Monitoring health and disease status using clonotype profiles
There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer, especially lymphoid neoplasms, such as lymphomas and leukemias. Provided herein are methods for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions. Identified biomarkers can be used to determine and/or monitor the disease state for a subject with an associated lymphoid disorder or autoimmune disease or other condition. In particular, the invention provides a sensitive method for monitoring lymphoid neoplasms that undergo clonal evolutions without the need to development alternative assays for the evolved or mutated clones serving as patient-specific biomarkers.
1. A method for quantifying a relative representation of tumor infiltrating T cells or B cells in a solid tissue tumor sample, comprising:
obtaining DNA templates from said solid tissue tumor sample;
amplifying rearranged T cell receptor or Ig CDR3-encoding region DNA molecules utilizing a plurality of V-segment oligonucleotide primers and a plurality of J-segment oligonucleotide primers in a single multiplex PCR from said DNA templates to produce a multiplicity of amplified rearranged DNA molecules, whose relative amounts are substantially the same as those in the DNA template population;
sequencing each of said multiplicity of amplified rearranged DNA molecules by high-throughput sequencing (HTS) to produce rearranged T cell receptor or Ig CDR3-encoding region sequence reads, wherein said sequencing is by (a) obtaining a nucleic acid sample from T cells or B cells of an individual; (b) spatially isolating individual molecules derived from said nucleic acid sample, the individual molecules each comprising nested sets of templates each generated from a nucleic acid in the sample and each containing a somatically rearranged region or a portion thereof, each nested set being capable of producing a plurality of sequence reads each extending in the same direction and each starting from a different position of the nucleic acid from which the nested set was generated; and (c) sequencing said spatially isolated individual molecules;
determining a number of rearranged T cell receptor or Ig CDR3-encoding region DNA molecules from said rearranged T cell receptor or Ig CDR3-encoding region sequence reads, wherein said number of T cell receptor or Ig CDR3-encoding region DNA molecules is proportional to a number of T cells or B cells in said sample;
determining a number of diploid genomes in the sample, wherein said number of diploid genomes represents a number of total cells in the sample, wherein said number of diploid genomes in said sample is determined by contacting said sample with a pair of control sequence primers and by amplifying a control sequence from said DNA templates, wherein said control sequence primers are capable of amplifying a control sequence present in all cells in said sample;
quantifying a ratio of the relative representation of tumor infiltrating T cells or B cells in said sample by comparing said number of T cells or B cells by said number of total cells in the sample; and
further comprising quantifying a number of unique sequence reads generated from said HTS, wherein each unique sequence read comprises a sequence distinct from the other sequence reads.
2. The method of claim 1 , wherein each V-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR V-encoding gene segment, and wherein said V-segment oligonucleotide primers specifically hybridize to at least 100,000 functional TCR V-encoding gene segments that are present in said sample.
3. The method of claim 1 , wherein each J-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR J-encoding gene segment, and wherein said J-segment oligonucleotide primers specifically hybridize to at least 100,000 functional TCR J-encoding gene segments that are present in said test sample.
4. The method of claim 1 , wherein each of said rearranged TCR encodes a T cell receptor (TCR) V-region polypeptide comprising a V gene recombination signal sequence (RSS) and a T cell receptor (TCR) J-region polypeptide comprising a J gene RSS, and wherein each rearranged DNA molecule comprises (i) at least 30, 40, 50 or 60 contiguous nucleotides of a sense strand of a TCR V-encoding gene segment, said at least 30, 40, 50 or 60 contiguous nucleotides being situated 5′ to said V gene RSS and (ii) at least 30, 40, 50 or 60 contiguous nucleotides of a sense strand of a TCR J-encoding gene segment, said at least 30, 40, 50 or 60 contiguous nucleotides being situated 3′ to said J gene RSS.
5. The method of claim 1 , wherein said comparing comprises dividing said number of T cells by a numerical factor.
6. The method of claim 1 , wherein said rearranged DNA molecules are selected from the group consisting of rearranged TCRαCDR3-encoding regions, TCRβ CDR3-encoding regions, TCRγCDR3-encoding regions, and TCRδ CDR3-encoding regions.