a sample containing mRNA from said subject;   evaluating the LPL/ADAM29 gene expression ratio; and   classifying the IgVH gene as:
 mutated if the LPL/ADAM29 ratio is less than one, or 
 unmutated if the LPL/ADAM29 ratio is greater than or equal to one 
   
     
     
         24 : The method according to  claim 23 , further comprising, prior to said determining, obtaining a specimen from said subject, wherein said specimen contains mRNA. 
     
     
         25 : The method according to  claim 24 , wherein said specimen contains at least one biological material selected from the group consisting of peripheral mononuclear blood cells, a tissue containing B cells, and extracted B cells. 
     
     
         26 : The method according to  claim 23 , wherein said classifying is confirmed by a method comprising
 determining the percentage of CD3+CD56+ cells present in a specimen obtained from said subject that are positive for ZAP-70 intracellular expression; and   classifying the IgVH gene sequence as:
 mutated if the percentage of CD19+CD3−CD56-cells present in said specimen that are positive for ZAP-70 intracellular expression is less than 20%, or 
 unmutated if the percentage of CD19+CD3−CD56-cells present in said specimen that are positive for ZAP-70 intracellular expression is greater than or equal to 20%. 
   
     
     
         27 - 28 . (canceled) 
     
     
         29 : A method of identifying a subject suspected of having a lymphocytic leukemia in a subject in need thereof, comprising:
 determining the gene expression levels of LPL and ADAM29 in a sample obtained from said subject; and   comparing said expression to a standard expression level of said genes in a normal subject.   
     
     
         30 : A method according to  claim 29 , wherein said gene expression level is determined by measuring mRNA, cDNA or protein expression level. 
     
     
         31 : A method according to  claim 29 , wherein the lymphocytic leukemia is a chronic lymphocytic leukemia. 
     
     
         32 : A kit for detecting lymphocytic leukemia in a subject suspected of having the same, comprising (a) primers that hybridize with the LPL gene, (b) primers that hybridize with the ADAM29 gene, (c) and at least one housekeeping gene. 
     
     
         33 : The kit according to  claim 32 , wherein said components are individually packaged. 
     
     
         34 : The kit according to  claim 32 , further comprising at least one component selected from the group consisting of primers specific for the housekeeping gene and reagents for amplification. 
     
     
         35 : The kit according to  claim 32 , further comprising instructions for using of the components contained in the kit. 
     
     
         36 : A method for determining whether a
 subject has an aggressive form of chronic lymphocytic leukemia or an indolent form of chronic lymphocytic leukemia comprising:   obtaining a specimen from said subject;   determining the gene expression levels of LPL and ADAM29 in said specimen;   designating the subject as having a significant probability of having:
 an aggressive form of chronic lymphocytic leukemia if the LPL gene is over-expressed and the ADAM29 gene is under-expressed; or 
 an indolent form of chronic lymphocytic leukemia if the LPL gene is under-expressed and the ADAM29 gene is over-expressed, 
 wherein over- and under-expression are measured by comparison with the expression level of the corresponding genes in a normal subject. 
   
     
     
         37 : A method for determining whether a subject has an aggressive form of chronic lymphocytic leukaemia or an indolent form of chronic lymphocytic leukaemia comprising:
 obtaining a specimen from said subject;   determining the gene expression levels of the LPL and ADAM29 genes in said specimen;   determining the LPL/ADAM29 gene expression ratio;   designating the subject as having a significant probability of having:
 an aggressive form of chronic lymphocytic leukaemia if the LPL gene is over-expressed; or 
 an indolent form of chronic lymphocytic leukaemia if the ADAM29 ratio is over-expressed. 
   
     
     
         38 : The method of  claim 36 , wherein over-expression of ADAM29 and/or LPL is determined by comparison to expression of GAPDH, wherein LPL is over-expressed when a ratio of LPL/GADPH>1.0 and ADAM29 is over-expressed when a ratio of ADAM29/GADPH is >2.8. 
     
     
         39 : The method according to  claim 36 , wherein said specimen contains at least one biological material selected from the group consisting of peripheral mononuclear blood cells, a tissue containing B cells, and B cells. 
     
     
         40 : The method of  claim 36 , in which gene expression of the LPL and ADAM29 genes is determined by RT-PCR.