Quantification of adaptive immune cell genomes in a complex mixture of cells
First Claim
1. A method for assessing the prognosis of a subject receiving therapeutic treatment for a solid tumor based on the relative quantity of and/or degree of clonality of tumor infiltrating lymphocytes in said solid tumor comprising:
- a) obtaining a first sample from a solid tumor from a subject at a time point prior to therapeutic treatment and a second sample from the solid tumor from a subject at a time point after to therapeutic treatment;
b) extracting DNA from said first and second samples;
c) amplifying in a multiplex PCR reaction substantially all rearranged TCR or Ig CDR3 encoding regions present in said sample samples using a plurality of V segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOS;
1-52, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 843-879 and a plurality of said J segment oligonucleotide primers comprising sequences selected from the group consisting of 53-63, 65, 215-220, 247, 638-639, 696-697 and 699-701 to produce a plurality of rearranged DNA amplicons;
d) sequencing said plurality of rearranged DNA amplicons using high throughput single molecule sequencing to produce a plurality of sequence reads; and
,e) determining the relative quantity of and/or degree of clonality of said plurality of sequence reads in step (d) thereby determining the relative quantity of and/or degree of clonality of tumor infiltrating lymphocytes in the first sample and the second sample wherein the relative quantity number of and/or degree of clonality of tumor infiltrating lymphocytes is an assessment of prognosis of a subject receiving therapeutic treatment for a solid tumor.
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Abstract
Compositions and methods are described for highly sensitive quantification of the relative representation of DNA from adaptive immune cells (e.g., T and/or B lymphocytes) in DNA extracted from complex mixtures of cells that include cells which are not adaptive immune cells. Included are methods for determining the relative presence in a tumor of tumor infiltrating lymphocytes (TIL), the relative presence of lymphocytes infiltrating a somatic tissue that is the target of an autoimmune disease, and the relative presence of lymphocytes infiltrating a transplanted organ.
287 Citations
11 Claims
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1. A method for assessing the prognosis of a subject receiving therapeutic treatment for a solid tumor based on the relative quantity of and/or degree of clonality of tumor infiltrating lymphocytes in said solid tumor comprising:
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a) obtaining a first sample from a solid tumor from a subject at a time point prior to therapeutic treatment and a second sample from the solid tumor from a subject at a time point after to therapeutic treatment; b) extracting DNA from said first and second samples; c) amplifying in a multiplex PCR reaction substantially all rearranged TCR or Ig CDR3 encoding regions present in said sample samples using a plurality of V segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOS;
1-52, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 843-879 and a plurality of said J segment oligonucleotide primers comprising sequences selected from the group consisting of 53-63, 65, 215-220, 247, 638-639, 696-697 and 699-701 to produce a plurality of rearranged DNA amplicons;d) sequencing said plurality of rearranged DNA amplicons using high throughput single molecule sequencing to produce a plurality of sequence reads; and
,e) determining the relative quantity of and/or degree of clonality of said plurality of sequence reads in step (d) thereby determining the relative quantity of and/or degree of clonality of tumor infiltrating lymphocytes in the first sample and the second sample wherein the relative quantity number of and/or degree of clonality of tumor infiltrating lymphocytes is an assessment of prognosis of a subject receiving therapeutic treatment for a solid tumor. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification