MONITORING CLONOTYPES OF PLASMA CELL PROLIFERATIVE DISORDERS IN PERIPHERAL BLOOD
The invention is directed to sequencing-based methods for monitoring a minimal residual disease of a plasma cell proliferative disorder, such as multiple myeloma and/or MGUS, by one or more clonotypes correlated with the disorder. In some embodiments, such methods comprise the following steps: (a) obtaining a sample of peripheral blood from the patient; (b) amplifying molecules of nucleic acid from the sample, the molecules of nucleic acid comprising recombined DNA sequences from immunoglobulin genes; (c) sequencing the amplified molecules of nucleic acid to form a clonotype profile; and (d) determining from the clonotype profile a presence, absence and/or level of one or more patient-specific clonotypes correlated with the plasma cell proliferative disorder and phylogenic clonotypes thereof.
1 . A method of monitoring a multiple myeloma residual disease in a patient, the method comprising the steps of:
(a) obtaining a sample of peripheral blood from the patient;
(b) amplifying molecules of nucleic acid from the sample, the molecules of nucleic acid comprising recombined DNA sequences from immunoglobulin genes;
(c) sequencing the amplified molecules of nucleic acid to form a clonotype profile; and
(d) determining from the clonotype profile a presence, absence and/or level of one or more patient-specific clonotypes correlated with the multiple myeloma and phylogenic clonotypes thereof.
2 . The method of claim 1 further including the step of repeating said steps (a) through (d) to monitor said multiple myeloma residual disease in the patient.
3 . The method of claim 1 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.
4 . The method of claim 1 wherein each of said clonotype profiles includes at least 10 4 clonotypes.
5 . The method of claim 1 wherein said one or more patient-specific clonotypes correlated with said multiple myeloma are determined from a bone marrow sample of the patient.
6 . The method of claim 5 wherein said one or more patient-specific clonotypes correlated with said multiple myeloma are determined by the steps of:
(a) obtaining a sample of bone marrow from said patient;
(b) amplifying molecules of nucleic acid from the sample of bone marrow, the molecules of nucleic acid comprising recombined DNA sequences from immunoglobulin genes;
(c) sequencing the amplified molecules of nucleic acid to form a clonotype profile; and
(d) associating said one or more patient-specific clonotypes correlated with said multiple myeloma with clonotypes of the clonotype profile having the highest frequencies.
7 . The method of claim 1 wherein said step of determining includes measuring said level of said one or more patient-specific clonotypes correlated with said multiple myeloma with a sensitivity of at least 10 −5 .
8 . The method of claim 7 wherein said step of determining includes measuring said level of said one or more patient-specific clonotypes correlated with said multiple myeloma with a sensitivity of at least 10 −6 .
9 . The method of claim 1 wherein said step of obtaining further includes depleting said sample of granulocytes.
10 . A method of monitoring a multiple myeloma in a patient, the method comprising the steps of:
(a) obtaining a peripheral blood sample from the patient comprising B-cells, plasma cells and/or cell-free nucleic acids from B-cells and/or plasma cells;
(b) extracting a nucleic acid sample from the peripheral blood sample;
(c) amplifying in a polymerase chain reaction molecules of nucleic acid from nucleic acid sample, the molecules of nucleic acid comprising a VDJ rearrangement of IgH;
(c) sequencing the amplified molecules of nucleic acid to form a clonotype profile; and
(d) determining from the clonotype profile a level of each of one or more patient-specific clonotypes correlated with the multiple myeloma, wherein such levels include phylogenic clonotypes of each of such one or more patient-specific clonotypes.
11 . The method of claim 10 wherein said step of sequencing includes generating sequence reads in a range of from 20 to 400 nucleotides for determining a sequence of each clonotype.
12 . The method of claim 10 further including a step of treating said patient by transplanting bone marrow based on said level of said one or more patient-specific clonotypes.
13 . A method of monitoring a multiple myeloma residual disease in a patient, the method comprising the steps of:
(a) obtaining a sample of peripheral blood from the patient;
(b) removing granulocytes from the sample to form a depleted sample;
(c) amplifying molecules of nucleic acid from the depleted sample, the molecules of nucleic acid comprising recombined DNA sequences from immunoglobulin genes;
(d) sequencing the amplified molecules of nucleic acid to form a clonotype profile; and
(e) determining from the clonotype profile a presence, absence and/or level of one or more patient-specific clonotypes correlated with the multiple myeloma and phylogenic clonotypes thereof.
14 . A method of monitoring a plasma cell proliferative disorder, the method comprising the steps of:
obtaining a sample of peripheral blood of an individual, the sample comprising recombined sequences each including at least a portion of a C gene segment of a B cell receptor;
generating an amplicon from the recombined sequences, each sequence of the amplicon including a portion of a C gene segment;
sequencing the amplicon to generate a clonotype profile of clonotypes that each comprise at least a portion of a VDJ region of a B cell receptor and at least a portion of a C gene segment; and
determining from the clonotype profile a presence, absence and/or level of one or more patient-specific clonotypes and their respective isotypes correlated with the plasma cell proliferative disorder and phylogenic clonotypes thereof.
15 . The method of claim 14 wherein said C gene segment is from a nucleotide sequence encoding an IgH chain of said B cell receptor.