DETECTING DISEASE-CORRELATED CLONOTYPES FROM FIXED SAMPLES
The invention is directed to a method for determining immunophenotypes of tissue-infiltrating lymphocytes in a solid tissue of a patient by (a) generating clonotype profiles from a sample of nucleic acid extracted from a fixed tissue sample from a solid tissue of the patient, where such tissue contains tissue-infiltrating lymphocytes; and (b) determining immunophenotypes of the tissue-infiltrating lymphocytes by (i) obtaining a sample of lymphocytes from peripheral blood of the patient; (ii) sorting the lymphocytes from peripheral blood into at least one subset based on different immunophenotypes of the lymphocytes; (iii) generating a clonotype profile for each of the at least one subset of lymphocytes; and (iv) determining immunophenotypes of lymphocytes in the fixed tissue sample by a correspondence between clonotypes of the fixed tissue sample and clonotypes of the at least one subset.
1 - 20 . (canceled)
21 . A method for determining immunophenotypes of tissue-infiltrating lymphocytes in a solid tissue of a patient, the method comprising the steps of:
(a) generating one or more clonotype profiles from a sample of nucleic acid extracted from a fixed tissue sample from a solid tissue of the patient, the fixed tissue sample comprising tissue-infiltrating lymphocytes and the clonotype profiles each comprising recombined DNA sequences from T-cell receptor genes or immunoglobulin genes;
(b) determining immunophenotypes of the tissue-infiltrating lymphocytes by
(i) obtaining a sample of lymphocytes from peripheral blood of the patient;
(ii) sorting the lymphocytes from peripheral blood into at least one subset based on different immunophenotypes of the lymphocytes;
(iii) generating a clonotype profile for each of the at least one subset of lymphocytes; and
(iv) determining immunophenotypes of lymphocytes in the fixed tissue sample by a correspondence between clonotypes of the fixed tissue sample and clonotypes of the at least one subset;
wherein the clonotype profiles are each generated from at least 1000 sequence reads each of at least 30 bp.
22 . The method of claim 21 wherein said solid tissue is a solid tumor.
23 . The method of claim 21 wherein said sample of said extracted nucleic acids is in an amount in the range of from 10 to 500 ng.
24 . The method of claim 21 wherein said sample of said extracted nucleic acids is in an amount in the range of from 1 to 50 μg.
25 . The method of claim 21 wherein each of said clonotype profiles includes every clonotype present at a frequency of 0.01 percent or greater with a probability of ninety-nine percent.
26 . The method of claim 21 wherein said recombined DNA sequences comprise a genomic rearrangement selected from the group consisting of a VDJ rearrangement of IgH, a DJ rearrangement of IgH, a VJ rearrangement of IgK, a VJ rearrangement of IgL, a VDJ rearrangement of TCR β, a DJ rearrangement of TCR β, a VJ rearrangement of TCR α, a VJ rearrangement of TCR γ, a VDJ rearrangement of TCR δ, and a VD rearrangement of TCR δ.
27 . The method of claim 21 wherein said steps of generating said clonotype profiles include amplifying said nucleic acid from said sample to form an amplicon and sequencing nucleic acids of the amplicon.
28 . The method of claim 21 wherein said subsets of said lymphocytes includes CD4 + T cells and CD8 + T cells.
29 . The method of claim 21 wherein said fixed tissue is bone marrow or a lymphoid tissue.