A method for detecting and quantificating minimal residual disease in patients with hematologic malignancies using a diversity of coding and signaling compounds in immunoglobulin receptor gene rearrangements

Claims

1. A method for detecting and quantifying minimal residual disease (MRD) in patients with hematological malignancies, which includes the following steps: a) identification of junctions of coding and signaling regions of genomic rearrangements of T-cell receptors (TCR) and B-cell receptors (BCR) specific for malignant clones in the initial patient sample, by: i) performing a multiplex polymerase chain reaction (PCR) using the isolated genomic DNA, wherein the PCR reaction includes oligonucleotide primers designed to amplify one or more of the following rearrangements: TRα / β / γ / δ, IGH / K / λ, DJ, DD, VD, kappa deletion element (KDE), in a single multiplex mixture; ii) conducting high-throughput sequencing of the resulting library; iii) extraction of coding and signal region junctions of complete and incomplete rearrangements by aligning putative rearrangements with a reference library containing V, D, and J gene segments, the KDE kappa chain deletion element, and the IGKC intron, using a semi-global alignment algorithm to identify flanking sequences, followed by a clustering algorithm to correct for PCR and sequencing errors, and assembly of complete and incomplete rearrangement clonotypes; iv) determination of the exact sizes of clonotypes in repertoires obtained for malignant cell samples; v) identification of clonotypes specific to the malignant clone; b) performing subsequent MRD monitoring by conducting at least four independent PCR reactions using isolated and quantified genomic DNA obtained at the monitoring point, wherein the concentration of the malignant clone is determined based on the proportion of independent PCR reactions in which a rearrangement characteristic of the malignant clone was observed.

2. The method according to claim 1, wherein the special library of oligonucleotide primers for multiplex PCR is designed with the possibility of reducing the formation of primer dimers and minimizing nucleotide diversity.

3. The method according to claim 1 or 2, wherein the oligonucleotide library is produced by a method comprising: partitioning target regions of genomic DNA into k-mers of a given length; clustering of target regions of genomic DNA using a Hamming distance of no more than 4, 5, 6 or 7 nucleotides; identification of clusters of k-mers with similar structures according to the selected Hamming distance.

4. The method according to claim 3, wherein the k-mers have a length of at least 16 bases.

5. The method according to claim 3, wherein from the identified clusters, tables of 5-mers in reverse orientation and 5-mers located at the 3' end of the sequences in direct orientation are formed.

6. The method according to paragraph 5, in which the 5-mer tables are intersected in pairs, and the set of primers with the least overlap of 5-mers is selected for further analysis.

7. The method according to any one of claims 4-6, wherein up to 14 additional nucleotides corresponding to the target region are added to the 5' end of the k-mers to balance the annealing temperatures of the primers.

8. The method according to paragraphs 5-7, in which the intersection procedure using 5-dimensional variables is repeated iteratively.

9. The method according to any one of claims 3-8, wherein at least 2 working sets of forward primers and at least 2 working sets of reverse primers are generated.

10. The method according to claim 9, wherein the set of reverse primers comprises primers to J genes, introns adjacent to the 3' ends of D genes, and the kappa chain deletion element KDE.

11. The method according to claim 9 or 10, wherein the set of forward primers comprises primers to V genes, introns adjacent to the 5' ends of D genes, and the C intron of IgK.

12. The method according to paragraphs 9-11, in which the set of forward primers contains primers to introns adjacent to the 5' ends of the J genes.

13. The method according to paragraphs 9-11, in which the set of reverse primers contains primers to introns adjacent to the 3' ends of the V genes.

14. The method according to any one of claims 9 to 13, in which the formed multiplex sets of primers are intended for separate amplification of junctions of the coding regions of complete VJ and / or VDJ rearrangements in the TRα, TRβ, TRγ, TRδ, IgH, IgK or IgL loci; junctions of the coding regions of incomplete DJ rearrangements in the TRβ, TRδ or IgH loci; junctions of the coding regions of VD and DD type rearrangements in the TRδ and TRβ loci; junctions of the coding regions of VD type rearrangements in the IgH locus; junctions of the coding regions of TRDV-TRAJ chimeric rearrangements; junctions of the coding regions of TRGV-TRBJ and TRBV-TRGJ chimeric rearrangements; junctions of the coding regions of IGHV-TRAJ and IGHV-TRDJ chimeric rearrangements; rearrangements involving the kappa deletion element (KDE); chromosome-retained signal region junctions of complete VJ rearrangements at the IgK, TRβ, TRδ loci and the chimeric loci TRGV-TRBJ, TRBV-TRGJ, TRDV-TRAJ, IGHV-TRAJ and IGHV-TRDJ;chromosome-retained junctions of signal regions of incomplete DD rearrangements at the TRβ and TRδ loci; chromosome-retained junctions of signal regions of incomplete DJ rearrangements at the IGH and TRB loci; chromosome-retained junctions of signal regions of incomplete VD rearrangements at the IgH, TRβ and TRδ loci.

15. The method according to claim 14, wherein the double indexing of each aliquot of the amplicon and the introduction of adapters for sequencing are carried out using step-out PCR.

16. The method according to any one of paragraphs 14, 15, in which the average length of the target amplicons in four multiplex primer mixtures is: Mix1 (Set2+Set3): ~120 p.p.; Mix2 (Set2+Set4): ~160 p.p.; Mix3 (Set1+Set3): ~240 p.p.; Mix4 (Set1+Set4): ~280 p.p.

17. The method according to any one of claims 9-16, wherein the amplicons obtained using one set of primers are not used as a template for PCR with a subsequent set of primers.

18. The method according to any one of claims 1-17, wherein the concentrations of primers in the multiplex mixture are optimized by analyzing the ratio of the frequencies of non-functional rearrangements of the TCR and BCR genes.

19. The method of claim 18, wherein an overamplification ratio (OAR) is determined for at least one rearrangement, and the concentration of the primer flanking the corresponding rearrangement is adjusted to eliminate amplification biases, including over- and underamplification.

20. The method of any one of claims 1-19, wherein the overamplification ratio (OAR) for at least one rearrangement is used to computationally correct for amplification biases, including over- and underamplification.

21. The method according to any one of claims 1-20, in which the ratio of weighted frequencies (proportion among all reads) to unweighted frequencies (proportion among all clonotypes) for V-, D- and J-genes is calculated.

22. The method according to claim 21, wherein a significant deviation of this ratio from unity indicates the presence of quantitative imbalances that arose at the amplification stage.

23. The method according to claim 22, wherein the concentrations of primers in the multiplex mixture are selected in such a way as to minimize the deviation of the ratio of weighted to unweighted frequencies from the expected value of 1.

24. The method according to any one of claims 1-23, wherein the sensitivity of detection of MRD is determined solely by the available amount of input DNA and the sequencing depth.

25. The method according to any of the preceding claims, wherein the junctions of the coding and signaling regions of the TRα / β / γ / δ, IgH / K / λ, DJ, DD, VD rearrangements and the kappa deletion element (KDE) are detected in a single multiplex oligonucleotide mixture.