Methods for cancer detection and monitoring
The invention provides methods for detecting single nucleotide variants in breast cancer, bladder cancer, or colorectal cancer. Additional methods and compositions, such as reaction mixtures and solid supports comprising clonal populations of nucleic acids, are provided. For example, provided here is a method for monitoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising generating a set of amplicons by performing a multiplex amplification reaction on nucleic acids isolated from a sample of blood or urine or a fraction thereof from a patient who has been treated for a breast cancer, bladder cancer, or colorectal cancer, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, wherein detection of one or more patient-specific single nucleotide variants is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.
1. A method for preparing a composition of amplified DNA from a sample of a patient who has been diagnosed with a breast cancer useful for identifying single-nucleotide variant loci indicative of minimal residual disease (MRD), comprising
(a) longitudinally collecting one or more plasma samples from the patient after the patient has been treated with chemotherapy, radiation therapy, immunotherapy, stem cell transplantation, bone marrow transplantation, photodynamic therapy, palliative treatment, surgery, neoadjuvant therapy, and/or adjuvant therapy and extracting cell-free DNA from the plasma sample or a fraction thereof;
(b) preparing a composition of amplified DNA by performing a multiplex amplification reaction on cell-free DNA extracted in (a) or DNA derived therefrom to obtain a set of amplicons, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of at least 16 patient-specific single nucleotide variant loci associated with the cancer, and wherein the set of at least 16 patient-specific single nucleotide variant loci each encompasses a somatic mutation identified in a tumor sample of the patient who has been diagnosed with the cancer; and
(c) analyzing the composition of amplified DNA obtained in (b) by performing high-throughput sequencing of at least some of the amplicons with a median depth of read of at least 100,000 per patient-specific single nucleotide variant locus to determine the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, and identifying at least two or more patient-specific single nucleotide variants above a confidence threshold of 0.97, wherein identification of two or more patient-specific single nucleotide variants above a confidence threshold of 0.97 from the sample is indicative of MRD with a specificity of at least 99.5% in identifying MRD of the breast cancer, wherein the breast cancer comprises HER2+, triple negative, or HR+/HER2− breast cancer.
2. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in at least 85% of patients having MRD.
3. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in at least 95% of patients having MRD of HER2+ breast cancer.
4. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in at least 95% of patients having MRD of triple negative breast cancer.
5. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in at least 80% of patients having MRD of HR+/HER2− breast cancer.
6. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in patients having MRD at least 200 days prior to clinical relapse or metastasis of cancer identifiable by imaging and/or at least at least 100 days prior to elevation of CA15-3 level.
7. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in patients having MRD of HER2+ breast cancer at least 150 days prior to clinical relapse or metastasis of HER2+ breast cancer identifiable by imaging and/or at least at least 100 days prior to elevation of CA15-3 level.
8. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in patients having MRD of triple negative breast cancer at least 200 days prior to clinical relapse or metastasis of triple negative breast cancer identifiable by imaging and/or at least at least 100 days prior to elevation of CA15-3 level.
9. The method of claim 1 , wherein the method identifies patient-specific single nucleotide variants in patients having MRD of HR+/HER2− breast cancer at least 250 days prior to clinical relapse or metastasis of HR+/HER2-breast cancer identifiable by imaging and/or at least at least 100 days prior to elevation of CA15-3 level.
10. The method of claim 1 , wherein the method does not identify patient-specific single nucleotide variants in at least 99% of patients lacking MRD.
11. The method of claim 1 , wherein the method does not identify patient-specific single nucleotide variants in at least 99% of patients lacking MRD of HER2+ breast cancer.
12. The method of claim 1 , wherein the method does not identify patient-specific single nucleotide variants in at least 99% of patients lacking MRD of triple negative breast cancer.
13. The method of claim 1 , wherein the method does not identify patient-specific single nucleotide variants in at least 99% of patients lacking MRD of HR+/HER2− breast cancer.
14. The method of claim 1 , wherein the method has a specificity of at least 99.5% in identifying MRD of HER2+ breast cancer when two or more patient-specific single nucleotide variants are identified above a confidence threshold of 0.97.
15. The method of claim 1 , wherein the method has a specificity of at least 99.5% in identifying MRD of triple negative breast cancer when two or more patient-specific single nucleotide variants are identified above a confidence threshold of 0.97.
16. The method of claim 1 , wherein the method has a specificity of at least 99.5% in identifying MRD of HR+/HER2− breast cancer when two or more patient-specific single nucleotide variants are identified above a confidence threshold of 0.97.
17. The method of claim 1 , wherein the method comprises selecting 16 to 50 patient-specific single nucleotide variant loci each encompassing a clonal mutation identified in the tumor sample and generating a set of amplicons each spanning at least one single nucleotide variant locus of the 16 to 50 patient-specific single nucleotide variant loci.
18. The method of claim 1 , wherein the median depth of read is at least 500,000 per patient-specific single nucleotide variant locus.
19. The method of claim 1 , wherein the set of at least 16 patient-specific single nucleotide variant loci associated with the cancer are identified by performing whole exome sequencing of a sample obtained from a primary tumor of the patient.