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EP-4737593-A2 - METHODS FOR CANCER DETECTION AND MONITORING BY MEANS OF PERSONALIZED DETECTION OF CIRCULATING TUMOR DNA

EP4737593A2EP 4737593 A2EP4737593 A2EP 4737593A2EP-4737593-A2

Abstract

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.

Inventors

  • ZIMMERMANN, BERNHARD
  • SALARI, Raheleh
  • SWENERTON, RYAN
  • WU, HSIN-TA
  • SETHI, Himanshu

Assignees

  • Natera, Inc.

Dates

Publication Date
20260506
Application Date
20190412

Claims (15)

  1. A method for amplifying and sequencing DNA in a plasma sample from a subject who has been diagnosed with bladder cancer or colorectal cancer, comprising: (a) sequencing cellular DNA extracted from a tumor biopsy sample of the subject to identify at least 16 cancer mutations; (b) collecting a blood sample from the subject after the subject has been treated with surgery or neoadjuvant therapy; (c) extracting cell-free DNA (cfDNA) from a plasma fraction of the blood sample; (d) performing a targeted multiplex amplification reaction on the extracted cfDNA or DNA derived therefrom with at least 16 different primer pairs to generate amplicons, wherein the primer pairs are each designed to amplify a subject-specific single nucleotide variant locus that encompasses at least one of the cancer mutations identified in the tumor biopsy sample of the subject; and (e) performing high-throughput sequencing on the amplicons to generate sequence reads, wherein the subject-specific single nucleotide variant loci in the amplicons are each sequenced with a depth of read of at least 100,000, and identifying at least two of the cancer mutations in the sequence reads.
  2. The method of claim 1, wherein the bladder cancer is stage 1, lb, or 2a bladder cancer, muscle-invasive bladder cancer or locally advanced muscle-invasive bladder cancer.
  3. The method of claim 1, wherein the colorectal cancer is stage I, stage II, Stage III or stage IV colorectal cancer.
  4. The method of any of claims 1-3, wherein the subject has been treated with surgery, first-line chemotherapy, and/or adjuvant therapy before collecting the blood sample.
  5. The method of any of claims 1-4, wherein step (a) comprises sequencing the cellular DNA extracted from the tumor biopsy sample to identify 50-2,000 different cancer mutations.
  6. The method of any of claims 1-5, wherein step (a) comprises sequencing the cellular DNA extracted from the tumor biopsy sample to identify 100-1,000 different cancer mutations.
  7. The method of any of claims 1-6, wherein step (a) comprises performing whole-exome sequencing on the cellular DNA extracted from the tumor biopsy sample.
  8. The method of any of claims 1-7, wherein step (d) comprises performing the targeted multiplex amplification reaction with 50-2,000 different primer pairs to generate amplicons, wherein the primer pairs are each designed to amplify a subject-specific single nucleotide variant locus that encompasses at least one of the cancer mutations identified in the tumor biopsy sample.
  9. The method of any of claims 1-8, wherein step (d) comprises performing the targeted multiplex amplification reaction with 100-1,000 different primer pairs to generate amplicons, wherein the primer pairs are each designed to amplify a subject-specific single nucleotide variant locus that encompasses at least one of the cancer mutations identified in the tumor biopsy sample.
  10. The method of any of claims 1-9, wherein step (d) comprises identifying at least two of the cancer mutations in the sequence reads with a confidence threshold of at least 90%, preferably wherein the confidence threshold is at least 95%.
  11. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer with a sensitivity of greater than 95% at 0.01% tumor fraction in the sample.
  12. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject diagnosed with bladder cancer, with a mean lead time of at least 128 days prior to imaging-based clinical relapse or metastasis.
  13. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject diagnosed with bladder cancer, with a lead time of 50-265 days prior to imaging-based clinical relapse or metastasis.
  14. The method claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject diagnosed with colorectal cancer, with a lead time of at least 9.13 months prior to imaging-based clinical relapse or metastasis.
  15. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject with colorectal cancer, with a lead time of at least 10.2 months prior to imaging-based clinical relapse or metastasis.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62/657,727 filed April 14, 2018; U.S. Provisional Application No. 62/669,330 filed May 9, 2018; U.S. Provisional Application No. 62/693,843 filed July 3, 2018; U.S. Provisional Application No. 62/715,143 filed August 6, 2018; U.S. Provisional Application No. 62/746,210 filed October 16, 2018; U.S. Provisional Application No. 62/777,973 filed December 11, 2018; and U.S. Provisional Application No. 62/804,566 filed February 12, 2019. Each of these applications cited above is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Detection of early relapse or metastasis of cancers has traditionally relied on imaging and tissue biopsy. The biopsy of tumor tissue is invasive and carries risk of potentially contributing to metastasis or surgical complications, while imaging-based detection is not sufficiently sensitive to detect relapse or metastasis in an early stage. Better and less invasive methods are needed for detecting relapse or metastasis of cancers. SUMMARY OF THE INVENTION One aspect of the invention described herein relates to a method for monitoring and detection of early relapse or metastasis of cancer (e.g., 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 cancer (e.g., 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 cancer (e.g., 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 (or two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more) patient-specific single nucleotide variants is indicative of early relapse or metastasis of cancer (e.g., breast cancer, bladder cancer, or colorectal cancer). In addition to breast cancer, bladder cancer, and colorectal cancer, the methods described herein can also be used for monitoring and detection of early relapse or metastasis of other types of cancer, such as: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant po