Methods for detection and diagnosis of a lymphoid malignancy using high throughput sequencing

Patent metadata

Field	Value
Publication number	US-2015218656-A1
Application number	US-201514604633-A
Country	US
Kind code	A1
Filing date	Jan 23, 2015
Priority date	Feb 3, 2014
Publication date	Aug 6, 2015
Grant date	—

Abstract

Methods and compositions are provided for detection and diagnosis of a lymphoid malignancy using high throughput sequencing of rearranged T cell receptor DNA sequences.

First claim

1 - 15 . (canceled)

16 . A method for diagnosing a lymphoid malignancy in a human subject, comprising:
obtaining a genomic DNA sample from the human subject; generating a profile of rearranged T-cell receptor (TCR) complementarity determining region-3 (CDR3) sequences, the profile comprising a frequency of each unique TCR CDR3 rearranged sequence; identifying a T cell clone with the highest frequency of occurrence in a total number of nucleated cells in the sample; and determining whether the T cell clone with the highest frequency of occurrence has a frequency of occurrence that is above or below a predetermined threshold for malignancy, wherein a frequency of occurrence above the predetermined threshold indicates a lymphoid malignancy in the subject.

17 . The method of claim 16 , further comprising diagnosing the subject with a lymphoid malignancy.

18 . The method of claim 17 , wherein diagnosis is at an early stage.

19 . The method of claim 16 , further comprising detecting a lymphoid malignancy in a subject in whom a lymphoid malignancy was not identified using another detection method.

20 . The method of claim 16 , wherein the predetermined threshold for malignancy is a threshold of 1 in 1000 total nucleated cells.

21 . The method of claim 16 , wherein the predetermined threshold for malignancy is equal to or greater than a threshold of 1 in 1000 total nucleated cells.

22 . The method of claim 16 , further comprising determining that the most frequent T cell clone has a frequency of occurrence that is at least one standard deviation above or below the predetermined threshold.

23 . The method of claim 16 , further comprising determining that the most frequent T cell clone has a frequency of occurrence that is statistically significantly different at p<0.05 from the predetermined threshold.

24 . The method of claim 16 , further comprising determining that the most frequent T cell clone has a frequency of occurrence that is statistically significantly different at p<0.01 from the predetermined threshold.

25 . The method of claim 16 , further comprising determining that the most frequent T cell clone has a frequency of occurrence that is statistically significantly different at p<0.001 from the predetermined threshold.

26 . The method of claim 16 , wherein the sample is obtained from a blood sample.

27 . The method of claim 16 , wherein generating a profile of rearranged T-cell receptor (TCR) complementarity determining region-3 (CDR3) sequences comprises:
amplifying the rearranged T-cell receptor (TCR) complementarity determining region-3 (CDR3) sequences in a single multiplex PCR using a plurality of V-segment primers and a plurality of J-segment primers to produce a plurality of amplicons representing the diversity of TCR genes in the sample; and sequencing the plurality of amplicons to produce a plurality of sequence reads.

28 . The method of claim 27 , further comprising correcting for amplification bias in the plurality of V-segment primers and plurality of J-segment primers.

29 . A kit for diagnosing a lymphoid malignancy in a human subject, comprising:
compositions for amplifying genomic DNA obtained from a sample from the human subject in a single multiplex PCR; and instructions for amplification of the genomic DNA and high throughput sequencing and instructions for determining whether a top T cell clone in the sample has a frequency of occurrence that is above or below a predetermined threshold for malignancy, wherein a frequency of occurrence above the predetermined threshold is indicates a lymphoid malignancy in the subject.

30 . The method of claim 16 , wherein the lymphoid malignancy is selected from: acute T-cell lymphoblastic leukemia (T-ALL), acute B-cell lymphoblastic leukemia (B-ALL), multiple myeloma, plasmacytoma, macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), multiple myeloma, plasmacytoma, macroglobulinemia, Hodgkins lymphoma, non-Hodgkins lymphoma, cutaneous T-cell lymphoma (CTCL), mantle cell lymphoma, peripheral T-cell lymphoma, hairy cell leukemia, T prolymphocytic lymphoma, angioimmunoblastic T-cell lymphoma, T lymphoblastic leukemia/lymphoma, peripheral T-cell lymphoma, adult T cell leukemia/lymphoma, mycosis fungoides, Sezary syndrome, T lymphoblastic leukemia, myeloproliferative neoplasm, and myelodysplastic syndrome.

31 - 84 . (canceled)

85 . The method of claim 30 , wherein the lymphoid malignancy is CTCL.

86 . The method of claim 16 , wherein the predetermined threshold is determined by:
determining a T cell clone with the highest frequency of occurrence in one or more samples from subjects with a non-malignant condition; determining the T cell clone with the highest frequency in one or more samples obtained from subjects previously diagnosed with a lymphoid malignancy; comparing the frequencies of occurrence of T cell clones with the highest frequency from subjects with a non-malignant condition with frequencies of occurrence of T cells clones from subjects previously diagnosed with a lymphoid malignancy and determining a threshold for malignancy based on the comparison.

87 . The method of claim 86 wherein determining the T cell clone with the highest frequency of occurrence comprises determining the frequency of the most frequent T cell clone a a fraction of a total number of nucleated cells in the sample.

88 . The method of claim 86 wherein determining the T cell clone with the highest frequency of occurrence in the sample comprises determining a percentage.

Assignees

• Adaptive Biotechnologies Corp
• Brigham & Womens Hospital

Inventors

• Kirsch Ilan
• Robins Harlan
• Clark Rachael
• Kupper Thomas

Classifications

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title