Method, reagents, and kits for detecting minimal residual disease

Abstract

The invention relates to the field of minimal residual disease (MRD) diagnostics, which is progressively more applied for the evaluation of treatment effectiveness in patients with a hematological malignancy, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell chronic lymphocytic leukemia (B-CLL), and multiple myeloma (MM). Provided are unique reagent compositions with carefully selected and thoroughly tested combinations of antibodies, for ≧8-color flow cytometric stainings as well as for 10-color and 12-color flow cytometric stainings, which can reach sensitivities of at least 10 −4 , even down to 10 −5 . Also provided are diagnostic kits and methods for detecting MRD.

1 . A reagent composition for flow cytometric detection of B-cell precursor ALL (BCP-ALL) in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies, the panel comprising at least antibodies against the core markers CD10, CD19, CD20, CD34 and CD45, and wherein the panel further comprises one or more antibodies selected from the group of antibodies against CD38 CD81, CyIgμ, and deoxynucleotidyl transferase (NuTdT). 
     
     
         2 . (canceled) 
     
     
         3 . Reagent composition according to  claim 1 , wherein the panel further comprises one or more sets of antibodies selected from
 (a) set of antibodies against CD66c and CD123:   (b) set of antibodies against CD304 and CD73: and   (c) set of antibodies against SmIgκ and SmIgλ.   
     
     
         4 . Reagent composition according to  claim 3 , wherein the antibodies within each set are conjugated to the same fluorochrome. 
     
     
         5 . Reagent composition according to  claim 1 , comprising distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers:
 (i) CD20, CD45, CD81, CD66c, CD123, CD34, CD19, CD10 and CD38, wherein the antibodies against CD66c and CD123 are conjugated to the same fluorochrome:   (ii) CD20, CD45, CD81, CD304, CD73, CD34, CD19, CD10 and CD38, wherein the antibodies against CD304 and CD73 are conjugated to the some fluorochrome:   (iii) CD20, CD45, NuTdT, SmIgκ, SmIgλ, CyIgμ, CD19, CD34 and CD10, wherein the antibodies against SmIgκ and SmIgλ are conjugated to the same fluorochrome.   
     
     
         6 . Reagent composition according to  claim 5 , comprising distinct fluorochrome-conjugated antibodies directed against the markers CD20, CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, CD34, CD19, CD10 and CD38, and one or more sets of antibodies selected from
 (a) set of antibodies against CD66c and CD123:   (b) set of antibodies against CD304 and CD73; and   (c) set of antibodies against SmIgκ and SmIgλ, wherein the antibodies within each set are conjugated to the same fluorochrome.   
     
     
         7 . Reagent composition according to  claim 6 , comprising distinct fluorochrome-conjugated antibodies directed against the markers CD20, CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, SmIgκ, SmIgλ, CyIgμ, CD34, CD19, CD10 and CD38, wherein the antibodies against each of the sets CD66c/CD123, CD304/CD73 and SmIgκ/SmIgλ are conjugated to the same fluorochrome. 
     
     
         8 . A reagent composition for flow cytometric detection of chronic lymphocytic leukemia (CLL) in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies, the panel comprising at least antibodies against the core markers CD5, CD27, CD79b, CD3, CD200, CD81 and CD19. 
     
     
         9 . Reagent composition according to  claim 8 , wherein CD22 and/or Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is used as additional marker(s), preferably in combination with CD43 and CD38. 
     
     
         10 . A reagent composition for flow cytometric detection of MM or PCD in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies, the panel comprising at least antibodies against the core markers CD138, CD38, CD56 and CD19, supplemented with at least four additional markers selected from the group consisting of CD27, CD117, CD81, CD229, CD45, CyIgκ and CyIgλ. 
     
     
         11 . Reagent composition according to  claim 10 , wherein CD45 is the fifth marker, preferably in combination with the markers CD27, CD117 and CD81, or in combination with the markers CD229, CyIgκ and CyIgλ. 
     
     
         12 . Reagent composition according to  claim 10 , comprising distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers:
 (iv) CD45, CD138, CD38, CD56, CD27, CD19, CD117 and CD81   (v) CD45, CD138, CD38, CD56, CD229, CD19, CyIgκ and CyIgλ   (vi) CD138, CD27, CD38, CD56, CD45, CD19, CD117 and CD81   (vii) CD138, CD27, CD38, CD56, CD229, CD19, CyIgκ and CyIgλ   (viii) CD138, CD27, CD38, CD56, CD45, CD19, CyIgκ and CyIgλ   
     
     
         13 . Reagent composition according to  claim 12 , comprising distinct fluorochrome-conjugated antibodies directed against the markers CD138, CD27, CD38, CD56, CD45, CD19, CD117, CD81 and one or both set(s) of antibodies selected from
 (a) set of antibodies against CD229 and CD28;   (b) set of antibodies against CyIgκ and CyIgλ.   
     
     
         14 . A diagnostic kit for flow cytometric detection of minimal residual disease (MRD), comprising at least one reagent compositions according to  claim 1 , optionally together with instructions for use, buffer, and/or control samples. 
     
     
         15 . A multi-color flow cytometric method for detecting minimal residual disease (MRD) in a biological sample comprising cells, preferably lymphocytes, comprising the steps of:
 (i) staining the sample with a reagent composition according to  claim 1 ,   (ii) subjecting the sample to flow cytometry;   (iii) gating on cells for expression of the selected markers detected by the antibodies present in the reagent composition; and   (iv) distinguishing between normal and malignant cells, based on the expression profile of the multiple markers.   
     
     
         16 . Method according to  claim 15 , wherein step iv) involves multivariate analysis, preferably principal component analysis (PCA).