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WO2025096532A1 - Surveillance d'adn tumoral circulant (ctadn) - Google Patents

Surveillance d'adn tumoral circulant (ctadn) Download PDF

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Publication number
WO2025096532A1
WO2025096532A1 PCT/US2024/053553 US2024053553W WO2025096532A1 WO 2025096532 A1 WO2025096532 A1 WO 2025096532A1 US 2024053553 W US2024053553 W US 2024053553W WO 2025096532 A1 WO2025096532 A1 WO 2025096532A1
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ctdna
subject
cancer
cfdna
sample
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Jacob J. CHABON
David M. KURTZ
Maximilian Diehn
Arash Ash Alizadeh
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Foresight Diagnostics Inc
Leland Stanford Junior University
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Foresight Diagnostics Inc
Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • CTDNA MONITORING CIRCULATING TUMOR DNA
  • the present disclosure relates in some aspects to methods for assessing the therapeutic response and the risk of relapse or recurrence in a subject that has been treated with cancer, by studying the levels of circulating tumor DNA (ctDNA).
  • the present disclosure also relates to methods of selecting a treatment for a subject by studying the levels of circulating tumor DNA (ctDNA).
  • Provided herein are methods for treatment, monitoring, and surveillance of a subject by studying the levels of ctDNA.
  • ctDNA Circulating tumor DNA
  • MRD minimal residual disease
  • Analysis of ctDNA has the potential to transform the detection and monitoring of tumors, as well as the detection of relapse and acquired drug resistance at an early stage for selection of treatments for tumors through the evaluation of tumor DNA without invasive tissue biopsy procedures.
  • the present invention addresses the need for methods which promote regular and more frequent monitoring and surveillance of disease prior and after treatment, as well as improvement of treatment regimens.
  • a method for determining the Minimal Residual Disease (MRD) status in a subject that has been diagnosed with cancer comprising:(a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA:cfDNA molecules in the sample; (d) determining the MRD status of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject has MRD if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject does not have MRD if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • MRD Minimal Residual Disease
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the ratio is below 1 : 10 6 ctDNA: cfDNA molecules and the subject is not responsive to the treatment if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the ratio is below a detection threshold ratio of ctDNA: cfDNA molecules and the subject is not responsive to the treatment if the ratio is above a detection threshold ratio of ctDNA: cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the levels of ctDNA are below a detection threshold and the subject is not responsive to the treatment if the levels of ctDNA are above a detection threshold.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA:cfDNA molecules in the sample; (d) assessing the risk of cancer recurrence or relapse in the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is at risk of cancer recurrence or relapse if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject is not at risk of cancer recurrence or relapse
  • a method for treating a subject with a second treatment for cancer wherein the subject has been administered a first treatment for cancer, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; and (d) administering a second treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of treating cancer in a subject in need thereof comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; and (d) administering an effective amount of a treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of treating cancer in a subject in need thereof, wherein the subject is assessed as a candidate for treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA:cfDNA molecules in the sample; and (d) identifying the subject as a candidate for treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules, and identifying the subject as a non-candidate for treatment for cancer if the ratio of ctDNA: cfDNA is below 1 : 10 6 ctDNA:cfDNA molecules; and (e) administer
  • a medicament for use in a method of treating cancer in a subject in need thereof comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA:cfDNA molecules in the sample; and (d) administering an effective amount of the medicament to the subject if the ratio of ctDNA: cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a cell therapy for the manufacture of a medicament for treating cancer in a subject in need thereof, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; and (d) administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer wherein after administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ratio of ctDNA: cfDNA molecules in the sample is determined, wherein the subject is responsive to the treatment to cancer if the ratio of ctDNA:cfDNA is below 1 : 10 6 ctDNA:cfDNA molecules.
  • a method for assessing the risk of cancer recurrence or relapse of a subject that has been administered a treatment for cancer wherein after administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ratio of ctDNA: cfDNA molecules in the sample is determined, wherein the subject is at risk of cancer recurrence or relapse if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a treatment for cancer, wherein prior to administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ct DNA: cf NA molecules in the sample is determined, wherein the subject is identified as a candidate for treatment of cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of treating cancer in a subject with higher-risk disease comprising administering to the subject an effective amount of a treatment for cancer, wherein prior to administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ctDNA: cfDNA molecules in the sample is determined, wherein the subject is identified as having high-risk disease if the ratio of ctDNA: cfDNA is above 1 : 10 6 ctDNA: cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • the cancer is a blood cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is large B-cell lymphoma (LBCL) or a Diffuse Large B-Cell Lymphoma (DLBCL). In some embodiments, the cancer is follicular lymphoma (FL).
  • LBCL large B-cell lymphoma
  • DLBCL Diffuse Large B-Cell Lymphoma
  • FL follicular lymphoma
  • the cancer is selected from acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), follicular lymphoma (FL), refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancy is selected from among acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CML), acute lymphoc
  • the cancer is a pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancers, CNS cancers, brain tumors, bone cancer, or soft tissue sarcoma.
  • the treatment or the medicament is a first-line therapy. In other embodiments, the treatment or the medicament is a second-line therapy. In some embodiments, the treatment comprises further monitoring the subject. In other embodiments, the treatment comprises radiographic imaging of the subject. In some embodiments, the treatment comprises computed tomography (CT) imaging, positron emission tomography (PET) imaging, and/or magnetic resonance imaging (MRI) of the subject.
  • CT computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the treatment comprises a cell therapy.
  • the treatment comprises a CAR-T cell therapy.
  • the CAR-T cell therapy is an anti-CD19 cell therapy.
  • the cell therapy comprises genetically engineered cells.
  • the genetically engineered cells are T cells.
  • the genetically engineered T cells comprise a Chimeric Antigen Receptor (CAR).
  • CAR Chimeric Antigen Receptor
  • the CAR specifically binds to an antigen associated with a disease or condition and/or is expressed by cells associated with a disease or condition.
  • the CAR specifically binds to two antigens associated with a disease or condition and/or is expressed by cells associated with a disease or condition.
  • the antigen is selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, a cancer-testes antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD 19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, carcino embryonic antigen (CEA), CE7, a cyclin, cyclin A2, c-Met, dual antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrinB2, erb- B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, estrogen receptor, Fetal AchR, folate
  • the antigen is CD 19. In other embodiments, the antigen is CD20.
  • the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an IT AM.
  • the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3Q chain.
  • the CAR further comprises a costimulatory signaling region.
  • the costimulatory signaling region comprises a signaling domain of CD28 or 4- IBB. In some embodiments, the costimulatory domain is a domain of 4- 1BB. In some embodiments, the T cells are CD4+ or CD8+.
  • the T cells are primary T cells obtained from a subject.
  • the genetically engineered cells are autologous to the subject.
  • the genetically engineered cells are allogeneic to the subject.
  • the subject is a human.
  • the subject has Stage I/II disease. In some embodiments, the subject has Stage III/IV disease. In some embodiments, the subject has Minimal Residual Disease (MRD).
  • MRD Minimal Residual Disease
  • the subject is refractory to treatment with one or more prior therapies for the cancer.
  • the subject achieved an insufficient response to one or more prior therapies for the cancer.
  • the subject achieves a durable response to the treatment.
  • the durable response is defined as an absence of relapse or remission of the cancer for up to 3 months, 6 months, or 12 months. In some embodiments, the subject has a higher rate of survival.
  • the subject’s disease baseline characteristics are determined.
  • the baseline characteristics comprise international prognosis index (IPI) score, serum lactate dehydrogenase (LDH), or sum of the product of diameters (SPD), disease stage, or any combination of the above.
  • the baseline characteristics are defined by Lugano 2014 criteria.
  • the subject is classified under an international prognosis index (IPI) score.
  • the subject is classified as low, low-intermediate, high-intermediate risk on the IPI score.
  • the subject has no risk factor or one risk factor and is considered to be in an IPI low risk group.
  • the subject has two risk factors and is considered to be in an IPI low- intermediate risk group.
  • the subject has three risk factors and is considered to be in an IPI high-intermediate risk group.
  • the subject has a higher or lower IPI score.
  • the volumetric measure of tumor burden of the subject is measured.
  • the volumetric measure of tumor burden in the subject is a sum of products of diameter (SPD).
  • the volumetric measure of tumor burden is measured using computed tomography (CT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI) of the subject.
  • CT computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the SPD threshold value is or is about 30 per cm 2 , is or is about 40 per cm 2 , is or is about 50 per cm 2 , is or is about 60 per cm 2 , or is or is about 70 per cm 2 .
  • the level of an inflammatory marker is measured in the subject. In some embodiments, the level of the inflammatory marker is or is about 300 units per liter, is or is about 400 units per liter, is or is about 500 units per liter or is or is about 600 units per liter. In some embodiments, the inflammatory marker is lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • the mutation is a point mutation, a deletion, or a frameshift mutation. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation is a Phased Variant (PV). In some embodiments, the mutation is a single nucleotide variant (SNV). In some embodiments, the mutation comprises SNV, indels, rearrangements, or a combination thereof.
  • the detection threshold ratio of ctDNA:cfDNA molecules is 1:10, 1:100, 1:1000, l:10 4 , l:10 5 , l:10 6 , l:10 7 , l:10 8 .
  • the presence of one or more mutations in one or more genes of the cfDNA is determined by genotyping the cfDNA. In some embodiments, the presence of one or more mutations in one or more genes of the cfDNA is determined by sequencing the cfDNA.
  • the sequencing comprises high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys), phased variant enrichment and detection sequencing (PhasED-Seq), CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), Duplex Sequencing (Duplex-Seq).
  • measuring the levels of ctDNA further comprises determining the absolute ctDNA concentration as mutant haploid genome equivalents per milliliter of plasma (hGE/mL).
  • measuring the levels of ctDNA further comprises determining the mean variant allele frequency (AF). In some embodiments, a mean variant allele frequency (AF) greater than a range that is indicative of a detectable ctDNA level.
  • the detection threshold is a percentage of ctDNA level at or below total cfDNA levels in the sample. In some embodiments, the detection threshold is a concentration of ctDNA level at or below total cfDNA levels in the sample. In some embodiments, the detection threshold is within 25%, within 20%, within 15%, within 11% or within 5% and/or is within a standard deviation above the median or mean level, amount or concentration of cfDNA in the biological sample obtained from the subject following administration of the treatment.
  • the ctDNA level is less than or equal to 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005%, or 0.00001% of the total cfDNA in the sample.
  • the detection further comprises determining the correlation of baseline ctDNA concentration (hGE/mL) with subject baseline characteristics.
  • the method detects levels of ctDNA at a sensitivity of at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% in the sample.
  • the method detects levels of ctDNA at a specificity of at least 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in the sample.
  • the genotyping assay comprises sequencing one or more genes to identify ctDNA.
  • the sample is obtained 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days following administration of the treatment for cancer.
  • the sample is obtained at Day 15 following administration of the treatment for cancer.
  • the sample is obtained at about 1, 2, 3, 4 ,5, 6, 7, 8, 9, 10, 11, or 12 months following administration of the treatment for cancer.
  • the sample is obtained at about 1, 2, 3, or 12 months following administration of the treatment for cancer.
  • the ctDNA is undetected at or at least at 2, 4, or 6 weeks following, or 3, 6, or 12, 18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5, or more years, following the administration of the treatment for cancer.
  • the sample comprises a fluid, cell, or tissue sample.
  • the sample is a blood, serum or a plasma sample.
  • FIG. 1 shows a comparison of the detection limit of ctDNA between CAPP-Seq and PhasED-Seq.
  • CAPP-Seq quantifies ctDNA by detecting single nucleotide variants (SNVs) and has an error rate of about 1 in 20,000 molecules.
  • PhasED-Seq quantifies ctDNA by detecting phased variants (PVs) and has an error rate of ⁇ 1 in 10,000,000 molecules ( ⁇ 0.00001%).
  • FIG. 2 shows genomic coordinates of phased variants in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL).
  • DLBCL diffuse large B cell lymphoma
  • FL follicular lymphoma
  • FIG. 3 shows correlation between ctDNA levels in follicular lymphoma patients and disease stage.
  • the y-axis shows the ctDNA levels as a tumor fraction; the x-axis indicates disease stage.
  • FIG. 4 shows the measured allelic frequency of ctDNA using either tumor (x-axis) or plasma (y-axis) genotyping of PVs. Prior to therapy, ctDNA was detectable in 94% of cases using tumor-derived PVs, and in 75% of cases using plasma-derived PVs.
  • FIG. 5 shows concordance of SNVs between tumor and plasma. The rate of shared mutations genotyped from tumor, plasma, or both compartments are shown. Genes with mutations in more than 10% of cases are shown. A higher rate of concordance is observed between tumor and plasma in mutations in genes such as TNFRSF14, EZH2, and CREBBP, than in other candidate driver mutations in FL such as KMT2D, CARD11, and ARID 1 A.
  • FIG. 6 shows the probability of progression free survival between patients with or without residual ctDNA after two cycles of therapy.
  • FIG. 7 shows ctDNA monitoring from three patients.
  • MRD was observed prior to clinical detection of relapse (vertical dotted line).
  • PAT-FL-024 MRD was detected 13 months prior to relapse with levels as low as 1:100,000; for PAT-FL-039, MRD was detected more than two years prior to relapse.
  • the y-axis indicates tumor fraction; the x-axis indicates the time from the pre-treatment sample in years; each dot indicates a sample timepoint.
  • Methods described herein evade the difficulty and expense of a tumor biopsy by instead operating with sequences from cell-free fragments of tumor DNA circulating in the bloodstream, known as circulating tumor DNA, or ctDNA.
  • Tumor biomarkers can be assessed non-invasively by examining ctDNA.
  • Patient biological samples can provide time-based measurements of the total tumor burden as well as identify specific mutations that arise during clinical therapy and interventions.
  • Diseased state i.e., cancer
  • sequence reads can be analyzed through genomic mapping to a genomic reference that includes normal, non-tumor genetic sequence from the patient.
  • a method for determining the Minimal Residual Disease (MRD) status in a subject that has been diagnosed with cancer comprising:(a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) determining the MRD status of the subject from the ratio of ctDNA:cfDNA molecules in the sample; wherein the subject has MRD if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject does not have MRD if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • MRD Minimal Residual Disease
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules and the subject is not responsive to the treatment if the ratio is above 1 : 10 6 ctDNA: cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the ratio is below a detection threshold ratio of ctDNA:cfDNA molecules and the subject is not responsive to the treatment if the ratio is above a detection threshold ratio of ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the levels of ctDNA are below a detection threshold and the subject is not responsive to the treatment if the levels of ctDNA are above a detection threshold.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the risk of cancer recurrence or relapse in the subject from the ratio of ctDNA:cfDNA molecules in the sample; wherein the subject is at risk of cancer recurrence or relapse if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject is not at risk of cancer recurrence or relapse
  • a method for treating a subject with a second treatment for cancer wherein the subject has been administered a first treatment for cancer, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA:cfDNA molecules in the sample; and (d) administering a second treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of treating cancer in a subject in need thereof comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA:cfDNA molecules in the sample; and (d) administering an effective amount of a treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of treating cancer in a subject in need thereof, wherein the subject is assessed as a candidate for treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; and (d) identifying the subject as a candidate for treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules, and identifying the subject as a non-candidate for treatment for cancer if the ratio of ctDNA: cfDNA is below 1 : 10 6 ctDNA:cfDNA molecules; and (e)
  • a method for assessing the likelihood of survival of a subject that has been administered a treatment for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is likely to survive if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of monitoring a subject being treated for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the therapeutic response of the subject from the ratio of ctDNA:cfDNA molecules in the sample; wherein the subject is responsive to the treatment if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules and the subject is not responsive to the treatment if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of assessing the likelihood of a durable response in a subject being treated for cancer comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) assessing the response of the subject from the ratio of ctDNA: cfDNA molecules in the sample; wherein the subject is likely to have a durable response to the treatment if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules and the subject is not likely to have a durable response to the treatment if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules.
  • a medicament for use in a method of treating cancer in a subject in need thereof comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; and (d) administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a cell therapy for the manufacture of a medicament for treating cancer in a subject in need thereof, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; and (d) administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the therapeutic response of a subject that has been administered a treatment for cancer wherein after administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ratio of ctDNA:cfDNA molecules in the sample is determined, wherein the subject is responsive to the treatment to cancer if the ratio of ctDNA:cfDNA is below 1 : 10 6 ctDNA:cfDNA molecules.
  • a method for assessing the risk of cancer recurrence or relapse of a subject that has been administered a treatment for cancer wherein after administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ratio of ctDNA:cfDNA molecules in the sample is determined, wherein the subject is at risk of cancer recurrence or relapse if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a treatment for cancer, wherein prior to administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ctDNA:cfDNA molecules in the sample is determined, wherein the subject is identified as a candidate for treatment of cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method of treating cancer in a subject with higher-risk disease comprising administering to the subject an effective amount of a treatment for cancer, wherein prior to administering the treatment for cancer, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ctDNA:cfDNA molecules in the sample is determined, wherein the subject is identified as having high-risk disease if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • a method for assessing the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer comprising (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) identifying cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more phased variant (PV)-containing cfDNA molecules; (c) determining a Minimal Residual Disease (MRD) status of the subject from the ctDNA in the sample; (d) assessing the risk of cancer recurrence or relapse in the subject from the MRD status of the subject; wherein the treatment comprises at least two cycles; and wherein the subject is at risk of cancer recurrence or relapse if MRD is detected after two cycles of the treatment.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • MRD Minimal Residual Disease
  • the cancer is a blood cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is large B-cell lymphoma (LBCL) or a Diffuse Large B-Cell Lymphoma (DLBCL). In some embodiments, the cancer is follicular lymphoma (FL).
  • LBCL large B-cell lymphoma
  • DLBCL Diffuse Large B-Cell Lymphoma
  • FL follicular lymphoma
  • the cancer is selected from acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), follicular lymphoma (FL), refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancy is selected from among acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CML), acute lymphoc
  • the cancer is a pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancers, CNS cancers, brain tumors, bone cancer, or soft tissue sarcoma.
  • the treatment or the medicament is a first-line therapy. In other embodiments, the treatment or the medicament is a second-line therapy. In some embodiments, the treatment comprises further monitoring the subject. In other embodiments, the treatment comprises radiographic imaging of the subject. In some embodiments, the treatment comprises computed tomography (CT) imaging, positron emission tomography (PET) imaging, and/or magnetic resonance imaging (MRI) of the subject.
  • CT computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the treatment comprises a cell therapy.
  • the treatment comprises a CAR-T cell therapy.
  • the CAR-T cell therapy is an anti-CD19 cell therapy.
  • the cell therapy comprises genetically engineered cells.
  • the genetically engineered cells are T cells.
  • the genetically engineered T cells comprise a Chimeric Antigen Receptor (CAR).
  • CAR Chimeric Antigen Receptor
  • the CAR specifically binds to an antigen associated with a disease or condition and/or is expressed by cells associated with a disease or condition.
  • the CAR specifically binds to two antigens associated with a disease or condition and/or is expressed by cells associated with a disease or condition.
  • the antigen is selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, a cancer-testes antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD 123, CD 138, CD171, carcinoembryonic antigen (CEA), CE7, a cyclin, cyclin A2, c-Met, dual antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrinB2, erb- B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, estrogen receptor, Fetal
  • the antigen is CD 19. In other embodiments, the antigen is CD20.
  • the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an IT AM.
  • the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3Q chain.
  • the CAR further comprises a costimulatory signaling region.
  • the costimulatory signaling region comprises a signaling domain of CD28 or 4-1BB. In some embodiments, the costimulatory domain is a domain of 4- 1BB. In some embodiments, the T cells are CD4+ or CD8+.
  • the T cells are primary T cells obtained from a subject.
  • the genetically engineered cells are autologous to the subject.
  • the genetically engineered cells are allogeneic to the subject.
  • the subject is a human.
  • the subject has Stage VII disease. In some embodiments, the subject has Stage III/IV disease. In some embodiments, the subject has Minimal Residual Disease (MRD).
  • MRD Minimal Residual Disease
  • the subject is refractory to treatment with one or more prior therapies for the cancer.
  • the subject achieved an insufficient response to one or more prior therapies for the cancer.
  • the subject achieves a durable response to the treatment.
  • the durable response is defined as an absence of relapse or remission of the cancer for up to 3 months, 6 months, or 12 months. In some embodiments, the subject has a higher rate of survival.
  • the subject is treatment-naive prior to administration of the treatment.
  • the subject’s disease baseline characteristics are determined.
  • the baseline characteristics comprise international prognosis index (IP I) score, serum lactate dehydrogenase (LDH), or sum of the product of diameters (SPD), disease stage, or any combination of the above.
  • IP I international prognosis index
  • LDH serum lactate dehydrogenase
  • SPD sum of the product of diameters
  • the baseline characteristics are defined by Lugano 2014 criteria.
  • the subject is classified under an international prognosis index (IP I) score.
  • the subject is classified as low, low-intermediate, high-intermediate risk on the IPI score.
  • the subject has no risk factor or one risk factor and is considered to be in an IPI low risk group.
  • the subject has two risk factors and is considered to be in an IPI low- intermediate risk group. In some embodiments, the subject has three risk factors and is considered to be in an IPI high-intermediate risk group. In some embodiments, the subject has a higher or lower IPI score.
  • the volumetric measure of tumor burden of the subject is measured.
  • the volumetric measure of tumor burden in the subject is a sum of products of diameter (SPD).
  • the volumetric measure of tumor burden is measured using computed tomography (CT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI) of the subject.
  • CT computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the SPD threshold value is or is about 30 per cm 2 , is or is about 40 per cm 2 , is or is about 50 per cm 2 , is or is about 60 per cm 2 , or is or is about 70 per cm 2 .
  • the level of an inflammatory marker is measured in the subject. In some embodiments, the level of the inflammatory marker is or is about 300 units per liter, is or is about 400 units per liter, is or is about 500 units per liter or is or is about 600 units per liter. In some embodiments, the inflammatory marker is lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • the mutation is a point mutation, a deletion, or a frameshift mutation. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation is a Phased Variant (PV). In some embodiments, the mutation is a single nucleotide variant (SNV). In some embodiments, the mutation comprises SNV, indels, rearrangements, or a combination thereof.
  • the detection threshold ratio of ctDNA:cfDNA molecules is 1 : 10, 1 : 100, 1 : 1000, L IO 4 , L IO 5 , L IO 6 , LIO 7 , L IO 8 .
  • the presence of one or more mutations in one or more genes of the cfDNA is determined by genotyping the cfDNA. In some embodiments, the presence of one or more mutations in one or more genes of the cfDNA is determined by sequencing the cfDNA.
  • the sequencing comprises high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys), phased variant enrichment and detection sequencing (PhasED-Seq), CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), Duplex Sequencing (Duplex-Seq).
  • measuring the levels of ctDNA further comprises determining the absolute ctDNA concentration as mutant haploid genome equivalents per milliliter of plasma (hGE/mL).
  • measuring the levels of ctDNA further comprises determining the mean variant allele frequency (AF). In some embodiments, a mean variant allele frequency (AF) greater than a range that is indicative of a detectable ctDNA level.
  • the detection threshold is a percentage of ctDNA level at or below total cfDNA levels in the sample. In some embodiments, the detection threshold is a concentration of ctDNA level at or below total cfDNA levels in the sample. In some embodiments, the detection threshold is within 25%, within 20%, within 15%, within 11% or within 5% and/or is within a standard deviation above the median or mean level, amount or concentration of cfDNA in the biological sample obtained from the subject following administration of the treatment.
  • the ctDNA level is less than or equal to 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005%, or 0.00001% of the total cfDNA in the sample.
  • the detection further comprises determining the correlation of baseline ctDNA concentration (hGE/mL) with subject baseline characteristics.
  • the method detects levels of ctDNA at a sensitivity of at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% in the sample.
  • the method detects levels of ctDNA at a specificity of at least 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in the sample.
  • the genotyping assay comprises sequencing one or more genes to identify ctDNA.
  • the sample is obtained 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days following administration of the treatment for cancer.
  • the sample is obtained at Day 15 following administration of the treatment for cancer.
  • the sample is obtained at about 1, 2, 3, 4 ,5, 6, 7, 8, 9, 10, 11, or 12 months following administration of the treatment for cancer.
  • the sample is obtained at about 1, 2, 3, or 12 months following administration of the treatment for cancer.
  • the ctDNA is undetected at or at least at 2, 4, or 6 weeks following, or 3, 6, or 12, 18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5, or more years, following the administration of the treatment for cancer.
  • the sample comprises a fluid, cell, or tissue sample.
  • the sample is a blood, serum or a plasma sample.
  • a method for assessing the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer comprising (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) identifying cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more phased variant (PV)-containing cfDNA molecules; (c) determining a Minimal Residual Disease (MRD) status of the subject from the ctDNA in the sample; (d) assessing the risk of cancer recurrence or relapse in the subject from the MRD status of the subject; wherein the treatment comprises at least two cycles; and wherein the subject is at risk of cancer recurrence or relapse if MRD is detected after two cycles of the treatment.
  • cfDNA cell-free DNA
  • ctDNA cell-free tumor DNA
  • MRD Minimal Residual Disease
  • the cancer is a blood cancer. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is follicular lymphoma.
  • the subject is a human. In some embodiments, the subject has Stage I/II disease. In some embodiments, the subject has Stage II/IV disease. In some embodiments, the subject was treatment-naive prior to administration of the treatment.
  • the treatment is a first-line therapy.
  • the treatment comprises Bendamustine/Rituximab or R-CHOP.
  • the induction phase comprises 6 cycles of Bendamustine/Rituximab or R-CHOP.
  • the sample is obtained on the first day of a treatment cycle.
  • the method further comprises obtaining multiple biological samples serially for at least 1 year, at least 2, years, or at least 3 years following administration of the treatment.
  • the sample is a blood sample, a serum sample, or a plasma sample.
  • the biological sample has a tumor fraction of less than 0.28%.
  • CTDNA CIRCULATING TUMOR DNA
  • ctDNA circulating tumor DNA
  • MRD Minimal Residual Disease
  • Circulating cell-free DNA refers to fragments of DNA that are no longer within a cell and are present in the circulatory system.
  • the term cfDNA can also be used to describe these DNA fragments following extraction and subsequent processing.
  • cfDNA can be released from a cell as a result of various processes, including both normal and abnormal apoptotic events, cellular excretions, necrosis, and the like.
  • Specific forms of cfDNA can be present in the circulatory system as a result of various medical conditions, disease states, pregnancy, and the like.
  • the accessibility of these genomic fragments in the circulatory system through a simple blood sample provides a low-risk opportunity to screen for various phenotypes, conditions, and the like.
  • cfDNA testing is an emerging diagnostic approach that allows for noninvasive, rapid, and real-time testing in research and clinical settings.
  • Surveillance modalities for cancer can be categorized into history and physical exams, tumor markers, diagnostic procedures, and imaging.
  • Surveillance for recurrent or secondary cancer is an important component of survivorship care. These methods detect signs of disease or check whether tumors or masses of cells have spread to areas such as the lymph nodes, chest, or lungs.
  • surveillance methods used can include x-rays, magnetic resonance imaging (MRI), computed tomography (CT) scans, computerized axial tomography (CAT) scans, positron emission tomography (PET), FDG-PET, PET/CT, histopathology, or the like.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • CAT computerized axial tomography
  • PET positron emission tomography
  • PET/CT histopathology
  • a liquid biopsy describes the testing of bodily fluids, including blood, urine, cerebrospinal fluid, and saliva, for biomolecular features that have the potential to indicate disease status.
  • Obtaining a liquid biopsy entails a non-invasive or minimally invasive procedure.
  • Different biological components can be identified in a liquid biopsy, including circulating rare cells, cfDNA, cfRNA, and extracellular vesicles (exosomes). With a half-life of approximately 16 minutes to 2.5 hours, cfDNA is relatively short-lived. cfDNA is cleared by nuclease action followed by renal excretion or absorbed by the liver and spleen followed by macrophage degradation.
  • the cfDNA fragments’ stability can increase by binding with cell membranes, extracellular vesicles, or proteins. See, e.g., Gao, et al., Circulating cell-free DNA for cancer early detection, Innovation (Camb.) 2016, and Neumann, et al., ctDNA and CTCs in Liquid Biopsy - Current Status and Where We Need to Progress, Comput Struct Biotechnol J. 2018, which are incorporated by reference herein in its entirety.
  • cfDNA a biomolecular marker that influences cancer diagnosis, prognosis, and monitoring.
  • concentration of cfDNA is low, approximately 100 ng/mL in healthy patients.
  • cfDNA concentrations can vary significantly in cancer patients, with reported ranges between zero and greater than 1000 ng/mL.
  • the increased number of cfDNA in cancer patients is linked to the shedding of molecules by tumors in the blood. Given the observed differences in cfDNA levels between healthy and diseased individuals, using cfDNA as a biomolecular marker can improve cancer diagnosis, prognosis, and monitoring.
  • cfDNA The origin of cfDNA is linked to both passive and active release mechanisms, including apoptosis, necrosis, and active cellular secretion. Moreover, it is thought that genomic instability can influence the active cellular release of cfDNA. These processes release the cellular content into the extracellular space. While cell release mechanisms and clearance of cfDNA requires further elucidation, certain cfDNA characteristics have provided clues regarding its origin. For example, apoptosis causes the cleavage of chromosomal DNA into -170 bp, corresponding to the number of base pairs wrapped around a nucleosome plus a small section that links two nucleosome cores together.
  • This apoptotic-specific size feature is reflected in the modal fragment size of cfDNA, which is -170 bp.
  • cfDNA apoptotic-specific size feature
  • Bronkhorst, et al. Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells, Tumour Biol 2018, and Bronkhorst, et al., The emerging role of cell-free DNA as a molecular marker for cancer management, Biomol Detect Quantif. 2019, which are incorporated by reference herein in its entirety.
  • ctDNA circulating tumor DNA
  • uncontrolled proliferation causes local nutrient depletion, hypoxia, inflammation, and metabolic stress, which leads to apoptosis and necrosis.
  • cfDNA originating from cells interacting with tumor cells known as the tumor microenvironment. It has been shown that the amount of ctDNA increases as the number of tumor cells increases. Additionally, there is growing evidence that patients with cancer have more fragmented and shorter cfDNA. For example, the amount of DNA fragments shorter than 150 bp positively correlated with the tumor DNA fraction in the plasma of patients with hepatocellular carcinoma.
  • tissue-specific processes such as specific nucleosome wrapping, or changes in the methylation levels that lead to less organized DNA regions more prone to nuclease digestion. See, e.g., Bettegowda, et al., Detection of circulating tumor DNA in early- and late-stage human malignancies, Sci Transl Med. 2014, Underhill, et al., Fragment Length of Circulating Tumor DNA, PLoS Genet. 2016, and Jiang, et al,, Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci USA 2015, which are incorporated by reference herein in its entirety.
  • Cancer-specific genetic and epigenetic alterations are reflected in the sequences of isolated ctDNAs. These alterations include mutations, copy number aberrations, genomic rearrangements, and methylation changes. Most ctDNA screening methods have focused on detecting tumor-specific mutations to analyze ctDNA levels, which provide information regarding tumor burden, for example, with high sensitivity and specificity. These mutations include base substitutions (i.e., single nucleotide variants (SNVs)) and insertions or deletions (indels). Mutations can be identified via an array of methods used to identify the composition of sequences.
  • SNVs single nucleotide variants
  • indels insertions or deletions
  • Methods include targeted approaches for single or low numbers of mutations that can be detected using highly sensitive techniques, including amplification-based assays like digital droplet PCR and quantitative PCR (qPCR).
  • amplification-based assays like digital droplet PCR and quantitative PCR (qPCR).
  • qPCR quantitative PCR
  • NGS-based methods have been developed to perform multi-gene analyses in parallel using targeted and untargeted approaches that can detect sequence mutations originating from a tumor (i.e., somatic mutations) versus mutations found in the germline. NGS-based methods can also quantify the fraction of ctDNA found in cfDNA and provide a strategy for screening, detecting, and monitoring cancers. ctDNA in the patient’s sample can be detected by identifying sequences with tumor-specific mutations.
  • Concentrations of ctDNA in plasma are expressed in haploid genome equivalents per mL (hGE / mL) that is obtained from the mean variant allele frequency (VAF) and the input concentration of cfDNA (pg / mL), considering that one hGE equals 3.3 pg of DNA.
  • the concentration of ctDNA in plasma can be low and account for less than 0.01 % of cfDNA, but ctDNA concentrations can constitute cfDNA with a wide range of ⁇ 0.1-89%.
  • Liquid biopsies inform a patient’s cancer progression by measuring ctDNA levels in the cfDNA mixture at different time points.
  • the low concentration of ctDNA in plasma presents a technical challenge for detecting ctDNA.
  • levels of ctDNA are considerably lower than the detection thresholds of many sequencing-based methods, such as in the case of early-stage cancers.
  • pre-cancer treatment ctDNA concentration is ⁇ 0.5% in the majority of patients with lung and colorectal carcinomas.
  • the low levels of ctDNA require methods with increased analytical sensitivity that distinguish background noise and detect true rare mutations.
  • UMIs unique molecular identifiers
  • CAPP-Seq Cancer Personalized Profiling by deep Sequencing
  • NSCLC non-small cell lung cancer
  • TEC-Seq Targeted-error correction sequencing
  • TEC-Seq is another UMLbased technique that measures 58 genes frequently mutated in colorectal, lung, ovarian, and breast cancers. It can detect 50.0%-70.0% of stage I or II cancers across those different cancer types. See, e.g., Newman, et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med.
  • PhasED-Seq Another targeted sequencing method, PhasED-Seq, focuses on phased variants (PVs), two SNVs that occur in cis (on the same strand of DNA). By identifying PVs, PhasED-Seq can identify mutations in these mutationally rich cancers with high confidence, as the probability that two (or more) mutations occurring due to chance on the same strand is extremely low. This technique has demonstrated a remarkably low limit of detection, in the parts-per-million (ppm) range.
  • ppm parts-per-million
  • Illustrative examples of biological fluids that are suitable sources from which to isolate cfDNA in particular embodiments include, but are not limited to amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebral-spinal fluid, ocular fluid, urine, saliva, mucous, and sweat.
  • the samples in some embodiments include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, amniotic fluid, body fluids, such as blood, plasma, serum, semen, lymphatic fluid, cerebrospinal fluid, ocular fluid, urine, saliva, mucous, and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample is peripheral blood. In some embodiments, the sample is processed within 1, 2, or 3 hours from collection time point. In some embodiments, processing comprises separating plasma. In some embodiments, the separation of plasma comprises using centrifugation. In some embodiments, the germline genomic DNA is isolated from the samples. In some embodiments, matched tumor DNA is isolated from the sample. In some embodiments, the genomic DNA is quantified. In some embodiments, circulating cfDNA is isolated from plasma. In some embodiments, the circulating cfDNA is isolated from 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mL of plasma. In some embodiments, the purified plasma DNA is quantified.
  • next-generation sequencing libraries are prepared from isolated plasma DNA. In some embodiments, the next-generation sequencing libraries are prepared from isolated shorn tumor genomic DNA. In some embodiments, the next-generation sequencing libraries are prepared from isolated germline genomic DNA. In some embodiments, the next-generation sequencing libraries are prepared from isolated cell line genomic DNA.
  • next-generation sequencing libraries are constructed using a DNA polymerase with strong 3’-5’ exonuclease activity and high fidelity. In some embodiments, the next-generation sequencing libraries are cleaned up. In some embodiments, the next-generation libraries are ligated with appropriate sequencing adapters. In some embodiments, the ligated next-generation libraries are amplified. In some embodiments, the amplified nextgeneration libraries are quantified.
  • the amplified next-generation sequencing libraries are captured with hybridization probes. In some embodiments, the captured next-generation sequencing libraries are amplified. In some embodiments, the amplified captured next-generation sequencing libraries are sequenced. See e.g., Newman, et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med. 2014, which is incorporated by reference herein in its entirety.
  • the biological fluid is blood or blood plasma.
  • the sample is blood or a blood-derived sample.
  • Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • PBMCs peripheral blood mononuclear cells
  • the sample is obtained on or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days following administration of the treatment for cancer.
  • that sample is obtained at or about 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33
  • the sample is obtained at or about 1, 2, 3, 4 ,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 months following administration of the treatment for cancer. In some embodiments, the sample is obtained at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years following administration of the treatment for cancer. In some embodiments, the sample is obtained at Day 15 following administration of the treatment for cancer.
  • a biomarker i.e., ctDNA
  • the sample is obtained from a subject, such as a subject having a cancer, e.g., a cancer described herein.
  • tNA tumor nucleic acids
  • cfNA cell-free nucleic acids
  • the method can comprise obtaining a cfNA sample, selecting the cfNA for sequences corresponding to a plurality of regions of mutations in a cancer described herein, sequencing the selected cfNA; determining the presence of somatic mutations, wherein the presence of the somatic mutations can be indicative of tumor cells present in the subject; and providing the subject with an assessment of the presence of tumor cells.
  • the cell-free nucleic acid can be cell-free DNA (cfDNA).
  • the cell-free nucleic acid can be cell-free RNA (cfRNA).
  • the cell-free nucleic acids can be a mixture of cell-free DNA (cfDNA) and cell-free RNA (cfRNA).
  • the tumor nucleic acid can be a nucleic acid originating from a tumor cell.
  • the tumor nucleic acid can be tumor-derived DNA (tDNA).
  • the tumor nucleic acid can be a circulating tumor DNA (ctDNA).
  • the tumor nucleic acid can be tumor-derived RNA (tRNA).
  • the tumor nucleic acid can be a circulating tumor RNA (ctRNA).
  • the tumor nucleic acids can be a mixture of tumor-derived DNA and tumor-derived RNA.
  • the tumor nucleic acids can be a mixture of ctDNA and ctRNA.
  • Determining the presence of somatic mutation can comprise the steps of a sequencing method as disclosed herein.
  • determining quantities of ctDNA can comprise determining absolute quantities of ctDNA.
  • Determining quantities of ctDNA can comprise determining relative quantities of ctDNA.
  • Determining quantities of ctDNA can be performed by counting sequence reads pertaining to the ctDNA.
  • Determining quantities of ctDNA can be performed by quantitative PCR.
  • Determining quantities of ctDNA can be performed by a sequencing method as disclosed herein. Determining quantities of ctDNA can comprise counting sequencing reads of the ctDNA.
  • determining the quantities of ctDNA can comprise detecting one or more mutations.
  • Determining the quantities of ctDNA can comprise detecting two or more different types of mutations.
  • the types of mutations include, but are not limited to, SNVs, indels, fusions, breakpoints, structural variants, variable number of tandem repeats, hypervariable regions, mini satellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, or a combination thereof in selected regions of the subject's genome.
  • Determining the quantities of ctDNA can comprise detecting one or more of single nucleotide variant (SNVs), phased variant (PVs), indels, copy number variants, and rearrangements in selected regions of the subject's genome.
  • Determining the quantities of ctDNA can comprise detecting two or more of SNVs, indels, copy number variants, and rearrangements in selected regions of the subject's genome.
  • Determining the quantities of ctDNA can comprise detecting at least one SNV, indel, copy number variant, and rearrangement in selected regions of the subject's genome.
  • detecting ctDNA can comprise detecting a mutation in wherein the mutation is a point mutation, a deletion, or a frameshift mutation.
  • the methods described herein detect a mutation that is a somatic mutation, a phased variant (PV), or a single nucleotide variant (SNV).
  • determining the quantities of ctDNA can comprise detecting one or more PVs. In some embodiments, determining the quantities of ctDNA can comprise detecting one or more SNVs. In some embodiments, determining the quantities of ctDNA can comprise detecting one or more somatic mutation. In some embodiments, determining the quantities of ctDNA can comprise detecting one or more point mutation. In some embodiments, determining the quantities of ctDNA can comprise detecting one or more deletion. In some embodiments, determining the quantities of ctDNA can comprise detecting one or more frameshift mutation.
  • determining the quantities of ctDNA can comprise detecting two or more mutations comprising SNVs, indels, rearrangements, or a combination thereof.
  • phased variants generally refers to two or more mutations (e.g., SNVs or indels) that occur in cis (i.e., on the same strand of a nucleic acid molecule) within a single cell-free nucleic acid molecule.
  • a cell-free nucleic acid molecule can be a cell-free deoxyribonucleic acid (cfDNA) molecule.
  • cfDNA cell-free deoxyribonucleic acid
  • a cfDNA molecule can be derived from a diseased tissue, such as a tumor (e.g., a circulating tumor DNA (ctDNA) molecule).
  • identifying a mutation in one or more genes in the cfDNA comprises sequencing the genes.
  • sequencing the genes comprises any appropriate sequencing technique that can include but is not limited to high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys), phased variant enrichment and detection sequencing (PhasED-Seq), CAncer Personalized Profiling by deep Sequencing (CAPP-Seq),
  • the methods described herein detect ctDNA at a sensitivity of at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%
  • the methods described herein detect ctDNA at a specificity of at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%
  • measuring the levels of ctDNA comprises determining the absolute ctDNA concentration as mutant haploid genome equivalents per milliliter of plasma (hGE/mL).
  • the ctDNA level was classified as haploid genome equivalents (hGE) per mL of plasma (hGE/mL) and calculated with the following formula: [(the mean VAF for all mutations detected) x cfDNA concentration (pg/mL of plasma)] - ⁇ 3.3, as previously published by Scherer et al., Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Transl Med. 2016.
  • the detection further comprises determining the correlation of baseline ctDNA concentration (hGE/mL) with subject baseline characteristics.
  • measuring the levels of ctDNA further comprises determining the mean variant allele frequency (VAF).
  • VAF mean variant allele frequency
  • the mean VAF is classified as the percentage of sequence reads observed matching a specific DNA variant divided by the overall coverage at that locus.
  • the methods described herein can comprise measuring a mean variant allele frequency (VAF) of greater than a range that is indicative of higher or lower total ctDNA level. See, e.g., Strom, S., Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology, Cancer Biol Med. 2016, which is incorporated by reference herein in its entirety.
  • the methods described herein detect ctDNA at a sensitivity of at least about 50% from cfDNA in a sample.
  • the detection threshold is a percentage of ctDNA level at or below total cfDNA levels in the sample. In some embodiments, the detection threshold is a concentration of ctDNA level at or below total cfDNA levels in the sample.
  • the detection threshold is within 25%, within 24%, within 23%, within 22%, within 21%, within 20%, within 19%, within 18%, within 17%, within 16%, within 15%, within 14%, within 13%, within 12%, within 11%, within 10%, within 9%, within 8%, within 7%, within 6%, or within 5% and/or is within a standard deviation above the median or mean level, amount or concentration of cfDNA in the biological sample obtained from the subject following administration of the treatment.
  • the ctDNA is undetected at or at least at 2, 3, 4, 5, or 6 weeks following, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 32, 34, or 36 months, or 1, 2, 3, 4, 5, or more years following the administration of the treatment for cancer. Detection of nucleic acids
  • a ctDNA of the disclosure is detected using any suitable method known in the art, such as a nucleic acid hybridization assay, an amplification-based assay (e.g., polymerase chain reaction, PCR), a PCR-RFLP assay, real-time PCR, sequencing (e.g., Sanger sequencing or next-generation sequencing), a screening analysis (e.g., using karyotype methods), fluorescence in situ hybridization (FISH), break away FISH, spectral karyotyping, multiplex-FISH, comparative genomic hybridization, in situ hybridization, single specific primer-polymerase chain reaction (SSP-PCR), high performance liquid chromatography (HPLC), or mass- spectrometric genotyping.
  • a nucleic acid hybridization assay e.g., an amplification-based assay (e.g., polymerase chain reaction, PCR), a PCR-RFLP assay, real-time PCR, sequencing (e.g., Sanger sequencing or next
  • Analytic methods can include generating and capturing genetic information.
  • Genetic information can include genetic sequence information, ploidy states, the identity of one or more genetic variants, as well as a quantitative measure of the variants.
  • quantitative measure refers to any measure of quantity including absolute and relative measures.
  • a quantitative measure can be, for example, a number (e.g., a count), a percentage, a frequency, a degree or a threshold amount.
  • Samples comprising genetic material can be collected from a subject at a plurality of time points, that is, serially.
  • the genetic material can be sequenced, e.g., using a high-throughput sequencing system. Sequencing can target loci of interest to detect genetic variants, such genes bearing somatic mutations, genes that undergo copy number variation, or genes involved in gene fusions, for example, in cancer.
  • a quantitative measure of the genetic variants found can be determined.
  • the quantitative measure can be the frequency or percentage of a genetic variant among polynucleotides mapping to a locus, or the absolute number of sequence reads or polynucleotides mapping to a locus.
  • a mutation or variant can comprise a genetic aberration that includes, but is not limited to a single base substitution, or small indels, transversions, translocations, inversion, deletions, truncations or gene truncations.
  • a mutation can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • a mutation can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the methods can be used to detect any number of genetic aberrations that can cause or result from cancers.
  • mutations can include but are not limited to mutations, mutations, indels, copy number variations, transversions, translocations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, abnormal changes in nucleic acid methylation infection and cancer.
  • the mutation is a single nucleotide variant (SNV).
  • the mutation is phased variant (PV).
  • a sample can comprise various amount of nucleic acid that contains genome equivalents.
  • a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cfDNA, about 200 billion (2x 1011) individual polynucleotide molecules.
  • a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.
  • a haploid human genome equivalent has about 3 picograms of DNA.
  • a sample of about 1 microgram of DNA contains about 300,000 haploid human genome equivalents.
  • Genotyping ctDNA and/or detection, identification and/or quantitation of the ctDNA can be done through sequencing. Sequencing can be accomplished using high-throughput systems.
  • Polynucleotides can be analyzed by any method known in the art.
  • the DNA sequencer will employ next generation sequencing (e.g., Illumina, 454, Ion torrent, SOLiD, Pacific Biosciences).
  • Next generation sequencing methods can be grouped into two major categories, sequencing by hybridization and sequencing by synthesis (SBS). See, e.g., Slatko, et al., Overview of Next Generation Sequencing Technologies, Curr Protoc Mol Biol. 2019, which is incorporated by reference herein in its entirety.
  • SBS methods incorporate repeated cycles of synthesis, imaging, and methods to add additional nucleotides in the growing chain and obtain a sequence readout by imaging the incorporated nucleotides.
  • SBS technologies typically rely on shorter reads, up to 300 - 500 bases. They generally have higher error rate and rely on high sequence coverage of millions to billions of short DNA sequence reads to obtain an accurate sequence based upon the identification of a consensus sequence.
  • Sequencing by hybridization hybridizes and washes away the non-hybridized DNA, making it possible to determine whether the hybridizing labeled fragments matched the sequence of the DNA probes on the filter. It was thus possible to build larger contiguous sequence information, based upon overlapping information from the probe hybridization spots. Sequencing by hybridization has been largely relegated to technologies that depend upon using specific probes to interrogate sequences, such as in diagnostic applications for identifying disease-related SNPs (single-nucleotide polymorphisms) in specific genes or identifying gross chromosome abnormalities (rearrangements, deletions, duplications, copy number variants, CNVs).
  • SNPs single-nucleotide polymorphisms
  • CNVs copy number variants
  • Sequence analysis can be performed by massively parallel sequencing, that is, simultaneously (or in rapid succession) sequencing any of at least 100,000, 1 million, 10 million, 100 million, or 1 billion polynucleotide molecules.
  • Sequencing methods can include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, singlemolecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS - Helicos), massively- parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam- Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys) or Nanopore (e.g., Oxford Nanopore) platforms and any other sequencing methods known in the art.
  • Illumina technology uses bridge amplification wherein DNA molecules of about 500 bp with adapters ligate on each end and are used as substrates for repeated amplification synthesis reactions on a solid support that contains oligonucleotide sequences complementary to a ligated adapter.
  • the oligonucleotides on the slide are spaced such that the DNA, which is then subjected to repeated rounds of amplification, creates clonal “clusters” consisting of about 1000 copies of each oligonucleotide fragment.
  • Each glass slide can support millions of parallel cluster reactions.
  • proprietary modified nucleotides corresponding to each of the four bases, each with a different fluorescent label, are incorporated and then detected.
  • a nucleotide sequence (e.g., DNA sequence) can refer to raw sequence reads or processed sequence reads, such as unique molecular counts inferred from raw sequence reads.
  • Sequence reads generated from sequencing are subject to analysis including, for example, identifying genetic variants. This can include identifying sequence variants and quantifying numbers of base calls at each locus. Quantifying can involve, for example, counting the number of reads mapping to a particular genetic locus. Different numbers of reads at different loci can indicate copy number variation (CNV).
  • CNV copy number variation
  • Methods of the present disclosure can be used in the detection of genetic variants (also referred to a “gene alterations”). Genetic variants are alternative forms at a genetic locus. Genetic variants include sequence variants, copy number variants and nucleotide modification variants. A sequence variant is a variation in a genetic nucleotide sequence. A copy number variant is a deviation from wild type in the number of copies of a portion of a genome.
  • Genetic variants include, for example, single nucleotide variations (SNPs), insertions, deletions, inversions, transversions, translocations, gene fusions, chromosome fusions, gene truncations, copy number variations (e.g., aneuploidy, partial aneuploidy, polyploidy, gene amplification), abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns and abnormal changes in nucleic acid methylation.
  • SNPs single nucleotide variations
  • insertions e.g., deletions, inversions, transversions, translocations
  • gene fusions e.g., chromosome fusions, gene truncations
  • copy number variations e.g., aneuploidy, partial aneuploidy, polyploidy, gene amplification
  • abnormal changes in nucleic acid chemical modifications e.g., aneuploidy, partial aneuploidy, polyploidy, gene
  • Genetic variants can be detected by comparing sequences from polynucleotides in a sample to a reference, e.g., to a reference genome sequence, to an index or to a database of known mutations.
  • the reference sequence is a publicly available reference sequence, such as the human genome sequence HG-19 or NCBI Build 37.
  • the reference sequence is a sequence in a non-public database.
  • the reference sequence is a germ line sequence of an organism inferred or determined from sequencing polynucleotides from the organism.
  • the statistical association analysis that is performed is a genome-wide association study (GWAS) statistical analysis (van der Sluis S, et al., PLOS Genetics 2013; 9: el003235; Visscher PM, et al., Am J Hum Genet 2012; 90: 7).
  • GWAS genome-wide association study
  • sequence variants can be detected from consensus sequences from multiple sequence reads, for example, from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least
  • a consensus sequence can be from sequence reads of a single strand polynucleotide.
  • a consensus sequence can also be from sequence reads of one strand of a double-stranded polynucleotide (e.g., pairing reads).
  • pairing reads allows one to identify with increased confidence the existence of a sequence variant in a molecule.
  • a somatic mutation or somatic alteration is a genetic variant that arises in a somatic cell.
  • Somatic mutations are distinguished from mutations that arise in the genome of a germ line cell (i.e., sperm or egg) or a zygote, of an individual.
  • Somatic mutations e.g., those found in cancer cells, are distinguishable from the germ line genome of a subject in which the cancer arose. They also can be detected by comparing the cancer genome with the germ line genome or with a reference genome.
  • Deep-targeted sequencing techniques have been developed to deal with the scarcity of mutated fragments and have increased sensitivity by incorporating gene panels targeting the most frequently mutated genes. These methods need to overcome technical noise that can impact the ability to distinguish single nucleotide variants (SNVs) signals from sequencing errors.
  • Examples of deep-targeted sequencing methods include cancer-personalized profiling by deep sequencing (CAPP-Seq), duplex sequencing (Duplex-Seq), and phased variant enrichment and detection sequencing (PhasED-Seq). See, e.g., Newman, et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med.
  • Phased variant enrichment and detection sequencing is a method to detect and quantify ctDNA.
  • PhasED-Seq lowers the error profile of mutation detection in sequencing data by requiring the concordant detection of two separate non-reference events in an individual DNA molecule. By detecting more than one mutation, PhasED-Seq can more accurately distinguish tumor-derived cell free DNA (i.e., ctDNA) from healthy cell free DNA - enabling detection of ctDNA at levels below one part-per-million ( ⁇ 0.0001%). PhasED-Seq has been extensively validated in hundreds of patients with B-cell lymphomas.
  • PhasED- Seq detects two or more single nucleotide variants from top or bottom strands of circulating DNA fragments.
  • This next-generation circulating-tumor DNA sequencing assay has a high molecule recovery rate and is less susceptible to sequencing errors, increasing the sensitivity of ctDNA detection. See, e.g., Kurtz, et al. Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA, Nat Biotechnol 2021, which is incorporated by reference herein in its entirety.
  • ctDNA as a biomarker before and after therapy is a promising prognostic tool, yet methods require improved sensitivity to universally detect ctDNA associated with residual disease. Methods lacking sufficient sensitivity yield ‘false-negative’ measurements, in which patients will be classified as having undetectable ctDNA but will experience eventual disease progression. PhasED-Seq’s improved sensitivity and detection of ctDNA increases the prognostic value of ctDNA as a biomarker. See, e.g., Kurtz, et al. Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA, Nat Biotechnol 2021, which is incorporated by reference herein in its entirety.
  • phased variant identification focuses on sets of variants less than 170 base pairs (bp) apart, the typical length of a ctDNA fragment.
  • Putative phased variants (PVs) were classified by analyzing 2,538 tumors of 24 cancer histologies to identify genomic regions of interest for detecting phased variants. Particularly, this methodology identified stereotyped genomic regions with increased PV distribution in lymphoid cancers.
  • the PhasED-Seq panel for lymphoma targets approximately 115 kb of genomic space that encompasses 0.0035% of the human genome but covers 26% of the phased variants detected by whole genome sequencing. This results in the ability of PhasED-Seq to detect tumor fractions as low as 1 part per 1,000,000. See e.g., Kurtz et al., Enhanced Detection of Minimal Residual Disease by Targeting Sequencing of Phased Variants in Circulating Tumor DNA, Nat. Biotechnol., 39(12): 1537-1547 (2021), which is incorporated by reference in its entirety herein.
  • An example process flow to perform clinal intervention and/or treatment on an individual based on detecting ctDNA sequences using PhasED-Seq has three main segments. The first is to obtain, prepare, and sequence cfDNA from an individual’s biological sample, using a capture sequencing approach across regions that have been shown to harbor a plurality of genetic variants occurring in phase. This is followed by the analysis of the cfDNA sequencing result to detect ctDNA, as determined by the detection of genetic variants occurring in phase. Lastly, a clinical intervention and/or treatment on the basis that the sequencing result indicates that the biological sample contains ctDNA is performed. See, e.g., U.S. Pat. No.11,447,833, which is incorporated by reference herein in its entirety.
  • the identification of phase variants uses read-level data, taking the paired-end reads and identifying the non-reference positions.
  • germ line sequencing of peripheral blood mononuclear cells PBMCs
  • SNPs patient-specific single nucleotide polymorphisms
  • a position was classified as non-reference if it had a variant allele fraction (VAF) above 40% with a read depth of at least 10, or a VAF of above 0.25% with at least 100 reads.
  • VAF variant allele fraction
  • read-level data from a sample of interest was used to classify PVs. A sample’s read-pair containing two or more non-reference positions was considered a somatic PV.
  • PVs containing putative germ -line SNPs were removed, ensuring that any remaining PVs were not seen in the germ line sample, a step important for sensitivity and specificity.
  • the number of deduplicated read-pairs that contain PVs were divided by the number of read-pairs spanning the genomic locus of the given PVs to calculate the VAF associated with each PV. See, e.g., Kurtz, et al. Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA, Nat Biotechnol 2021, which is incorporated by reference herein in its entirety.
  • PhasED-Seq is used for cancer screening and biopsy-free tumor genotyping, where a patient ctDNA sample is analyzed without reference to a biopsy sample.
  • the detection of ctDNA via PhasED-Seq is prognostic of event- free survival.
  • PhasED-Seq offers greater sensitivity for residual disease detection than other ctDNA detection assays.
  • PhasED-Seq can be used to detect MRD in curative settings.
  • PhasED-Seq is an important step in overcoming technical noise and improving the sensitivity of MRD detection.
  • PhasED-Seq provides the potential of ctDNA-based assays to accurately monitor residual disease and to inform clinical decision-making in low tumor burden settings.
  • the increased sensitivity of PhasED-Seq can allow for earlier detection of treatment failure, allowing to offer patients alternative therapies while the tumor burden is low and the disease is still within the curative window.
  • improved sensitivity of PhasED-Seq can detect cancer during and after treatment and can find use in guiding treatment decisions, where physicians can de-escalate therapy when ctDNA levels become undetectable via PhasED-Seq, sparing patients toxic and costly therapies.
  • some methods using PhasED-Seq can be performed in conjunction with tumor imaging methods, e.g. PET/CT scans and the like. b. CAPP-Seq
  • CAncer Personalized Profiling by deep Sequencing is a method for quantifying ctDNA.
  • CAPP-Seq combines optimized library preparation methods for low DNA input masses with a multi-phase bioinformatics approach to design a “selector” consisting of biotinylated DNA oligonucleotides that target recurrently mutated regions in the cancer of interest.
  • the selector is applied to tumor DNA to identify a patient’s cancerspecific genetic aberrations and then directly to circulating DNA to quantify them.
  • CAPP-Seq can detect single nucleotide variants (SNVs) on circulating DNA fragments.
  • This next-generation circulating-tumor DNA sequencing assay allows for a high molecule recovery rate but is susceptible to sequencing error. See, e.g., Newman, et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med. 2014, which is incorporated by reference herein in its entirety.
  • Designing a CAPP-Seq selector can be done for any cancer for which recurrent mutations have been identified. Exons with recurrent mutations can be selected using whole exome sequencing to maximize the number of missense mutations per patient while minimizing the selector size. For example, for non-small cell lung cancer (NSCLC), selected design targets cover a region of approximately 125 kb.
  • NSCLC non-small cell lung cancer
  • optimization of library preparation is important when DNA amounts are limited, as in the case of DNA derived from plasma.
  • libraries for CAPP-Seq can be constructed from as little as 4 ng.
  • the detection limit of CAPP-Seq can be affected by (i) the input amount and recovery rate of circulating DNA molecule, (ii) allelic bias in the capture reagent, and (iii) PCR or sequencing errors.
  • CAPP-Seq is used for cancer screening and biopsy-free tumor genotyping, where a patient ctDNA sample is analyzed without reference to a biopsy sample.
  • the methods include providing a therapy appropriate for the target.
  • CAPP-Seq combines optimized library preparation methods for low DNA input masses with a multi-phase bioinformatics approach to design a “selector” consisting of biotinylated DNA oligonucleotides that target recurrently mutated regions in the cancer of interest.
  • the selector is applied to tumor DNA to identify a patient’s cancerspecific genetic aberrations and then directly to circulating DNA to quantify them.
  • CAPP-Seq can be performed in conjunction with tumor imaging methods, e.g. PET/CT scans and the like.
  • Duplex-Seq is a method for detecting and quantifying ctDNA that relies on identifying a single nucleotide variant (SNVs) on both strands of a ctDNA molecule, thereby reducing the probability of sequencing errors.
  • Duplex-Seq detects single nucleotide variants (SNVs) on both DNA strands from circulating DNA fragments. This next-generation circulatingtumor DNA sequencing assay decreases the impact of sequencing error on identifying SNVs, but the efficiency of recovering both the top and bottom strands can be low, leading to a low molecule recovery rate. See, e.g., Schmitt, et al., Detection of ultra-rare mutations by nextgeneration sequencing, Proc Natl Acad Sci USA 2012, which is incorporated by reference herein in its entirety.
  • Duplex-Seq is a desirable approach for tag-based error correction, which reduces or eliminates artifactual mutations arising from DNA damage, PCR errors, and sequencing errors.
  • Duplex-Seq allows rare variants in heterogeneous populations to be detected with sensitivity and capitalizes on the redundant information stored in complexed double-stranded DNA.
  • the methods disclosed herein can comprise amplification of cell-free DNA (cfDNA) and/or of circulating tumor DNA (ctDNA).
  • Amplification can comprise PCR-based amplification.
  • amplification can comprise non-PCR-based amplification.
  • a ctDNA as described herein is detected using an amplification-based method.
  • a sample of nucleic acids such as a sample obtained from an individual or from a tumor
  • an amplification reaction e.g., Polymerase Chain Reaction (PCR)
  • PCR Polymerase Chain Reaction
  • oligonucleotides or primers e.g., such as one or more oligonucleotides or primers provided herein.
  • the presence of a ctDNA of the disclosure in the sample can be determined based on the presence or absence of an amplification product.
  • Quantitative amplification methods are also known in the art and can be used according to the methods provided herein. Methods of measurement of DNA copy number at microsatellite loci using quantitative PCR analysis are known in the art. The known nucleotide sequence for genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR can also be used. In fluorogenic quantitative PCR, quantitation is based on the amount of fluorescence signals, e.g., TaqMan and SYBR green.
  • LCR ligase chain reaction
  • transcription amplification e.g., transcription amplification
  • self-sustained sequence replication e.g., transcription amplification
  • dot PCR e.g., transcription amplification
  • linker adapter PCR e.g., linker adapter PCR
  • a ctDNA disclosed herein is detected using an array-based method, such as array-based comparative genomic hybridization (CGH) methods.
  • CGH comparative genomic hybridization
  • a first sample of nucleic acids e.g., from a sample, such as from a tumor
  • a second sample of nucleic acids e.g., a control, such as from a healthy sample
  • equal quantities of the two samples are mixed and co-hybridized to a DNA microarray of several thousand evenly spaced cloned DNA fragments or oligonucleotides, which have been spotted in triplicate on the array.
  • Array-based CGH can also be performed with single-color labeling.
  • a cancer provided herein i.e., LBCL and/or DLBCL
  • methods for determining a disease state of a cancer in a subject can comprise obtaining a quantity of circulating tumor DNA (ctDNA) in a sample from the subject; and determining a disease state of a cancer in the subject based on a ratio of the quantity of ctDNA to cfDNA.
  • a therapy e.g., a cell therapy
  • the methods comprise detecting a ctDNA provided herein in a sample obtained from the subject. In some embodiments, the methods comprise acquiring knowledge of the presence of a ctDNA provided herein in a sample obtained from the subject. In some embodiments, the presence of the ctDNA in the sample identifies the subject as one who can benefit from a treatment, e.g., a cancer treatment. In some embodiments, the presence of the ctDNA in the sample identifies the subject as one who can benefit from a treatment, e.g., an anti-cancer therapy, for example, a cell therapy, provided herein.
  • detection of the ctDNA in the sample identifies the subject as one who can benefit from a treatment comprising a cell therapy provided herein. In some embodiments, responsive to knowledge of the ctDNA in the sample, the subject is identified as one who can benefit from a treatment comprising the cell therapy provided herein.
  • the sample is a sample described herein.
  • the sample comprises a biological sample from the subject diagnosed with a cancer, such as a blood, serum or a plasma sample.
  • the methods of treating or delaying progression of a cancer of the disclosure in a subject comprise administering to the individual a therapeutically effective amount of a treatment such as a cancer treatment and/or a cell therapy provided herein.
  • the methods of treating or delaying progression of a cancer of the disclosure in an individual comprise administering to the individual an effective amount of a cancer treatment provided herein, responsive to knowledge of the presence of the ctDNA in a sample obtained from the subject.
  • the methods of treating or delaying progression of a cancer of the disclosure in an individual comprise administering to the individual an effective amount of a treatment such as a cell therapy provided herein, responsive to knowledge of the presence of the ctDNA in a sample obtained from the subject.
  • the method comprises obtaining a biological sample and isolating cfDNA from the biological sample.
  • the method comprises identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • the subject is administered a treatment for cancer if levels of ctDNA in the sample are below a detection threshold.
  • the subject is administered a treatment for cancer if levels of ctDNA in the sample are above a detection threshold.
  • the method comprises administering to the subject an effective amount of a treatment for cancer, wherein prior to administering the treatment for cancer, the subject is identified as a candidate for receiving the treatment for cancer.
  • the method comprises obtaining a biological sample and isolating cell-free DNA (cfDNA) from the biological sample.
  • the method comprises identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • the method comprises determining a ratio of ctDNA:cfDNA molecules.
  • the method comprises administering to the subject an effective amount of a treatment for cancer, wherein prior to administering the treatment for cancer, the subject is identified as a candidate for receiving the treatment for cancer.
  • the method comprises obtaining a biological sample and isolating cell-free DNA (cfDNA) from the biological sample.
  • the method comprises identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • the method comprises treating cancer in a subject by administering to the subject an effective amount of a treatment for cancer.
  • the levels of ctDNA in a sample from the subject are measured and levels of ctDNA in the sample that are above a detection threshold indicate that the subject is a candidate for administration of the treatment for cancer.
  • the method comprises treating cancer in a subject with higher-risk disease by administering to the subject an effective amount of a treatment for cancer.
  • the subject prior to administering the treatment for cancer, the subject is identified as having higher-risk disease by measurement of the levels of ctDNA in a sample from the subject.
  • levels of ctDNA in the sample that are above a detection threshold indicate that the subject has higher-risk disease. In other embodiments, levels of ctDNA that are above a detection threshold indicate that the subject is a candidate for a treatment for cancer. In some embodiments, levels of ctDNA in the sample that are below a detection threshold indicate that the subject does not have higher-risk disease. In other embodiments, levels of ctDNA that are below a detection threshold indicate that the subject is not a candidate for a treatment for cancer.
  • the detection threshold is a ratio of ctDNA: cfDNA that is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1.0: 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1: 10 8 above ctDNA:cfDNA molecules.
  • the detection threshold is a ratio of ctDNA: cfDNA that is or is about 1 : 10, 1 : 100, 1 : 10 3 , 1 : 10 4 , 1 : 10 5 , 1 : 10 6 , 1 : 10 7 , 1 : 10 8 , 1 : 10 9 , 1 : 10 10 , l:10 n , or 1 : 10 12 .
  • the detection threshold is a ratio of ctDNA:cfDNA that is or is about 5: 10, 5: 100, 5 : 10 3 , 5 : 10 4 , 5 : 10 5 , 5 : 10 6 , 5 : 10 7 , 5 : 10 8 , 5 : 10 9 , 5 : 10 10 , 5 : 10 11 , or 5 : 10 12 .
  • the detection threshold is a ratio of ctDNA: cfDNA that is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1.0: 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1: 10 8 above ctDNA:cfDNA molecules.
  • the method comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free tumor DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA:cfDNA molecules in the sample, and administering an effective amount of a treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the methods provides administering an effective amount of a treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules. In some embodiments, the methods provides for administering an effective amount of a treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 4 , l : 10 5 , l : 10 6 , l :10 7 , l : 10 8 , l : 10 9 , l :10 10 , l : 10 n , or l :10 12 ctDNA:cfDNA molecules.
  • the methods provides administering an effective amount of a treatment for cancer if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1.0: 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • a method for treating a subject with a second treatment for cancer comprising, isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA: cfDNA molecules in the sample, and administering a second treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the methods provides for administering a second treatment for cancer if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1.0: 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1 : 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1 : 10 7 , from at or about 1 : 10 6 to at or about 1 : 10 8 is above ctDNA:cfDNA molecules.
  • a method of treating cancer in a subject in need thereof is assessed as a candidate for treatment for cancer.
  • the method comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA: cfDNA molecules in the sample, and identifying the subject as a candidate for treatment for cancer if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules, and administering to the subject an effective amount of a treatment for cancer. In some embodiments, identifying the subject as a non-candidate for treatment for cancer if the ratio of ctDNA:cfDNA is below 1 : 10 6 ctDNA:cfDNA molecules.
  • the method provides identifying the subject as a non-candidate for treatment for cancer if the ratio of ctDNA:cfDNA is below 1 : 10 6 ctDNA:cfDNA molecules. In some embodiments, the method provides for identifying the subject as a non-candidate for treatment for cancer if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 :
  • 10 5 to at or about 1.0: 10 7 from at or about 1 : 10 5 to at or about 1 : 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • the method provides for identifying the subject as a candidate for treatment for cancer if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1 : 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5:
  • 10 6 to at or about 1.0: 10 8 from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • a method for treating cancer in a subject with higher-risk disease is provided herein.
  • the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ctDNA: cfDNA molecules in the sample is determined.
  • the subject is identified as having high-risk disease if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is identified as having higher-risk disease if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules. In other embodiments, the subject is identified as having higher-risk disease if the ratio of ctDNA:cfDNA is above 1 : 10 4 , 1 : 10 5 , 1 : 10 6 , 1 :10 7 , 1 : 10 8 , 1 :10 9 , 1 : 10 10 , l: 10 n , or ! :10 12 ctDNA:cfDNA molecules.
  • the method comprises isolating cell- free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free tumor DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA:cfDNA molecules in the sample; and administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the methods provides administering an effective amount of the medicament if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules. In some embodiments, the methods provides for administering an effective amount of the medicament if the ratio of ctDNA: cfDNA is above l :10 4 , l :10 5 , l : 10 6 , l : 10 7 , l : 10 8 , l : 10 9 , l :10 10 , l : 10 n , or 1 : 10 12 ctDNA:cfDNA molecules.
  • the method provides administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules. In some embodiments, the method provides administering an effective amount of the medicament to the subject if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1 : 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • the method comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA:cfDNA molecules in the sample, and administering an effective amount of the medicament to the subject if the ratio of ctDNA: cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the use provides for administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the method provides for administering an effective amount of the medicament if the ratio of ctDNA:cfDNA is above l:10 4 , l:10 5 , l:10 6 , l:10 7 , l:10 8 , l:10 9 , 1: IO 10 , l:10 n , or 1: 10 12 ctDNA:cfDNA molecules.
  • the use provides for administering an effective amount of the medicament to the subject if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the method provides administering an effective amount of the medicament to the subject if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1 : 10 6 , from at or about 1: 10 6 to at or about 1.0: 10 8 , from at or about 1: 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • the ratio of ctDNA:cfDNA molecules is a detection threshold ratio of ctDNA:cfDNA molecules. In other embodiments, the ratio of ctDNA:cfDNA molecules is a ratio of ctDNA:cfDNA molecules in a sample of the subject. In some embodiments, the ratio of ctDNA:cfDNA molecules is 1:10, 1:100, 1:1000, l:10 4 , l:10 5 , l:10 6 , l:10 7 , l:10 8 .
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 10 6 to at or about 50: 10 6 , from at or about 1 : 10 6 to at or about 40: 10 6 , at or about 1 : 10 6 to at or about 30: 10 6 , at or about 1 : 10 6 to at or about 20: 10 6 , at or about 1 : 10 6 to at or about 10: 10 6 , at or about 1 : 10 6 to at or about 5 : 10 6 , at or about 1 : 10 6 to at or about 2.5 : 10 6 , from at or about 2.5 : 10 6 to at or about 50: 10 6 , from at or about 2.5: 10 6 to at or about 40: 10 6 , at or about 2.5: 10 6 to at or about 30: 10 6 , at or about 2.5: 10 6 to at or about 20: 10 6 , at or about 2.5: 10 6 to at or about 10: 10 6 , at or about 2.5:10 6 to at or about 5 : 10
  • the ratio of ctDNA:cfDNA molecules is at least or at least about 0.2:10 6 , 0.3:10 6 , 0.4:10 6 , 0.5:10 6 , 0.6:10 6 , 0.7:10 6 , 0.8:10 6 , 0.9:10 6 , 1: 10 6 , l.l:10 6 , 1.2: 10 6 , 1.3 : 10 6 , 1.4: 10 6 , 1.5: 10 6 , 1.6: 10 6 , 1.7: 10 6 , 1.8: 10 6 , 1.9: 10 6 , 2:10 6 , 2.1 : 10 6 , 2.2:10 6 , 2.3:10 6 , 2.4:10 6 , 2.5:10 6 , 2.6:10 6 , 2.7:10 6 , 2.8:10 6 , 2.9:10 6 , 3 : 10 6 , 3.1 : 10 6 , 3.2:10 6 , 3.3 : 10 6 , 3.4
  • the ratio of ctDNA:cfDNA molecules is 1 : 10, 1 : 100, 1 : 1000, 1 : 10 4 , 1 : 10 5 , l :10 6 , 1 : 10 7 , l :10 8 .
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 1000000 to at or about 1 : 50000000, at or about 1 : 1000000 to at or about 1 :45000000, at or about 1 : 1000000 to at or about 1 :40000000, at or about 1 : 1000000 to at or about 1 :35000000, at or about 1 : 1000000 to at or about 1 :30000000, at or about 1 : 1000000 to at or about 1 :25000000, at or about 1 : 1000000 to at or about 1 :20000000, at or about 1 : 1000000 to at or about 1 : 15000000, at or about 1 : 1000000 to at or about 1 : 10000000, at or about 1 : 1000000 to at or about 1 : 5000000, at or about 1 : 1000000 to at or about 1 :4000000, at or about 1 : 1000000 to at or about 1 :3000000, at or about 1 : 1000000 to at
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 1000000 to at or about 1 : 50000000, at or about 1 : 1000000 to at or about 1 :45000000, at or about 1 : 1000000 to at or about 1 :40000000, at or about 1 : 1000000 to at or about 1 :35000000, at or about 1 : 1000000 to at or about 1 :30000000, at or about 1 : 1000000 to at or about 1 :25000000, at or about 1 : 1000000 to at or about 1 :20000000, at or about 1 : 1000000 to at or about 1 : 15000000, at or about 1 : 1000000 to at or about 1 : 10000000, at or about 1 : 1000000 to at or about 1 : 5000000, at or about 1 : 1000000 to at or about 1 :4000000, at or about 1 : 1000000 to at or about 1 :3000000, at or about 1 : 1000000 to at
  • the ratio of ctDNA:cfDNA molecules is from at or about 1:10 to about 1 : 100, 1 : 10 to about 1 : 1000, 1 : 10 to about 1 : 10000, 1 : 10 to about 1 :20000, 1 : 10 to about 1:30000, 1:10 to about 1:40000, 1:10 to about 1:50000, 1:10 to about 1:60000, 1:10 to about 1:70000, 1:10 to about 1:80000, 1:10 to about 1:90000, 1:10 to about 1:100000, from 2:10 to about 2:100, 2:10 to about 2: 1000, 2:10 to about 2:10000, 2:10 to about 2:20000, 2:10 to about 2:30000, 2:10 to about 2:40000, 2:10 to about 2:50000, 2:10 to about 2:60000, 2:10 to about 2:70000, 2:10 to about 2:80000, 2:10 to about 2:90000, 2:10 to about 2:100000, from 3:10 to about 3:100, 3
  • the ratio of ctDNA:cfDNA molecules is from at or about 1:10 to about 1 : 100, 1 : 10 to about 1 : 1000, 1 : 10 to about 1 : 10000, 1 : 10 to about 1 :20000, 1 : 10 to about 1:30000, 1:10 to about 1:40000, 1:10 to about 1:50000, 1:10 to about 1:60000, 1:10 to about 1:70000, 1:10 to about 1:80000, 1:10 to about 1:90000, 1:10 to about 1:100000, from 2:10 to about 2:100, 2:10 to about 2: 1000, 2:10 to about 2:10000, 2:10 to about 2:20000, 2:10 to about 2:30000, 2:10 to about 2:40000, 2:10 to about 2:50000, 2:10 to about 2:60000, 2:10 to about 2:70000, 2:10 to about 2:80000, 2:10 to about 2:90000, 2:10 to about 2:100000, from 3:10 to about 3:100, 3
  • the ratio of ctDNA:cfDNA molecules is at least or at least about 2:10000, 3:10000, 4:10000, 5:10000, 6:10000, 7:10000, 8:10000, 9:10000, 1:15000, 2:15000, 3:15000, 4:15000, 5:15000, 6:15000, 7:15000, 8:15000, 9:15000, 1:100000, 2:100000, 3:100000, 4:100000, 5:100000, 6:100000, 7:100000, 8:100000, 9:100000, 1:150000, 2:150000, 3:150000, 4:150000, 5:150000, 6:150000, 7:150000, 8:150000, 9:150000, 1:500000, 2:500000, 3:500000, 4:500000, 5:500000, 6:500000, 7:500000, 8:500000, 9:500000, 1:750000, 2:750000, 3:750000, 4:750000, 5:750000, 6:750
  • the ratio of ctDNA:cfDNA molecules is at least or at least about 2:10000, 3:10000, 4:10000, 5:10000, 6:10000, 7:10000, 8:10000, 9:10000, 1:15000, 2:15000, 3:15000, 4:15000, 5:15000, 6:15000, 7:15000, 8:15000, 9:15000, 1:100000, 2:100000, 3:100000, 4:100000, 5:100000, 6:100000, 7:100000, 8:100000, 9:100000, 1:150000, 2:150000, 3:150000, 4:150000, 5:150000, 6:150000, 7:150000, 8:150000, 9:150000, 1:500000, 2:500000, 3:500000, 4:500000, 5:500000, 6:500000, 7:500000, 8:500000, 9:500000, 1:750000, 2:750000, 3:750000, 4:750000, 5:750000, 6:750
  • the method comprises treating the subject with a second treatment for cancer by administering to the subject an effective amount of a first treatment for cancer and a second treatment for cancer.
  • the subject is identified as a candidate for a second treatment for cancer by measurement of the levels of ctDNA in a sample from the subject.
  • levels of ctDNA in the sample that are above a detection threshold indicate that the subject is a candidate for a second treatment for cancer.
  • At least 30%, at least 35%, at least 40% or at least 50% of subjects treated according to the method achieve complete remission (CR); and/or at least about 40%, at least about 50%, at least about 60% or at least about 70% of the subjects treated according to the method achieve an objective response (OR).
  • at least or at least about 50% of subjects, at least or at least about 60% of the subjects, at least or at least about 70% of the subjects, at least or at least about 80% of the subjects or at least or at least about 90% of the subjects treated according to the method achieve CR and/or achieve an objective response (OR).
  • criteria assessed for effective treatment includes overall response rate (ORR; also known in some cases as objective response rate), complete response (CR); also known in some cases as complete remission), duration of response (DOR), progression-free survival (PFS), and/or overall survival (OS).
  • ORR overall response rate
  • CR complete response
  • DOR duration of response
  • PFS progression-free survival
  • OS overall survival
  • At least 40% or at least 50% of subjects treated according to the methods provided herein achieve complete remission (CR; also known in some cases as complete response), exhibit progression- free survival (PFS) and/or overall survival (OS) of greater than at or about 3 months, 6 months or 12 months or greater than 13 months or approximately 14 months; on average, subjects treated according to the method exhibit a median PFS or OS of greater than at or about 6 months, 12 months, or 18 months; and/or the subject exhibits PFS or OS following therapy for at least at or about 6, 12, 18 or more months or longer.
  • CR complete remission
  • PFS progression- free survival
  • OS overall survival
  • Lugano criteria include evaluation by imaging, tumor bulk measurements, and assessments of spleen, liver, and bone marrow involvement.
  • the response assessed using the Lugano criteria involves the use of positron emission tomography (PET)-computed tomography (CT) and/or CT as appropriate for imaging evaluation.
  • PET positron emission tomography
  • CT computed tomography
  • PET-CT evaluations can further comprise the use of fluorodeoxyglucose (FDG), to assess FDG uptake, in FDG-avid lymphomas.
  • FDG fluorodeoxyglucose
  • a 5-point scale can be used.
  • the 5-point scale comprises the following criteria: 1, no uptake above background; 2, uptake ⁇ mediastinum; 3, uptake > mediastinum but ⁇ liver; 4, uptake moderately > liver; 5, uptake markedly higher than liver and/or new lesions; X, new areas of uptake unlikely to be related to lymphoma.
  • a complete response at the end of treatment involves a complete metabolic response and a complete radiologic response at various measurable sites.
  • these sites include lymph nodes and extra lymphatic sites, wherein a CR is described as a score of 1, 2, or 3 with or without a residual mass on the 5- point scale, when PET-CT is used.
  • uptake can be greater than normal mediastinum and/or liver.
  • complete metabolic response can be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake.
  • a response is assessed in the lymph nodes using CT, wherein a CR is described as no extra lymphatic sites of disease and target nodes/nodal masses must regress to ⁇ 1.5 cm in longest transverse diameter of a lesion (LDi).
  • Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate a lack of evidence of FDG-avid disease in marrow and a CT-based assessment should indicate a normal morphology, which if indeterminate should be H4C negative. Further sites of assessment can include assessment of organ enlargement, which should regress to normal.
  • CR rate can be defined as the incidence of a CR per the Lugano Classification.
  • a complete response as described using the Lugano criteria involves a complete metabolic response and a complete radiologic response at various measurable sites.
  • these sites include lymph nodes and extra-lymphatic sites, wherein a CR is described as a score of 1, 2, or 3 with or without a residual mass on the 5-point scale, when PET-CT is used.
  • uptake in Waldeyer’s ring or extra-nodal sites with high physiologic uptake or with activation within spleen or marrow (e.g., with chemotherapy or myeloid colony-stimulating factors), uptake can be greater than normal mediastinum and/or liver. In this circumstance, complete metabolic response can be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake. In some aspects, a response is assessed in the lymph nodes using CT, wherein a CR is described as no extralymphatic sites of disease and target nodes/nodal masses must regress to ⁇ 1.5 cm in longest transverse diameter of a lesion (LDi).
  • CT wherein a CR is described as no extralymphatic sites of disease and target nodes/nodal masses must regress to ⁇ 1.5 cm in longest transverse diameter of a lesion (LDi).
  • PET-CT-based assessment can indicate a lack of evidence of FDG-avid disease in marrow and a CT-based assessment should indicate a normal morphology, which if indeterminate should be IHC negative.
  • Further sites can include assessment of organ enlargement, which should regress to normal.
  • non-measured lesions and new lesions are assessed, which in the case of CR should be absent (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).
  • a partial response (PR; also known in some cases as partial remission) as described using the Lugano criteria involves a partial metabolic and/or radiological response at various measurable sites.
  • these sites include lymph nodes and extralymphatic sites, wherein a PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used.
  • PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used.
  • findings can indicate responding disease.
  • At the end of treatment such findings can indicate residual disease.
  • response is assessed in the lymph nodes using CT, wherein a PR is described as >50% decrease in SPD of up to 6 target measurable nodes and extra-nodal sites.
  • 5 mm x 5 mm is assigned as the default value; if the lesion is no longer visible, the value is 0 mm x 0 mm; for a node >5 mm x 5 mm, but smaller than normal, actual measurements are used for calculation.
  • Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate residual uptake higher than uptake in normal marrow but reduced compared with baseline (diffuse uptake compatible with reactive changes from chemotherapy allowed).
  • consideration should be given to further evaluation with MRI or biopsy, or an interval scan.
  • further sites can include assessment of organ enlargement, where the spleen must have regressed by >50% in length beyond normal.
  • non-measured lesions and new lesions are assessed, which in the case of PR should be absent/normal, regressed, but no increase.
  • No response/ stable disease (SD) or progressive disease (PD) can also be measured using PET-CT and/or CT based assessments.
  • progression-free survival is described as the length of time during and after the treatment of a disease, such as cancer, that a subject lives with the disease but it does not get worse.
  • objective response is described as a measurable response.
  • objective response rate is described as the proportion of patients who achieved CR or PR.
  • overall survival is described as the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that subjects diagnosed with the disease are still alive.
  • event-free survival is described as the length of time after treatment for a cancer ends that the subject remains free of certain complications or events that the treatment was intended to prevent or delay. These events can include the return of the cancer or the onset of certain symptoms, such as bone pain from cancer that has spread to the bone, or death.
  • the measure of duration of response includes the time from documentation of tumor response to disease progression.
  • the parameter for assessing response can include durable response, e.g., response that persists after a period of time from initiation of therapy.
  • durable response is indicated by the response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months after initiation of therapy.
  • the response is durable for greater than 1 week, 2 weeks, three weeks, four weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or greater than 6 months.
  • the Response Evaluation Criteria in Solid Tumors is used to determine objective tumor response; in some aspects, in solid tumors.
  • the RECIST criteria is used to determine objective tumor response for target lesions.
  • a complete response as determined using RECIST criteria is described as the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) must have reduction in short axis to ⁇ 10 mm.
  • a partial response as determined using RECIST criteria is described as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
  • progressive disease is described as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (in some aspects, the appearance of one or more new lesions is also considered progression).
  • stable disease is described as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
  • Objective Response Rate as the incidence of either a CR or a partial response (PR) per the Lugano Classification
  • Duration of Response DOR, for participants who experience an objective response and is the time from the first objective response to disease progression per the Lugano Classification
  • Progression-Free Survival PFS, as the time from the infusion date to the date of disease progression per Lugano Classification
  • Overall survival is defined as the proportion of subjects who achieve a response of PR or CR, prior to initiation of subsequent therapy.
  • Time to response is defined among responders, as the time between first dose (from day 1, cycle 1) of the treatment such as a CD19-CAR-T therapy and the initial documentation of PR or CR.
  • Duration of response is defined among responders, as the time from the initial documentation of PR or CR to the date of disease progression or death, whichever occurs earlier.
  • Progression-free survival is defined as the time from the first dosing date (day 1, cycle 1) of the treatment such as a CD19-CAR-T therapy and the date of disease progression or death, whichever occurs earlier.
  • OS Overall survival
  • TTNT Time to next anti- lymphoma therapy
  • exemplary ctDNA can be assessed or analyzed with respect to assessment of likelihood of durable response after administration of a cell therapy.
  • the subject is likely to achieve a durable response if the ratio of ctDNA:cfDNA is below a detection threshold ratio of ctDNA:cfDNA molecules and/or the subject is not likely to achieve a durable response if the ratio of ctDNA:cfDNA is above a detection threshold ratio of ctDNA:cfDNA molecules.
  • the durable response is or comprises a complete response (CR) or partial response (PR) that is durable for at or greater than 3 months, 4 months, 5 months, or 6 months. In some embodiments, the durable response is or comprises a CR or PR that is durable for at least 3 months. In some aspects, increased levels of ctDNA in a biological sample from a subject obtained prior to administration of a cell therapy (pre-treatment), can be associated with not achieving durable response, such as a CR or PR that is durable for at least 3 months.
  • undetected or decreased levels of ctDNA, in a biological sample from a subject obtained prior to or subsequent to administration of a cell therapy (pre-treatment), can be associated with achieving durable response, such as a CR or PR that is durable for at least 3 months.
  • methods provided herein generally reduce or prevent the expansion or burden of the disease or condition in the subject.
  • the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable cancer and/or improve prognosis or survival or other symptom associated with tumor burden.
  • Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis.
  • tumor cells can be detected and/or quantified in the blood or bone marrow in the context of certain hematological malignancies.
  • Disease burden can include, in some embodiments, the mass of a tumor, the number or extent of metastases and/or the percentage of blast cells present in the bone marrow.
  • a subject has leukemia.
  • the extent of disease burden can be determined by assessment of residual leukemia in blood or bone marrow.
  • the method reduces the burden of the disease or condition, e.g., number of tumor cells, size of tumor, duration of patient survival or event-free survival, to a greater degree and/or for a greater period of time as compared to the reduction that would be observed without the method.
  • the burden of a disease or condition in the subject is detected, assessed, or measured.
  • Disease burden can be detected in some aspects by detecting the total number of disease or disease-associated cells, e.g., tumor cells, in the subject, or in an organ, tissue, or bodily fluid of the subject, such as blood or serum.
  • survival of the subject survival within a certain time period, extent of survival, presence or duration of event-free or symptom-free survival, or relapse-free survival, is assessed.
  • any symptom of the disease or condition is assessed.
  • the measure of disease or condition burden is specified.
  • the event-free survival rate or overall survival rate of the subject is improved by the methods.
  • event-free survival rate or probability for subjects treated by the methods at 6 months following the treatment or cell therapy is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%.
  • overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%.
  • the subject treated with the methods exhibits event-free survival, relapse-free survival, or survival to at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the time to progression is improved, such as a time to progression of greater than at or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the probability of relapse is reduced as compared to other methods, for example, methods in which the subject receives one or more alternative therapeutic agents and/or one in which the subject does not receive the cell therapy in accord with the provided methods.
  • the probability of relapse at 6 months following the treatment is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.
  • MRD negativity rate is defined as the proportion of subjects with at least 1 undetectable MRD result according to the specific threshold, prior to initiation of subsequent therapy.
  • a subject can exhibit complete remission, but a small proportion of morphologically undetectable (by light microscopy techniques) residual leukemic cells are present.
  • a subject is said to exhibit minimum residual disease (MRD) if the subject exhibits less than 5% blasts in the bone marrow and exhibits molecularly detectable cancer.
  • IP I International Prognostic Index
  • IP I International Prognostic Index
  • LDH serum lactate dehydrogenase
  • SC Eastern Cooperative Oncology Group
  • NCCN-IPI An enhanced International Prognostic Index
  • the IPI system can identify four risk categories as low, low-intermediate, high-intermediate.
  • the revised IPI score uses the same prognostic variables but re-distributes the stratification into 3 prognostic groups: very good (94% 4-year OS), good (79% 4-year OS) and poor (55% 4-year OS).
  • NCCN-IPI International Prognostic Index National Comprehensive Cancer Network
  • the NCCN-IPI score was developed to improve risk stratification of patients with DLBCL. Five variables (age, LDH, stage, extra-nodal disease and performance status) were found to significantly associate with prognosis.
  • the score stratifies patients into four risk groups: low (0-1), low-intermediate (2- 3), high-intermediate (4-5) and high (6-8).
  • the NCCN-IPI is able to discriminate between low and high risk groups (5-year OS 96% vs 33%) than the IPI (5-year OS 90% vs 54%).
  • survival rates in subjects are based on scoring systems of IPI.
  • IPI score is based on the independent prognostic roles of age, histology, cancer stage, and serum LDH levels.
  • scores can be used to characterize or predict overall survival rates of subjects with cancer described herein.
  • the present disclosure provides a cancer, such as DLBCL, risk assessment using the International Prognostic Index (IPI) for predicting outcomes.
  • IPI International Prognostic Index
  • a patient with zero or one risk factor is considered to be in an IPI low risk group.
  • a patient with two risk factors is considered to be in an IPI low- intermediate risk group.
  • a patient with three risk factors is considered to be in an IPI high-intermediate risk group.
  • a patient with four or five risk factors is considered to be in an IPI high risk group.
  • a higher IPI score is predictive of a worse outcome compared to a lower IPI score, and treatment of patients with higher IPI scores typically is less successful than treating patients with lower IPI scores.
  • parameters such as attributes, factors, characteristic of the patient and/or the disease or condition, and/or expression of the ctDNA described herein, are assessed prior to administration of the therapy, e.g., cell therapy.
  • the parameters such as attributes, factors, characteristic of the patient and/or the disease or condition, and/or expression of the ctDNA described, are assessed after administration of the therapy, e.g., cell therapy.
  • the parameters include levels or measurements, e.g., peak levels, of attributes, factors, characteristic of the patient and/or the disease or condition, and/or expression of the ctDNA described, that can be assessed after administration of the therapy, e.g., cell therapy.
  • the parameter is SPD.
  • a volumetric measure of tumor burden is measured and the volumetric measure is a sum of the products of diameters (SPD).
  • tumor burden is correlated with the SPD value that is above a threshold value. In some embodiments, higher or lower disease and/or tumor burden is correlated with SPD.
  • the volumetric measure is SPD and the threshold value is or is about 30 per cm 2 , is or is about 40 per cm 2 , is or is about 50 per cm 2 , is or is about 60 per cm 2 , or is or is about 70 per cm 2 .
  • the volumetric measure is SPD, and the threshold value is or is about 30 per cm 2 , is or is about 40 per cm 2 , is or is about 50 per cm 2 , is or is about 60 per cm 2 , or is or is about 70 per cm 2 . In some embodiments, the volumetric measure is SPD and the threshold value is or is about 30 per cm 2 , is or is about 40 per cm 2 , is or is about 50 per cm 2 , is or is about 60 per cm 2 , or is or is about 70 per cm 2 .
  • the parameter is an inflammatory marker such as LDH.
  • the LDH is assessed using a colorimetric test or an in vitro enzyme-linked immunosorbent assay.
  • LDH levels can be assessed alone and/or in combination with another pre-treatment parameter, such as another measure or indicator of disease burden, such as a volumetric tumor measurement such as sum of product dimensions (SPD).
  • the one or more parameters include LDH and/or a volumetric tumor measurement.
  • the parameter is SPD and/or LDH.
  • elevated levels or measure of a sum of the products of diameters (SPD), LDH in a biological sample from a subject obtained prior to administration of a cell therapy (pre-treatment), can be associated with a higher risk of selecting, treating or delaying progression of cancer.
  • the inflammatory marker is LDH and the threshold value is or is about 200 units per liter, is or is about 300 units per liter, is or is about 400 units per liter, is or is about 500 units per liter or is or is about 600 units per liter.
  • the level, concentration and/or number of LDH is a surrogate for disease burden, e.g., for tumors or cancers, and can be useful for potential neurotoxicity risk assessment and/or risk-adapted dosing or adjustment of treatment of certain subjects.
  • LDH levels can be assessed alone and/or in combination with another pre-treatment parameter, such as another measure or indicator of disease burden, such as a volumetric tumor measurement such as sum of product dimensions (SPD) or other CT-based or MRI-based volumetric measurement of disease burden, such as any described herein.
  • one or more parameters indicative of disease burden are assessed, and in some contexts can indicate the presence, absence or degree of risk of developing neurotoxicity following the T cell therapy.
  • the one or more parameters include LDH and/or a volumetric tumor measurement.
  • the parameter is SPD and/or LDH.
  • the presence, absence or level, amount, concentration and/or other measure of LDH is detected or determined in a sample.
  • Various methods of detecting or determining LDH are known. For example, an assay which measures LDH conversion of lactate to pyruvate through NAD+ reduction to NADH can be used to detect LDH in the sample.
  • the sample is contacted with lactate in the presence of coenzyme NAD which, as a measure of LDH in the sample, results in NADH that is then oxidized in the presence of an electron transfer agent.
  • the NADH interacts with a probe or dye precursor that is detectable by measuring absorption in a visible light range.
  • diaphorase uses the NADH to reduce tetrazolium salt (INT) to a red formazan product and the product is measured. Therefore, in some embodiments, the amount of colored product formed is directly proportional to the LDH activity in the sample.
  • INT tetrazolium salt
  • administration of a cell therapy described herein can result in changes in parameters, e.g., reduced volumetric measures, e.g., SPD, or expression of inflammatory markers, e.g., LDH.
  • reduced volumetric measures e.g., SPD
  • expression of inflammatory markers e.g., LDH.
  • Disclosed herein are methods for diagnosing, assessing, screening, or monitoring a subject that has cancer, or has been treated for cancer. In some aspects provided herein are methods of detecting or assessing the progression of a cancer, such as a cancer provided herein, in a subject.
  • provided herein are methods for assessing or predicting the therapeutic response of a subject that has been administered a treatment for cancer, methods for assessing or predicting the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer, methods for predicting survival of a subject that has been administered a treatment for cancer, methods of identifying Minimal Residual Disease (MRD) in a subject that has been diagnosed with cancer, and methods of monitoring a subject being treated for cancer.
  • MRD Minimal Residual Disease
  • provided herein are methods of assessing the likelihood of a durable response of a subject that has been administered a treatment for cancer, and methods of assessing the tumor burden of a subject that has been administered a treatment for cancer.
  • Provided herein are also methods for treating a subject with a second treatment for cancer, wherein the subject has been administered a first treatment for cancer.
  • the methods comprise acquiring knowledge of the presence or absence of a ctDNA provided herein in a sample obtained from the subject. In some embodiments, the methods comprise detecting a ctDNA provided herein in a sample obtained from the individual. In some embodiments, the methods further comprise providing an assessment of the levels of ctDNA. In some embodiments, the diagnosis or assessment identifies the presence or absence of the ctDNA in the sample. In some embodiments, the diagnosis or assessment identifies the cancer, such as a cancer provided herein, as likely to respond to a treatment, such as a cell therapy provided herein. In some embodiments, the sample is a sample described herein.
  • the subject has a cancer, is suspected of having a cancer, is being tested for a cancer, is being treated for a cancer, or is being tested for a susceptibility to a cancer described herein.
  • methods of diagnosing or assessing a cancer in a subject comprise detecting a ctDNA provided herein in a sample obtained from the subject, e.g., a sample comprising cells from the cancer.
  • provided herein is a method for assessing or predicting the therapeutic response of a subject that has been administered a treatment for cancer.
  • the method comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the treatment for cancer.
  • a method for predicting the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject during administration of the treatment for cancer.
  • the method comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the treatment for cancer.
  • provided herein is a method for predicting survival of a subject that has been administered a treatment for cancer.
  • the method comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the treatment for cancer.
  • ctDNA circulating tumor DNA
  • the method comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the treatment for cancer.
  • the method comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the treatment for cancer.
  • a method for treating a subject with a second treatment for cancer wherein the subject has been administered a first treatment for cancer.
  • the method comprises detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the first treatment for cancer.
  • a method for treating a subject with a second treatment or a third treatment for cancer wherein the subject has been administered a first treatment for cancer.
  • the method comprising detecting the levels of circulating tumor DNA (ctDNA) in the subject after administration of the first treatment for cancer.
  • the sample is a sample described herein.
  • the sample comprises a biological sample from the subject diagnosed with a cancer, such as a blood, serum or a plasma sample.
  • levels of ctDNA in the sample that are above a detection threshold indicate that the subject has higher-risk disease. In other embodiments, levels of ctDNA in the sample that are above a detection threshold indicate that the subject has Minimal Residual Disease (MRD). In other embodiments, levels of ctDNA that are above a detection threshold indicate that the subject has a risk of recurrence or relapse. In other embodiments, levels of ctDNA that are above a detection threshold indicate that the subject has a low chance of survival. In other embodiments, levels of ctDNA that are above a detection threshold indicate that the subject is not responsive to treatment.
  • MRD Minimal Residual Disease
  • the detection threshold is a ratio of ctDNA: cfDNA that is or is about 1 : 10, 1 : 100, 1 : 10 3 , 1 : 10 4 , 1 : 10 5 , 1 : 10 6 , 1 : 10 7 , 1 : 10 8 , 1 : 10 9 , 1 : 10 10 , IMO 11 , or 1 : 10 12 .
  • the detection threshold is a ratio of ctDNA:cfDNA that is or is about 5: 10, 5: 100, 5 : 10 3 , 5 : 10 4 , 5 : 10 5 , 5 : 10 6 , 5 : 10 7 , 5 : 10 8 , 5 : 10 9 , 5 : 10 10 , 5 : 10 11 , or 5 : 10 12 .
  • a method for determining the Minimal Residual Disease (MRD) status in a subject that has been diagnosed with cancer comprising:(a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; (c) determining the ratio of ctDNA: cfDNA molecules in the sample; (d) determining the MRD status of the subject from the ratio of ctDNA:cfDNA molecules in the sample; wherein the subject has MRD if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject does not have MRD if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • MRD Minimal Residual Disease
  • the subject has MRD if the ratio is above l : 10 4 , l : 10 5 , l :10 6 , l : 10 7 , l :10 8 , l : 10 9 , l : 10 10 , 1M0 11 , or l : 10 12 ctDNA: cfDNA molecules and the subject does not have MRD if the ratio is below l : 10 4 , l : 10 5 , l : 10 6 , l :10 7 , l : 10 8 , l : 10 9 , l : 10 10 , 1M0 11 , or l :10 12 ctDNA:cfDNA molecules.
  • the method comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject and measuring the levels of cfDNA in the sample and measuring the levels of cell- free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA, determining the ratio of ctDNA:cfDNA molecules in the sample and determining the MRD status of the subject from the ratio of ctDNA:cfDNA molecules in the sample.
  • the subject has MRD if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject does not have MRD if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • the method comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free tumor DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA: cfDNA molecules in the sample and assessing the therapeutic response of the subject from the ratio of ctDNA:cfDNA molecules in the sample.
  • the subject is responsive to the treatment if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules and the subject is not responsive to the treatment if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is responsive to the treatment if the ratio is below l : 10 4 , l : 10 5 , l : 10 6 , l : 10 7 , l : 10 8 , l : 10 9 , l :10 10 , l:I0 n , or l:10 12 ctDNA:cfDNA molecules and the subject is not responsive to the treatment if the ratio is above l : 10 4 , l : 10 5 , l :10 6 , l : 10 7 , l :10 8 , l : 10 9 , l : 10 10 , IMO 11 , or l : 10 12 ctDNA:cfDNA molecules.
  • the subject is responsive to the treatment if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules. In some embodiments, the subject is not responsive to the treatment if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is responsive to the treatment if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1.0: 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • the method comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the method provides for determining the ratio of ctDNA: cfDNA molecules in the sample and assessing the risk of cancer recurrence or relapse in the subject from the ratio of ctDNA:cfDNA molecules in the sample.
  • the subject is at risk of cancer recurrence or relapse if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules and the subject is not at risk of cancer recurrence or relapse if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is at risk of cancer recurrence or relapse if the ratio is above 1 : 10 6 ctDNA:cfDNA molecules. In some embodiments, the subject is not at risk of cancer recurrence or relapse if the ratio is below 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is at risk of cancer recurrence or relapse if the ratio is above 1 : 10 4 , l : 10 5 , l : 10 6 , l :10 7 , l : 10 8 , l :10 9 , l : 10 10 , lilO 11 , or l :10 12 ctDNA:cfDNA molecules.
  • the subject is at risk of cancer recurrence or relapse if the ratio is above 1 : 10 4 , 1 : 10 5 , 1 : 10 6 , 1 :10 7 , 1 : 10 8 , 1 :10 9 , 1 : IO 10 , lilO 11 , or l :10 12 ctDNA:cfDNA molecules.
  • the subject is at risk of cancer recurrence or relapse if the ratio is from at or about 1 : 10 5 to at or about 1 : 10 8 , from at or about 1 : 10 5 to at or about 1.0: 10 7 , from at or about 1 : 10 5 to at or about 1.0: 10 6 , from at or about 1 : 10 6 to at or about 1.0: 10 8 , from at or about 1 : 10 6 to at or about 1.0: 10 7 , from at or about 5: 10 6 to at or about 1.0: 10 8 , from at or about 5: 10 6 to at or about 1.0: 10 7 , from at or about 10: 10 6 to at or about 1.0: 10 8 is above ctDNA:cfDNA molecules.
  • kits for predicting or assessing survival of a subject having a cancer are provided herein.
  • the levels of cell-free DNA (cfDNA) and cell- free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ctDNA:cfDNA molecules in the sample is determined.
  • the subject is identified as having a low chance of survival if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is identified as having a high chance of survival if the ratio of ctDNA:cfDNA is above 1 : 10 6 ctDNA:cfDNA molecules.
  • the subject is identified as having a low chance of survival if the ratio of ctDNA:cfDNA is above 1 : 10 4 , 1 : 10 5 , 1 : 10 6 , 1 : 10 7 , 1 : 10 8 , 1 : 10 9 , l :10 10 , l : 10 n , or 1 : 10 12 ctDNA:cfDNA molecules.
  • the method comprises administering to the subject an effective amount of a treatment for cancer.
  • the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ratio of ctDNA:cfDNA molecules in the sample is determined.
  • the method for assessing the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer comprises isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the levels of cfDNA in the sample and measuring the levels of cell-free tumor DNA (ctDNA) in the sample.
  • cfDNA cell-free DNA
  • the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA.
  • the mutation is a phased variant.
  • the ctDNA is used to determine a Minimal Residual Disease (MRD) status of the subject.
  • MRD Minimal Residual Disease
  • the subject is determined to be at risk of cancer recurrence or relapse if MRD is detected. In some embodiments, the subject is determined to be at risk of cancer recurrence or relapse if MRD is detected after two cycles of an induction phase of the treatment.
  • the subject is being treated with a cell therapy described herein.
  • the methods comprise acquiring knowledge of a ctDNA provided herein in a sample from the subject.
  • the methods comprise detecting a ctDNA in a sample from the subject.
  • responsive to acquiring knowledge of a ctDNA in the sample the subject is predicted to have longer survival after treatment with a cell therapy provided herein, for example, as compared to a subject whose cancer does not exhibit a ctDNA.
  • the subject responsive to detecting a ctDNA provided herein in the sample, the subject is predicted to have longer survival after treatment with cell therapy provided herein, for example, as compared to a subject whose cancer does not exhibit the ctDNA.
  • the methods further comprise providing a diagnosis or an assessment.
  • the diagnosis or assessment identifies the presence or absence of the ctDNA in the sample.
  • the diagnosis or assessment identifies the subject as being predicted to have longer survival after treatment with the cell therapy provided herein, for example, as compared to a subject whose cancer does not exhibit the ctDNA.
  • the sample is a sample described herein.
  • provided herein are methods of screening a subject having cancer, suspected of having cancer, being tested for cancer, being treated for cancer, or being tested for a susceptibility to cancer provided herein.
  • the subject is being treated, or has been treated with a cell therapy described herein.
  • the subject responsive to acquiring knowledge of ctDNA provided herein in the sample, the subject is predicted to have increased risk of cancer recurrence, aggressive cancer, anti-cancer therapy resistance, or poor prognosis, for example, as compared to a subject whose cancer does not exhibit ctDNA.
  • the subject responsive to detecting ctDNA in the sample, is predicted to have increased risk of cancer recurrence, aggressive cancer, anti-cancer therapy resistance, or poor prognosis, for example, as compared to a subject whose cancer does not ctDNA.
  • the methods further comprise providing a diagnosis or an assessment.
  • the diagnosis or assessment identifies the presence or absence of the ctDNA in the sample.
  • the diagnosis or assessment identifies the subject as being predicted to have increased risk of cancer recurrence, aggressive cancer, anti-cancer therapy resistance, or poor prognosis, for example, as compared to a subject whose cancer does not exhibit the ctDNA.
  • the method comprises identifying Minimal Residual Disease (MRD) in a subject that has been diagnosed with cancer.
  • the method comprises detecting the levels of ctDNA in the subject.
  • the detecting comprises obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample and identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • levels of ctDNA in the sample that are above a detection threshold indicate that the subject has MRD.
  • the method comprises assessing the therapeutic response of a subject that has been administered a treatment for cancer.
  • the method comprises detecting the levels of ctDNA in the subject after administration of the treatment for cancer.
  • the detecting comprises obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample and identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • levels of ctDNA in the sample that are below a detection threshold indicate that the subject is responsive to the treatment for cancer.
  • the method comprises assessing the risk of cancer recurrence or relapse in a subject that has been administered a treatment for cancer.
  • the method comprises detecting the levels of ctDNA in the subject during administration of the treatment for cancer.
  • the method comprises detecting the levels of ctDNA in the subject after administration of the treatment for cancer.
  • the detecting comprises obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample and identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • levels of ctDNA in the sample that are above a detection threshold indicate that the subject is at risk of cancer recurrence or relapse.
  • the ratio of ctDNA:cfDNA molecules is a detection threshold ratio of ctDNA:cfDNA molecules. In other embodiments, the ratio of ctDNA:cfDNA molecules is a ratio of ctDNA:cfDNA molecules in a sample of the subject. In some embodiments, the ratio of ctDNA:cfDNA molecules is 1 : 10, 1 : 100, 1 : 1000, l : 10 4 , l : 10 5 , l : 10 6 , l : 10 7 , l : 10 8 .
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 10 6 to at or about 50: 10 6 , from at or about 1 : 10 6 to at or about 40: 10 6 , at or about 1 : 10 6 to at or about 30: 10 6 , at or about 1 : 10 6 to at or about 20: 10 6 , at or about 1 : 10 6 to at or about 10: 10 6 , at or about 1 : 10 6 to at or about 5 : 10 6 , at or about 1 : 10 6 to at or about 2.5 : 10 6 , from at or about 2.5 : 10 6 to at or about 50: 10 6 , from at or about 2.5: 10 6 to at or about 40: 10 6 , at or about 2.5: 10 6 to at or about 30: 10 6 , at or about 2.5: 10 6 to at or about 20: 10 6 , at or about 2.5: 10 6 to at or about 10: 10 6 , at or about 2.5: 10 6 to at or about 5 : 10
  • the ratio of ctDNA:cfDNA molecules is at least or at least about 0.2: 10 6 , 0.3: 10 6 , 0.4: 10 6 , 0.5: 10 6 , 0.6:10 6 , 0.7: 10 6 , 0.8: 10 6 , 0.9: 10 6 , 1 : 10 6 , l.
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 1000000 to at or about 1 : 50000000, at or about 1 : 1000000 to at or about 1 :45000000, at or about 1 : 1000000 to at or about 1 :40000000, at or about 1 : 1000000 to at or about 1 :35000000, at or about 1 : 1000000 to at or about 1 :30000000, at or about 1 : 1000000 to at or about 1 :25000000, at or about 1 : 1000000 to at or about 1 :20000000, at or about 1 : 1000000 to at or about 1 : 15000000, at or about 1 : 1000000 to at or about 1 : 10000000, at or about 1 : 1000000 to at or about 1 : 5000000, at or about 1 : 1000000 to at or about 1 :4000000, at or about 1 : 1000000 to at or about 1 :3000000, at or about 1 : 1000000 to at
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 1000000 to at or about 1 : 50000000, at or about 1 : 1000000 to at or about 1 :45000000, at or about 1 : 1000000 to at or about 1 :40000000, at or about 1 : 1000000 to at or about 1 :35000000, at or about 1 : 1000000 to at or about 1 :30000000, at or about 1 : 1000000 to at or about 1 :25000000, at or about 1 : 1000000 to at or about 1 :20000000, at or about 1 : 1000000 to at or about 1 : 15000000, at or about 1 : 1000000 to at or about 1 : 10000000, at or about 1 : 1000000 to at or about 1 : 5000000, at or about 1 : 1000000 to at or about 1 :4000000, at or about 1 : 1000000 to at or about 1 :3000000, at or about 1 : 1000000 to at
  • the ratio of ctDNA:cfDNA molecules is from at or about 1 : 10 to about 1 : 100, 1 : 10 to about 1 : 1000, 1 : 10 to about 1 : 10000, 1 : 10 to about 1 :20000, 1 : 10 to about 1 :30000, 1 : 10 to about 1 :40000, 1 : 10 to about 1 :50000, 1 : 10 to about 1 :60000, 1 : 10 to about 1 :70000, 1 : 10 to about 1 :80000, 1 : 10 to about 1 :90000, 1 : 10 to about 1 : 100000, from 2: 10 to about 2: 100, 2: 10 to about 2: 1000, 2: 10 to about 2: 10000, 2: 10 to about 2:20000, 2: 10 to about 2:30000, 2: 10 to about 2:40000, 2: 10 to about 2:50000, 2: 10 to about 2:60000, 2: 10 to about 2:70000
  • the ratio of ctDNA:cfDNA molecules is from at or about 1:10 to about 1 : 100, 1 : 10 to about 1 : 1000, 1 : 10 to about 1 : 10000, 1 : 10 to about 1 :20000, 1 : 10 to about 1:30000, 1:10 to about 1:40000, 1:10 to about 1:50000, 1:10 to about 1:60000, 1:10 to about 1:70000, 1:10 to about 1:80000, 1:10 to about 1:90000, 1:10 to about 1:100000, from 2:10 to about 2:100, 2:10 to about 2: 1000, 2:10 to about 2:10000, 2:10 to about 2:20000, 2:10 to about 2:30000, 2:10 to about 2:40000, 2:10 to about 2:50000, 2:10 to about 2:60000, 2:10 to about 2:70000, 2:10 to about 2:80000, 2:10 to about 2:90000, 2:10 to about 2:100000, from 3:10 to about 3:100, 3
  • the ratio of ctDNA:cfDNA molecules is at least or at least about 2:10000, 3:10000, 4:10000, 5:10000, 6:10000, 7:10000, 8:10000, 9:10000, 1:15000, 2:15000, 3:15000, 4:15000, 5:15000, 6:15000, 7:15000, 8:15000, 9:15000, 1:100000, 2:100000, 3:100000, 4:100000, 5:100000, 6:100000, 7:100000, 8:100000, 9:100000, 1:150000, 2:150000, 3:150000, 4:150000, 5:150000, 6:150000, 7:150000, 8:150000, 9:150000, 1:500000, 2:500000, 3:500000, 4:500000, 5:500000, 6:500000, 7:500000, 8:500000, 9:500000, 1:750000, 2:750000, 3:750000, 4:750000, 5:750000, 6:750
  • the ratio of ctDNA:cfDNA molecules is at least or at least about 2:10000, 3:10000, 4:10000, 5:10000, 6:10000, 7:10000, 8:10000, 9:10000, 1:15000, 2:15000, 3:15000, 4:15000, 5:15000, 6:15000, 7:15000, 8:15000, 9:15000, 1:100000, 2:100000, 3:100000, 4:100000, 5:100000, 6:100000, 7:100000, 8:100000, 9:100000, 1:150000, 2:150000, 3:150000, 4:150000, 5:150000, 6:150000, 7:150000, 8:150000, 9:150000, 1:500000, 2:500000, 3:500000, 4:500000, 5:500000, 6:500000, 7:500000, 8:500000, 9:500000, 1:750000, 2:750000, 3:750000, 4:750000, 5:750000, 6:750
  • the method comprises assessing the tumor burden of a subject that has been administered a treatment for cancer.
  • the method comprises detecting the levels of ctDNA in the subject after administration of the treatment for cancer.
  • the detecting comprises obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample and identifying a mutation in one or more genes in the cfDNA, wherein the identification of the mutation is indicative of the presence of ctDNA in the sample and measuring the levels of ctDNA in the sample.
  • levels of ctDNA in the sample that are above a detection threshold indicate that the subject is predicted to have a high tumor burden.
  • Quantitative levels of ctDNA can be measured in haploid genome equivalents per milliliter (hGE/mL), determined as the product of total cell-free DNA concentration and the mean allele fraction of somatic mutations, expressed in log scale (log hGE/mL).
  • ctDNA levels are measured over time to determine a rate of change (e.g., a rate of decrease) in ctDNA over time. For instance, the rate at which ctDNA decreases over time after one or more treatments (or during a treatment regimen) may be determined. In some instances, the rate of change is determined on a linear scale.
  • the rate of change is determined on a log-fold scale (e.g., log-fold change in variant allele frequency).
  • the rate at which ctDNA decreases after or during a course of treatment may be indicative/prognostic of progression-free survival or overall survival of the subject.
  • the methods of the invention can be used to treat a cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor from a subject, prevent relapse of a tumor, prevent tumor metastasis, induce remission in a subject, or any combination thereof.
  • the methods induce a complete response. In other embodiments, the methods induce a partial response.
  • the cancer in the method provided herein is a blood cancer. In some embodiments, the cancer is a B cell malignancy.
  • the method can be used to treat a tumor, wherein the tumor is a lymphoma or a leukemia.
  • Lymphoma and leukemia are cancers of the blood that specifically affect lymphocytes. All leukocytes in the blood originate from a single type of multipotent hematopoietic stem cell found in the bone marrow. This stem cell produces both myeloid progenitor cells and lymphoid progenitor cell, which then give rise to the various types of leukocytes found in the body.
  • Leukocytes arising from the myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells.
  • Leukocytes arising from the lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias can affect one or more of these cell types in a patient.
  • Lymphomas can be divided into at least two sub-groups: Hodgkin lymphoma and non-Hodgkin lymphoma.
  • Non-Hodgkin Lymphoma (NHL) is a heterogeneous group of cancers originating in B lymphocytes, T lymphocytes or natural killer cells.
  • Diffuse large B cell lymphoma is the most common subtype of NHL, accounting for approximately 30% of NHL cases. It is classified as an aggressive lymphoma with the majority of patients cured with conventional chemotherapy (NCCN guidelines NHL 2014).
  • the cancer includes but are not limited to leukemia, lymphoma, e.g., acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancy is selected from among acute lympho
  • AML acute myeloid (or my
  • the method can be used to treat a lymphoma, wherein the lymphoma is a B cell malignancy.
  • the lymphoma is selected from small cell lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms (e.g., plasma cell myeloma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), Epstein-Barr virus- positive DLBCL, lymphomatoid granulomatosis
  • lymphoma is selected from small cell
  • the lymphoma is large B cell lymphoma (LBCL). In some embodiments, the lymphoma is diffuse large B cell lymphoma (DLBCL). In some embodiments, the cancer is follicular lymphoma (FL).
  • the cancer is refractory to or the cancer has relapsed following one or more of chemotherapy, radiotherapy, immunotherapy (including a T cell therapy and/or treatment with an antibody or antibody-drug conjugate), an autologous stem cell transplant, or any combination thereof.
  • the cancer is refractory diffuse large B cell lymphoma.
  • the subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • the individual or subject is a human.
  • the subject is a human.
  • the subject is a patient, e.g., a human patient having a cancer described herein.
  • the present disclosure relates to anti-cancer therapies.
  • a number of treatments for cancer can be performed, including (but not limited to) surgery, resection, chemotherapy, radiation therapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, and blood transfusion.
  • an anti-cancer and/or chemotherapeutic agent is administered, including (but not limited to) alkylating agents, platinum agents, taxanes, vinca agents, antiestrogen drugs, aromatase inhibitors, ovarian suppression agents, endocrine/hormonal agents, bisphophonate therapy agents and targeted biological therapy agents.
  • Medications include (but are not limited to) cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, zoledronate, t
  • the treatment is a first-line therapy. In some embodiments, the treatment is a second-line therapy. In some embodiments, the treatment comprises further monitoring the subject. In some embodiments, the treatment comprises a cell therapy. In some embodiments, the treatment comprises a CAR-T cell therapy and the CAR-T cell therapy is an anti-CD19 cell therapy. In some embodiments, the cell therapy comprises genetically engineered cells and the genetically engineered cells are T cells. In some embodiments, the genetically engineered T cells comprise a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR specifically binds to an antigen associated with a disease or condition and/or is expressed by cells associated with a disease or condition.
  • CAR Chimeric Antigen Receptor
  • the antigen is selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, a cancer-testes antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD 19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD 123, CD 138, CD171, carcinoembryonic antigen (CEA), CE7, a cyclin, cyclin A2, c-Met, dual antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG- 40), EPHa2, ephrinB2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, estrogen receptor, Fetal AchR, fo
  • the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an IT AM.
  • the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3Q chain.
  • the CAR further comprises a costimulatory signaling region.
  • the cell therapy comprises genetically engineered cells and the genetically engineered cells that can bind to multiple antigens.
  • improved selectivity and specificity is achieved through strategies targeting multiple antigens.
  • Such strategies generally involve multiple antigen-binding domains, which typically are present on distinct genetically engineered antigen receptors and specifically bind to distinct antigens.
  • the cells express multi-specific binding molecules.
  • the cells express multiple binding molecules, antigen-binding domains, each of which can target one antigen or multiple antigens, e.g., one binding domain that targets CD20, such as any described herein, and another binding domain that targets another antigen, e.g., CD 19.
  • the antigen receptor comprises a plurality of binding domains, which bind to different antigens, each expressed in or on the disease or condition to be targeted with the cells or tissues or cells thereof.
  • multi-specific cells such as those containing one or more of any of the recombinant receptors or cells provided herein.
  • the cells express a bispecific recombinant receptor, e.g. a CAR.
  • the costimulatory signaling region comprises a signaling domain of CD28 or 4-1BB. In some embodiments, the costimulatory domain is a domain of 4- 1BB.
  • the T cells are CD4+ or CD8+. In some embodiments, the T cells are primary T cells obtained from a subject. In some embodiments, the genetically engineered cells are autologous to the subject. In some embodiments, the genetically engineered cells are allogeneic to the subject. In some embodiments, the subject is refractory to treatment with one or more prior therapies for the cancer. In some embodiments, the subject achieved an insufficient response to one or more prior therapies for the cancer.
  • the treatment comprises radiographic imaging of the subject.
  • the treatment comprises computed tomography (CT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI) of the subject.
  • CT computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the treatment comprises further monitoring the subject.
  • an anti-cancer therapy of the disclosure includes one or more therapeutic agents, e.g., for treating a disease, disorder, or injury associated with a ctDNA described herein, such as a cancer provided herein.
  • the anti-cancer therapy is a small molecule inhibitor, an antibody, a cellular therapy (i.e., a cell-based therapy), or a nucleic acid.
  • the anti-cancer therapy is a chemotherapeutic agent, an anti-hormonal agent, an antimetabolite chemotherapeutic agent, a kinase inhibitor, a peptide, a gene therapy, a vaccine, a platinum-based chemotherapeutic agent, an immunotherapy, an antibody, or a checkpoint inhibitor.
  • the anti-cancer therapy is a CD19-targeted therapy.
  • the anti-cancer therapy comprises a second agent, such as a second anti-cancer agent.
  • the anti-cancer therapy is administered in combination with a second anti-cancer therapy or agent.
  • the anti-cancer therapy comprises a heat shock protein (HSP) inhibitor, a MYC inhibitor, an HD AC inhibitor, an immunotherapy, a neoantigen, a vaccine, or a cellular therapy.
  • HSP heat shock protein
  • the second anti-cancer agent includes one or more of an immune checkpoint inhibitor, a chemotherapy, a VEGF inhibitor, an Integrin P3 inhibitor, a statin, an EGFR inhibitor, an mTOR inhibitor, a PI3K inhibitor, a MAPK inhibitor, or a CDK4/6 inhibitor.
  • the anti-cancer therapy comprises a kinase inhibitor.
  • the methods provided herein comprise administering to the individual a kinase inhibitor, e.g., in combination with another anti-cancer therapy.
  • the kinase inhibitor is crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, ensartinib (X-396), repotrectinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP-37440, belizatinib (TSR- 011), ASP3026, KRCA-0008, TQ-B3139, TPX-0131, or TAE684 (NVP-TAE684).
  • the anti-cancer therapy comprises a cancer immunotherapy, such as a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy.
  • a cancer immunotherapy such as a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy.
  • the cancer immunotherapy comprises a small molecule, nucleic acid, polypeptide, carbohydrate, toxin, cell-based agent, or cell- binding agent.
  • cancer immunotherapies are described in greater detail herein but are not intended to be limiting.
  • the cancer immunotherapies of the present disclosure are contemplated for use as monotherapies, or in combination approaches comprising two or more in any combination or number, subject to medical judgement. Any of the cancer immunotherapies (optionally as monotherapies or in combination with another cancer immunotherapy or other therapeutic agent described herein) can find use in any of the methods described herein.
  • the cancer immunotherapy comprises a cell-based therapy. In some embodiments, the cancer immunotherapy comprises a T cell-based therapy. In some embodiments, the cancer immunotherapy comprises an adoptive therapy, e.g., an adoptive T cellbased therapy. In some embodiments, the T cells are autologous or allogeneic to the recipient. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells.
  • adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) cancer cells.
  • the immune response results in inhibition of tumor and/or metastatic cell growth and/or proliferation, and in related embodiments, results in neoplastic cell death and/or resorption.
  • the immune cells can be derived from a different organism/host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells).
  • the immune cells e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, or NKT cells) can be genetically engineered to express antigen receptors such as engineered TCRs and/or chimeric antigen receptors (CARs).
  • the host cells e.g., autologous or allogeneic T-cells
  • TCR T cell receptor
  • NK cells are engineered to express a TCR.
  • the NK cells can be further engineered to express a CAR. Multiple CARs and/or TCRs, such as to different antigens, can be added to a single cell type, such as T cells or NK cells.
  • the anti-cancer therapy comprises a nucleic acid molecule, such as a dsRNA, an siRNA, or an shRNA.
  • the methods provided herein comprise administering to the individual a nucleic acid molecule, such as a dsRNA, an siRNA, or an shRNA, e.g., in combination with another anti-cancer therapy.
  • dsRNAs having a duplex structure are effective at inducing RNA interference (RNAi).
  • the anti-cancer therapy comprises a small interfering RNA molecule (siRNA).
  • siRNAs small interfering RNA molecule
  • dsRNAs and siRNAs can be used to silence gene expression in mammalian cells (e.g., human cells).
  • the treatment in some embodiments is co-administered with one or more additional cancer treatments.
  • exemplary combination therapies and methods are described in published international applications WO 2018/085731, WO 2018/102785, WO 2019/213184, WO 2018/071873, WO 2018/102786, WO 2018/204427, WO 2019/152743, which are incorporated by reference in their entirety.
  • the treatment is administered in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cancer treatment is co-administered with another treatment sufficiently close in time such that the treatments enhance the effect of one or more additional therapeutic agents or vice versa.
  • the cancer treatment is administered prior to one or more additional cancer treatments.
  • the cancer treatment is administered after one or more additional therapeutic agents.
  • the treatment is administered as a follow-on treatment.
  • one or more additional therapeutic agents include a cytokine, such as IL-2, for example, to enhance persistence.
  • the cancer treatment comprises the administration of a chemotherapeutic agent.
  • the cancer treatment comprises the administration of a chemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, for example, to reduce tumor burden.
  • the cancer treatment comprises an induction phase.
  • the induction phase comprises two or more cycles of administration of the chemotherapeutic agent.
  • the induction phase comprises 2, 3, 4, 5, 6, or more cycles of administration of the chemotherapeutic agent.
  • the cancer treatment can comprise an administration of a kinase inhibitor, such as a BTK inhibitor (e.g. ibrutinib or acalibrutinib); an inhibitor or a tryptophan metabolism and/or kynurenine pathway, such as an inhibitor of indoleamine 2,3- dioxygenase-1 (IDO1) (e.g. epacadostat); an immunomodulatory agent, such as an immunomodulatory imide drug (IMiD), including a thalidomide or thalidomide derivative (e.g. lenalidomide or pomnalidomide); or a check point inhibitor, such as an anti-PD-Ll antibody (e.g. durvalumumab).
  • a BTK inhibitor e.g. ibrutinib or acalibrutinib
  • IDO1 indoleamine 2,3- dioxygenase-1
  • IDO1 indoleamine 2,3- dioxygenase-1
  • the cancer treatment is administered as part of a follow-on treatment. In some embodiments, the cancer treatment is administered as a first-line therapy. In some embodiments, the cancer treatment is administered as a second-line therapy. In some embodiments, following the administration of the first-line therapy, the subject is monitored to determine the appropriate type of second-line therapy to be administered to the subject and the appropriate second-line therapy is administered to the subject in need thereof. In some embodiments, the presence or level of a ctDNA disclosed herein can be used to select a candidate treatment.
  • the presence or levels of a ctDNA disclosed herein can be used to determine the success during the course of or after treatment of the first-line, second-line, or both the first and second-line of therapy.
  • the ratio of ctDNA:cfDNA molecules can be used to select a candidate treatment.
  • the ratio of ctDNA:cfDNA molecules can be used to determine the success during the course of or after treatment of the first-line, second-line, or both the first and second-line of therapy.
  • the cancer treatment is administered as a third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy. In some embodiments, the cancer treatment is administered prior to one or more additional therapeutic agents. In some embodiments, the treatment is administered after one or more additional therapeutic agents.
  • the cancer treatment is a cell therapy.
  • the cell therapy e.g., T cell therapy
  • the cell therapy includes administering engineered cells expressing recombinant receptors (e.g., Chimeric Antigen Receptor CAR) designed to recognize and/or specifically bind to antigens associated with the disease or condition, such as a cancer as described herein.
  • the antigen that is bound or recognized by the recombinant receptor e.g., CAR
  • the antigen that is bound or recognized by the recombinant receptor is CD 19.
  • the antigen that is bound or recognized by the recombinant receptor is CD20.
  • binding to the antigen results in a response, such as an immune response against such antigens.
  • the cells contain or are engineered to contain the recombinant receptor, such as a chimeric antigen receptor (CAR).
  • the recombinant receptor such as a CAR, generally includes an extracellular antigen (or ligand) binding domain specific to the antigen that is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s).
  • the engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subjects, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
  • the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids.
  • gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
  • chimeric receptors such as a chimeric antigen receptors, contain one or more domains that combine a ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity for a desired antigen (e.g., CD 19) with intracellular signaling domains.
  • the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or a primary signal.
  • the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions.
  • chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.
  • Exemplary antigen receptors including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S.
  • the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
  • the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also W02014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, and US Patent No.: 8,389,282.
  • the chimeric receptors such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
  • VH variable heavy
  • VL variable light
  • the antigen targeted by the receptor is a polypeptide.
  • the antigen target is CD 19.
  • the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues.
  • the CAR is constructed with a specificity for the particular antigen, such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type.
  • the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigenbinding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules.
  • the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
  • scFv single-chain antibody fragment
  • VH variable heavy
  • VL variable light chains of a monoclonal antibody
  • the antibody or antigen-binding portion thereof is expressed on cells as part of a recombinant receptor, such as a chimeric receptor (e.g. CAR), that binds, such as specifically binds, to the antigen (e.g. CD 19).
  • a recombinant receptor such as a chimeric receptor (e.g. CAR)
  • the antigen targeted by the chimeric receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy.
  • diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
  • cancers and tumors including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
  • the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes the antigen, such as an intact antigen, expressed on the surface of a cell.
  • an antigen-binding fragment e.g. scFv
  • the disease or condition is a B cell malignancy, such as a large B cell lymphoma (e.g., DLBCL) and the antigen is CD19.
  • a B cell malignancy such as a large B cell lymphoma (e.g., DLBCL) and the antigen is CD19.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rlgG fragment antigen binding
  • VH variable heavy chain
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody.
  • the heavy and light chains of an antibody can be full-length or can be an antigenbinding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (scFv)).
  • the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl).
  • the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
  • antibody fragments refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and singledomain VH single antibodies; and multispecific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CDR-H1, CDR-H2, CDR-H3 three CDRs in each heavy chain variable region
  • CDR-L1, CDR-L2, CDR-L3 three CDRs in each light chain variable region
  • “Framework regions” and “FR” are known, in some cases, to refer to the non-CDR portions of the variable regions of the heavy and light chains.
  • FR-H1, FR-H2, FR-H3, and FR-H4 there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.
  • Table 1 lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth.
  • CDR complementary determining region
  • individual specified CDRs e.g., CDR-H1, CDR-H2, CDR-H3
  • CDR-H1, CDR-H2, CDR-H3 individual specified CDRs
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence
  • a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes.
  • specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.
  • FR or individual specified FR(s) e.g., FR-H1, FR-H2, FR-H3, FR-H4
  • FR-H1, FR-H2, FR-H3, FR-H4 FR-H1, FR-H2, FR-H3, FR-H4
  • FR-H1, FR-H2, FR-H3, FR-H4 FR-H4, FR-H3, FR-H4
  • the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method, or other known schemes.
  • the particular amino acid sequence of a CDR or FR is given.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody.
  • the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are or may not be produced by enzyme digestion of a naturally occurring intact antibody.
  • the antibody fragments are scFvs.
  • a “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the CDR residues are derived
  • the antigen or antigen binding domain is CD 19.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19.
  • the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1.
  • the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
  • the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain.
  • the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain comprising an immunoreceptor tyrosinebased activation motif (IT AM).
  • a primary signaling domain a signaling domain that is capable of inducing a primary activation signal in a T cell
  • TCR T cell receptor
  • IT AM immunoreceptor tyrosinebased activation motif
  • the antibody portion of the recombinant receptor e.g., CAR
  • an immunoglobulin constant region such as a hinge region, e.g., an IgG4 hinge region, and/or a CHI/CL and/or Fc region.
  • the constant region or portion is of a human IgG, such as IgG4 or IgGl.
  • the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer.
  • Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US2014/0271635.
  • the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain.
  • the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
  • the intracellular signaling domain comprises an ITAM.
  • the antigen recognition domain e.g. extracellular domain
  • the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain.
  • the antigenbinding component e.g., antibody
  • the antigenbinding component is linked to one or more transmembrane and intracellular signaling domains.
  • a transmembrane domain that naturally is associated with one of the domains in the receptor e.g., CAR
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the linkage is by linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28.
  • the extracellular domain and transmembrane domain can be linked directly or indirectly.
  • the extracellular domain and transmembrane are linked by a spacer, such as any described herein.
  • the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.
  • intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
  • a short oligo- or polypeptide linker for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • secondary cytoplasmic signaling sequences those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal.
  • the CAR includes one or both of such signaling components.
  • the receptor e.g., the CAR
  • the CAR generally includes at least one intracellular signaling component or components.
  • the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.
  • cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
  • the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain.
  • the antigen-binding portion is linked to one or more cell signaling modules.
  • cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains.
  • the receptor e.g., CAR
  • the receptor further includes a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD16.
  • the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor y and CD8, CD4, CD25 or CD 16.
  • the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR.
  • the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors.
  • a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal.
  • the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
  • TCR T cell receptor
  • full activation In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal.
  • a component for generating secondary or co-stimulatory signal is also included in the CAR.
  • the CAR does not include a component for generating a costimulatory signal.
  • an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
  • the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule.
  • the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4- IBB, 0X40, DAP10, and ICOS.
  • the same CAR includes both the activating and costimulatory components.
  • the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain.
  • the T cell costimulatory molecule is CD28 or 41BB.
  • the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain.
  • the intracellular signaling domain comprises a chimeric CD28 and CD137 (4- IBB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
  • the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion.
  • exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
  • the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor.
  • a surrogate marker such as a cell surface marker
  • the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR).
  • the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A.
  • a linker sequence such as a cleavable linker sequence, e.g., T2A.
  • a marker, and optionally a linker sequence can be any as disclosed in published patent application No. W02014031687.
  • the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.
  • the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
  • the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.
  • the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered.
  • the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
  • CARs are referred to as first, second, and/or third generation CARs.
  • a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding;
  • a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD 137;
  • a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.
  • the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.
  • the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.
  • the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge- only spacer.
  • an Ig molecule such as a human Ig molecule
  • an Ig hinge e.g. an IgG4 hinge, such as a hinge- only spacer.
  • the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.
  • an antibody such as an antibody fragment, including scFvs
  • a spacer such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain
  • the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
  • the recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated.
  • the receptor Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an IT AM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition.
  • the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
  • Subjects with relapsed or refractory Large B-Cell Lymphoma were administered therapeutic cell compositions of engineered CD4+ T cells and engineered CD8+ T cells, each expressing the same anti-CD19 chimeric antigen receptor (CAR).
  • Eligible subjects were administered therapeutic T cell compositions containing engineered cells expressing antiCD 19 CARs.
  • the therapeutic T cell compositions administered had been generated by a process including immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples from the individual subjects to be treated.
  • Isolated CD4+ and CD8+ T cells were separately activated and independently transduced with a viral vector (e.g., lentiviral vector) encoding an anti-CD19 CAR followed by separate expansion and cryopreservation of the engineered cell populations in a low volume.
  • the CAR contained an anti-CD19 scFv derived from a murine antibody (variable region derived from FMC63, VL- linker-VH orientation), an immunoglobulin-derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD3-zeta intracellular signaling domain.
  • the viral vector further contained sequences encoding a truncated receptor, which served as a surrogate marker for CAR expression; separated from the CAR sequence by a T2A ribosome skip sequence.
  • the cryopreserved cell compositions were thawed prior to intravenous administration.
  • the therapeutic T cell dose was administered as a defined cell composition by administering a formulated CD4+ CAR+ cell population and a formulated CD8+ CAR+ population administered at a target ratio of approximately 1 : 1.
  • Subjects were administered a single dose of CAR-expressing T cells (each single dose via separate infusions of CD4+ CAR-expressing T cells and CD8+ CAR-expressing T cells, respectively) as follows: a single dose of dose level 1 (DL-1) containing 5 x 10 7 total CAR- expressing T cells or a single dose of dose level 2 (DL-2) containing 1 x 10 8 total CAR- expressing T cells.
  • DL-1 dose level 1
  • DL-2 single dose level 2
  • Exemplary antigen receptors e.g., CARs
  • CARs also include the CARs of FDA-approved products BREYANZI® (lisocabtagene maraleucel), TECARTUSTM (brexucabtagene autoleucel), KYMRIAHTM (tisagenlecleucel), and YESCARTATM (axicabtagene ciloleucel).
  • the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel), TECARTUSTM (brexucabtagene autoleucel), KYMRIAHTM (tisagenlecleucel), YESCARTATM (axicabtagene ciloleucel).
  • the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel, see Sehgal et al., 2020, Journal of Clinical Oncology 38: 15_suppl, 8040; Teoh et al., 2019, Blood 134(Supplement_l):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852).
  • the CAR is the CAR of TECARTUSTM (brexucabtagene autoleucel, see Mian and Hill, 2021, Expert Opin Biol Ther; 21 (4):435-441 ; and Wang et al., 2021, Blood 138(Supplement 1):744).
  • the CAR is the CAR of KYMRIAHTM (tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980- 4991; and Fowler et al., 2022, Nature Medicine 28:325-332).
  • KYMRIAHTM tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980- 4991; and Fowler et al.,
  • the CAR is the CAR of YESCARTATM (axicabtagene ciloleucel, see Neelapu et al., 2017, N Engl J Med 377(26):2531-2544; Jacobson et al., 2021, The Lancet 23(1):P91 -103 ; and Locke et al., 2022, N Engl J Med 386:640-654).
  • kits for detecting a ctDNA molecule of the disclosure comprises a reagent (e.g., one or more oligonucleotides, primers, probes or baits of the present disclosure) for detecting a ctDNA provided herein.
  • the kit comprises a reagent (e.g., one or more oligonucleotides, primers, probes or baits of the present disclosure) for detecting a ctDNA from cfDNA provided herein in a sample.
  • tumor-specific or “tumor-related” in reference to cfDNA refers to differences in DNA sequences of cfDNA in a subject whose cancer formed a tumor, such as a lung cancer patient, when compared to reference DNA, such as when cfDNA is compared to control DNA (gDNA) from a cell that is not a tumor, as described herein.
  • reference DNA such as when cfDNA is compared to control DNA (gDNA) from a cell that is not a tumor, as described herein.
  • tumor- specific can relate to pre-treatment cfDNA when compared to cfDNA collected during or after treatment.
  • biomarker generally refers to any measurable substance taken as a sample from a subject whose presence, absence and/or amount is indicative of some phenomenon. Non-limiting examples of such phenomenon can include a disease state, a condition, or exposure to a compound or environmental condition. In various embodiments described herein, biomarkers can be used for diagnostic purposes (e.g., to diagnose a disease state, a health state, an asymptomatic state, a symptomatic state, etc.). The term “biomarker” can be used interchangeably with the term “marker.”
  • a “subject” is a mammal, such as a human or other animal, and typically is human.
  • the subject e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human.
  • the primate is a monkey or an ape.
  • the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • the subject is a non-primate mammal, such as a rodent.
  • treatment refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
  • “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. In some embodiments, sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late-stage cancer, such as development of metastasis, can be delayed.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that can be predisposed to the disease but has not yet been diagnosed with the disease.
  • the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.
  • an “effective amount” of an agent refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
  • a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment.
  • the therapeutically effective amount can vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
  • the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of lower tumor burden, the prophylactically effective amount in some aspects will be higher than the therapeutically effective amount.
  • a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker.
  • a surface marker refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
  • FMO fluorescence minus one
  • a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker.
  • a surface marker refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
  • FMO fluorescence minus one
  • Example 1 [0386] In the absence of tumor tissue, several studies have shown the utility of plasma cell- free DNA (cfDNA) for blood-based noninvasive genotyping of diverse human tumors, including aggressive lymphomas. Whether these same cfDNA advantages also apply to follicular lymphoma (FL) has not been investigated, and if they could inform noninvasive minimal residual disease (MRD) monitoring in the context of modern regimens inducing deep FL remissions.
  • MRD noninvasive minimal residual disease
  • MRD was analyzed by PhasED-Seq (Foresight Diagnostics). Phased variants (PVs) were genotyped either using baseline blood plasma, or alternatively from FFPE tumor tissue; matched leukocytes were used as a source of constitutional DNA to censor germline variants and clonal hematopoiesis of indeterminate potential (CHIP). Baseline PV genotypes from each baseline source were then used to longitudinally assess MRD serially during IL therapy. Blood timepoints included baseline (pre-treatment), Cycle 2 Day 1 (C2D1), C3D1, C4D1, C5D1, C6D1, at End of Induction (EOI), and serially thereafter. Levels of ctDNA were compared to known FL prognostic factors, including radiographic responses, POD24, and progression-free survival (PFS).
  • PFS progression-free survival
  • PVs were successfully identified from tumor FFPE samples in all patients (61/61), enabling ctDNA MRD assessment by PhasED-Seq.
  • the median number of PVs identified was 530 (IQR 285-1152).
  • ctDNA was detectable in 94% of cases (57/61) using tumor derived PVs, and in 75% of cases using plasma derived PVs.
  • DLBCL diffuse large B-cell lymphoma
  • Circulating tumor DNA analysis with PhasED-Seq is feasible in FL, with baseline ctDNA levels significantly lower than those observed in DLBCL.
  • plasma DNA can frequently be used as a surrogate for tissue samples.
  • ctDNA detection during treatment is strongly associated with eventual treatment responses, and serial disease surveillance with ctDNA can anticipate clinical relapse.

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Abstract

L'invention concerne des procédés de surveillance, d'identification, d'évaluation d'ADN tumoral circulant (ADNct) pour traiter un sujet ou surveiller un sujet atteint d'un cancer, ainsi que des procédés associés et des utilisations associées. Dans certains modes de réalisation, la maladie ou l'affection est un lymphome à cellules B (LCB) récidivant ou réfractaire.
PCT/US2024/053553 2023-10-31 2024-10-30 Surveillance d'adn tumoral circulant (ctadn) Pending WO2025096532A1 (fr)

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WO2022029688A1 (fr) * 2020-08-05 2022-02-10 Inivata Ltd. Méthode hautement sensible de détection d'adn de cancer dans un échantillon
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US20210002728A1 (en) * 2018-02-27 2021-01-07 Cornell University Systems and methods for detection of residual disease
US20190316184A1 (en) * 2018-04-14 2019-10-17 Natera, Inc. Methods for cancer detection and monitoring
WO2022029688A1 (fr) * 2020-08-05 2022-02-10 Inivata Ltd. Méthode hautement sensible de détection d'adn de cancer dans un échantillon
US20220356530A1 (en) * 2021-04-22 2022-11-10 Natera, Inc. Methods for determining velocity of tumor growth

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