Monitoring circulating tumor DNA (ctDNA)

Abstract

Provided herein are methods for monitoring, identifying, evaluating circulating tumor DNA (ctDNA) for treating a subject or monitoring a subject having cancer, and related methods and uses thereof. In some embodiments, the disease or disorder is relapsed or refractory B-cell lymphoma (BCL).

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 546,721, filed October 31, 2023, and U.S. Provisional Application No. 63 / 607,421, filed December 7, 2023, which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates in some respects to methods for assessing treatment response and the risk of relapse or recurrence in subjects treated for cancer by studying the levels of circulating tumor DNA (ctDNA). This disclosure also relates to methods for selecting treatment for subjects by studying the levels of circulating tumor DNA (ctDNA). This document provides methods for treating, monitoring, and surveillance subjects by studying the levels of ctDNA. Background Technology

[0003] Non-invasive methods for assessing disease progression, such as cancer, remain a challenge in the clinical setting. Circulating tumor DNA (ctDNA) represents a promising biomarker for assessing minimal residual disease (MRD) in monitoring disease burden and predicting clinical treatment outcomes. ctDNA analysis has the potential to transform tumor detection and monitoring, as well as early detection of recurrence and acquired resistance, by evaluating tumor DNA to select appropriate treatment methods without the need for invasive tissue biopsy procedures. This invention addresses the need for methods that facilitate regular and more frequent monitoring and surveillance of disease before and after treatment, and for improved treatment regimens. Summary of the Invention

[0004] In some respects, this article provides a method for determining the minimal residual disease (MRD) status of a subject diagnosed with cancer, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) determining the subject's MRD status based on the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule ratio is less than 1:10, then the subject has MRD, and if this ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject does not have MRD.

[0005] In some respects, this article provides a method for assessing the treatment response of a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule is present, then the subject has responded to the treatment, and if this ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject will not respond to the treatment.

[0006] In some respects, this article provides a method for assessing treatment response in a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is below a detection threshold ratio of ctDNA:cfDNA molecules, the subject is responsive to the treatment, and if the ratio is above a detection threshold ratio of ctDNA:cfDNA molecules, the subject is unresponsive to the treatment.

[0007] In other respects, this article provides a method for assessing treatment response in a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ctDNA level is below a detection threshold, the subject is responsive to the treatment, and if the ctDNA level is above a detection threshold, the subject is unresponsive to the treatment.

[0008] In some respects, this article provides a method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the risk of cancer recurrence or relapse in the subject based on the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule is present, the subject is at risk of cancer recurrence or relapse, and if this ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject is not at risk of cancer recurrence or relapse.

[0009] In some respects, this article provides a method for treating a subject with a second cancer treatment, wherein the subject has received a first cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 ctDNA:cfDNA molecules, then a second cancer treatment is administered.

[0010] In some respects, this article provides a method for treating cancer in a subject of need, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 ctDNA:cfDNA molecules, then an effective amount of cancer treatment is administered.

[0011] In some respects, this article provides a method for treating cancer in a subject in need, wherein the subject is evaluated as a candidate for cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA ratio is less than 1:10, the subject is identified as a candidate for cancer treatment. 6 (e) If the ctDNA:cfDNA molecule is used, the subject is identified as a candidate for non-cancer treatment; and (e) an effective amount of cancer treatment is administered to the subject.

[0012] In some respects, this article provides a medicament in a method for treating cancer in a subject of need, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

[0013] In some respects, this article provides the use of cell therapy in the manufacture of a medicament for treating cancer in a subject of need, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

[0014] In some respects, this article provides a method for evaluating the treatment response of a subject who has received cancer treatment, wherein, after administration of cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject has responded to the cancer treatment.

[0015] In some respects, this article provides a method for assessing the risk of cancer recurrence or relapse in subjects who have received cancer treatment, wherein, after administration of cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject is at risk of cancer recurrence or relapse.

[0016] In some respects, this article provides a method for treating cancer in a subject in need, comprising administering an effective amount of cancer treatment to the subject, wherein, prior to administering the cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are used, the subject is identified as a candidate for cancer treatment.

[0017] In some respects, this article provides a method for treating cancer in a subject with a high-risk disease, comprising administering an effective amount of cancer treatment to the subject, wherein, prior to administering the cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are detected, the subject is identified as having a high-risk disease.

[0018] In some embodiments, 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 diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is follicular lymphoma (FL).

[0019] In some implementations, the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute 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), and the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

[0020] In some implementation schemes, cancer is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, stomach cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, central nervous system cancer (CNS cancer), brain tumor, bone cancer, or soft tissue sarcoma.

[0021] In some implementations, the treatment or drug is a first-line therapy. In other implementations, the treatment or drug is a second-line therapy. In some implementations, the treatment includes further monitoring of the subject. In other implementations, the treatment includes radiographic imaging of the subject. In some implementations, the treatment includes computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI) of the subject.

[0022] In some embodiments, the treatment includes cell therapy. In some embodiments, the treatment includes CAR-T cell therapy. In some embodiments, the CAR-T cell therapy is anti-CD19 cell therapy. In some embodiments, the cell therapy includes genetically engineered cells. In some embodiments, the genetically engineered cells are T cells.

[0023] In some embodiments, the genetically engineered T cells include chimeric antigen receptors (CARs). In some embodiments, the CAR specifically binds to an antigen associated with a disease or condition and / or is expressed by cells associated with the disease or condition. In other embodiments, the CAR specifically binds to two antigens associated with a disease or condition and / or is expressed by cells associated with the disease or condition.

[0024] In some implementations, the antigens are selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer-testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, dual antigens, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrinB2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR. vIII, estrogen receptor, fetal AchR, folate receptor α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Ra2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), cancer-fetal antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptor, BAFF-R, VEGF-R2, nephroblastoma 1 (WT-1), and pathogen-specific antigens.

[0025] In some embodiments, the antigen is CD19. In other embodiments, the antigen is CD20.

[0026] In some implementations, the CAR includes an extracellular antigen recognition domain that specifically binds to the antigen, and an intracellular signal transduction domain that includes ITAM.

[0027] In some embodiments, the intracellular signaling domain includes an intracellular domain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CAR further includes a co-stimulatory signaling region.

[0028] In some embodiments, the co-stimulatory signaling region includes a CD28 or 4-1BB signaling domain. In some embodiments, the co-stimulatory domain is a 4-1BB domain. In some embodiments, the T cell is CD4+ or CD8+.

[0029] In some embodiments, the T cells are primary T cells obtained from the 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 a human being.

[0030] In some implementations, the subject has stage I / II disease. In some implementations, the subject has stage III / IV disease. In some implementations, the subject has minimal residual disease (MRD).

[0031] In some implementations, the subject is refractory to one or more prior therapies for the cancer. In some implementations, the subject achieves an inadequate response to one or more prior therapies for the cancer. In some implementations, the subject achieves a durable response to the treatment. In some implementations, a durable response is defined as no cancer recurrence or remission for up to 3 months, 6 months, or 12 months. In some implementations, the subject has a high survival rate.

[0032] In some implementations, the subject's baseline disease characteristics are determined.

[0033] In some implementations, the baseline characteristic includes the International Prognostic Index (IPI) score, serum lactate dehydrogenase (LDH), or sum of diameter products (SPD), disease stage, or any combination thereof. In some implementations, the baseline characteristic is defined by the Lugano 2014 criteria. In some implementations, the subject is categorized based on the International Prognostic Index (IPI) score. In some implementations, the subject is categorized as low-risk, low-intermediate-risk, or high-intermediate-risk based on the IPI score. In some implementations, the subject has no risk factors or one risk factor and is considered to be in the low-risk IPI group. In some implementations, the subject has two risk factors and is considered to be in the low-intermediate IPI group. In some implementations, the subject has three risk factors and is considered to be in the high-intermediate IPI group. In some implementations, the subject has a high or low IPI score.

[0034] In some embodiments, the volumetric measurement of the subject's tumor burden is measured. In some embodiments, the volumetric measurement of the subject's tumor burden is the sum of diameter products (SPD). In some embodiments, the volumetric measurement of the tumor burden is measured using the subject's computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI). In some embodiments, the SPD threshold is or approximately 30 / cm², or approximately 40 / cm², or approximately 50 / cm², or approximately 60 / cm², or approximately 70 / cm².

[0035] In some embodiments, the level of an inflammatory marker in the subject is measured. In some embodiments, the level of the inflammatory marker is 300 units / L, 400 units / L, 500 units / L, or 600 units / L. In some embodiments, the inflammatory marker is lactate dehydrogenase (LDH).

[0036] In some embodiments, the mutation is a point mutation, deletion, or frameshift mutation. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation is a phase variation (PV). In some embodiments, the mutation is a single nucleotide variation (SNV). In some embodiments, the mutation includes SNVs, indels, rearrangements, or combinations thereof.

[0037] In some implementations, the detection threshold ratio of the ctDNA:cfDNA molecules is 1:10, 1:100, 1:1000, or 1:10. 4 1:10 5 1:10 6 1:10 7 1:10 8 In some embodiments, 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.

[0038] In some implementations, the sequencing includes high-throughput sequencing, pyrosequencing, sequencing synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), massively parallel sequencing, cloned 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 personalization analysis via deep sequencing (CAPP-Seq), and duplex sequencing (Duplex-Seq).

[0039] In some implementations, measuring the level of ctDNA also includes determining the absolute ctDNA concentration, expressed as mutant haploid genomic equivalents per milliliter of plasma (hGE / mL).

[0040] In some embodiments, measuring the ctDNA level further includes determining the mean variant allele frequency (AF). In some embodiments, the mean variant allele frequency (AF) is greater than a range indicating a detectable ctDNA level. In some embodiments, the detection threshold is a percentage of the ctDNA level equal to or lower than the total cfDNA level in the sample. In some embodiments, the detection threshold is a concentration of the ctDNA level equal to or lower than the total cfDNA level in the sample. In some embodiments, the detection threshold is within 25%, 20%, 15%, 11%, or 5% of the median or mean level, amount, or concentration of cfDNA obtained from a biological sample obtained from a subject after administration of the treatment, and / or within one standard deviation above that median or mean level, amount, or concentration. In some implementations, 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.

[0041] In some embodiments, the assay further includes determining the correlation between baseline ctDNA concentration (hGE / mL) and baseline characteristics of the subject. In some embodiments, the method detects ctDNA levels in the sample with 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 some embodiments, the method detects ctDNA levels in the sample with 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%.

[0042] In some embodiments, the genotyping assay includes sequencing one or more genes to identify ctDNA. In some embodiments, the sample is obtained at 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after cancer treatment. In some embodiments, the sample is obtained on day 15 after cancer treatment. In some embodiments, the sample is obtained approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after cancer treatment. In some embodiments, the sample is obtained approximately 1, 2, 3, or 12 months after cancer treatment. In some embodiments, ctDNA is not detected at or at least 2, 4, or 6 weeks after cancer treatment, or 3, 6, 12, 18, 24, 30, or 36 months after cancer treatment, or 1, 2, 3, 4, 5 years, or longer.

[0043] In some embodiments, the sample includes a fluid, cell, or tissue sample. In some embodiments, the sample is a blood, serum, or plasma sample. Attached Figure Description

[0044] Figure 1 This comparison shows the limits of ctDNA detection between CAPP-Seq and PhasED-Seq. CAPP-Seq quantifies ctDNA by detecting single nucleotide variants (SNVs), with an error rate of approximately 1 in 20,000 molecules. PhasED-Seq quantifies ctDNA by detecting phased variants (PVs), with an error rate of less than 1 in 10,000,000 molecules (<0.00001%).

[0045] Figure 2The genomic coordinates of phasing variants in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are displayed. Whole-genome sequencing data from the PCAWG cohort were reanalyzed, identifying PVs and placing them within 1,000-base-pair genomic regions. Data show that PVs are common and occur in stereotyped regions of the FL genome. The y-axis shows the fraction of DLBCL and FL cases with PVs in the indicated genes; the x-axis shows the genomic coordinates of the PVs.

[0046] Figure 3 The correlation between ctDNA levels and disease stage in patients with follicular lymphoma is shown. The y-axis represents ctDNA levels as tumor score; the x-axis represents disease stage. For stage I / II FL, the median pre-treatment ctDNA level was 0.04%; for stage III / IV FL, the median pre-treatment ctDNA level was 0.53% (P=0.006).

[0047] Figure 4 The allele frequencies of ctDNA obtained by genotyping PV using tumor (x-axis) or plasma (y-axis) are shown. Before treatment, ctDNA was detectable in 94% of cases using tumor-derived PV and in 75% of cases using plasma-derived PV.

[0048] Figure 5 This demonstrates the concordance of SNVs between tumor and plasma. It shows the proportion of shared mutations from tumor, plasma, or both compartments when genotyping is performed. It shows genes with mutations in more than 10% of cases. In mutations of genes such as TNFRSF14, EZH2, and CREBBP, the observed concordance rate between tumor and plasma is higher than that of other candidate driver mutations in FL, such as KMT2D, CARD11, and ARID1A.

[0049] Figure 6 This shows the probability of progression-free survival between patients with and without residual ctDNA after two cycles of treatment.

[0050] Figure 7 The ctDNA monitoring data from three patients is shown. MRD was observed in patients PAT-FL-024 and PAT-FL-039 prior to clinical detection of recurrence (vertical dashed line). For PAT-FL-024, MRD was detected 13 months before recurrence at levels as low as 1:100,000; for PAT-FL-039, MRD was detected more than two years before recurrence. The y-axis represents tumor fraction; the x-axis represents time (in years) since the pre-treatment sample; each point represents a single sample time point. Detailed Implementation

[0051] The method described in this article circumvents the difficulties and costs of tumor biopsies by utilizing sequences of cell-free fragments of tumor DNA circulating in the bloodstream (called circulating tumor DNA or ctDNA). Tumor biomarkers can be non-invasively assessed by detecting ctDNA. Patient biosamples can provide time-based measurements of total tumor burden, as well as the identification of specific mutations that occur during clinical treatment and intervention. By sequencing patient samples containing ctDNA, for example, by analyzing the resulting sequence reads, disease status (i.e., cancer) can be detected or monitored. Such sequence reads can be analyzed by genomic localization to genomic references that include sequences of normal, non-tumor genes from the patient.

[0052] In some respects, this article provides a method for determining the minimal residual disease (MRD) status of a subject diagnosed with cancer, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) determining the subject's MRD status based on the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule ratio is less than 1:10, then the subject has MRD, and if this ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject does not have MRD.

[0053] In some respects, this article provides a method for assessing the treatment response of a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule is present, then the subject has responded to the treatment, and if this ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject will not respond to the treatment.

[0054] In some respects, this article provides a method for assessing treatment response in a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is below a detection threshold ratio of ctDNA:cfDNA molecules, the subject is responsive to the treatment, and if the ratio is above the detection threshold ratio of ctDNA:cfDNA molecules, the subject is unresponsive to the treatment.

[0055] In other respects, this article provides a method for assessing treatment response in a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ctDNA level is below a detection threshold, the subject is responsive to the treatment, and if the ctDNA level is above a detection threshold, the subject is unresponsive to the treatment.

[0056] In some respects, this article provides a method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the risk of cancer recurrence or relapse in the subject based on the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule is present, the subject is at risk of cancer recurrence or relapse, and if this ratio is less than 1:10... 6If ctDNA:cfDNA molecules are present, then the subject is not at risk of cancer recurrence or relapse.

[0057] In some respects, this article provides a method for treating a subject with a second cancer treatment, wherein the subject has received a first cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 ctDNA:cfDNA molecules, then a second cancer treatment is administered.

[0058] In some respects, this article provides a method for treating cancer in a subject of need, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 ctDNA:cfDNA molecules, then an effective amount of cancer treatment is administered.

[0059] In some respects, this article provides a method for treating cancer in a subject in need, wherein the subject is evaluated as a candidate for cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA ratio is less than 1:10, the subject is identified as a candidate for cancer treatment. 6 (e) If the ctDNA:cfDNA molecule is used, the subject is identified as a candidate for non-cancer treatment; and (e) an effective amount of cancer treatment is administered to the subject.

[0060] In some respects, this article provides a method for assessing the survival probability of a subject who has received cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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; and (c) determining the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject may survive.

[0061] In other respects, this article provides a method for monitoring a subject undergoing cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule is present, then the subject has responded to the treatment, and if this ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject will not respond to the treatment.

[0062] In some respects, this article provides a method for assessing the likelihood of a durable response in a subject undergoing cancer treatment, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) assessing the subject's response by the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule is present, the subject is likely to have a durable response to the treatment, and if the ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject is unlikely to have a durable response to the treatment.

[0063] In some respects, this article provides a medicament in a method for treating cancer in a subject of need, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

[0064] In some respects, this article provides the use of cell therapy in the manufacture of a medicament for treating cancer in a subject of need, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) if the ctDNA:cfDNA ratio is greater than 1:10 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

[0065] In some respects, this article provides a method for evaluating the treatment response of a subject who has received cancer treatment, wherein, after administration of cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject has responded to the cancer treatment.

[0066] In some respects, this article provides a method for assessing the risk of cancer recurrence or relapse in subjects who have received cancer treatment, wherein, after administration of cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject is at risk of cancer recurrence or relapse.

[0067] In some respects, this article provides a method for treating cancer in a subject in need, comprising administering an effective amount of cancer treatment to the subject, wherein, prior to administering the cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are used, the subject is identified as a candidate for cancer treatment.

[0068] In some respects, this article provides a method for treating cancer in a subject with a high-risk disease, comprising administering an effective amount of cancer treatment to the subject, wherein, prior to administering the cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are detected, the subject is identified as having a high-risk disease.

[0069] In some respects, this article provides a method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, the method 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 cfDNA molecules containing a phase variation (PV); (c) determining the subject's minimal residual disease (MRD) status from the ctDNA in the sample; and (d) assessing the subject's risk of cancer recurrence or relapse based on the subject's MRD status; wherein the treatment comprises at least two cycles; and wherein if MRD is detected after two cycles of the treatment, the subject is at risk of cancer recurrence or relapse.

[0070] In some implementations, the cancer is a blood cancer. In some implementations, the cancer is a solid tumor. In some implementations, the cancer is a B-cell malignancy. In some implementations, the cancer is leukemia or lymphoma. In some implementations, the cancer is large B-cell lymphoma (LBCL) or diffuse large B-cell lymphoma (DLBCL). In some implementations, the cancer is follicular lymphoma (FL).

[0071] In some implementations, the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute 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). The B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

[0072] In some implementation schemes, cancer is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, stomach cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer, or soft tissue sarcoma.

[0073] In some implementations, the treatment or drug is a first-line therapy. In other implementations, the treatment or drug is a second-line therapy. In some implementations, the treatment includes further monitoring of the subject. In other implementations, the treatment includes radiographic imaging of the subject. In some implementations, the treatment includes computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI) of the subject.

[0074] In some embodiments, the treatment includes cell therapy. In some embodiments, the treatment includes CAR-T cell therapy. In some embodiments, the CAR-T cell therapy is anti-CD19 cell therapy. In some embodiments, the cell therapy includes genetically engineered cells. In some embodiments, the genetically engineered cells are T cells.

[0075] In some embodiments, the genetically engineered T cells include chimeric antigen receptors (CARs). In some embodiments, the CAR specifically binds to an antigen associated with a disease or condition and / or is expressed by cells associated with the disease or condition. In other embodiments, the CAR specifically binds to two antigens associated with a disease or condition and / or is expressed by cells associated with the disease or condition.

[0076] In some implementations, the antigens are selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer-testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, dual antigens, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrinB2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR. vIII, estrogen receptor, fetal AchR, folate receptor α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Ra2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), cancer-fetal antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptor, BAFF-R, VEGF-R2, nephroblastoma 1 (WT-1), and pathogen-specific antigens.

[0077] In some implementations, the antigen is CD19. In other implementations, the antigen is CD20.

[0078] In some implementations, the CAR includes an extracellular antigen recognition domain that specifically binds to the antigen, and an intracellular signal transduction domain that includes ITAM.

[0079] In some embodiments, the intracellular signal transduction domain includes an intracellular domain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CAR further includes a co-stimulatory signal transduction region.

[0080] In some embodiments, the co-stimulatory signaling region includes a CD28 or 4-1BB signaling domain. In some embodiments, the co-stimulatory domain is a 4-1BB domain. In some embodiments, the T cell is CD4+ or CD8+.

[0081] In some embodiments, the T cells are primary T cells obtained from the 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 human.

[0082] In some implementations, the subject has stage I / II disease. In some implementations, the subject has stage III / IV disease. In some implementations, the subject has minimal residual disease (MRD).

[0083] In some implementations, the subject is refractory to one or more prior therapies for the cancer. In some implementations, the subject achieves an inadequate response to one or more prior therapies for the cancer. In some implementations, the subject achieves a durable response to the treatment. In some implementations, a durable response is defined as no cancer recurrence or remission for up to 3 months, 6 months, or 12 months. In some implementations, the subject has a high survival rate.

[0084] In some implementations, the subject is treatment-naïve prior to the administration of the treatment.

[0085] In some implementations, the subject's baseline disease characteristics are determined.

[0086] In some implementations, baseline characteristics include International Prognostic Index (IPI) score, serum lactate dehydrogenase (LDH), or sum of diameter products (SPD), disease stage, or any combination thereof. In some implementations, baseline characteristics are defined by the Lugano 2014 criteria. In some implementations, the subject is categorized based on the International Prognostic Index (IPI) score. In some implementations, the subject is categorized as low-risk, low-intermediate-risk, or high-intermediate-risk based on the IPI score. In some implementations, the subject has no risk factors or one risk factor and is considered to be in the low-risk IPI group. In some implementations, the subject has two risk factors and is considered to be in the low-intermediate IPI group. In some implementations, the subject has three risk factors and is considered to be in the high-intermediate IPI group. In some implementations, the subject has a high or low IPI score.

[0087] In some embodiments, the volumetric measurement of the subject's tumor burden is measured. In some embodiments, the volumetric measurement of the subject's tumor burden is the sum of diameter products (SPD). In some embodiments, the volumetric measurement of the tumor burden is measured using the subject's computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI). In some embodiments, the SPD threshold is 30 / cm² or approximately 40 / cm², 50 / cm² or approximately 60 / cm², or 70 / cm².

[0088] In some implementations, the levels of inflammatory markers in the subject are measured. In some implementations, the levels of the inflammatory markers are 300 units / L, 400 units / L, 500 units / L, or 600 units / L. In some implementations, the inflammatory marker is lactate dehydrogenase (LDH).

[0089] In some embodiments, the mutation is a point mutation, deletion, or frameshift mutation. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation is a phase variation (PV). In some embodiments, the mutation is a single nucleotide variation (SNV). In some embodiments, the mutation includes SNVs, insertions / deletions, rearrangements, or combinations thereof.

[0090] In some implementations, the detection threshold ratio of ctDNA:cfDNA molecules is 1:10, 1:100, 1:1000, or 1:10. 4 1:10 5 1:10 6 1:10 7 1:10 8 In some embodiments, 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.

[0091] In some implementations, the sequencing includes high-throughput sequencing, pyrosequencing, sequencing synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), massively parallel sequencing, cloned 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 personalization analysis via deep sequencing (CAPP-Seq), and duplex sequencing (Duplex-Seq).

[0092] In some implementations, measuring ctDNA levels also includes determining the absolute ctDNA concentration, expressed as mutant haploid genomic equivalents per milliliter of plasma (hGE / mL).

[0093] In some embodiments, measuring ctDNA levels further includes determining the mean variant allele frequency (AF). In some embodiments, the mean variant allele frequency (AF) is greater than a range indicating detectable ctDNA levels. In some embodiments, the detection threshold is a percentage of the ctDNA level equal to or lower than the total cfDNA level in the sample. In some embodiments, the detection threshold is a concentration of the ctDNA level equal to or lower than the total cfDNA level in the sample. In some embodiments, the detection threshold is within 25%, 20%, 15%, 11%, or 5% of the median or mean level, amount, or concentration of cfDNA obtained from a biological sample obtained from a subject after administration of the treatment, and / or within one standard deviation above that median or mean level, amount, or concentration. In some implementations, 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.

[0094] In some embodiments, the assay further includes determining the correlation between baseline ctDNA concentration (hGE / mL) and baseline characteristics of the subject. In some embodiments, the method detects ctDNA levels in the sample with 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 some embodiments, the method detects ctDNA levels in the sample with 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%.

[0095] In some embodiments, the genotyping assay includes sequencing one or more genes to identify ctDNA. In some embodiments, the sample is obtained at 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after cancer treatment. In some embodiments, the sample is obtained on day 15 after cancer treatment. In some embodiments, the sample is obtained approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after cancer treatment. In some embodiments, the sample is obtained approximately 1, 2, 3, or 12 months after cancer treatment. In some embodiments, ctDNA is not detected at or at least 2, 4, or 6 weeks after cancer treatment, or 3, 6, 12, 18, 24, 30, or 36 months after cancer treatment, or 1, 2, 3, 4, 5 years, or longer.

[0096] In some embodiments, the sample includes a fluid, cell, or tissue sample. In some embodiments, the sample is a blood, serum, or plasma sample.

[0097] In some respects, this article provides a method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, the method 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 cfDNA molecules containing a phase variation (PV); (c) determining the subject's minimal residual disease (MRD) status from the ctDNA in the sample; and (d) assessing the subject's risk of cancer recurrence or relapse based on the subject's MRD status; wherein the treatment comprises at least two cycles; and wherein if MRD is detected after two cycles of the treatment, the subject is at risk of cancer recurrence or relapse.

[0098] In some implementations, the cancer is a blood cancer. In some implementations, the cancer is a B-cell malignancy. In some implementations, the cancer is leukemia or lymphoma. In some implementations, the cancer is follicular lymphoma.

[0099] In some embodiments, the subject is a human being. 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 untreated prior to administration of the treatment.

[0100] In some implementations, this treatment is a first-line therapy. In some implementations, the treatment includes bendamustine / rituximab or R-CHOP. In some implementations, the induction phase includes 6 cycles of bendamustine / rituximab or R-CHOP.

[0101] In some embodiments, the sample is obtained on the first day of the treatment cycle. In some embodiments, the method further includes obtaining multiple biological samples consecutively over a period of at least one year, at least two years, or at least three years after the administration of the treatment.

[0102] In some embodiments, the sample is a blood sample, serum sample, or plasma sample. In some embodiments, the tumor fraction of the biological sample is less than 0.28%.

[0103] All publications cited in this application, including patent documents, scientific articles, and databases, are incorporated herein by reference in their entirety for all purposes, as if each individual publication were individually incorporated by reference. Where any definition presented herein conflicts with or is otherwise inconsistent with definitions presented in patents, applications, published applications, or other publications incorporated herein by reference, the definitions set forth herein shall prevail and supersede those incorporated by reference.

[0104] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0105] I. Circulating tumor DNA (ctDNA) and methods for detecting ctDNA This article provides methods for monitoring disease progression and / or treatment response, including methods for highly sensitive analysis of circulating tumor DNA (ctDNA), such as DNA sequences derived from tumor cells present in individual patient samples. The presence and level of ctDNA are correlated with tumor burden and minimal residual disease (MRD) and can be used to monitor response to therapy, monitor residual lesions, monitor the presence of metastases, monitor total tumor burden, etc. The presence and / or level of ctDNA also provide information for treatment paradigms and therapies.

[0106] Circulating cell-free DNA (cfDNA) refers to DNA fragments that are no longer located within cells and exist in the circulatory system. The term cfDNA can also be used to describe these DNA fragments after extraction and subsequent processing. cfDNA can be released from cells as a result of various processes, including normal and abnormal apoptosis, cell secretion, necrosis, etc. Specific forms of cfDNA can be present in the circulatory system as a result of various medical conditions, disease states, pregnancy, etc. These genomic fragments in the circulatory system can be obtained from simple blood samples, providing a low-risk opportunity for screening various phenotypes, conditions, etc. Therefore, cfDNA testing is an emerging diagnostic method that allows for non-invasive, rapid, and real-time detection in research and clinical settings.

[0107] Cancer surveillance can be categorized into medical history and physical examination, tumor markers, diagnostic procedures, and imaging studies. Surveillance of recurrent or secondary cancers is a crucial component of survivor care. These methods detect signs of disease or examine whether tumors or cell masses have spread to areas such as lymph nodes, the chest cavity, or the lungs. In some implementations, surveillance methods may include X-rays, magnetic resonance imaging (MRI), computed tomography (CT), computed axial computed tomography (CAT), positron emission tomography (PET), FDG-PET, PET / CT, and histopathology. The clinical application of these screening tools may be limited due to their poor sensitivity and specificity, high invasiveness, and poor compliance. See, e.g., Merkow et al., Quality of Cancer Surveillance Clinical Practice Guidelines: Specificity and Consistency of Recommendations, JAMA Intern Med. 2017; Aykan et al., Objective response rate assessment in oncology: Current situation and future expectations, World J Clin Oncol. 2020; Gurcan et al., HistopathologicalImage Analysis: A Review, IEEE Rev Biomed Eng. 2010; and Griffeth, K., Use ofPET / CT scanning in cancer patients: technical and practical considerations, Proc (Bayl Univ Med Cent) 2005.

[0108] Liquid biopsy describes the detection of biomolecular features in bodily fluids, including blood, urine, cerebrospinal fluid, and saliva, that have the potential to indicate disease states. Obtaining a liquid biopsy requires a non-invasive or minimally invasive procedure. Different biological components can be identified in liquid biopsies, including circulating rare cells, cell-free DNA (cfDNA), cell-free RNA (cfRNA), and extracellular vesicles (exosomes). cfDNA has a relatively short half-life of approximately 16 minutes to 2.5 hours. cfDNA is cleared by nuclease action followed by renal excretion, or by absorption by the liver and spleen followed by degradation by macrophages. The stability of cfDNA fragments can be increased by binding to cell membranes, extracellular vesicles, or proteins. See, for example, 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 StructBiotechnol J. 2018, which are incorporated herein by reference in their entirety.

[0109] Certain physiological and pathological conditions, including exercise, inflammation, and cancer, can affect the amount of cfDNA. Typically, under normal conditions, cfDNA concentrations are low, around 100 ng / mL in healthy patients. However, cfDNA concentrations in cancer patients can vary significantly, reported ranging from 0 to greater than 1000 ng / mL. Increased cfDNA levels in cancer patients are associated with tumor-shedded molecules into the bloodstream. Given the observed differences in cfDNA levels between healthy and diseased individuals, using cfDNA as a biomolecular marker could improve cancer diagnosis, prognosis, and surveillance. See, for example, Kustanovich et al., Life and death of circulating cell-free DNA, Cancer Biol Ther 2019; and Stejskal et al., Circulating tumor nucleic acids: biology, release mechanisms, and clinical relevance, Mol Cancer 2023, which are incorporated herein by reference in their entirety.

[0110] The origin of cell-free DNA (cfDNA) is associated with both passive and active release mechanisms, including apoptosis, necrosis, and active cellular secretion. Furthermore, genomic instability is thought to influence the active cellular release of cfDNA. These processes release cellular contents into the extracellular space. While the mechanisms of cellular release and the clearance of cfDNA require further elucidation, certain cfDNA characteristics have provided clues to their origin. For example, apoptosis results in the cleavage of chromosomal DNA into fragments of approximately 170 bp, corresponding to the number of base pairs surrounding the nucleosome plus a small fragment connecting the two nucleosome cores. The modal fragment size of cfDNA (approximately 170 bp) reflects this apoptosis-specific size characteristic. See, for example, 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 amolecular marker for cancer management, Biomol Detect Quantif. 2019, which are incorporated herein by reference in their entirety.

[0111] cfDNA produced by cancer cells undergoing apoptosis, necrosis, or active secretion is classified as circulating tumor DNA (ctDNA). In cancer, uncontrolled proliferation leads to local nutrient depletion, hypoxia, inflammation, and metabolic stress, thereby inducing apoptosis and necrosis. There is also evidence that cfDNA originates from cells interacting with tumor cells, i.e., the tumor microenvironment. Studies have shown that the amount of ctDNA increases with the number of tumor cells. Furthermore, mounting evidence suggests that cfDNA in cancer patients is more fragmented and shorter. For example, the number of DNA fragments shorter than 150 bp is positively correlated with the fraction of tumor DNA in the plasma of hepatocellular carcinoma patients. Possible factors associated with this interpopulation-observed difference in fragment size could be tissue-specific processes (such as specific nucleosome encapsulation) or changes in methylation levels, which cause DNA regions to become less ordered and more easily digested by nucleases. See, for example, 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 herein by reference in their entirety.

[0112] Cancer-specific genetic and epigenetic alterations are reflected in the sequence of isolated ctDNA. These alterations include mutations, copy number aberrations, genomic rearrangements, and methylation changes. Most ctDNA screening methods focus on detecting tumor-specific mutations to analyze ctDNA levels, thereby providing information about tumor burden with high sensitivity and specificity, for example. These mutations include base substitutions (i.e., single nucleotide variants (SNVs)) and insertions or deletions (insertions and deletions). Mutations can be identified using a range of methods for identifying sequence composition. Methods include targeted approaches for single or small numbers of mutations that can be detected using highly sensitive techniques, including amplification-based assays such as digital droplet PCR and quantitative PCR (qPCR). Several ctDNA-based diagnostic tests using targeted approaches have been approved for clinical use. The first FDA-approved method for detecting specific mutations in plasma-derived ctDNA is cobas EGFR Mutation Test v2, which uses real-time PCR to detect predefined mutations in exons 18, 19, 20, and 21 of the EGFR gene in NSCLC patients. Other FDA-approved targeted methods using cfDNA to detect specific mutations include Qiagen for breast cancer. therascreenThe PIK3CA RGQ PCR Kit and the EpiproColon assay for colorectal cancer. See, for example, Heitzer et al., Circulating tumor DNA as a liquid biopsy for cancer, Clin Chem. 2015; and Keller et al., Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond, Br J Cancer 2021, which are incorporated herein by reference in their entirety. See also CISION PR NEWSWIRE “Roche receives FDA approval for the cobasEGFR Mutation Test v2 as the first companion diagnostic test for expandedEGFR TKI therapies in patients with non-small cell lung cancer,” published on October 29, 2020. Retrieved from: https: / / www.prnewswire.com / news-releases / roche-receives-fda-approval-for-the-cobas-egfr-mutation-test-v2-as-the-first-companion-diagnostic-test-for-expanded-egfr-tki-therapies-in-patients-with-non-small-cell-lung-cancer-301163170.html

[0113] Next-generation sequencing (NGS)-based methods have been developed to perform multigene analysis using both targeted and non-targeted approaches. These methods can detect sequence mutations originating from tumors (i.e., somatic mutations) as well as mutations found in germlines. NGS-based methods can also quantify the ctDNA fraction within cfDNA and provide strategies for screening, detecting, and monitoring cancer. ctDNA in patient samples can be detected by identifying sequences with tumor-specific mutations. The concentration of ctDNA in plasma is expressed as haplogenous genomic equivalents per milliliter (hGE / mL), a value obtained from the mean variant allele frequency (VAF) and the input concentration of cfDNA (pg / mL) (considering that 1 hGE equals 3.3 pg DNA). The concentration of ctDNA in plasma may be low, accounting for less than 0.01% of cfDNA, but ctDNA concentrations can constitute a wide range of approximately 0.1-89% of cfDNA. Several parameters considered to influence tumor growth dynamics, including cell doubling time, proliferating cell number, and tumor cell loss, can affect the ctDNA levels seen in different cancer types and stages. See, for example, Bourbon et al., How to Obtain a High Quality ctDNA in Lymphoma Patients: Preanalytical Tips and Tricks, Pharmaceuticals (Basel) 2021, which is incorporated herein by reference in its entirety.

[0114] The FDA has approved the liquid biopsy tests Guardant360 CDx and FoundationOne Liquid CDx for any solid tumor, but not for blood cancers. These methods use NGS-based gene panels to detect ctDNA mutations and guide the selection of targeted therapies. FoundationOne Liquid CDx uses an NGS-based panel of 300 genes to analyze cfDNA in the blood, while Guardant360 CDx uses a panel of more than 60 genes.

[0115] Liquid biopsy provides information about a patient's cancer progression by measuring ctDNA levels in a mixture of cfDNA at different time points. However, the low concentration of ctDNA in plasma presents a technical challenge for its detection. In many patients, ctDNA levels are significantly below the detection threshold of many sequencing-based methods, such as in the case of early-stage cancer. For example, in most lung and colorectal cancer patients, the pre-treatment ctDNA concentration is less than 0.5%. Low levels of ctDNA necessitate methods with higher analytical sensitivity to distinguish background noise and detect truly rare mutations. Sequencing methods have used unique molecular identifiers (UMIs) (barcodes attached to the ends of DNA fragments in the library) to reduce noise. The addition of UMIs allows bioinformatics analysis to distinguish true mutations from PCR errors and minimizes errors in subsequent analyses.

[0116] Targeted sequencing methods enhance sensitivity by incorporating gene panels targeting the most frequently mutated genes and combining them with UMI (Unified Biometrics). Cancer Personalized Analysis via Deep Sequencing (CAPP-Seq) is one such targeted method for quantifying ctDNA. It utilizes the most common genetic alterations in a given cancer, along with sophisticated wet-lab methods and customized bioinformatics analyses, to establish a sensitive ctDNA detection strategy. In one case, CAPP-Seq assessed mutations in 139 frequently mutated genes in non-small cell lung cancer (NSCLC) with a detection limit of 0.02% in cfDNA. Subsequent iterations of CAPP-Seq further reduced the detection limit to approximately 0.002% by introducing two key innovations—molecular barcoding and background trimming—to suppress errors caused by oxidative damage during library preparation. Targeted Error Correction Sequencing (TEC-Seq) is another UMI-based technique that measures 58 frequently mutated genes in colorectal cancer, lung cancer, ovarian cancer, and breast cancer. It can detect 50.0%–70.0% of stage I or II cancers in these different cancer types. See, for example, Newman et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med. 2014; Newman et al., Integrated digital error suppression for improved detection of circulating tumor DNA, Nat Biotechnol. 2016; Semenkovich et al., Genomic approaches to cancer and minimal residual disease detection using circulating tumor DNA, J Immunother Cancer 2023; and Phallen et al., Direct detection of early-stage cancers using circulating tumor DNA, Sci Transl Med. 2017, which are incorporated herein by reference in their entirety.

[0117] Another targeted sequencing method, PhasED-Seq, focuses on phased variants (PVs), which are two single-mode mutations (SNVs) occurring in cis (on the same DNA strand). By identifying PVs, PhasED-Seq can identify mutations in these mutation-rich cancers with high confidence because the probability of two (or more) mutations occurring by chance on the same strand is extremely low. This technology has shown extremely low detection limits, reaching parts per million (ppm).

[0118] While methods have improved in their limits of detection for ctDNA, cases with low ctDNA concentrations (e.g., where ctDNA concentrations typically decrease after treatment) require even lower detection thresholds. Methods with improved sensitivity will reduce false negative rates, increase detection rates, and potentially reduce treatment costs. Methods with higher specificity will improve false positive rates. See, for example, 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 herein by reference in its entirety. See also the disclosure in U.S. Patent No. 11,447,833, which is incorporated herein by reference in its entirety.

[0119] In a particular implementation, illustrative examples of biological fluids suitable for isolating cfDNA include, but are not limited to, amniotic fluid, blood, plasma, serum, semen, lymph, cerebrospinal fluid, eye discharge, urine, saliva, mucus, and sweat.

[0120] Therefore, in some implementations, samples include tissues, fluids, and other samples taken directly from the subject, as well as samples generated by one or more processing steps (e.g., separation, centrifugation, washing, and / or incubation). Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, amniotic fluid, bodily fluids (e.g., blood, plasma, serum, semen, lymph, cerebrospinal fluid, eye discharge, urine, saliva, mucus, and sweat), tissue and organ samples, including processed samples derived therefrom.

[0121] In some embodiments, the sample is peripheral blood. In some embodiments, the sample is processed within 1, 2, or 3 hours after collection. In some embodiments, processing includes plasma separation. In some embodiments, plasma separation includes centrifugation. In some embodiments, germline genomic DNA is separated from the sample. In some embodiments, matched tumor DNA is separated from the sample. In some embodiments, the genomic DNA is quantified. In some embodiments, circulating cfDNA is separated from the plasma. In some embodiments, circulating cfDNA is separated 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.

[0122] In some embodiments, next-generation sequencing libraries are prepared from isolated plasma DNA. In some embodiments, next-generation sequencing libraries are prepared from isolated shorn tumor genomic DNA. In some embodiments, next-generation sequencing libraries are prepared from isolated germline genomic DNA. In some embodiments, next-generation sequencing libraries are prepared from isolated cell line genomic DNA.

[0123] In some implementations, next-generation sequencing libraries are constructed using DNA polymerases with strong 3'-5' exonuclease activity and high fidelity. In some implementations, the next-generation sequencing libraries are cleaned. In some implementations, the next-generation libraries are ligated with suitable sequencing adapters. In some implementations, the ligated next-generation libraries are amplified. In some implementations, the amplified next-generation libraries are quantified.

[0124] In some implementations, a hybridization probe is used to capture the amplified next-generation sequencing library. In some implementations, the captured next-generation sequencing library is amplified. In some implementations, the amplified captured next-generation sequencing library is sequenced. See, for example, Newman et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med. 2014, which is incorporated herein by reference in its entirety.

[0125] In certain embodiments, the biofluid is blood or plasma. In other embodiments, 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, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived therefrom.

[0126] In some implementations, the sample is obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the administration of cancer treatment, or approximately at that number of days. In some implementations, the sample is obtained at or approximately at weeks 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52. In some embodiments, the sample is obtained at or approximately 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 after cancer treatment, or approximately at that number of months. In some embodiments, the sample is obtained at or approximately 10 years after cancer treatment, or approximately at that number of years. In some embodiments, the sample is obtained on day 15 after cancer treatment.

[0127] In some aspects, this document provides methods for detecting the presence of the biomarkers described herein (i.e., ctDNA), for example, in a sample. In some embodiments, the sample is obtained from a subject, such as a subject with cancer (e.g., the cancer described herein). This document discloses a method for determining the presence of tumor nucleic acids (tNA) in a cell-free nucleic acid (cfNA) sample from an individual by detecting somatic mutations. The method may include: obtaining a cfNA sample, selecting cfNAs for sequences corresponding to multiple mutated regions in the cancer described herein, sequencing the selected cfNAs; determining the presence of somatic mutations, wherein the presence of somatic mutations may indicate the presence of tumor cells in the subject; and providing the subject with an assessment of the presence of tumor cells. The cell-free nucleic acid may be cell-free DNA (cfDNA). The cell-free nucleic acid may be cell-free RNA (cfRNA). The cell-free nucleic acid may be a mixture of cell-free DNA (cfDNA) and cell-free RNA (cfRNA). The tumor nucleic acid may be a nucleic acid derived from tumor cells. The tumor nucleic acid may be tumor-derived DNA (tDNA). The tumor nucleic acid may be circulating tumor DNA (ctDNA). The tumor nucleic acid may be tumor-derived RNA (tRNA). The tumor nucleic acid may be circulating tumor RNA (ctRNA). The tumor nucleic acid can be a mixture of tumor-derived DNA and tumor-derived RNA. It can also be a mixture of ctDNA and ctRNA.

[0128] Determining the presence of a somatic mutation may include the steps of the sequencing methods disclosed herein. In some embodiments of the methods provided herein, determining the amount of ctDNA may include determining the absolute amount of ctDNA. Determining the amount of ctDNA may include determining the relative amount of ctDNA. Determining the amount of ctDNA can be done by counting sequence reads associated with the ctDNA. Determining the amount of ctDNA can be done by quantitative PCR. Determining the amount of ctDNA can be done using the sequencing methods disclosed herein. Determining the amount of ctDNA may include counting sequencing reads of the ctDNA.

[0129] In some embodiments of the methods provided herein, determining the amount of ctDNA may include detecting one or more mutations. Determining the amount of ctDNA may include detecting two or more different types of mutations. Mutation types include, but are not limited to: SNVs, insertions / deletions, fusions, breakpoints, structural variations, variable-number tandem repeats, hypervariable regions, microsatellite sequences, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, or combinations thereof, in selected regions of the subject's genome. Determining the amount of ctDNA may include detecting one or more of single nucleotide variants (SNVs), phasing variants (PVs), insertions / deletions, copy number variations, and rearrangements in selected regions of the subject's genome. Determining the amount of ctDNA may include detecting two or more of SNVs, insertions / deletions, copy number variations, and rearrangements in selected regions of the subject's genome. Determining the amount of ctDNA may include detecting at least one SNV, insertion / deletion, copy number variation, or rearrangement in selected regions of the subject's genome.

[0130] In some embodiments of the methods provided herein, detecting ctDNA may include detecting mutations, wherein the mutation is a point mutation, deletion, or frameshift mutation. In some embodiments, the mutations detected by the methods described herein are somatic mutations, phased variants (PVs), or single nucleotide variants (SNVs).

[0131] In some embodiments, determining the amount of ctDNA may include detecting one or more PVs. In some embodiments, determining the amount of ctDNA may include detecting one or more SNVs. In some embodiments, determining the amount of ctDNA may include detecting one or more individual cell mutations. In some embodiments, determining the amount of ctDNA may include detecting one or more point mutations. In some embodiments, determining the amount of ctDNA may include detecting one or more deletions. In some embodiments, determining the amount of ctDNA may include detecting one or more frameshift mutations.

[0132] In some implementations, determining the amount of ctDNA may include detecting two or more mutations, including SNVs, insertions / deletions, rearrangements, or combinations thereof.

[0133] In some implementations, as used interchangeably herein, “phased variation” or “PV” typically refers to two or more cis- (i.e., SNVs or insertions / deletions) mutations occurring within a single cell-free nucleic acid molecule. In some cases, the cell-free nucleic acid molecule may be a cell-free deoxyribonucleic acid (cfDNA) molecule. In some cases, the cfDNA molecule may be derived from diseased tissue, such as tumors (e.g., circulating tumor DNA (ctDNA) molecules).

[0134] In some embodiments of the methods provided herein, identifying mutations in one or more genes in cfDNA involves sequencing that gene. In some embodiments of the methods provided herein, sequencing that gene includes any suitable sequencing technology, which may include, but is not limited to: high-throughput sequencing, pyrosequencing, sequencing by synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule sequencing by synthesis (SMSS), massively parallel sequencing, cloned 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 personalization analysis via deep sequencing (CAPP-Seq), and duplex sequencing (Duplex-Seq).

[0135] In some embodiments, the method described herein uses 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 7... Sensitivity of 5%, 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%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or higher for detecting ctDNA from cfDNA in a sample.

[0136] In some embodiments, the method described herein uses 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 7... 5%, 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%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or higher specificity for detecting ctDNA from cfDNA in a sample.

[0137] In some implementations, measuring ctDNA levels involves determining the absolute ctDNA concentration, expressed as mutant haploid genomic equivalents (hGE / mL) per milliliter of plasma. ctDNA levels are categorized as haploid genomic equivalents (hGE / mL) per milliliter of plasma and calculated using the following formula: [(average VAF of all detected mutations) × cfDNA concentration (pg / mL plasma)] ÷ 3.3, as described in the previous article by Scherer et al., Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Transl Med. 2016.

[0138] In some implementations, the assay also includes determining the correlation between baseline ctDNA concentration (hGE / mL) and the subject's baseline characteristics.

[0139] In some embodiments, measuring ctDNA levels also includes determining the mean variant allele frequency (VAF). The mean VAF is categorized as the percentage of observed sequence reads matching a particular DNA variant divided by the total coverage of that site. In some embodiments, the methods described herein may include measuring a mean variant allele frequency (VAF) greater than a certain range that indicates higher or lower total ctDNA levels. See, for example, Strom, S., Evaluating the analytical validity of circulating tumor DNA sequencing assays for precisiononcology, Cancer Biol Med. 2016, which is incorporated herein by reference in its entirety.

[0140] In some implementations, the method described herein detects ctDNA from cfDNA in a sample with a sensitivity of at least about 50%.

[0141] In some embodiments, the detection threshold is a percentage of the total cfDNA level or lower than the ctDNA level in the sample. In some embodiments, the detection threshold is a concentration of the total cfDNA level or lower than the ctDNA level in the sample. In some embodiments, the detection threshold is within 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% of the median or average level, amount, or concentration of cfDNA in the biological sample obtained from the subject after administration of the treatment, and / or within one standard deviation above the median or average level, amount, or concentration of cfDNA in the biological sample obtained from the subject after administration of the treatment.

[0142] In some implementations, 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%, 0.00075%, 0.0005%, 0.00025%, 0.0001%, or 0.00001% of the total cfDNA in the sample.

[0143] In some implementations, the methods disclosed herein may include detecting SNVs, CNVs, insertions, deletions, and / or rearrangements at specific regions in the genome. These specific genomic regions may include sequences from one or more genes.

[0144] In some implementations, for the methods disclosed herein, ctDNA was not detected at least 2, 3, 4, 5, or 6 weeks after administration of cancer treatment, or at 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 after administration, or at 1, 2, 3, 4, 5 years, or longer. Nucleic acid detection.

[0145] This document provides methods for detecting the ctDNA molecules of this disclosure. In some embodiments, the ctDNA of this disclosure is detected using any suitable method known in the art, such as nucleic acid hybridization assays, amplification-based assays (e.g., polymerase chain reaction, PCR), PCR-RFLP assays, real-time PCR, sequencing (e.g., Sanger sequencing or next-generation sequencing), screening analyses (e.g., using karyotyping methods), fluorescence in situ hybridization (FISH), breakage FISH, spectroscopic karyotyping, multiplex FISH, comparative genomic hybridization, in situ hybridization, single-specific primer polymerase chain reaction (SSP-PCR), high-performance liquid chromatography (HPLC), or mass spectrometry genotyping.

[0146] Other methods are provided for determining and quantifying when circulating tumor DNA is above background detectable levels, such as methods that integrate information content and mutation category into a detection index.

[0147] Analytical methods may include generating and capturing genetic information. Genetic information may include genetic sequence information, ploidy status, the identity of one or more genetic variations, and a quantitative measurement of that variation. The term "quantitative measurement" refers to the measurement of any quantity, including absolute and relative measurements. Quantitative measurements can be, for example, numbers (e.g., counts), percentages, frequencies, degrees, or threshold quantities.

[0148] Samples containing genetic material (e.g., cfDNA) can be collected from subjects at multiple time points, i.e., sequentially. This genetic material can be sequenced, for example, using a high-throughput sequencing system. Sequencing can target loci of interest (LOCIs) to detect genetic variations, such as genes carrying somatic mutations, genes undergoing copy number variations, or genes involved in gene fusions, as in cancer. At each time point, a quantitative measurement of the identified genetic variation can be determined. For example, in the case of cfDNA, the quantitative measurement could be the frequency or percentage of genetic variations in polynucleotides aligned to a locus, or the absolute number of sequence reads or polynucleotides aligned to a locus.

[0149] Mutations or variations can include genetic aberrations, including but not limited to single-base substitutions, small insertions or deletions, transversions, translocations, inversions, deletions, truncations, or gene truncations. In some cases, the mutation length can be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20 nucleotides. In other cases, the mutation length can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20 nucleotides. This method can be used to detect any number of genetic aberrations that can cause or be caused by cancer. These can include, but are not limited to, mutations, insertions / deletions, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, altered chromosomal structure, gene fusions, gene truncation, gene amplification, gene duplication, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, abnormal changes in nucleic acid methylation, infections, and cancer. In some implementations, the mutation is a single nucleotide variant (SNV). In some implementations, the mutation is a phased variant (PV).

[0150] In some implementations, the sample may contain various amounts of nucleic acids, including genomic equivalents. For example, a sample of about 30 ng of DNA may contain about 10,000 (10^3 ng) of nucleic acids. 4 One haploid human genome equivalent, in the case of cfDNA, contains approximately 200 billion (2 × 10¹¹) individual polynucleotide molecules. Similarly, a sample of approximately 100 ng of DNA can contain approximately 30,000 haploid human genome equivalents, and in the case of cfDNA, approximately 600 billion individual molecules. One haploid human genome equivalent has approximately 3 picograms of DNA. A sample of approximately 1 microgram of DNA contains approximately 300,000 haploid human genome equivalents.

[0151] 1. Sequencing methods Genotyping and / or detection, identification, and / or quantification of ctDNA can be performed using sequencing. Sequencing can be performed using high-throughput systems.

[0152] Polynucleotides can be analyzed using any method known in the art. Typically, DNA sequencers will employ next-generation sequencing (e.g., Illumina, 454, Ion Torrent, SOLiD, Pacific Biosciences). Next-generation sequencing methods can be broadly categorized into two classes: sequencing by hybridization and sequencing by synthesis (SBS). See, for example, Slatko et al., Overview of Next Generation Sequencing Technologies, Curr Protoc Mol Biol. 2019, which is incorporated herein by reference in its entirety.

[0153] SBS methods involve a repetitive cycle of synthesis, imaging, and adding additional nucleotides to the growing strand and obtaining sequence reads by imaging the incorporated nucleotides. SBS techniques typically rely on short reads, at most 300-500 bases long. They generally have high error rates and depend on high sequence coverage of millions to billions of short DNA sequence reads to obtain accurate sequences based on the identification of common sequences.

[0154] Hybridization sequencing hybridizes and washes away unhybridized DNA, allowing the determination of whether the hybridized marker fragment matches the DNA probe sequence on the filter. Therefore, based on overlap information from probe hybridization sites, a larger contiguous sequence can be constructed. Hybridization sequencing has largely been categorized as a technique that relies on using specific probes to probe sequences, such as in diagnostic applications for identifying disease-related SNPs (single nucleotide polymorphisms) in specific genes or for identifying gross chromosomal abnormalities (rearrangements, deletions, duplications, copy number variations, CNVs).

[0155] Sequence analysis can be performed through massively parallel sequencing, i.e., simultaneous (or rapid sequential) sequencing 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, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule sequencing by synthesis (SMSS-Helicos), massively parallel sequencing, cloning single-molecule arrays (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 method known in the art.

[0156] Illumina technology uses bridging amplification, where approximately 500 bp DNA molecules with adapters at both ends serve as substrates. Repeated amplification synthesis reactions are performed on a solid support containing oligonucleotide sequences complementary to the attached adapters. The oligonucleotides on the slide are spaced apart, allowing the DNA to undergo multiple rounds of amplification, producing clonal "clusters" of approximately 1000 copies of each oligonucleotide fragment. Each glass slide can support millions of parallel cluster reactions. During the synthesis reaction, proprietary modified nucleotides corresponding to each of the four bases (each with a different fluorescent label) are incorporated, followed by detection. Compared to camera-based imaging, fluorescence detection improves detection speed due to direct imaging.

[0157] Nucleotide sequences (such as DNA sequences) can refer to raw sequence reads or processed sequence reads, such as unique molecular counts inferred from raw sequence reads.

[0158] The sequence reads generated from sequencing need to be analyzed, including, for example, identifying genetic variations. This can include identifying sequence variations and quantifying the number of bases detected at each site. Quantification can involve, for example, counting the number of reads aligned to a specific genetic site. Different numbers of reads at different sites can indicate copy number variations (CNVs).

[0159] The methods disclosed herein can be used to detect genetic variations (also known as “genetic alterations”). Genetic variations are substitutions at genetic loci. Genetic variations include sequence variations, copy number variations, and nucleotide modification variations. Sequence variations are variations in the genetic nucleotide sequence. Copy number variations are deviations in the copy number of a portion of the genome relative to the wild type. Genetic variations include, for example, single nucleotide variations (SNPs), insertions, deletions, inversions, translocations, translocations, gene fusions, chromosome fusions, gene truncations, copy number variations (e.g., aneuploidy, partial aneuploidy, polyploidy, gene amplification), aberrant changes in nucleic acid chemical modifications, aberrant changes in epigenetic patterns, and aberrant changes in nucleic acid methylation.

[0160] Genetic variations can be detected by comparing sequences of polynucleotides from a sample with a reference (e.g., with a reference genome sequence, an index, or a database of known mutations). In one embodiment, the reference sequence is a publicly available reference sequence, such as the human genome sequence HG-19 or NCBI Build 37. In another embodiment, the reference sequence is a sequence from a non-public database. In yet another embodiment, the reference sequence is a germline sequence of an organism inferred or determined from sequencing polynucleotides from that organism. As genetic variation and phenotypic data from new subjects are added over time, the disclosed methods and systems support large-scale, automated statistical analyses of genetic variation-phenotype associations in a rolling manner. For example, in one aspect, the statistical association analysis performed is a genome-wide association study (GWAS) statistical analysis (van der Sluis S et al., PLOS Genetics 2013; 9: e1003235; Visscher PM et al., Am J Hum Genet 2012; 90: 7). In a GWAS analysis, which genes or genetic variations are associated with the phenotype of interest are identified.

[0161] In some implementations, the methods disclosed herein can be used to detect sequence variations, such as SNVs. For example, sequence variations can be detected by a common sequence from multiple sequence reads, such as 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, and 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 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88 At least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1500, at least 2 000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, at least 10000, at least 15000, at least 20000, at least 25000, at least 30000, at least 35000, at least 40000, at least 45000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000 or more sequence reads.

[0162] The shared sequence can originate from a sequence read of a single-stranded polynucleotide. It can also originate from a sequence read of one strand of a double-stranded polynucleotide (e.g., a paired read). In one exemplary approach, paired reads allow for the identification of the presence of sequence variations in a molecule with higher confidence.

[0163] Somatic mutations or alterations are genetic variations that occur in somatic cells. Somatic mutations are distinct from mutations that occur in the genome of an individual's germline cells (i.e., sperm or egg cells) or zygote. Somatic mutations (such as those found in cancer cells) are distinguishable from the germline genome of a subject who has developed cancer. They can also be detected by comparing the cancer genome with the germline genome or with a reference genome.

[0164] To address the scarcity of mutated fragments, deep targeted sequencing technologies have been developed, with enhanced sensitivity achieved by incorporating gene panels targeting the most frequently mutated genes. These methods require overcoming technical noise that can affect the differentiation of single nucleotide variant (SNV) signals from sequencing errors. Examples of deep targeted sequencing methods include Cancer Personalization Analysis via Deep Sequencing (CAPP-Seq), Duplex Sequencing (Duplex-Seq), and Phased Variant Enrichment and Detection Sequencing (PhasED-Seq). See, for example, Newman et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med. 2014; Schmitt et al., Detection of ultra-rare mutations by next-generation sequencing, Proc Natl Acad Sci USA 2012; and Kurtz et al., Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA, Nat Biotechnol 2021, all of which are incorporated herein by reference in their entirety.

[0165] a.PhasED-Seq Phased variant enrichment and detection sequencing (PhasED-Seq) is a method for detecting and quantifying ctDNA. PhasED-Seq reduces the error spectrum of mutation detection in sequencing data by requiring the consistent detection of two independent non-reference events in a single DNA molecule. By detecting more than one mutation, PhasED-Seq can more accurately distinguish tumor-derived cell-free DNA (ctDNA) from healthy cell-free DNA—enabling the detection of ctDNA at levels below one part per million (<0.0001%). PhasED-Seq has been extensively validated in hundreds of B-cell lymphoma patients. Ultimately, PhasED-Seq detects two or more single nucleotide variants from the top or bottom strand of circulating DNA fragments. This next-generation circulating tumor DNA sequencing assay offers high molecular recovery and is less susceptible to sequencing errors, thus improving the sensitivity of ctDNA detection. See, for example, 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 herein by reference in its entirety.

[0166] In clinical settings, ctDNA detection has been limited by the relatively low DNA yield from typical blood collections and the background error rate associated with current sequencing technologies. Approximately 5 mL of plasma can be isolated from a whole blood collection tube (about 10 mL). In cancer patients, this plasma is expected to contain approximately 50 ng of DNA (about 10 ng / mL), which is equivalent to approximately 15,000 haploidentical genomic equivalents. At a VAF of 0.1% (consistent with localized malignancies or minimal residual disease (MRD) after treatment), this equates to only 15 tumor DNA molecules. See, for example, Semenkovich et al., Genomic approaches to cancer and minimal residual disease detection using circulating tumor DNA, J ImmunotherCancer 2023, which is incorporated herein by reference in its entirety.

[0167] Using ctDNA as a biomarker before and after treatment is a promising prognostic tool; however, these methods require improved sensitivity to universally detect ctDNA associated with residual lesions. Methods lacking sufficient sensitivity can produce “false negative” measurements, where patients are classified as having undetectable ctDNA even as disease progression occurs. The improved sensitivity of PhasED-Seq and ctDNA detection enhance the prognostic value of ctDNA as a biomarker. See, for example, 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 herein by reference in its entirety.

[0168] Using PhasED-Seq to detect phasing variants (two or more mutations occurring in cis (i.e., on the same DNA strand) eliminates the need to detect two consistent events on separate strands, as is the case with double-strand sequencing. Identification of phasing variants focuses on variant groups that are less than 170 base pairs (bp) apart (typical length of cfDNA fragments). By analyzing 2538 tumors from 24 cancer histologies, putative phasing variants (PVs) were classified to identify target genomic regions for phasing variant detection. Specifically, this method identified phasing genomic regions with increased PV distribution in lymphomas. The PhasED-Seq panel for lymphoma targets approximately 115 kb of genomic space, representing 0.0035% of the human genome, but covering 26% of phasing variants detected by whole-genome sequencing. This allows PhasED-Seq to detect tumor fractions as low as one in a million (1 / 1,000,000). See, for example, 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 herein by reference in its entirety.

[0169] An exemplary procedure for clinical intervention and / or treatment of an individual based on the detection of ctDNA sequences using PhasED-Seq comprises three main parts. First, cfDNA from a biological sample from the individual is obtained, prepared, and sequenced using capture sequencing methods across regions shown to contain multiple genetic variations occurring in a phased manner. Subsequently, the cfDNA sequencing results are analyzed to detect ctDNA, which is determined by detecting genetic variations occurring in a phased manner. Finally, based on the sequencing results indicating the presence of ctDNA in the biological sample, clinical intervention and / or treatment are performed. See, for example, U.S. Patent No. 11,447,833, which is incorporated herein by reference in its entirety.

[0170] Specifically, phasing variant identification uses read-level data, taking paired-end reads and identifying non-reference sites. First, germline sequencing is performed on peripheral blood mononuclear cells (PBMCs) to identify patient-specific single nucleotide polymorphisms (SNPs). A site is classified as non-reference if it has a variant allele fraction (VAF) greater than 40% and a read depth of at least 10, or a VAF greater than 0.25% and at least 100 reads. Next, PVs are classified using read-level data from samples of interest. Sample read pairs containing two or more non-reference sites are considered somatic PVs. PVs containing putative germline SNPs are removed to ensure that no remaining PVs are present in the germline samples; this is a crucial step for both sensitivity and specificity. The number of deduplicated read pairs containing PVs is divided by the number of read pairs spanning the genomic locus for a given PV to calculate the VAF associated with each PV. See, for example, 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 herein by reference in its entirety.

[0171] In some implementations, PhasED-Seq is used for cancer screening and biopsy-free tumor genotyping, where patient ctDNA samples are analyzed in the absence of a reference biopsy sample. In some implementations, ctDNA detection by PhasED-Seq is a prognostic indicator of event-free survival. In some implementations, PhasED-Seq has higher sensitivity for detecting residual disease than other ctDNA assays. In some implementations, PhasED-Seq can be used to detect MRD in curative settings. In some implementations, PhasED-Seq is an important step in overcoming technical noise and improving the sensitivity of MRD detection. In some implementations, PhasED-Seq offers the potential for accurate monitoring of residual disease using ctDNA-based assays and informing clinical decision-making in low tumor burden settings. In some implementations, the improved sensitivity of PhasED-Seq can allow for earlier detection of treatment failure, thus providing alternative therapies to patients with low tumor burden and while the disease is still within the curative window. In some implementations, the enhanced sensitivity of PhasED-Seq can detect cancer during and after treatment and can be used to guide treatment decisions. When ctDNA levels obtained via PhasED-Seq become undetectable, physicians can downgrade treatment, sparing patients from toxic and costly therapies. In some implementations, methods using PhasED-Seq can be combined with tumor imaging methods such as PET / CT scans.

[0172] b.CAPP-Seq Cancer Personalized Analysis via Deep Sequencing (CAPP-Seq) is a method for quantifying circulating tumor DNA (ctDNA). CAPP-Seq combines optimized library preparation methods for low-quality DNA input with multi-stage bioinformatics approaches to design "selectors" composed of biotinylated DNA oligonucleotides that target regions of recurrent mutation in cancers of interest. To monitor ctDNA, this selector is applied to tumor DNA to identify patient-specific genetic aberrations, and then directly to circulating DNA for quantification. Ultimately, CAPP-Seq can detect single nucleotide variants (SNVs) on fragments of circulating DNA. This next-generation circulating tumor DNA sequencing assay allows for high molecular recovery rates but is susceptible to sequencing errors. See, for example, Newman et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nat Med. 2014, which is incorporated herein by reference in its entirety.

[0173] For any cancer with identified recurrent mutations, a CAPP-Seq selector can be designed. Whole-exome sequencing can be used to select exons with recurrent mutations to maximize the number of missense mutations per patient while minimizing the selector size. For example, for non-small cell lung cancer (NSCLC), the selected design target covers a region of approximately 125 kb.

[0174] When DNA quantities are limited (such as in the case of DNA derived from plasma), library preparation optimization is crucial. Optimized library preparation allows for the construction of CAPP-Seq libraries from as little as 4 ng of DNA. For preparing next-generation sequencing libraries from low-input circulating DNA, it is important to optimize adapter ligation efficiency, reduce the number of PCR cycles after adapter ligation, preserve the native size distribution of circulating DNA fragments, and minimize variability in sequencing coverage depth across all captured genomic regions. Overall, the detection limit of CAPP-Seq can be affected by: (i) the input amount and recovery rate of circulating DNA molecules, (ii) allelic bias in the capture reagent, and (iii) PCR or sequencing errors.

[0175] In some implementations, CAPP-Seq is used for cancer screening and biopsy-free tumor genotyping, where a patient's ctDNA sample is analyzed in the absence of a reference biopsy sample. In some such implementations, when CAPP-Seq identifies mutations in clinically actionable targets from the ctDNA sample, these methods include providing a therapy suitable for that target. In some implementations, CAPP-Seq combines optimized library preparation methods for low DNA input quality with multi-stage bioinformatics methods to design "selectors" composed of biotinylated DNA oligonucleotides that target recurrently mutated regions in cancers of interest. To monitor ctDNA, this selector is applied to tumor DNA to identify patient-specific genetic aberrations and then directly to circulating DNA for quantification. In some implementations, some methods using CAPP-Seq can be combined with tumor imaging methods such as PET / CT scans.

[0176] c.Duplex-Seq Duplex-Seq is a method for detecting and quantifying ctDNA that relies on identifying single nucleotide variants (SNVs) on both strands of the ctDNA molecule, thereby reducing the probability of sequencing errors. Duplex-Seq detects SNVs on double-stranded DNA derived from circulating DNA fragments. This next-generation circulating tumor DNA sequencing assay reduces the impact of sequencing errors on SNV identification, but may also have lower efficiency in recovering the top and bottom strands, resulting in low molecule recovery rates. See, for example, Schmitt et al., Detection of ultra-rare mutations by next-generation sequencing, Proc Natl Acad Sci USA 2012, which is incorporated herein by reference in its entirety.

[0177] In some implementations, Duplex-Seq is an ideal tag-based error correction method that reduces or eliminates artificial mutations caused by DNA damage, PCR errors, and sequencing errors. In some implementations, Duplex-Seq allows for the sensitive detection of rare variants in heterogeneous populations and utilizes redundant information stored in complex double-stranded DNA.

[0178] 2. Amplification Methods The methods disclosed herein may include the amplification of cell-free DNA (cfDNA) and / or circulating tumor DNA (ctDNA). Amplification may include PCR-based amplification. Alternatively, amplification may include non-PCR-based amplification.

[0179] In some embodiments, amplification-based methods are used to detect the ctDNA described herein. As is known in the art, in such amplification-based methods, a nucleic acid sample (e.g., a sample obtained from an individual or from a tumor) is used as a template in an amplification reaction (e.g., polymerase chain reaction (PCR)) using one or more oligonucleotides or primers, such as the one or more oligonucleotides or primers provided herein. The presence of the ctDNA disclosed herein in the sample can be determined based on the presence or absence of the amplification product. Quantitative amplification methods are also known in the art and can be used according to the methods provided herein. Methods for measuring DNA copy number at microsatellite sites using quantitative PCR analysis are known in the art. The known nucleotide sequence of a gene is sufficient to enable those skilled in the art to routinely select primers to amplify any part of the gene. Real-time fluorescence PCR can also be used. In real-time fluorescence PCR, quantification is based on the amount of fluorescence signal, such as TaqMan and SYBR Green.

[0180] Other amplification methods suitable for use with respect to the methods provided herein include, for example, ligase chain reaction (LCR), transcriptional amplification, self-sustaining sequence replication, spot PCR, and adapter PCR.

[0181] 3. Array-based methods In some embodiments, array-based methods, such as array-based comparative genomic hybridization (CGH) methods, are used to detect the ctDNA disclosed herein. In array-based CGH methods, a first nucleic acid sample (e.g., from a sample such as a tumor) is labeled with a first marker, while a second nucleic acid sample (e.g., a control such as from a healthy sample) is labeled with a second marker. In some embodiments, equal volumes of the two samples are mixed and co-hybridized onto a DNA microarray, which is a microarray of thousands of uniformly spaced clonal DNA fragments or oligonucleotides spotted onto the array in triplicate. After hybridization, the relative fluorescence intensity of each hybridized fluorophore is captured and quantified using a digital imaging system. The resulting ratio of fluorescence intensities is proportional to the ratio of the copy numbers of the DNA sequences in the two samples. In some embodiments, when chromosomal deletions or polyploidization are present, a difference in the signal ratio from the two markers is detected, and this ratio provides a measure of copy number. Array-based CGH can also be performed using monochromatic labeling.

[0182] II. Treatment methods This document provides methods for treating individual cancers or delaying the progression of individual cancers (such as the cancers described herein, namely LBCL and / or DLBCL) in subjects. This document discloses methods for determining the disease status of cancer in subjects. These methods may include: obtaining the amount of circulating tumor DNA (ctDNA) in a sample from the subject; and determining the disease status of the cancer in the subject based on the ratio of the amounts of ctDNA to cfDNA. In some aspects, this document provides methods for identifying subjects with cancer (such as the cancers described herein) who could benefit from the therapies (e.g., cell therapies) described herein. In some embodiments, these methods include detecting the ctDNA described herein in a sample obtained from the subject. In some embodiments, these methods include knowing the presence of the ctDNA described herein in a sample obtained from the subject. In some embodiments, the presence of ctDNA in the sample identifies the subject as a subject who could benefit from a treatment (e.g., cancer treatment). In some embodiments, the presence of ctDNA in the sample identifies the subject as a subject who could benefit from a treatment (e.g., an anticancer therapy, such as cell therapy) described herein. In some embodiments, the detection of ctDNA in the sample identifies the subject as a subject who could benefit from a treatment comprising the cell therapy described herein. In some implementations, in response to the knowledge of ctDNA in a sample, the subject is identified as a subject who can benefit from treatment containing the cell therapy provided herein.

[0183] In some embodiments, the sample is the sample described herein. In some embodiments, the sample comprises a biological sample, such as a blood, serum, or plasma sample, from a subject diagnosed with cancer.

[0184] In some embodiments, methods of treating the cancer of this disclosure or delaying the progression of the cancer of this disclosure in a subject include administering a therapeutically effective amount of treatment, such as the cancer treatment and / or cell therapy provided herein, to the individual. In some embodiments, methods of treating the cancer of this disclosure or delaying the progression of the cancer of this disclosure in an individual (e.g., an individual containing ctDNA provided herein) include administering an effective amount of the cancer treatment provided herein to the individual in response to knowing the presence of ctDNA in a sample obtained from the subject. In some embodiments, methods of treating the cancer of this disclosure or delaying the progression of the cancer of this disclosure in an individual (e.g., by detecting ctDNA provided herein) include administering an effective amount of treatment, such as the cell therapy provided herein, to the individual in response to knowing the presence of ctDNA in a sample obtained from the subject.

[0185] In some aspects, the method includes obtaining a biological sample and isolating cell-free DNA (cfDNA) from the biological sample. In some embodiments, the method includes identifying mutations in one or more genes of the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample. In some embodiments, if the level of ctDNA in the sample is below a detection threshold, cancer treatment is administered to the subject. In some embodiments, if the level of ctDNA in the sample is above a detection threshold, cancer treatment is administered to the subject. In some embodiments, the method includes administering an effective amount of cancer treatment to the subject, wherein the subject is identified as a candidate for receiving the cancer treatment prior to administration of the cancer treatment. In some embodiments, the method includes obtaining a biological sample and isolating cell-free DNA (cfDNA) from the biological sample. In some embodiments, the method includes identifying mutations in one or more genes of the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample. In some embodiments, the method includes determining the ctDNA:cfDNA molecule ratio.

[0186] In some embodiments, the method includes administering an effective amount of cancer treatment to a subject, wherein the subject is identified as a candidate for receiving the cancer treatment prior to administration. In some embodiments, the method includes obtaining a biological sample and isolating cell-free DNA (cfDNA) from the biological sample. In some embodiments, the method includes identifying mutations in one or more genes within the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample.

[0187] In some aspects, the method includes treating a subject's cancer by administering an effective amount of cancer treatment to the subject. In some embodiments, prior to administering the cancer treatment, the level of ctDNA in a sample from the subject is measured, and a ctDNA level in the sample above a detection threshold indicates that the subject is a candidate for administering the cancer treatment. In some embodiments, the method includes treating a subject with a high-risk disease's cancer by administering an effective amount of cancer treatment to the subject. In some embodiments, prior to administering the cancer treatment, the subject is identified as having a high-risk disease by measuring the level of ctDNA in a sample from the subject.

[0188] In some embodiments, ctDNA levels above a detection threshold in a sample indicate that the subject has a high-risk disease. In other embodiments, ctDNA levels above a detection threshold indicate that the subject is a candidate for cancer treatment. In some embodiments, ctDNA levels below a detection threshold in a sample indicate that the subject does not have a high-risk disease. In other embodiments, ctDNA levels below a detection threshold indicate that the subject is not a candidate for cancer treatment.

[0189] In some implementations, the detection threshold is the ctDNA:cfDNA ratio, which is from 1:10 or approximately. 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1:10. 8 The above are ctDNA:cfDNA molecules.

[0190] In some implementations, the detection threshold is the ctDNA:cfDNA ratio, which is 1:10, 1:100, 1:10³, or approximately 1:10. 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 1:10¹¹ or 1:10¹². In other embodiments, the detection threshold is the ctDNA:cfDNA ratio, which is 5:10, 5:100, 5:10³, or 5:10. 4 5:10 5 5:10 6 5:10 7 5:10 8 5:10 9 5:10¹0 5:10¹¹ or 5:10¹².

[0191] In some implementations, the detection threshold is the ctDNA:cfDNA ratio, which is from 1:10 or approximately. 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 、or from or approximately 10:10 6 The ratio is approximately 1:10. 8 The above are ctDNA:cfDNA molecules.

[0192] In some aspects, this document provides a method for treating cancer in a subject of need. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In other embodiments, the method specifies: determining the ctDNA:cfDNA molecule ratio in the sample, and if the ctDNA:cfDNA ratio is greater than 1:10... 6 ctDNA:cfDNA molecules, then an effective amount of cancer treatment is administered.

[0193] In some implementations, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA molecule ratio is greater than 1:10, then an effective amount of cancer treatment is administered. In some implementations, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:109 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then an effective amount of cancer treatment is administered.

[0194] In some implementations, the method specifies that if the ratio is 1:10 or approximately 1:10 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 If the above ctDNA:cfDNA molecules are present, then an effective amount of cancer treatment will be administered.

[0195] In some aspects, this document provides a method for treating a subject with a second cancer treatment. In some embodiments, the subject has received a first cancer treatment. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In other embodiments, the method specifies: determining the ctDNA:cfDNA molecule ratio in the sample, and if the ctDNA:cfDNA ratio is greater than 1:10... 6 ctDNA:cfDNA molecules, then a second cancer treatment is administered.

[0196] In some implementations, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA molecule ratio is greater than 1:10, then a second cancer treatment is administered. In some implementations, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10... 4 1:10 51:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then a second cancer treatment is administered.

[0197] In some implementations, the method specifies that if the ratio is 1:10 or approximately 1:10 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1:10. 7 The ratio is approximately 1:10. 6 The ratio is approximately 1:10. 8 The above ctDNA:cfDNA molecules are then used for a second cancer treatment.

[0198] In some aspects, this article provides a method for treating cancer in a subject in need. In some embodiments, the subject is evaluated as a candidate for cancer treatment. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. The method specifies: determining the ctDNA:cfDNA molecule ratio in the sample, and if the ctDNA:cfDNA ratio is greater than 1:10... 6 If the ctDNA:cfDNA ratio is found to be less than 1:10, the subject is identified as a candidate for cancer treatment, and an effective dose of cancer treatment is administered to the subject. In some implementations, if the ctDNA:cfDNA ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are used, the subject is identified as a candidate for non-cancer treatment.

[0199] In some implementations, the method specifies that if the ctDNA:cfDNA ratio is less than 1:10 6 If the ctDNA:cfDNA molecule is detected, the subject is identified as a candidate for non-cancer treatment. In some embodiments, the method specifies that if the ratio is 1:10 or approximately 1:10... 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 The above ctDNA:cfDNA molecules identify the subject as a candidate for non-cancer treatment.

[0200] In other implementations, the method specifies that: if the ratio is 1:10 or approximately 1:10 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 The above ctDNA:cfDNA molecules identify the subject as a candidate for cancer treatment.

[0201] In some aspects, this article provides a method for treating cancer in subjects with a high-risk disease. In some embodiments, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined. In some embodiments, if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are detected, the subject is identified as having a high-risk disease.

[0202] In some implementations, if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA molecule ratio is high, the subject is identified as having a high-risk disease. In other implementations, if the ctDNA:cfDNA ratio is greater than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², the subject is identified as having a higher risk disease.

[0203] In some aspects, this document provides a medicament in a method for treating cancer in a subject of need. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In some embodiments, the method specifies: determining the ctDNA:cfDNA molecule ratio in the sample; and if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

[0204] In some implementations, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA molecule ratio is greater than 1:10, then an effective amount of the drug is administered. In some embodiments, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10... 4 1:10 5 1:10 6 1:10 7 1:108 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then an effective amount of the drug is administered.

[0205] In some implementations, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA molecule is identified, then an effective amount of the drug is administered to the subject. In some embodiments, the method specifies that if the ratio is 1:10 or approximately 1:10... 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 If the above ctDNA:cfDNA molecules are present, an effective amount of the drug will be administered to the subject.

[0206] In some aspects, this article provides the use of cell therapy in the manufacture of a medicament for treating cancer in a subject of need. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In some embodiments, the method specifies: determining the ctDNA:cfDNA molecule ratio in the sample, and if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

[0207] In some implementations, this usage specifies that if the ctDNA:cfDNA ratio is greater than 1:10 6If the ctDNA:cfDNA molecule ratio is positive, then an effective amount of the drug is administered to the subject. In other embodiments, the method specifies that if the ctDNA:cfDNA ratio is greater than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then an effective amount of the drug is administered.

[0208] In some implementations, this usage specifies that if the ctDNA:cfDNA ratio is greater than 1:10 6 If the ctDNA:cfDNA molecule is identified, then an effective amount of the drug is administered to the subject. In some embodiments, the method specifies that if the ratio is 1:10 or approximately 1:10... 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 If the above ctDNA:cfDNA molecules are present, an effective amount of the drug will be administered to the subject.

[0209] In some embodiments, the ctDNA:cfDNA molecule ratio is a detection threshold ratio of ctDNA:cfDNA molecules. In other embodiments, the ctDNA:cfDNA molecule ratio is the ratio of ctDNA:cfDNA molecules in the subject sample. In some embodiments, the ctDNA:cfDNA molecule ratio is 1:10, 1:100, 1:1000, or 1:10. 4 1:10 5 1:10 6 1:10 7Or 1:10 8 .

[0210] In some implementations, the ctDNA:cfDNA molecule ratio is 1:10 or approximately 1:10. 6 The ratio is approximately 50:10 6 The ratio is approximately 1:10. 6 The ratio is approximately 40:10 6 The ratio is approximately 1:10. 6 The ratio is approximately 30:10 6 The ratio is approximately 1:10. 6 It is approximately 20:10 6 The ratio is approximately 1:10. 6 It is at most or approximately 10:10 6 The ratio is approximately 1:10. 6 It is at most or approximately 5:10 6 The ratio is approximately 1:10. 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 50:10 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 40:10 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 30:10 6 The ratio is approximately 2.5:10. 6 It is approximately 20:10 6 The ratio is approximately 2.5:10. 6 It is at most or approximately 10:10 6 The ratio is approximately 2.5:10. 6 It is at most or approximately 5:10 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 50:10 6 From or approximately 5:10 6 The ratio is approximately 50:10 6 From or approximately 5:10 6 The ratio is approximately 40:10 6 , or approximately 5:10 6 The ratio is approximately 30:10 6 , or approximately 5:10 6 It is approximately 20:10 6 , or approximately 5:10 6 It is at most or approximately 10:10 6 From or approximately 10:10 6 The ratio is approximately 50:10 6From or approximately 10:10 6 The ratio is approximately 40:10 6 The ratio is approximately 10:10. 6 The ratio is approximately 30:10 6 The ratio is approximately 10:10. 6 It is approximately 20:10 6 、From or approximately 20:10 6 The ratio is approximately 50:10 6 、From or approximately 20:10 6 The ratio is approximately 40:10 6 , or approximately 20:10 6 The ratio is approximately 30:10 6 、From or approximately 30:10 6 The ratio is approximately 50:10 6 、From or approximately 30:10 6 The ratio is approximately 40:10 6 ,or from or approximately 40:10 6 The ratio is approximately 50:10 6 The above are ctDNA:cfDNA molecules.

[0211] In some embodiments, the ctDNA:cfDNA molecule ratio 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 1.1:10 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 2.7:106 2.8:10 6 2.9:10 6 3:10 6 3.1:10 6 3.2:10 6 3.3:10 6 3.4:10 6 3.5:10 6 3.6:10 6 3.7:10 6 3.8:10 6 3.9:10 6 4:10 6 4.1:10 6 4.2:10 6 4.3:10 6 4.4:10 6 4.5:10 6 4.6:10 6 4.7:10 6 4.8:10 6 4.9:10 6 5:10 6 5.1:10 6 5.2:10 6 5.3:10 6 5.4:10 6 5.5:10 6 5.6:10 6 5.7:10 6 5.8:10 6 5.9:10 6 Or 6:10 6 The above are ctDNA:cfDNA molecules.

[0212] In some implementations, the ctDNA:cfDNA molecule ratio is 1:10, 1:100, 1:1000, or 1:10. 4 1:10 5 1:10 6 1:10 7 Or 1:10 8 .

[0213] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from approximately 1:1,000,000 to approximately 1:50,000,000; approximately 1:1,000,000 to approximately 1:45,000,000; approximately 1:1,000,000 to approximately 1:40,000,000; approximately 1:1,000,000 to approximately 1:35,000,000; approximately 1:1,000,000 to approximately 1:30,000,000; approximately 1:1,000,000 to approximately 1:25,000,000; approximately 1:1,000,000 to approximately 1:20,000,000; and approximately 1:1,000,000. Up to or approximately 1:15,000,000; up to or approximately 1:1,000,000; up to or approximately 1:1,000,000; up to or approximately 1:5,000,000; up to or approximately 1:1,000,000; up to or approximately 1:4,000,000; up to or approximately 1:1,000,000; up to or approximately 1:3,000,000; up to or approximately 1:1,000,000; up to or approximately 1:2,000,000; from up to or approximately 2:1,000,000; up to or approximately 2:5,000,000; up to or approximately 2:1,000,000; up to or approximately 2:4,500,000; up to or approximately 2:1,000,000; up to or approximately 2:4,000,000. The ratio is approximately 2:1,000,000 to approximately 2:35,000,000; approximately 2:1,000,000 to approximately 2:30,000,000; approximately 2:1,000,000 to approximately 2:25,000,000; approximately 2:1,000,000 to approximately 2:20,000,000; approximately 2:1,000,000 to approximately 2:15,000,000; approximately 2:1,000,000 to approximately 2:1,000,000; approximately 2:1,000,000 to approximately 2:500,000; approximately 2:1,000,000 to approximately 2:4,000,000; approximately 2:1,000,000 to approximately 2:100,000,000. Or approximately 2:3000000, or approximately 2:1000000 to approximately 2:2000000, or approximately 3:1000000 to approximately 3:50000000, from or approximately 3:1000000 to approximately 3:45000000, or approximately 3:1000000 to approximately 3:40000000, or approximately 3:1000000 to approximately 3:35000000, or approximately 3:1000000 to approximately 3:30000000, or approximately 3:1000000 to approximately 3:25000000, or approximately 3:1000000 to approximately 3:20000000.The ratio is approximately 3:1000000 to approximately 3:15000000, approximately 3:1000000 to approximately 3:10000000, approximately 3:10000000 to approximately 3:5000000, approximately 3:1000000 to approximately 3:4000000, approximately 3:1000000 to approximately 3:3000000, approximately 3:1000000 to approximately 3:2000000, from approximately 4:1000000 to approximately 4:50000000, approximately 4:1000000 to approximately 4:45000000, approximately 4:1000000 to or approximately 4:40000000, 4:1000000 to or approximately 4:35000000, 4:1000000 to or approximately 4:30000000, 4:1000000 to or approximately 4:25000000, 4:1000000 to or approximately 4:20000000, 4:1000000 to or approximately 4:15000000, 4:1000000 to or approximately 4:10000000, 4:10000000 to or approximately 4:5000000, 4:1 000000 to approximately 4:4000000, approximately 4:1000000 to approximately 4:3000000, approximately 4:1000000 to approximately 4:2000000, approximately 5:1000000 to approximately 5:50000000, approximately 5:1000000 to approximately 5:45000000, approximately 5:1000000 to approximately 5:40000000, approximately 5:1000000 to approximately 5:35000000, approximately 5:1000000 to approximately 5:30000000, approximately 5:1000 000 to approximately 5:25000000, approximately 5:1000000 to approximately 5:20000000, approximately 5:1000000 to approximately 5:15000000, approximately 5:1000000 to approximately 5:1000000, approximately 5:10000000, approximately 5:10000000, approximately 5:5000000, approximately 5:1000000 to approximately 5:4000000, approximately 5:1000000 to approximately 5:3000000, approximately 5:1000000 to approximately 5:2000000 and above ctDNA:cfDNA molecules.

[0214] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from approximately 1:1,000,000 to approximately 1:50,000,000; approximately 1:1,000,000 to approximately 1:45,000,000; approximately 1:1,000,000 to approximately 1:40,000,000; approximately 1:1,000,000 to approximately 1:35,000,000; approximately 1:1,000,000 to approximately 1:30,000,000; approximately 1:1,000,000 to approximately 1:25,000,000; approximately 1:1,000,000 to approximately 1:20,000,000; and approximately 1:1,000,000. Up to or approximately 1:15,000,000; up to or approximately 1:1,000,000; up to or approximately 1:1,000,000; up to or approximately 1:5,000,000; up to or approximately 1:1,000,000; up to or approximately 1:4,000,000; up to or approximately 1:1,000,000; up to or approximately 1:3,000,000; up to or approximately 1:1,000,000; up to or approximately 1:2,000,000; from up to or approximately 2:1,000,000; up to or approximately 2:5,000,000; up to or approximately 2:1,000,000; up to or approximately 2:4,500,000; up to or approximately 2:1,000,000; up to or approximately 2:4,000,000. The ratio is approximately 2:1,000,000 to approximately 2:35,000,000; approximately 2:1,000,000 to approximately 2:30,000,000; approximately 2:1,000,000 to approximately 2:25,000,000; approximately 2:1,000,000 to approximately 2:20,000,000; approximately 2:1,000,000 to approximately 2:15,000,000; approximately 2:1,000,000 to approximately 2:1,000,000; approximately 2:1,000,000 to approximately 2:500,000; approximately 2:1,000,000 to approximately 2:4,000,000; approximately 2:1,000,000 to approximately 2:100,000,000. Or approximately 2:3000000, or approximately 2:1000000 to approximately 2:2000000, or approximately 3:1000000 to approximately 3:50000000, from or approximately 3:1000000 to approximately 3:45000000, or approximately 3:1000000 to approximately 3:40000000, or approximately 3:1000000 to approximately 3:35000000, or approximately 3:1000000 to approximately 3:30000000, or approximately 3:1000000 to approximately 3:25000000, or approximately 3:1000000 to approximately 3:20000000.The ratio is approximately 3:1000000 to approximately 3:15000000, approximately 3:1000000 to approximately 3:10000000, approximately 3:10000000, approximately 3:1000000 to approximately 3:5000000, approximately 3:1000000 to approximately 3:4000000, approximately 3:1000000 to approximately 3:3000000, approximately 3:1000000 to approximately 3:2000000, from approximately 4:1000000 to approximately 4:50000000, approximately 4:1000000 to approximately 4 :45000000, or approximately 4:1000000 to or approximately 4:40000000, or approximately 4:1000000 to or approximately 4:35000000, or approximately 4:1000000 to or approximately 4:30000000, or approximately 4:1000000 to or approximately 4:25000000, or approximately 4:1000000 to or approximately 4:20000000, or approximately 4:1000000 to or approximately 4:15000000, or approximately 4:1000000 to or approximately 4:10000000, or approximately 4:10000000, or approximately 4:1 000000 to approximately 4:5000000, approximately 4:1000000 to approximately 4:4000000, approximately 4:1000000 to approximately 4:3000000, approximately 4:1000000 to approximately 4:2000000, approximately 5:1000000 to approximately 5:50000000, approximately 5:1000000 to approximately 5:45000000, approximately 5:1000000 to approximately 5:40000000, approximately 5:1000000 to approximately 5:350000 00, or approximately 5:1000000 to approximately 5:30000000, or approximately 5:1000000 to approximately 5:25000000, or approximately 5:1000000 to approximately 5:20000000, or approximately 5:1000000 to approximately 5:15000000, or approximately 5:1000000 to approximately 5:10000000, or approximately 5:10000000 to approximately 5:3000000, or approximately 5:1000000 to approximately 5:2000000 ctDNA:cfDNA molecules.

[0215] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from or approximately 1:10 to approximately 1:100, 1:10 to approximately 1:1000, 1:10 to approximately 1:10000, 1:10 to approximately 1:20000, 1:10 to approximately 1:30000, 1:10 to approximately 1:40000, 1:10 to approximately 1:50000, 1:10 to approximately 1:60000, 1:10 to approximately 1:70000, 1:10 to approximately 1:80000, 1:10 to approximately 1:90000, 1:10 to approximately 1:100000, from 2:10 to approximately 2:100, 2:10 to approximately 2:1 000, 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, 3:10 to about 3:1000, 3:10 to about 3:10000, 3:10 to about 3:20000, 3:10 to about 3:30000, 3:10 to about 3: 40000, 3:10 to approximately 3:50000, 3:10 to approximately 3:60000, 3:10 to approximately 3:70000, 3:10 to approximately 3:80000, 3:10 to approximately 3:90000, 3:10 to approximately 3:100000, from 4:10 to approximately 4:100, 4:10 to approximately 4:1000, 4:10 to approximately 4:10000, 4:10 to approximately 4:20000, 4:10 to approximately 4:30000, 4:10 to approximately 4:40000, 4:10 to approximately 4:50000, 4:10 to approximately 4:60000, 4:10 to approximately 4:70000, 4:10 to Approximately 4:80000, 4:10 to approximately 4:90000, 4:10 to approximately 4:100000, from 5:10 to approximately 5:100, 5:10 to approximately 5:1000, 5:10 to approximately 5:10000, 5:10 to approximately 5:20000, 5:10 to approximately 5:30000, 5:10 to approximately 5:40000, 5:10 to approximately 5:50000, 5:10 to approximately 5:60000, 5:10 to approximately 5:70000, 5:10 to approximately 5:80000, 5:10 to approximately 5:90000, 5:10 to approximately 5:100000 and above ctDNA:cfDNA molecules.

[0216] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from or approximately 1:10 to approximately 1:100, 1:10 to approximately 1:1000, 1:10 to approximately 1:10000, 1:10 to approximately 1:20000, 1:10 to approximately 1:30000, 1:10 to approximately 1:40000, 1:10 to approximately 1:50000, 1:10 to approximately 1:60000, 1:10 to approximately 1:70000, 1:10 to approximately 1:80000, 1:10 to approximately 1:90000, 1:10 to approximately 1:100000, from 2:10 to approximately 2:100, 2:10 to approximately 2:1 000, 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, 3:10 to about 3:1000, 3:10 to about 3:10000, 3:10 to about 3:20000, 3:10 to about 3:30000, 3:10 to about 3: 40000, 3:10 to approximately 3:50000, 3:10 to approximately 3:60000, 3:10 to approximately 3:70000, 3:10 to approximately 3:80000, 3:10 to approximately 3:90000, 3:10 to approximately 3:100000, from 4:10 to approximately 4:100, 4:10 to approximately 4:1000, 4:10 to approximately 4:10000, 4:10 to approximately 4:20000, 4:10 to approximately 4:30000, 4:10 to approximately 4:40000, 4:10 to approximately 4:50000, 4:10 to approximately 4:60000, 4:10 to approximately 4:70000, 4:10 to ctDNA:cfDNA molecules below approximately 4:80000, 4:10 to approximately 4:90000, 4:10 to approximately 4:100000, from 5:10 to approximately 5:100, 5:10 to approximately 5:1000, 5:10 to approximately 5:10000, 5:10 to approximately 5:20000, 5:10 to approximately 5:30000, 5:10 to approximately 5:40000, 5:10 to approximately 5:50000, 5:10 to approximately 5:60000, 5:10 to approximately 5:70000, 5:10 to approximately 5:80000, 5:10 to approximately 5:90000, 5:10 to approximately 5:100000.

[0217] In some embodiments, the ctDNA:cfDNA molecule ratio 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, 7:15 000, 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, 6:15 0000, 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 ctDNA:cfDNA molecules with the following counts: 750000, 6:750000, 7:750000, 8:750000, 9:750000, 1:1000000, 2:1000000, 3:1000000, 4:1000000, 5:1000000, 6:1000000, 7:1000000, 8:1000000, 9:1000000 and above.

[0218] In some embodiments, the ctDNA:cfDNA molecule ratio 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, 7:15 000, 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, 6:15 0000, 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 ctDNA and cfDNA molecules with values ​​below 750000, 6:750000, 7:750000, 8:750000, 9:750000, 1:1000000, 2:1000000, 3:1000000, 4:1000000, 5:1000000, 6:1000000, 7:1000000, 8:1000000, and 9:1000000.

[0219] In some embodiments, the method includes treating the subject with the second cancer treatment by administering an effective amount of the first cancer treatment and the second cancer treatment. In some embodiments, the subject is identified as a candidate for the second cancer treatment by measuring the level of ctDNA in a sample from the subject after administration of the first cancer treatment and before administration of the second cancer treatment. In some embodiments, a ctDNA level in the sample above a detection threshold indicates that the subject is a candidate for the second cancer treatment.

[0220] a. Response, therapeutic effect, and survival outcome In some implementations, at least 30%, at least 35%, at least 40%, or at least 50% of subjects treated according to this method achieve complete remission (CR); and / or at least approximately 40%, at least approximately 50%, at least approximately 60%, or at least approximately 70% of subjects treated according to this method achieve an objective response (OR). In some implementations, at least or at least approximately 50%, at least or at least approximately 60%, at least or at least approximately 70%, at least or at least approximately 80%, or at least or at least approximately 90% of subjects treated according to this method achieve CR and / or achieve an objective response (OR). In some implementations, the criteria for evaluating effective treatment include overall response rate (ORR; also referred to as objective response rate in some cases), complete response (CR; also referred to as complete remission in some cases), duration of response (DOR), progression-free survival (PFS), and / or overall survival (OS). In some implementations, at least 40% or at least 50% of subjects treated according to the methods provided herein achieve complete remission (CR; also referred to in some cases as complete response), demonstrating progression-free survival (PFS) and / or overall survival (OS) greater than or greater than about 3 months, 6 months, or 12 months, or greater than 13 months or about 14 months; on average, subjects treated according to the methods demonstrate median PFS or OS greater than or greater than about 6 months, 12 months, or 18 months; and / or the subject demonstrates at least or at least about 6, 12, 18 months or longer of PFS or OS after treatment.

[0221] In some implementations, clinical response to the methods and / or therapies described herein may be assessed using the Lugano criteria (Cheson et al., JCO September 20, 2014 vol. 32 no. 27 3059-3067; Johnson et al., (2015), Imaging for staging and response assessment in lymphoma. Radiology 2:323-338). The Lugano criteria include assessment by imaging, tumor volume measurement, and assessment of spleen, liver, and bone marrow involvement. In some implementations, response assessed using the Lugano criteria may include imaging assessment using positron emission tomography (PET-CT) and / or CT, as appropriate. PET-CT assessment may also include assessment of FDG uptake in FDG-avid lymphomas using fluorodeoxyglucose (FDG). In some aspects, a 5-point scale may be used when assessing the histological response to FDG-avid lymphomas using PET-CT. In some respects, the 5-point scale includes the following criteria: 1. No uptake above background; 2. Uptake ≤ mediastinum; 3. Uptake > mediastinum but ≤ liver; 4. Uptake moderately > liver; 5. Uptake significantly higher than liver and / or new lesions; X, new uptake areas are unlikely to be associated with lymphoma.

[0222] In some embodiments, complete response at the end of treatment, as described using the Lugano criteria, includes complete metabolic response and complete radiographic response at various measurable sites. In some embodiments, these sites include lymph nodes and extranodal sites, where, when using PET-CT, CR is described as a score of 1, 2, or 3 on a 5-point scale, with or without residual mass. In some embodiments, uptake may be higher than in normal mediastinum and / or liver in cases of Waldeyer's ring or extranodal sites with high physiological uptake, or in cases of activation in the spleen or bone marrow (e.g., due to chemotherapy or myeloid colony-stimulating factor). In some embodiments, complete metabolic response can be inferred even if the initial affected site has high physiological uptake, provided that uptake is not higher than that of the surrounding normal tissue. In some embodiments, lymph node response is assessed using CT, where CR is described as in disease-free extranodal sites, and the target lymph node / lymph node mass must regress to a lesion's longest transverse diameter (LDi) < 1.5 cm. Other sites for assessment include the bone marrow, where PET-CT-based assessment should indicate no evidence of FDG hyperuptake disease and CT-based assessment should indicate normal morphology, or, if indeterminate, IHC negativity. Other sites for assessment may include assessment of organ enlargement, which should regress to normal.

[0223] In some implementations, non-measurable lesions and new lesions are assessed, and these lesions should be absent in the case of CR. The primary outcome measure is the complete response (CR) rate. The CR rate can be defined as the incidence of CR according to the Lugano classification. In some aspects, a complete response described using the Lugano criteria includes complete metabolic response and complete radiographic response at various measurable sites. In some aspects, these sites include lymph nodes and extranodal sites, where CR is described as a score of 1, 2, or 3 on a 5-point scale when using PET-CT, with or without residual mass. In some aspects, in cases of Waldeyer's ring or extranodal sites with high physiological uptake, or in cases of activation in the spleen or bone marrow (e.g., due to chemotherapy or myeloid colony-stimulating factor), uptake may be higher than in the normal mediastinum and / or liver. In such cases, a complete metabolic response can be inferred if the uptake at the initially affected site is not higher than that of the surrounding normal tissue, even if the tissue has high physiological uptake. In some respects, CT is used to assess the responsiveness of lymph nodes, where CR is described as a disease-free extralymphatic site and the target lymph node / lymph node mass must be regressed to the longest transverse diameter (LDi) of the lesion ≤1.5 cm. Other sites for assessment include the bone marrow, where PET-CT-based assessment can indicate no evidence of FDG-avid disease in the bone marrow and CT-based assessment should indicate normal morphology, or IHC negative if uncertain. Other sites may include assessment of organ enlargement, which should be regressed to normal. In some respects, non-measurable lesions and new lesions are assessed, which should be absent in the case of CR (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323–338; Cheson, BD (2015) Chin Clin Oncol 4(1):5).

[0224] In some respects, a partial response (PR; also referred to in some cases as partial remission) as described using the Lugano criteria includes partial metabolic and / or radiographic responses at various measurable sites. In some respects, these sites include lymph nodes and extranodal sites, where, when using PET-CT, PR is described as a reduction in uptake compared to baseline with a residual mass (any size) score of 4 or 5. In the intermediate stage, such findings may indicate reactive disease. At the end of treatment, such findings may indicate residual lesions. In some respects, lymph node response is assessed using CT, where PR is described as a reduction in SPD ≥50% at up to 6 target measurable lymph nodes and extranodal sites. If a lesion is too small to be measured on CT, a default value of 5 mm × 5 mm is assigned; if a lesion is no longer visible, a value of 0 mm × 0 mm is assigned; for lymph nodes >5 mm × 5 mm but smaller than normal, the actual measurement is used for calculation. Other sites for assessment include the bone marrow, where PET-CT-based assessment should indicate residual uptake higher than in normal bone marrow but lower than baseline (diffuse uptake consistent with chemotherapy-responsive changes is permissible). In some cases, if persistent focal changes are present in the bone marrow in the presence of lymph node response, further evaluation by MRI or biopsy, or sectional scanning, should be considered. In some cases, other sites may include assessment of organ enlargement, where spleen length must be reduced by >50% from normal. In some cases, non-measurable lesions and new lesions should be assessed; in the case of PR, these lesions should be absent / normal, reduced but not increased. PET-CT-based and / or CT-based assessments may also be used to measure unresponsive / stable disease (SD) or disease progression (PD). (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323–338; Cheson, BD (2015) Chin Clin Oncol 4(1):5), are incorporated herein by reference in their entirety.

[0225] In some contexts, progression-free survival (PFS) is described as the length of time a subject lives with a disease (e.g., cancer) without its disease worsening during and after treatment. In some contexts, objective response (OR) is described as a measurable response. In some contexts, the objective response rate (ORR; sometimes also called the overall response rate) is described as the proportion of patients who achieve complete remission (CR) or partial response (PR). In some contexts, overall survival (OS) is described as the length of time a subject diagnosed with a disease (e.g., cancer) remains alive from the date of diagnosis or the start of treatment. In some contexts, event-free survival (EFS) is described as the length of time a subject does not experience certain complications or events that the treatment was designed to prevent or delay after the completion of cancer treatment. These events may include cancer recurrence or the occurrence of certain symptoms (e.g., bone pain caused by cancer spreading to the bones), or death.

[0226] In some embodiments, the duration of response (DOR) is measured from the time from recording the tumor response to disease progression. In some embodiments, parameters assessing the response may include a durable response, such as a response that persists for a period of time after the start of therapy. In some embodiments, a 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 the start of therapy. In some embodiments, the response lasts longer than 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer than 6 months.

[0227] In some respects, the Evaluation Criteria for Response to Solid Tumors (RECIST) criteria are used to determine objective tumor response; in others, they are used for solid tumors. (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247.) In some respects, the RECIST criteria are used to determine objective tumor response to the target lesion. In some respects, a complete response determined using the RECIST criteria is described as the disappearance of all target lesions and a reduction in the short axis of any pathological lymph nodes (whether target or non-target) to <10 mm. In other respects, a partial response determined using the RECIST criteria is described as a reduction of at least 30% in the total diameter of the target lesions, with the total diameter at baseline as a reference. In other respects, disease progression (PD) is described as an increase of at least 20% in the total diameter of the target lesions, with the minimum total in the study as a reference (the baseline total is included if it is the minimum total in the study). In addition to a relative increase of 20%, the total must also show an absolute increase of at least 5 mm (in some respects, the appearance of one or more new lesions is also considered progression). In other respects, stable disease (SD) is described as having neither a sufficient reduction to meet PR nor a sufficient increase to meet PD, with the minimum total diameter in the study as a reference.

[0228] Objective response rate (ORR, the incidence of CR or partial response (PR) according to Lugano classification); duration of response (DOR, for participants who experienced an objective response, the time from the first objective response to disease progression according to Lugano classification); progression-free survival (PFS, the time from the date of infusion to the date of disease progression according to Lugano classification); and overall survival.

[0229] The endpoint was defined as follows: Overall response rate (ORR), which is the proportion of subjects who achieved a partial or complete response before the initiation of subsequent therapy.

[0230] Time to response (TTR) is defined in responders as the time from the first dose of treatment (e.g., CD19-CAR-T therapy) (day 1 of cycle 1) to the first recorded partial response (PR) or complete remission (CR). Duration of response (DOR) is defined in responders as the time from the first recorded PR or CR to the date of disease progression or death (whichever occurs earlier). Progression-free survival (PFS) is defined as the time from the date of the first dose of treatment (e.g., CD19-CAR-T therapy) (day 1 of cycle 1) to the date of disease progression or death (whichever occurs earlier).

[0231] Overall survival (OS) is defined as the time from the date of first administration of CD19-CAR-T therapy (day 1 of cycle 1) to the date of death. Time to next anti-lymphoma therapy (TTNT) is defined as the number of days from day 1 of cycle 1 to the first recorded administration of subsequent anti-lymphoma therapy.

[0232] In some aspects, exemplary ctDNAs can be evaluated or analyzed to assess the likelihood of achieving a durable response after administration of cell therapy. In some embodiments, for any of the aforementioned ctDNAs, if the ctDNA:cfDNA ratio is below a detection threshold ratio of ctDNA:cfDNA molecules, the subject is likely to achieve a durable response; and / or if the ctDNA:cfDNA ratio is above a detection threshold ratio of ctDNA:cfDNA molecules, the subject is unlikely to achieve a durable response.

[0233] In some embodiments, a durable response is or includes a complete response (CR) or partial response (PR) lasting 3 months, 4 months, 5 months, or 6 months or longer. In some embodiments, a durable response is or includes a CR or PR lasting at least 3 months. In some aspects, elevated ctDNA levels in biological samples obtained from a subject prior to administration of cell therapy (pre-treatment) may be associated with not achieving a durable response (e.g., a CR or PR lasting at least 3 months). In some aspects, undetectable or decreased ctDNA levels in biological samples obtained from a subject prior to or after administration of cell therapy (pre-treatment) may be associated with achieving a durable response (e.g., a CR or PR lasting at least 3 months).

[0234] In some respects, the methods described herein typically reduce or prevent the spread or burden of disease or condition in a subject. For example, when the disease or condition is a tumor, these methods typically reduce tumor size, volume, metastasis, percentage of blast cells in the bone marrow, or detectable cancer at the molecular level, and / or improve prognosis or survival or other symptoms associated with tumor burden. Disease burden can include the total number of disease cells in the subject's body or in the subject's organs, tissues, or body fluids (e.g., the organ or tissue or other location of the tumor, such as a site that may indicate metastasis). For example, in the context of certain hematologic malignancies, tumor cells can be detected and / or quantified in the blood or bone marrow. In some embodiments, disease burden can include the quality of the tumor, the number or extent of metastases, and / or the percentage of blast cells present in the bone marrow.

[0235] In some implementations, the subject has leukemia. The extent of the disease burden can be determined by assessing residual leukemia in the blood or bone marrow.

[0236] In some embodiments, the method reduces the burden of disease or condition (e.g., number of tumor cells, tumor size, patient survival duration, or event-free survival) to a greater extent and / or for a longer duration compared to the reduction observable without the method. In some embodiments, the burden of disease or condition is detected, assessed, or measured in a subject. In some aspects, the disease burden can be detected by measuring the total number of disease- or disease-related cells (e.g., tumor cells) in the subject's body or in the subject's organs, tissues, or bodily fluids (e.g., blood or serum). In some aspects, the subject's survival, survival over a specific time period, degree of survival, presence or duration of event-free or symptom-free survival, or recurrence-free survival is assessed. In some embodiments, any symptoms of the disease or condition are assessed. In some embodiments, a measure of the burden of disease or condition is specified.

[0237] In some implementations, this method improves event-free survival or overall survival in subjects. For example, in some implementations, at 6 months after treatment or cell therapy, subjects treated with this method have an event-free survival or probability greater than about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some aspects, overall survival is greater than about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some implementations, subjects treated with this method demonstrate event-free survival, recurrence-free survival, or survival for at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some implementations, time to progression is improved, for example, time to progression is greater than or equal to about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0238] In some implementations, the probability of relapse is reduced by this method compared to other methods (e.g., methods in which the subject receives one or more alternative treatments and / or methods in which the subject does not receive cell therapy according to the provided method). For example, in some implementations, the probability of relapse at 6 months post-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%.

[0239] The MRD negative rate is defined as the proportion of subjects who have at least one undetectable MRD result according to a specific threshold before starting subsequent treatment. In some implementations, subjects may demonstrate complete remission but still have a small percentage of morphologically undetectable (by optical microscopy) residual leukemia cells. A subject is considered to have minimal residual disease (MRD) if they show less than 5% blast cells in their bone marrow and exhibit molecularly detectable cancer.

[0240] b. International Prognostic Index (IPI) The International Prognostic Index (IPI) is a well-established prognostic index system. It uses a combination of five clinical variables to identify four independent risk groups for patients. These five variables include age, serum lactate dehydrogenase (LDH) level, tumor stage, Eastern Cooperative Oncology Group (ECOG) performance status (PS), and extranodal lesion location (Zhou Z et al., An enhanced International Prognostic Index (NCCN-IPI) for patients with diffuse large B-cell lymphoma treated in the rituximab era. Blood. 2014 Feb 6;123(6):837-42). The IPI is widely used in clinical practice and is a standard and practical prognostic tool for DLBCL patients. The IPI system can identify four risk categories: low risk, low-intermediate risk, high-intermediate risk, and high risk. The revised IPI score uses the same prognostic variables but re-stratifies patients into three prognostic groups: excellent (94% 4-year OS), good (79% 4-year OS), and poor (55% 4-year OS). The Enhanced International Prognostic Index National Comprehensive Cancer Network (NCCN-IPI) is used for patients with diffuse large B-cell lymphoma (DLBCL). The NCCN-IPI score was developed to improve risk stratification in DLBCL patients. Five variables (age, LDH, stage, extranodal lesions, and performance status) were found to be significantly associated with prognosis. The score categorizes patients into four risk groups: low risk (0-1), low-intermediate risk (2-3), high-intermediate risk (4-5), and high risk (6-8). Compared to the IPI (90% 5-year OS vs. 54%), the NCCN-IPI is able to distinguish between the low-risk and high-risk groups (96% 5-year OS vs. 33%).

[0241] In some respects, the survival of subjects (e.g., those with the cancers described herein) is based on the IPI scoring system. In some respects, the IPI score is based on the independent prognostic role of age, histology, cancer stage, and serum LDH levels. In some respects, the score can be used to characterize or predict the overall survival of subjects with the cancers described herein.

[0242] In some implementations, this disclosure provides for using the International Prognostic Index (IPI) for cancer (e.g., DLBCL) risk assessment to predict outcomes. In some implementations, patients with no risk factors or one risk factor are considered to be in the low-risk IPI group. In some implementations, patients with two risk factors are considered to be in the low--intermediate-risk IPI group. In some implementations, patients with three risk factors are considered to be in the high--intermediate-risk IPI group. In some implementations, patients with four or five risk factors are considered to be in the high-risk IPI group. In some implementations, a higher IPI score predicts a worse outcome compared to a lower IPI score, and treatment of patients with higher IPI scores generally has a lower success rate than treatment of patients with lower IPI scores.

[0243] In some embodiments, parameters such as attributes, factors, characteristics, and / or ctDNA expression described herein are assessed prior to the administration of a therapy (e.g., cell therapy). In some embodiments, parameters such as attributes, factors, characteristics, and / or ctDNA expression described herein are assessed after the administration of a therapy (e.g., cell therapy). In some embodiments, parameters include levels or measurements of attributes, factors, characteristics, and / or ctDNA expression described herein that can be assessed after the administration of a therapy (e.g., cell therapy), such as peak levels.

[0244] In some embodiments, the parameter is SPD (Sum of Diameter Products). In some embodiments, a volumetric measurement of tumor burden is measured, and this volumetric measurement is the sum of diameter products (SPD). In some embodiments, tumor burden is associated with an SPD value above a threshold. In some embodiments, higher or lower disease and / or tumor burden is associated with SPD. In some embodiments, the volumetric measurement is SPD, and the threshold is or approximately 30 / cm², or approximately 40 / cm², or approximately 50 / cm², or approximately 60 / cm², or approximately 70 / cm². In some embodiments, the volumetric measurement is SPD, and the threshold is or approximately 30 / cm², or approximately 40 / cm², or approximately 50 / cm², or approximately 60 / cm², or approximately 70 / cm². In some implementations, the volume measurement is SPD, and the threshold is or is about 30 / cm², or about 40 / cm², or about 50 / cm², or about 60 / cm², or about 70 / cm².

[0245] In some embodiments, the parameter is an inflammatory marker, such as LDH. In some embodiments, LDH is assessed using a colorimetric test or an in vitro enzyme-linked immunosorbent assay (ELISA). In some aspects, LDH levels can be assessed alone and / or in conjunction with another pretreatment parameter, such as another measure or indicator of disease burden, like a tumor volume measurement, such as the sum of diameter products (SPD)). In some aspects, the one or more parameters include LDH and / or a tumor volume measurement. In some embodiments, the parameter is SPD and / or LDH.

[0246] In some respects, elevated levels or measurements of the sum of diameter products (SPD) and LDH in biological samples obtained from subjects prior to administration of cell therapy (pre-treatment) may be associated with a higher risk of cancer selection, treatment, or delayed progression. In some implementations, the inflammatory marker is LDH, and the threshold is or approximately 200 units / L, or approximately 300 units / L, or approximately 400 units / L, or approximately 500 units / L, or approximately 600 units / L.

[0247] In some embodiments, the level, concentration, and / or quantity of LDH are surrogate indicators of disease burden (e.g., the disease burden of a tumor or cancer) and can be used for risk assessment of potential neurotoxicity risk in certain subjects and / or for risk-based dose or treatment adjustments. In some aspects, LDH levels can be assessed alone and / or in conjunction with another pretreatment parameter, such as another measure or indicator of disease burden, like a tumor volume measurement, such as the sum of diameter products (SPD) or other CT- or MRI-based measures of disease burden volume, such as any of the measurements described herein. In some aspects, one or more parameters indicative of disease burden are assessed, and in certain cases, these parameters can indicate the presence, absence, or extent of risk of neurotoxicity following T-cell therapy. In some aspects, the one or more parameters include LDH and / or a tumor volume measurement. In some embodiments, the parameter is SPD and / or LDH.

[0248] In some implementations, the presence, level, amount, concentration, and / or other measurements of LDH are detected or determined in a sample. Various methods for detecting or determining LDH are known. For example, LDH can be measured to convert lactic acid to pyruvate (via NAD). + The method involves the determination of LDH in a sample using a method that reduces LDH to NADH. In some embodiments, the sample is contacted with lactate in the presence of the coenzyme NAD, which serves as a measure of LDH in the sample, generating NADH, which is then oxidized in the presence of an electron transfer agent. In some embodiments, NADH interacts with a probe or dye precursor that can be detected by measuring absorption in the visible light range. In some examples, diaphorase uses NADH to reduce tetrazolium salt (INT) to a red formazan product, and this product is measured. Therefore, in some embodiments, the amount of colored product formed is proportional to the LDH activity in the sample.

[0249] In some implementations, administration of the cell therapy described herein can lead to changes in parameters, such as a decrease in volume measurements (e.g., SPD) or a change in the expression of inflammatory markers (e.g., LDH).

[0250] III. Diagnostic, assessment, screening, monitoring, or predictive methods This document discloses methods for diagnosing, assessing, screening, or monitoring subjects who have cancer or have received cancer treatment. In some aspects, this document provides methods for detecting or assessing the progression of cancer (such as the cancers described herein) in subjects. In other aspects, this document provides methods for assessing or predicting treatment response in subjects who have received cancer treatment, methods for assessing or predicting the risk of cancer recurrence or relapse in subjects who have received cancer treatment, methods for predicting the survival of subjects who have received cancer treatment, methods for identifying minimal residual disease (MRD) in subjects who have been diagnosed with cancer, and methods for monitoring subjects who are receiving cancer treatment.

[0251] In some respects, this article provides methods for assessing the likelihood of a durable response in subjects who have received cancer treatment, and methods for assessing the tumor burden in subjects who have received cancer treatment. This article also provides methods for treating subjects who have already received a first cancer treatment with a second cancer treatment.

[0252] In some embodiments, these methods include determining the presence or absence of the ctDNA described herein in a sample obtained from a subject. In some embodiments, these methods include detecting the ctDNA described herein in a sample obtained from an individual. In some embodiments, these methods further include providing an assessment of the ctDNA level. In some embodiments, the diagnosis or assessment identifies the presence or absence of ctDNA in the sample. In some embodiments, the diagnosis or assessment identifies a cancer (such as the cancer described herein) as likely to respond to treatment (such as the cell therapy described herein). In some embodiments, the sample is the sample described herein.

[0253] In some implementations, the subject has cancer, is suspected of having cancer, is undergoing cancer testing, is receiving cancer treatment, or is undergoing testing for susceptibility to the cancer described herein. In some aspects, this document provides methods for diagnosing or assessing cancer in a subject. In some implementations, methods for diagnosing or assessing cancer include detecting ctDNA, as described herein, in a sample obtained from the subject (e.g., a sample containing cells derived from cancer).

[0254] In some aspects, this document provides a method for assessing or predicting treatment response in a subject who has received cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in the subject after administration of cancer treatment. In some aspects, this document provides a method for predicting the risk of cancer recurrence or relapse in a subject who has received cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in the subject during administration of cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in the subject after administration of cancer treatment. In some aspects, this document provides a method for predicting the survival of a subject who has received cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in the subject after administration of cancer treatment. In some aspects, this document provides a method for monitoring a subject who is receiving cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in the subject after administration of cancer treatment. In some aspects, this document provides a method for assessing the likelihood of a subject who has received cancer treatment achieving a durable response.

[0255] In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in a subject after administration of cancer treatment. In some aspects, this document provides a method for treating a subject with a second cancer treatment, wherein the subject has received a first cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in a subject after administration of the first cancer treatment. In some aspects, this document provides a method for treating a subject with a second or third cancer treatment, wherein the subject has received a first cancer treatment. In some embodiments, the method includes detecting the level of circulating tumor DNA (ctDNA) in a subject after administration of the first cancer treatment.

[0256] In some embodiments, the sample is the sample described herein. In some embodiments, the sample comprises a biological sample, such as a blood, serum, or plasma sample, from a subject diagnosed with cancer.

[0257] In some embodiments, ctDNA levels above a detection threshold in a sample indicate that the subject has a higher-risk disease. In other embodiments, ctDNA levels above a detection threshold in a sample indicate that the subject has minimal residual disease (MRD). In other embodiments, ctDNA levels above a detection threshold indicate that the subject has a risk of recurrence or relapse. In other embodiments, ctDNA levels above a detection threshold indicate that the subject has a low chance of survival. In other embodiments, ctDNA levels above a detection threshold indicate that the subject is unresponsive to treatment.

[0258] In some embodiments, ctDNA levels below a detection threshold in the sample indicate that the subject does not have a high-risk disease. In other embodiments, ctDNA levels below a detection threshold in the sample indicate that the subject does not have minimal residual disease (MRD). In other embodiments, ctDNA levels below a detection threshold indicate that the subject does not have a risk of recurrence or relapse. In other embodiments, ctDNA levels below a detection threshold indicate that the subject has a high chance of survival. In other embodiments, ctDNA levels below a detection threshold indicate that the subject is responding to treatment.

[0259] In some implementations, the detection threshold is the ctDNA:cfDNA ratio, which is 1:10, 1:100, 1:10³, or approximately 1:10. 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 1:10¹¹ or 1:10¹². In other embodiments, the detection threshold is the ctDNA:cfDNA ratio, which is 5:10, 5:100, 5:10³, or 5:10. 4 5:10 5 5:10 6 5:10 7 5:10 8 5:10 9 5:10¹ 0 5:10¹¹ or 5:10¹².

[0260] In some respects, this article provides a method for determining the minimal residual disease (MRD) status in a subject diagnosed with cancer, the method comprising: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; (b) measuring the level of cfDNA in the sample and measuring the level 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 ctDNA:cfDNA molecule ratio in the sample; and (d) determining the subject's MRD status based on the ctDNA:cfDNA molecule ratio in the sample; wherein if the ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule ratio is less than 1:10, then the subject has MRD, and if this ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule ratio is high, then the subject does not have MRD. In other embodiments, if the ratio is higher than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then the subject has MRD, and if the ratio is less than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then the subject does not have MRD.

[0261] In some aspects, this document provides a method for determining the minimal residual disease (MRD) status in a subject who has been diagnosed with cancer. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject; measuring the level of cfDNA in the sample; and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA; determining the ctDNA:cfDNA molecule ratio in the sample; and determining the subject's MRD status based on the ctDNA:cfDNA molecule ratio in the sample. In some embodiments, if the ratio is greater than 1:10... 6If the ctDNA:cfDNA molecule ratio is less than 1:10, then the subject has MRD, and if this ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject does not have MRD.

[0262] In some aspects, this document provides a method for assessing the treatment response of a subject who has received cancer treatment. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In some embodiments, the method specifies: determining the ctDNA:cfDNA molecule ratio in the sample, and assessing the subject's treatment response by the ctDNA:cfDNA molecule ratio in the sample. In some embodiments, if the ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule is present, then the subject has responded to the treatment, and if this ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule ratio is less than 1:10, then the subject is unresponsive to the treatment. In other implementations, if the ratio is less than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then the subject has responded to the treatment, and if the ratio is higher than 1:10¹¹, then the subject has responded to the treatment. 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then the subject will not respond to the treatment.

[0263] In some implementations, if the ratio is less than 1:10 6 If the ctDNA:cfDNA molecule is present, then the subject has responded to the treatment. In some implementations, if this ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule is present, then the subject is unresponsive to the treatment. In some implementations, if the ratio is 1:10 or approximately 1:10... 5The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 If the above ctDNA:cfDNA molecules are present, then the subject has responded to the treatment.

[0264] In some aspects, this document provides a method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In other embodiments, the method specifies: determining the ctDNA:cfDNA molecule ratio in the sample, and assessing the subject's risk of cancer recurrence or relapse based on the ctDNA:cfDNA molecule ratio in the sample. In some embodiments, if the ratio is greater than 1:10... 6 If the ctDNA:cfDNA molecule is present, the subject is at risk of cancer recurrence or relapse, and if this ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject is not at risk of cancer recurrence or relapse.

[0265] In some implementations, if the ratio is higher than 1:10 6 If the ctDNA:cfDNA molecule is present, the subject is at risk of cancer recurrence or relapse. In some implementations, if this ratio is less than 1:10... 6 If the ctDNA:cfDNA molecule is present, then the subject is not at risk of cancer recurrence or relapse. In some implementations, if this ratio is higher than 1:10... 4 1:10 5 1:106 1:10 7 1:10 8 1:10 9 1:10¹ 0 A ctDNA:cfDNA ratio of 1:10¹¹ or 1:10¹² indicates that the subject is at risk of cancer recurrence or relapse. In some implementations, if the ratio is higher than 1:10¹¹, the risk is lower. 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², then the subject is at risk of cancer recurrence or relapse.

[0266] In some implementations, if the ratio is 1:10 or approximately 1:10 5 The ratio is approximately 1:10. 8 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 7 The ratio is approximately 1:10. 5 The ratio is approximately 1.0:10. 6 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 8 The ratio is approximately 1:10. 6 The ratio is approximately 1.0:10. 7 From or approximately 5:10 6 The ratio is approximately 1.0:10. 8 From or approximately 5:10 6 The ratio is approximately 1.0:10. 7 From or approximately 10:10 6 The ratio is approximately 1.0:10. 8 If the above ctDNA:cfDNA molecules are present, the subject is at risk of cancer recurrence or relapse.

[0267] In some aspects, this article provides methods for predicting or assessing the survival of subjects with cancer. In some embodiments, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined. In some embodiments, if the ctDNA:cfDNA ratio is greater than 1:10... 6If the ctDNA:cfDNA molecule ratio is high, the subject is identified as having a low chance of survival. In some implementations, if the ctDNA:cfDNA ratio is higher than 1:10... 6 If the ctDNA:cfDNA molecule ratio is high, the subject is identified as having a high chance of survival. In other implementations, if the ctDNA:cfDNA ratio is greater than 1:10... 4 1:10 5 1:10 6 1:10 7 1:10 8 1:10 9 1:10¹ 0 If the ratio of ctDNA to cfDNA molecules is 1:10¹¹ or 1:10¹², the subject is identified as having a low chance of survival.

[0268] In some embodiments, the method includes administering an effective amount of cancer treatment to the subject. In some embodiments, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample from the subject are measured, and the ctDNA:cfDNA molecule ratio in the sample is determined.

[0269] In some aspects, this document provides a method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment. In some embodiments, the method includes: isolating cell-free DNA (cfDNA) from a biological sample obtained from the subject, measuring the level of cfDNA in the sample, and measuring the level of cell-free tumor DNA (ctDNA) in the sample. In some embodiments, the ctDNA is identified by the presence of one or more mutations in one or more genes of the cfDNA. In some embodiments, the mutation is a phasing variant. In some embodiments, the ctDNA is used to determine the subject's minimal residual disease (MRD) status. In some embodiments, if MRD is detected, the subject is determined to be at risk of cancer recurrence or relapse. In some embodiments, if MRD is detected after two cycles of the induction phase of treatment, the subject is determined to be at risk of cancer recurrence or relapse.

[0270] In some embodiments, the subject is receiving the cell therapy described herein. In some embodiments, these methods include knowing the presence of the ctDNA provided herein in a sample from the subject. In some embodiments, these methods include detecting the ctDNA in a sample from the subject. In some embodiments, in response to knowing the ctDNA in the sample, the subject is predicted to have longer survival after treatment with the cell therapy provided herein (e.g., compared to a subject whose cancer does not express ctDNA). In some embodiments, in response to detecting the ctDNA provided herein in the sample, the subject is predicted to have longer survival after treatment with the cell therapy provided herein (e.g., compared to a subject whose cancer does not express ctDNA). In some embodiments, these methods further include providing a diagnosis or assessment. In some embodiments, the diagnosis or assessment identifies the presence or absence of ctDNA in the sample. In some embodiments, the diagnosis or assessment identifies the subject (e.g., compared to a subject whose cancer does not express ctDNA) as predicted to have longer survival after treatment with the cell therapy provided herein. In some embodiments, the sample is the sample described herein.

[0271] In some aspects, this document provides methods for screening subjects who have cancer, are suspected of having cancer, are undergoing cancer testing, are receiving cancer treatment, or are undergoing susceptibility testing for the cancers described herein. In some embodiments, the subject is receiving or has received the cell therapy treatments described herein. In some embodiments, in response to the discovery of ctDNA as described herein in a sample, the subject is predicted to have an increased risk of cancer recurrence, invasive cancer, anticancer therapy resistance, or poor prognosis (e.g., compared to subjects whose cancer does not express ctDNA). In some embodiments, in response to the detection of ctDNA in a sample, the subject is predicted to have an increased risk of cancer recurrence, invasive cancer, anticancer therapy resistance, or poor prognosis (e.g., compared to subjects whose cancer does not express ctDNA). In some embodiments, these methods further include providing a diagnosis or assessment. In some embodiments, the diagnosis or assessment identifies the presence or absence of ctDNA in the sample. In some embodiments, the diagnosis or assessment identifies the subject (e.g., compared to subjects whose cancer does not express ctDNA) as having a predicted increased risk of cancer recurrence, invasive cancer, anticancer therapy resistance, or poor prognosis.

[0272] In some aspects, the method includes identifying minimal residual disease (MRD) in a subject who has been diagnosed with cancer. In some embodiments, the method includes detecting the level of ctDNA in the subject. In some embodiments, the detection includes: obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample, identifying mutations in one or more genes in the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample. In some embodiments, a ctDNA level in the sample above a detection threshold indicates that the subject has MRD.

[0273] In some aspects, the method includes assessing the treatment response of a subject who has received cancer treatment. In some embodiments, the method includes detecting the level of ctDNA in the subject after administration of cancer treatment. In some embodiments, the detection includes: obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample, identifying mutations in one or more genes within the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample. In some embodiments, a ctDNA level below a detection threshold in the sample indicates that the subject has responded to cancer treatment.

[0274] In some aspects, the method includes assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment. In some embodiments, the method includes detecting the level of ctDNA in the subject during cancer treatment administration. In some embodiments, the method includes detecting the level of ctDNA in the subject after cancer treatment administration. In some embodiments, the detection includes: obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample, identifying mutations in one or more genes within the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample. In some embodiments, a ctDNA level in the sample above a detection threshold indicates that the subject is at risk of cancer recurrence or relapse.

[0275] In some embodiments, the ctDNA:cfDNA molecule ratio is a detection threshold ratio of ctDNA:cfDNA molecules. In other embodiments, the ctDNA:cfDNA molecule ratio is the ratio of ctDNA:cfDNA molecules in the subject sample. In some embodiments, the ctDNA:cfDNA molecule ratio is 1:10, 1:100, 1:1000, or 1:10. 4 1:10 5 1:10 6 1:10 7 Or 1:108 .

[0276] In some implementations, the ctDNA:cfDNA molecule ratio is 1:10 or approximately 1:10. 6 The ratio is approximately 50:10 6 The ratio is approximately 1:10. 6 The ratio is approximately 40:10 6 The ratio is approximately 1:10. 6 The ratio is approximately 30:10 6 The ratio is approximately 1:10. 6 It is approximately 20:10 6 The ratio is approximately 1:10. 6 It is at most or approximately 10:10 6 The ratio is approximately 1:10. 6 It is at most or approximately 5:10 6 The ratio is approximately 1:10. 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 50:10 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 40:10 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 30:10 6 The ratio is approximately 2.5:10. 6 It is approximately 20:10 6 The ratio is approximately 2.5:10. 6 It is at most or approximately 10:10 6 The ratio is approximately 2.5:10. 6 It is at most or approximately 5:10 6 The ratio is approximately 2.5:10. 6 The ratio is approximately 50:10 6 From or approximately 5:10 6 The ratio is approximately 50:10 6 From or approximately 5:10 6 The ratio is approximately 40:10 6 , or approximately 5:10 6 The ratio is approximately 30:10 6 , or approximately 5:10 6 It is approximately 20:10 6 , or approximately 5:10 6 It is at most or approximately 10:10 6 From or approximately 10:10 6 The ratio is approximately 50:10 6 From or approximately 10:106 The ratio is approximately 40:10 6 The ratio is approximately 10:10. 6 The ratio is approximately 30:10 6 The ratio is approximately 10:10. 6 It is approximately 20:10 6 、From or approximately 20:10 6 The ratio is approximately 50:10 6 、From or approximately 20:10 6 The ratio is approximately 40:10 6 , or approximately 20:10 6 The ratio is approximately 30:10 6 、From or approximately 30:10 6 The ratio is approximately 50:10 6 、From or approximately 30:10 6 The ratio is approximately 40:10 6 Or from a value of approximately 40:10 6 The ratio is approximately 50:10 6 The above are ctDNA:cfDNA molecules.

[0277] In some embodiments, the ctDNA:cfDNA molecule ratio 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 1.1:10 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 62.8:10 6 2.9:10 6 3:10 6 3.1:10 6 3.2:10 6 3.3:10 6 3.4:10 6 3.5:10 6 3.6:10 6 3.7:10 6 3.8:10 6 3.9:10 6 4:10 6 4.1:10 6 4.2:10 6 4.3:10 6 4.4:10 6 4.5:10 6 4.6:10 6 4.7:10 6 4.8:10 6 4.9:10 6 5:10 6 5.1:10 6 5.2:10 6 5.3:10 6 5.4:10 6 5.5:10 6 5.6:10 6 5.7:10 6 5.8:10 6 5.9:10 6 Or 6:10 6 The above are ctDNA:cfDNA molecules.

[0278] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from approximately 1:1,000,000 to approximately 1:50,000,000; approximately 1:1,000,000 to approximately 1:45,000,000; approximately 1:1,000,000 to approximately 1:40,000,000; approximately 1:1,000,000 to approximately 1:35,000,000; approximately 1:1,000,000 to approximately 1:30,000,000; approximately 1:1,000,000 to approximately 1:25,000,000; approximately 1:1,000,000 to approximately 1:20,000,000; and approximately 1:1,000,000. Up to or approximately 1:15,000,000; up to or approximately 1:1,000,000; up to or approximately 1:1,000,000; up to or approximately 1:5,000,000; up to or approximately 1:1,000,000; up to or approximately 1:4,000,000; up to or approximately 1:1,000,000; up to or approximately 1:3,000,000; up to or approximately 1:1,000,000; up to or approximately 1:2,000,000; from up to or approximately 2:1,000,000; up to or approximately 2:5,000,000; up to or approximately 2:1,000,000; up to or approximately 2:4,500,000; up to or approximately 2:1,000,000; up to or approximately 2:4,000,000. The ratio is approximately 2:1,000,000 to approximately 2:35,000,000; approximately 2:1,000,000 to approximately 2:30,000,000; approximately 2:1,000,000 to approximately 2:25,000,000; approximately 2:1,000,000 to approximately 2:20,000,000; approximately 2:1,000,000 to approximately 2:15,000,000; approximately 2:1,000,000 to approximately 2:1,000,000; approximately 2:1,000,000 to approximately 2:500,000; approximately 2:1,000,000 to approximately 2:4,000,000; approximately 2:1,000,000 to approximately 2:100,000,000. Or approximately 2:3000000, or approximately 2:1000000 to approximately 2:2000000, or approximately 3:1000000 to approximately 3:50000000, from or approximately 3:1000000 to approximately 3:45000000, or approximately 3:1000000 to approximately 3:40000000, or approximately 3:1000000 to approximately 3:35000000, or approximately 3:1000000 to approximately 3:30000000, or approximately 3:1000000 to approximately 3:25000000, or approximately 3:1000000 to approximately 3:20000000.The ratio is approximately 3:1000000 to approximately 3:15000000, approximately 3:1000000 to approximately 3:10000000, approximately 3:10000000 to approximately 3:5000000, approximately 3:1000000 to approximately 3:4000000, approximately 3:1000000 to approximately 3:3000000, approximately 3:1000000 to approximately 3:2000000, from approximately 4:1000000 to approximately 4:50000000, approximately 4:1000000 to approximately 4:45000000, approximately 4:1000000 to or approximately 4:40000000, 4:1000000 to or approximately 4:35000000, 4:1000000 to or approximately 4:30000000, 4:1000000 to or approximately 4:25000000, 4:1000000 to or approximately 4:20000000, 4:1000000 to or approximately 4:15000000, 4:1000000 to or approximately 4:10000000, 4:10000000 to or approximately 4:5000000, 4:1 000000 to approximately 4:4000000, approximately 4:1000000 to approximately 4:3000000, approximately 4:1000000 to approximately 4:2000000, approximately 5:1000000 to approximately 5:50000000, approximately 5:1000000 to approximately 5:45000000, approximately 5:1000000 to approximately 5:40000000, approximately 5:1000000 to approximately 5:35000000, approximately 5:1000000 to approximately 5:30000000, approximately 5:1000 000 to approximately 5:25000000, approximately 5:1000000 to approximately 5:20000000, approximately 5:1000000 to approximately 5:15000000, approximately 5:1000000 to approximately 5:1000000, approximately 5:10000000, approximately 5:10000000, approximately 5:5000000, approximately 5:1000000 to approximately 5:4000000, approximately 5:1000000 to approximately 5:3000000, approximately 5:1000000 to approximately 5:2000000 and above ctDNA:cfDNA molecules.

[0279] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from approximately 1:1,000,000 to approximately 1:50,000,000; approximately 1:1,000,000 to approximately 1:45,000,000; approximately 1:1,000,000 to approximately 1:40,000,000; approximately 1:1,000,000 to approximately 1:35,000,000; approximately 1:1,000,000 to approximately 1:30,000,000; approximately 1:1,000,000 to approximately 1:25,000,000; approximately 1:1,000,000 to approximately 1:20,000,000; and approximately 1:1,000,000. Up to or approximately 1:15,000,000; up to or approximately 1:1,000,000; up to or approximately 1:1,000,000; up to or approximately 1:5,000,000; up to or approximately 1:1,000,000; up to or approximately 1:4,000,000; up to or approximately 1:1,000,000; up to or approximately 1:3,000,000; up to or approximately 1:1,000,000; up to or approximately 1:2,000,000; from up to or approximately 2:1,000,000; up to or approximately 2:5,000,000; up to or approximately 2:1,000,000; up to or approximately 2:4,500,000; up to or approximately 2:1,000,000; up to or approximately 2:4,000,000. The ratio is approximately 2:1,000,000 to approximately 2:35,000,000; approximately 2:1,000,000 to approximately 2:30,000,000; approximately 2:1,000,000 to approximately 2:25,000,000; approximately 2:1,000,000 to approximately 2:20,000,000; approximately 2:1,000,000 to approximately 2:15,000,000; approximately 2:1,000,000 to approximately 2:1,000,000; approximately 2:1,000,000 to approximately 2:500,000; approximately 2:1,000,000 to approximately 2:4,000,000; approximately 2:1,000,000 to approximately 2:100,000,000. Or approximately 2:3000000, or approximately 2:1000000 to approximately 2:2000000, or approximately 3:1000000 to approximately 3:50000000, from or approximately 3:1000000 to approximately 3:45000000, or approximately 3:1000000 to approximately 3:40000000, or approximately 3:1000000 to approximately 3:35000000, or approximately 3:1000000 to approximately 3:30000000, or approximately 3:1000000 to approximately 3:25000000, or approximately 3:1000000 to approximately 3:20000000.The ratio is approximately 3:1000000 to approximately 3:15000000, approximately 3:1000000 to approximately 3:10000000, approximately 3:10000000, approximately 3:1000000 to approximately 3:5000000, approximately 3:1000000 to approximately 3:4000000, approximately 3:1000000 to approximately 3:3000000, approximately 3:1000000 to approximately 3:2000000, from approximately 4:1000000 to approximately 4:50000000, approximately 4:1000000 to approximately 4 :45000000, or approximately 4:1000000 to or approximately 4:40000000, or approximately 4:1000000 to or approximately 4:35000000, or approximately 4:1000000 to or approximately 4:30000000, or approximately 4:1000000 to or approximately 4:25000000, or approximately 4:1000000 to or approximately 4:20000000, or approximately 4:1000000 to or approximately 4:15000000, or approximately 4:1000000 to or approximately 4:10000000, or approximately 4:10000000, or approximately 4:1 000000 to approximately 4:5000000, approximately 4:1000000 to approximately 4:4000000, approximately 4:1000000 to approximately 4:3000000, approximately 4:1000000 to approximately 4:2000000, approximately 5:1000000 to approximately 5:50000000, approximately 5:1000000 to approximately 5:45000000, approximately 5:1000000 to approximately 5:40000000, approximately 5:1000000 to approximately 5:350000 00, or approximately 5:1000000 to approximately 5:30000000, or approximately 5:1000000 to approximately 5:25000000, or approximately 5:1000000 to approximately 5:20000000, or approximately 5:1000000 to approximately 5:15000000, or approximately 5:1000000 to approximately 5:10000000, or approximately 5:10000000 to approximately 5:3000000, or approximately 5:1000000 to approximately 5:2000000 ctDNA:cfDNA molecules.

[0280] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from approximately 1:10 to approximately 1:100, 1:10 to approximately 1:1000, 1:10 to approximately 1:10000, 1:10 to approximately 1:20000, 1:10 to approximately 1:30000, 1:10 to approximately 1:40000, 1:10 to approximately 1:50000, 1:10 to approximately 1:60000, 1:10 to approximately 1:70000, 1:10 to approximately 1:80000, 1:10 to approximately 1:90000, 1:10 to approximately 1:100000, from 2:10 to approximately 2:100, 2:10 to approximately 2: 1000, 2:10 to approximately 2:10000, 2:10 to approximately 2:20000, 2:10 to approximately 2:30000, 2:10 to approximately 2:40000, 2:10 to approximately 2:50000, 2:10 to approximately 2:60000, 2:10 to approximately 2:70000, 2:10 to approximately 2:80000, 2:10 to approximately 2:90000, 2:10 to approximately 2:100000, from 3:10 to approximately 3:100, 3:10 to approximately 3: 1000, 3:10 to approximately 3:10000, 3:10 to approximately 3:20000, 3:10 to approximately 3:30000, 3:10 to approximately 3:40000, 3:10 to approximately 3:50000, 3:10 to approximately 3:60000, 3:10 to approximately 3:70000, 3:10 to approximately 3:80000, 3:10 to approximately 3:90000, 3:10 to approximately 3:100000, from 4:10 to approximately 4:100, 4:10 to approximately 4: 1000, 4:10 to approximately 4:10000, 4:10 to approximately 4:20000, 4:10 to approximately 4:30000, 4:10 to approximately 4:40000, 4:10 to approximately 4:50000, 4:10 to approximately 4:60000, 4:10 to approximately 4:70000, 4:10 to approximately 4:80000, 4:10 to approximately 4:90000, 4:10 to approximately 4:100000, from 5:10 to approximately 5:100, 5:10 to Approximately 5:1000, 5:10 to approximately 5:10000, 5:10 to approximately 5:20000, 5:10 to approximately 5:30000, 5:10 to approximately 5:40000, 5:10 to approximately 5:50000, 5:10 to approximately 5:60000, 5:10 to approximately 5:70000, 5:10 to approximately 5:80000, 5:10 to approximately 5:90000, 5:10 to approximately 5:100000 and above ctDNA:cfDNA molecules.

[0281] In some embodiments, the ctDNA:cfDNA molecule ratio ranges from approximately 1:10 to approximately 1:100, 1:10 to approximately 1:1000, 1:10 to approximately 1:10000, 1:10 to approximately 1:20000, 1:10 to approximately 1:30000, 1:10 to approximately 1:40000, 1:10 to approximately 1:50000, 1:10 to approximately 1:60000, 1:10 to approximately 1:70000, 1:10 to approximately 1:80000, 1:10 to approximately 1:90000, 1:10 to approximately 1:100000, from 2:10 to approximately 2:100, 2:10 to approximately 2: 1000, 2:10 to approximately 2:10000, 2:10 to approximately 2:20000, 2:10 to approximately 2:30000, 2:10 to approximately 2:40000, 2:10 to approximately 2:50000, 2:10 to approximately 2:60000, 2:10 to approximately 2:70000, 2:10 to approximately 2:80000, 2:10 to approximately 2:90000, 2:10 to approximately 2:100000, from 3:10 to approximately 3:100, 3:10 to approximately 3: 1000, 3:10 to approximately 3:10000, 3:10 to approximately 3:20000, 3:10 to approximately 3:30000, 3:10 to approximately 3:40000, 3:10 to approximately 3:50000, 3:10 to approximately 3:60000, 3:10 to approximately 3:70000, 3:10 to approximately 3:80000, 3:10 to approximately 3:90000, 3:10 to approximately 3:100000, from 4:10 to approximately 4:100, 4:10 to approximately 4: 1000, 4:10 to approximately 4:10000, 4:10 to approximately 4:20000, 4:10 to approximately 4:30000, 4:10 to approximately 4:40000, 4:10 to approximately 4:50000, 4:10 to approximately 4:60000, 4:10 to approximately 4:70000, 4:10 to approximately 4:80000, 4:10 to approximately 4:90000, 4:10 to approximately 4:100000, from 5:10 to approximately 5:100, 5:10 to ctDNA:cfDNA molecules with ratios of approximately 5:1000, 5:10 to approximately 5:10000, 5:10 to approximately 5:20000, 5:10 to approximately 5:30000, 5:10 to approximately 5:40000, 5:10 to approximately 5:50000, 5:10 to approximately 5:60000, 5:10 to approximately 5:70000, 5:10 to approximately 5:80000, 5:10 to approximately 5:90000, and 5:10 to approximately 5:100000.

[0282] In some implementations, the ctDNA:cfDNA molecule ratio 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, 7:15 000, 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, 6:15 0000, 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 ctDNA:cfDNA molecules with the following counts: 750000, 6:750000, 7:750000, 8:750000, 9:750000, 1:1000000, 2:1000000, 3:1000000, 4:1000000, 5:1000000, 6:1000000, 7:1000000, 8:1000000, 9:1000000 and above.

[0283] In some implementations, the ctDNA:cfDNA molecule ratio 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, 7:15 000, 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, 6:15 0000, 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 ctDNA and cfDNA molecules with values ​​below 750000, 6:750000, 7:750000, 8:750000, 9:750000, 1:1000000, 2:1000000, 3:1000000, 4:1000000, 5:1000000, 6:1000000, 7:1000000, 8:1000000, and 9:1000000.

[0284] In some aspects, the method includes assessing the tumor burden of a subject who has received cancer treatment. In some embodiments, the method includes detecting the level of ctDNA in the subject after administration of cancer treatment. In some embodiments, the detection includes: obtaining a biological sample from the subject, isolating cell-free DNA (cfDNA) from the biological sample, identifying mutations in one or more genes in the cfDNA, wherein the identification of the mutation indicates the presence of ctDNA in the sample, and measuring the level of ctDNA in the sample. In some embodiments, a ctDNA level in the sample above a detection threshold indicates that the subject is predicted to have a high tumor burden.

[0285] Quantitative levels of ctDNA can be measured per milliliter of haplogenous genomic equivalents (hGE / mL), determined as the product of total cell-free DNA concentration and the mean allele fraction of somatic mutations, expressed on a logarithmic scale (log hGE / mL). In some embodiments, ctDNA levels are measured over time to determine the rate of change (e.g., rate of decline) of ctDNA over time. For example, the rate of decline of ctDNA over time after one or more treatments (or during a treatment regimen) can be determined. In some cases, the rate of change is determined on a linear scale. In some embodiments, the rate of change is determined on a logarithmic fold-down scale (e.g., a logarithmic fold change in the frequency of variant alleles). In some cases, the rate of decline of ctDNA after or during a treatment cycle can indicate / predict progression-free survival or overall survival in a subject.

[0286] IV. Cancer Treatment and Cell Therapy A. Cancer The methods of the present invention can be used to treat cancer in subjects, reduce tumor size, kill tumor cells, inhibit tumor cell proliferation, inhibit tumor growth, eliminate tumors from subjects, prevent tumor recurrence, prevent tumor metastasis, induce remission in subjects, or any combination thereof. In some embodiments, these methods induce a complete response. In other embodiments, these methods induce a partial response.

[0287] In some implementations, the cancer in the methods provided herein is a blood cancer. In some implementations, the cancer is a B-cell malignancy.

[0288] In some implementations, this method can be used to treat tumors, where the tumor is lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of pluripotent hematopoietic stem cell found in the bone marrow. These stem cells produce myeloid progenitor cells and lymphoid progenitor cells, which then produce various types of white blood cells found in the body. White blood cells derived from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells derived from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia can affect one or more of these cell types in a patient.

[0289] Lymphoma can be divided into at least two subgroups: Hodgkin lymphoma and non-Hodgkin lymphoma. Non-Hodgkin lymphoma (NHL) is a heterogeneous group of cancers that originate from B lymphocytes, T lymphocytes, or natural killer cells.

[0290] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. It is classified as an aggressive lymphoma, and most patients are cured with conventional chemotherapy (NCCN Guidelines for NHL 2014).

[0291] Therefore, in some implementations, cancer includes, but is not limited to, leukemia and lymphoma, such as acute myeloid (or myeloid cell) leukemia (AML), chronic myeloid (or myeloid cell) leukemia (CML), acute lymphoblastic (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, and Hodgkin's lymphoma. Chigkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM). B-cell malignancies are selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

[0292] In some implementations, this method can be used to treat lymphoma, wherein the lymphoma is a B-cell malignancy. In some implementations, the lymphoma is selected from small cell lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenström macroglobulinemia), splenic marginal zone lymphoma, plasma cell tumor (e.g., plasma cell myeloma (i.e., multiple myeloma) or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., MALT lymphoma), intranodal marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicular center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), EBV-positive DL... BCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary exudative lymphoma, large B-cell lymphoma in HHV8-associated multicentric Castleman's disease, Burkitt lymphoma, adult T-cell lymphoma, extranodal K / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatocellular and splenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sezary syndrome This includes primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic lymphoma, B-lymphoblastic lymphoma with recurrent genetic abnormalities, T-lymphoblastic lymphoma, and Hodgkin's lymphoma. In some implementations, the cancer is refractory to one or more prior therapies, and / or the cancer has relapsed after one or more prior therapies.

[0293] In some implementations, the lymphoma is large B-cell lymphoma (LBCL). In some implementations, the lymphoma is diffuse large B-cell lymphoma (DLBCL). In some implementations, the cancer is follicular lymphoma (FL).

[0294] In some implementation schemes, cancer is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, stomach cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer, or soft tissue sarcoma.

[0295] In some implementations, the cancer is refractory to one or more of chemotherapy, radiotherapy, immunotherapy (including T-cell therapy and / or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof, or the cancer has relapsed after the aforementioned treatments. In one specific implementation, the cancer is refractory diffuse large B-cell lymphoma.

[0296] In some embodiments, the subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human. In some embodiments, the subject is a human. In other embodiments, the subject is a patient, such as a human patient suffering from the cancer described herein.

[0297] B. Cancer Treatment a. General cancer treatment In some respects, this disclosure relates to anticancer therapies. A variety of cancer treatments can be performed, including (but not limited to) surgery, resection, chemotherapy, radiation therapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplantation, and blood transfusion.

[0298] In some implementations, anticancer agents and / or chemotherapeutic agents are administered, including (but not limited to) alkylating agents, platinum-based drugs, taxanes, vinca derivatives, antiestrogenic drugs, aromatase inhibitors, ovarian suppressants, endocrine / hormone drugs, bisphosphonate therapy drugs, and targeted biological therapy drugs. 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, ixaprilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mitomycin, capecitabine, capecitabine, anastrozole, exemestane, letrozole, leuprorelin, abaricicliz, busherin, goserelin, megestrol acetate. Ketoconazole, Rifelodronate, Pamidronate, Ibandronate, Alendronate, Zoledronic Acid, Tykerb, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Penoxorubicin, Mitoxantrone, Bendamustine, Bevacizumab, Cetuximab, Ipilimumab, Trastuzumab emtansine, Afatinib, Interleukin, Alectinib, Alendronate, Atezolizumab, Avelumab, Axitinib, Belimumab, Belistat, Bevacizumab, Bonatumab, Bortezomib, Bosutinib, Brentuximab velituximab, Brigadiib, Carbohydrate Botinib, Canatumab, Carfilzomib, Ceritinib, Cetuximab, Cobitinib, Crizotinib, Dabrafenib, Daratumumab, Dasatinib, Denosumab, Denosumab, Duvalimumab, Erotocilizumab, Ensidipine, Erlotinib, Everolimus, Gefitinib, Tiimomab, Ibrutinib, Adelalipi, Imatinib, Ipilimumab, Ixazomib, Lapatinib, Lenvatinib, Midotutolin, Nexituzumab, Lenatinib, Nilotinib, Niraparib, Nivolumab, Obituzumab, Ofamumab, Olaparib The following drugs are included: irinotecan, osimertinib, palbociclib, panitumumab, pembrolizumab, pertuzumab, ponatinib, ramucirumab, regorafenib, ribociclib, rituximab, romidesin, rucaparib, ruxolitinib, sutuximab, sipuleucel-T, sorafenib, tesiromoxetine, tocilizumab, tofacitinib, tosimomumab, trametinib, trastuzumab, vandetanib, vemurafenib, veneclax, vemodega, vorinostat, and aflibercept. Depending on the specific implementation plan, subjects may be treated with a single drug or combination of drugs described herein (e.g., R-CHOP).

[0299] In some embodiments, the treatment is a first-line therapy. In some embodiments, the treatment is a second-line therapy. In some embodiments, the treatment includes further monitoring of the subject. In some embodiments, the treatment includes cell therapy. In some embodiments, the treatment includes CAR-T cell therapy, and the CAR-T cell therapy is an anti-CD19 cell therapy. In some embodiments, the cell therapy includes genetically engineered cells, and the genetically engineered cells are T cells. In some embodiments, the genetically engineered T cells include chimeric antigen receptors (CARs). In some embodiments, the CAR specifically binds to an antigen associated with the disease or condition and / or is expressed by cells associated with the disease or condition. In some implementations, the antigens are selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer-testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, dual antigens, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrinB2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR. vIII, estrogen receptor, fetal AchR, folate receptor α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Ra2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), cancer-fetal antigen, melanoma preferentially expressed antigen (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptor, BAFF-R, VEGF-R2, nephroblastoma 1 (WT-1), and pathogen-specific antigens. In some embodiments, the antigen is CD19. In other embodiments, the antigen is CD20.

[0300] In some embodiments, the CAR includes an extracellular antigen recognition domain that specifically binds to the antigen, and an intracellular signaling domain including ITAM. In some embodiments, the intracellular signaling domain includes an intracellular domain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CAR further includes a co-stimulatory signaling region.

[0301] In some embodiments, cell therapy includes genetically engineered cells that can bind multiple antigens. In some embodiments, improved selectivity and specificity are achieved through strategies targeting multiple antigens. Such strategies typically involve multiple antigen-binding domains that are typically present on different genetically engineered antigen receptors and specifically bind to different antigens. For example, in some embodiments, cells express multispecific binding molecules. In some embodiments, cells express multiple binding molecules, antigen-binding domains, where each can target one or more antigens, such as a binding domain targeting CD20 (e.g., any binding domain described herein) and another binding domain targeting another antigen (e.g., CD19). In some aspects, antigen receptors include multiple binding domains that bind different antigens, each expressed in a disease or condition or its cells or tissues targeted by the cells to be used.

[0302] Multispecific cells are also provided, such as cells containing one or more of the recombinant receptors or cells provided herein. In some embodiments, the cells express bispecific recombinant receptors, such as CARs.

[0303] In some embodiments, the co-stimulatory signaling region includes a CD28 or 4-1BB signaling domain. In some embodiments, the co-stimulatory domain is a 4-1BB domain. In some embodiments, the T cells are CD4+ or CD8+. In some embodiments, the T cells are primary T cells obtained from the 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 one or more prior therapies for the cancer. In some embodiments, the subject has an inadequate response to one or more prior therapies for the cancer.

[0304] In some embodiments, the treatment includes radiological imaging of the subject. In some embodiments, the treatment includes computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI) of the subject. In some embodiments, the treatment includes further monitoring of the subject.

[0305] Certain aspects of this disclosure relate to anticancer therapies. In some embodiments, the anticancer therapies of this disclosure include one or more therapeutic agents, such as those used to treat diseases, conditions, or injuries associated with ctDNA described herein (e.g., the cancers provided herein). In some embodiments, the anticancer therapy is a small molecule inhibitor, antibody, cell therapy (i.e., cell-based therapy), or nucleic acid. In some embodiments, the anticancer therapy is a chemotherapy agent, an antihormonal agent, an antimetabolite chemotherapy agent, a kinase inhibitor, peptide, gene therapy, vaccine, platinum-based chemotherapy agent, immunotherapy, antibody, or checkpoint inhibitor. In some embodiments, the anticancer therapy is a CD19-targeted therapy. In some embodiments, the anticancer therapy includes a second agent, such as a second anticancer agent. In some embodiments, the anticancer therapy is administered in combination with a second anticancer therapy or agent. In some embodiments, the anticancer therapy includes a heat shock protein (HSP) inhibitor, a MYC inhibitor, an HDAC inhibitor, immunotherapy, a neoantigen, a vaccine, or cell therapy.

[0306] In some implementations, the second anticancer agent includes one or more of the following: immune checkpoint inhibitors, chemotherapy drugs, VEGF inhibitors, integrin β3 inhibitors, statins, EGFR inhibitors, mTOR inhibitors, PI3K inhibitors, MAPK inhibitors, or CDK4 / 6 inhibitors.

[0307] In some implementations, the anticancer therapy includes a kinase inhibitor. In some implementations, the methods provided herein include administering a kinase inhibitor to an individual, for example, in combination with another anticancer therapy. In some implementations, the kinase inhibitor is crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, ensartinib (X-396), repotinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP-37440, bezatinib (TSR-011), ASP3026, KRCA-0008, TQ-B3139, TPX-0131, or TAE684 (NVP-TAE684).

[0308] In some embodiments, anticancer therapies include cancer immunotherapies, such as checkpoint inhibitors, cancer vaccines, cell-based therapies, T-cell receptor (TCR)-based therapies, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy. In some embodiments, the methods provided herein include administering a cancer immunotherapy to an individual, such as a checkpoint inhibitor, cancer vaccine, cell-based therapies, T-cell receptor (TCR)-based therapies, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy, for example, in combination with another anticancer therapy. In some embodiments, cancer immunotherapies include small molecules, nucleic acids, peptides, carbohydrates, toxins, cell-based agents, or cell binders. Examples of cancer immunotherapies are described in more detail herein, but these examples are not intended to be limiting. The cancer immunotherapies disclosed herein are intended for use as a monotherapy or, based on medical judgment, in combination (including two or more methods in any combination or number of ways). Any cancer immunotherapy (optionally as a monotherapy or in combination with another cancer immunotherapy or other therapeutic agents described herein) may be used in any of the methods described herein.

[0309] In some embodiments, cancer immunotherapy includes cell-based therapies. In some embodiments, cancer immunotherapy includes T-cell-based therapies. In some embodiments, cancer immunotherapy includes adoptive therapy, such as adoptive T-cell-based therapies. 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 treatment method for cancer or infectious diseases in which immune cells are administered to a host with the aim of enabling these cells to directly or indirectly mediate specific immunity against cancer cells (i.e., triggering an immune response against cancer cells). In some embodiments, this immune response results in the inhibition of tumor and / or metastatic cell growth and / or proliferation, and in related embodiments, results in tumor cell death and / or resorption. Immune cells may be derived from a different organism / host (exogenous immune cells) or may be cells obtained from a subject's organism (autologous immune cells). In some embodiments, immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or γδ T cells), NK cells, inert NK cells, or NKT cells) can be genetically engineered to express antigen receptors, such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, host cells (e.g., autologous or allogeneic T cells) are modified to express antigen-specific T cell receptors (TCRs) against cancer antigens. In some embodiments, NK cells are engineered to express TCRs. NK cells can be further engineered to express CARs. Multiple CARs and / or TCRs (e.g., targeting different antigens) can be added to a single cell type, such as T cells or NK cells.

[0310] In some embodiments, the anticancer therapy includes nucleic acid molecules such as dsRNA, siRNA, or shRNA. In some embodiments, the methods provided herein include administering nucleic acid molecules, such as dsRNA, siRNA, or shRNA, to an individual, for example, in combination with another anticancer therapy. As is known in the art, dsRNA, having a double-stranded structure, is effective in inducing RNA interference (RNAi). In some embodiments, the anticancer therapy includes small interfering RNA molecules (siRNA). dsRNA and siRNA can be used to silence gene expression in mammalian cells, such as human cells.

[0311] In some implementations, the treatment is administered in combination 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, and WO 2019 / 152743, which are incorporated herein by reference in their entirety.

[0312] In other embodiments, the treatment is administered in combination with another therapeutic intervention, simultaneously or sequentially in any order. In some embodiments, the cancer treatment is administered in combination with another treatment sufficiently close in time such that these treatments enhance the effects of one or more additional therapeutic agents, or vice versa. In some embodiments, the cancer treatment is administered prior to one or more additional cancer treatments. In some embodiments, the cancer treatment is administered after one or more additional therapeutic agents. In some embodiments, the treatment is administered as a follow-up treatment.

[0313] In some embodiments, one or more additional therapeutic agents include cytokines, such as IL-2, to enhance persistence. In some embodiments, the cancer treatment includes the administration of chemotherapy agents.

[0314] In some embodiments, the cancer treatment includes the administration of chemotherapy agents (e.g., opsonizing chemotherapy agents) to reduce tumor burden, for example. In some embodiments, the cancer treatment includes an induction phase. In some embodiments, the induction phase includes two or more cycles of chemotherapy agent administration. In some embodiments, the induction phase includes 2, 3, 4, 5, 6, or more cycles of chemotherapy agent administration.

[0315] In some implementations, the cancer treatment may include administration of kinase inhibitors, such as BTK inhibitors (e.g., ibrutinib or acalatinib); inhibitors of tryptophan metabolism and / or the kynurenine pathway, such as indoleamine 2,3-dioxygenase-1 (IDO1) inhibitors (e.g., icardolstat); immunomodulators, such as immunomodulatory imide drugs (IMiDs), including thalidomide or thalidomide derivatives (e.g., lenalidomide or pomalidomide); or checkpoint inhibitors, such as anti-PD-L1 antibodies (e.g., durvalumab).

[0316] In some embodiments, the cancer treatment is administered as part of a follow-up 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, after administration of 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. In some embodiments, the presence or level of the ctDNA disclosed herein can be used to select candidate treatments. In some embodiments, the presence or level of the ctDNA disclosed herein can be used to determine the success of first-line therapy, second-line therapy, or both first-line and second-line therapy during or after treatment. In some embodiments, the ctDNA:cfDNA molecule ratio can be used to select candidate treatments. In other embodiments, the ctDNA:cfDNA molecule ratio can be used to determine the success of first-line therapy, second-line therapy, or both first-line and second-line therapy during or after treatment.

[0317] In some embodiments, the cancer treatment is administered as a third-line, fourth-line, fifth-line, or sixth-line therapy. In some embodiments, the cancer treatment is administered before one or more other therapeutic agents. In some embodiments, the treatment is administered after one or more other therapeutic agents.

[0318] b. Cell therapy and chimeric antigen receptors In some aspects, this cancer treatment is a cell therapy. In some embodiments, the cell therapy (e.g., T-cell therapy) methods disclosed herein include administering engineered cells expressing a recombinant receptor (e.g., a chimeric antigen receptor CAR), said recombinant receptor being designed to recognize and / or specifically bind to an antigen associated with a disease or condition (e.g., the cancer described herein). In a specific embodiment, the antigen bound or recognized by the recombinant receptor (e.g., CAR) is CD19. In other embodiments, the antigen bound or recognized by the recombinant receptor is CD20. In some embodiments, binding to the antigen leads to a reaction, such as an immune response against such an antigen. In some embodiments, the cells contain or are engineered to contain a recombinant receptor, such as a chimeric antigen receptor (CAR). Recombinant receptors (e.g., CARs) typically include an extracellular antigen (or ligand) binding domain specific to a particular antigen, which in some aspects is linked to one or more intracellular signaling components via a linker and / or transmembrane domain. In some aspects, the engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subject (e.g., for adoptive cell therapy). It also provides treatment methods for administering cells and compositions to subjects (e.g., patients).

[0319] In some embodiments, the cells include one or more nucleic acids introduced through genetic engineering and thus express recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is achieved by first stimulating the cells, for example by binding them to a stimulus that induces a response (e.g., proliferation, survival, and / or activation, measured, for example, by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in a culture to a number sufficient for clinical use.

[0320] In some embodiments of the provided methods and uses, the chimeric receptor (e.g., a chimeric antigen receptor) contains one or more domains that combine a ligand-binding domain (e.g., an antibody or antibody fragment) specific for a desired antigen (e.g., CD19) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a stimulatory or activating portion of the intracellular domain, such as a T cell stimulation or activation domain, providing a primary activation signal or primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to promote effector function. In some embodiments, the chimeric receptor, when genetically engineered into immune cells, can modulate T cell activity and, in certain cases, T cell differentiation or homeostasis, thereby generating genetically engineered cells with improved in vivo lifespan, survival, and / or durability, for example, for adoptive cell therapy approaches.

[0321] Exemplary antigen receptors (including CARs) and methods for engineering such receptors and introducing them into cells include, for example, those described in the following documents: International Patent Application Publications Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061; and U.S. Patent Application Publications Nos. US2002131960, US2013287748. US20130149337, US Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. 2013 April; 3(4): 388–398; Davila et al. (2013) PLoSONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, antigen receptors include those described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1. Examples of CARs include those published in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US 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 WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US Patent No. 7,446,190 and US Patent No. 8,389,282.

[0322] Chimeric receptors (such as CARs) typically include an extracellular antigen-binding domain, such as a portion of an antibody molecule, usually a variable heavy chain (V) of the antibody. H ) region and / or variable light chain (V L (e.g., scFv antibody fragments)

[0323] In some embodiments, the receptor-targeted antigen is a peptide. In a specific embodiment, the antigen target is CD19. In some embodiments, the antigen is selectively expressed or overexpressed on cells of disease or condition (e.g., tumor or pathogenic cells) compared to normal or non-target cells or tissues.

[0324] In some embodiments, the CAR is constructed to be specific to a particular antigen, such as an antigen expressed in a specific cell type targeted by adoptive therapy (e.g., a cancer biomarker), and / or an antigen intended to induce a damping response (e.g., an antigen expressed on normal or disease-free cell types). Therefore, the CAR typically includes one or more antigen-binding molecules in its extracellular portion, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR includes one or more antigen-binding portions of an antibody molecule, such as a variable heavy chain (V) derived from a monoclonal antibody (mAb). H ) and variable light chains (V L ) single-chain antibody fragments (scFv).

[0325] In some implementations, the antibody or its antigen-binding portion is expressed on the cell as part of a recombinant receptor (such as a chimeric receptor, e.g., CAR) that binds (e.g., specifically binds) to an antigen (e.g., CD19). Chimeric receptor-targeted antigens include those expressed in the context of a disease, condition, or cell type that will be targeted by adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and conditions, including cancers and tumors, including blood cancers, immune system cancers such as lymphoma, leukemia, and / or myeloma, such as B-cell, T-cell, and myeloid leukemia, lymphoma, and multiple myeloma.

[0326] In some implementations, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen (e.g., the complete antigen) expressed on the cell surface.

[0327] In some implementations, the disease or condition is a B-cell malignancy, such as large B-cell lymphoma (e.g., DLBCL), and the antigen is CD19.

[0328] The term "antibody" is used in the broadest sense in this article, including polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including antigen-binding fragments (Fab), F(ab')2, Fab', Fv, recombinant IgG (rIgG) fragments, and variable heavy chains (V) capable of specifically binding antigens. H This term encompasses single-chain antibody fragments (including single-chain variable fragments (scFv)) and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). It covers genetically engineered and / or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific (e.g., bispecific) antibodies, biantibodies, triantibodies and tetraantibodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass its functional antibody fragment. This term also covers complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0329] In some embodiments, antigen-binding proteins, antibodies, and their antigen-binding fragments specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full-length or may be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragments (scFv)). In other embodiments, the constant region of the antibody heavy chain is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly from, for example, IgG1, IgG2, IgG3, and IgG4, and more particularly IgG1 (e.g., human IgG1). In yet another embodiment, the constant region of the antibody light chain is selected from, for example, κ or λ, particularly κ.

[0330] The provided antibodies include antibody fragments. An "antibody fragment" refers to a molecule that is different from the complete antibody but contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; and variable heavy chains (V...). H ) region, single-chain antibody molecules (such as scFv) and single-domain V H Monoclonal antibodies; and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0331] The terms “complementarity-determining region” and “CDR” are synonymous with “hypervariant region” or “HVR” and are known in some cases to refer to the discontinuous amino acid sequence within the antibody variable region that confers antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms “frame region” and “FR” are known in some cases to refer to the non-CDR portions of the heavy and light chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0332] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” DevComp Immunol, 2003. Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0333] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number based on the length of the most common antibody region sequence, accommodating insertions by inserting letters (e.g., "30a"), and deletings in some antibodies. These two schemes place certain insertions and deletions ("insertion-deletion") in different positions, leading to numbering differences. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways. The AbM scheme is a compromise between the Kabat and Chothia definitions used by the OxfordMolecular AbM antibody modeling software.

[0334] Table 1 below lists exemplary positional boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using the Kabat and Chothia numbering schemes. FRs are located between CDRs; for example, FR-L1 precedes CDR-L1, FR-L2 is between CDR-L1 and CDR-L2, FR-L3 is between CDR-L2 and CDR-L3, and so on. It should be noted that because the Kabat numbering scheme shown places the insertion at H35A and H35B, when numbering using the Kabat numbering convention shown, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length.

[0335]

[0336] 1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD 2 - Al-Lazikani et al., (1997) JMB 273, 927-948.

[0337] Therefore, unless otherwise stated, a “CDR” or “complementarity-determining region” or individually designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or its region (e.g., its variable region) should be understood to encompass one (or a specific) complementarity-determining region as defined by any of the foregoing schemes or other known schemes. For example, when stating that a particular CDR (e.g., CDR-H3) contains a given V H or V L When specifying the amino acid sequence of the corresponding CDR within the variable region, it should be understood that such a CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within that variable region as defined by any of the foregoing schemes or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibody are described using various numbering schemes, but it should be understood that the provided antibody may include CDRs according to any of the other foregoing numbering schemes or other numbering schemes known to those skilled in the art.

[0338] Similarly, unless otherwise stated, a FR for a given antibody or its region (e.g., its variable region) or a separately specified FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4) should be understood to cover a (or specific) frame region defined by any known protocol. In some cases, a protocol for identifying a particular CDR, FR, or multiple FRs or multiple CDRs is specified, such as a CDR defined by the Kabat, Chothia, AbM, or Contact methods or other known protocols. In other cases, a specific amino acid sequence of the CDR or FR is given.

[0339] The term "variable region" or "variable domain" refers to the structural domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V L They typically have similar structures, with each domain containing four conserved frame regions (FRs) and three core regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single V H or V L The structural domain may be sufficient to confer antigen-binding specificity. Furthermore, V-terminal molecules derived from antibodies that bind to specific antigens can be used. H or V L The structural domains are selected to screen complementary V. L or V HA library of domains is used to isolate antibodies that bind to the antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0340] A single-domain antibody is an antibody fragment containing all or a portion of the variable domain of the heavy chain or the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody. In some embodiments, the CAR contains an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or cell surface antigen of the cell or disease to be targeted (e.g., tumor cells or cancer cells), such as any target antigen described herein or known.

[0341] Antibody fragments can be prepared using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and production via recombinant host cells. In some embodiments, the antibody is a recombinantly generated fragment, such as a fragment containing a non-naturally occurring arrangement, such as a fragment having two or more antibody regions or chains linked by synthetic linkers (e.g., peptide linkers), and / or a fragment that is not or may not be generated by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0342] A “humanized” antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR, and all or substantially all of the FR amino acid residues are derived from human FR. A humanized antibody may optionally contain 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 humanized variant of a non-human antibody, typically designed to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody derived from CDR residues) to restore or improve antibody specificity or affinity, for example.

[0343] In some embodiments, the antigen or antigen-binding domain is CD19. In some embodiments, the scFv comprises a V derived from an antibody or antibody fragment specific to CD19. H and V L In some embodiments, the antibody or antibody fragment binding to CD19 is a murine antibody, such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, such as that described in U.S. Patent Publication No. US 2016 / 0152723.

[0344] In some embodiments, 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 an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes scFv. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain capable of inducing primary activation signals in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immune receptor tyrosine activation motif (ITAM).

[0345] In some embodiments, the antibody portion of the recombinant receptor (e.g., CAR) also includes at least a portion of an immunoglobulin constant region, such as a hinge region (e.g., the IgG4 hinge region) and / or C. H 1 / C L And / or the Fc region. In some embodiments, the constant region or portion is of human IgG, such as IgG4 or IgG1. In some aspects, the constant region portion serves as a spacer between an antigen recognition component (e.g., scFv) and a transmembrane domain. The length of the spacer can provide an increase in cellular reactivity after antigen binding compared to the absence of a spacer. Exemplary spacers include, but are not limited to, Hudecek et al. (2013). Clin. Cancer Res Those described in ., 19:3153, International Patent Application Publication No. WO2014031687, U.S. Patent No. 8,822,647 or Publication No. US2014 / 0271635.

[0346] In some embodiments, the antigen receptor includes an intracellular domain directly or indirectly linked to an extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain includes an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is typically linked to one or more intracellular signaling components, such as signaling components that mimic activation via an antigen receptor complex (e.g., a TCR complex) in the case of CAR, and / or components that transduce signals via another cell surface receptor. In some embodiments, the chimeric receptor includes a transmembrane domain connected or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0347] In one implementation, a transmembrane domain naturally associated with a domain in the receptor (e.g., CAR) is used. In some cases, transmembrane domains are selected or modified by amino acid substitutions to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0348] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. If the source is natural, the domain is in some respects derived from any membrane-binding or transmembrane protein. The transmembrane region includes those derived from (i.e., containing at least a transmembrane region) the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some respects, the synthetic transmembrane domain primarily contains hydrophobic residues, such as leucine and valine. In some respects, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In some embodiments, linkages are made via linkers, spacer regions, and / or the transmembrane domain itself. In some respects, the transmembrane domain contains the transmembrane portion of CD28.

[0349] In some embodiments, the extracellular domain and the transmembrane domain may be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane domain are connected via a spacer region (such as any spacer region described herein). In some embodiments, the receptor comprises the extracellular portion of a molecule that derives the transmembrane domain, such as the extracellular portion of CD28.

[0350] Intracellular signal transduction domains include those that mimic or approximate signals transmitted through natural antigen receptors, signals transmitted through combinations of such receptors and co-stimulatory receptors, and / or signals transmitted solely through co-stimulatory receptors. In some embodiments, short oligopeptide or polypeptide linkers (e.g., linkers of 2 to 10 amino acids in length, such as linkers containing glycine and serine, like glycine-serine duplexes) are present and form a connection between the transmembrane domain and the cytoplasmic signal transduction domain of the CAR.

[0351] In some respects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some respects, a CAR includes one or both of these signaling components.

[0352] Receptors (such as CARs) typically include at least one or more intracellular signaling components. In some aspects, CARs include primary cytoplasmic signaling sequences that regulate primary activation of the TCR complex. Primary cytoplasmic signaling sequences that function in a stimulatory manner may contain signaling motifs known as immune receptor tyrosine activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from the CD3ζ chain, FcRγ, CD3γ, CD3δ, and CD3ε. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0353] In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as the TCR CD3 chain, e.g., the CD3ζ chain, which mediates T cell activation and cytotoxicity. Therefore, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor (e.g., CAR) also comprises a portion of one or more additional molecules (e.g., Fc receptor γ, CD8, CD4, CD25, or CD16). For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0354] In some embodiments, upon attachment of a CAR or other chimeric receptor, a cytoplasmic domain or intracellular signaling domain of the receptor activates at least one normal effector function or response of an immune cell (e.g., T cells engineered to express a CAR). For example, in some cases, CAR induces T cell functions such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used instead of the complete immune stimulation chain, for example, if it is possible to transduce effector function signals. In some embodiments, one or more intracellular signaling domains include a cytoplasmic sequence of a T cell receptor (TCR) and, in some respects, also include a cytoplasmic sequence of co-receptors that, under natural conditions, interact with the receptor to initiate signal transduction upon antigen receptor binding.

[0355] In the case of a natural TCR, full activation typically requires not only signal transduction via the TCR but also co-stimulatory signals. Therefore, in some embodiments, the CAR includes components for generating secondary or co-stimulatory signals to promote full activation. In other embodiments, the CAR does not include components for generating co-stimulatory signals. In some aspects, additional CARs are expressed in the same cell, providing components for generating secondary or co-stimulatory signals.

[0356] In some embodiments, the chimeric antigen receptor includes an intracellular domain of a T-cell co-stimulatory molecule. In some embodiments, the CAR includes a signaling domain and / or transmembrane portion of a co-stimulatory receptor (e.g., CD28, 4-1BB, OX40, DAP10, and ICOS). In some aspects, the same CAR includes both an activating component and a co-stimulatory component. In some embodiments, the chimeric antigen receptor includes an intracellular domain derived from a T-cell co-stimulatory molecule or a functional variant thereof, e.g., located between the transmembrane domain and the intracellular signaling domain. In some aspects, the T-cell co-stimulatory molecule is CD28 or 41BB.

[0357] In some embodiments, the intracellular signaling domain includes a CD28 transmembrane and signaling domain connected to the intracellular domain of CD3 (e.g., CD3-ζ). In some embodiments, the intracellular signaling domain includes a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domain connected to the intracellular domain of CD3ζ.

[0358] In some implementations, the CAR includes one or more (e.g., two or more) co-stimulatory domains and an activation domain (e.g., a primary activation domain) in its cytoplasmic portion. Exemplary CARs include intracellular components of CD3-ζ, CD28, and 4-1BB.

[0359] In some embodiments, the antigen receptor further comprises a biomarker, and / or cells expressing CAR or other antigen receptors also comprise alternative biomarkers, such as cell surface biomarkers, which can be used to confirm cell transduction or engineering to express the receptor. In some aspects, the biomarker includes all or part (e.g., truncated forms) of CD34, NGFR, or epidermal growth factor receptors, such as truncated versions of such cell surface receptors (e.g., tEGFR). In some embodiments, the nucleic acid encoding the biomarker is operatively linked to a polynucleotide encoding an adapter sequence (e.g., a cleavable adapter sequence, such as T2A). For example, the biomarker and optional adapter sequence can be any biomarker and adapter sequence disclosed in published patent application number WO2014031687. For example, the biomarker can be a truncated EGFR (tEGFR) optionally linked to an adapter sequence, such as the T2A cleavable adapter sequence.

[0360] In some embodiments, the biomarker is a molecule (e.g., a cell surface protein) that is not naturally present on or on the surface of T cells. In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that is not recognized as "self" by the host immune system to which the cell will be adopted.

[0361] In some embodiments, the biomarker has no therapeutic function and / or produces no effect other than being used as a biomarker for genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the biomarker may be a therapeutic molecule or a molecule that otherwise exerts certain desired effects, such as a ligand for cells encountered in vivo, such as a co-stimulatory or immune checkpoint molecule, to enhance and / or inhibit cellular responses upon adoptive transfer and encounter with the ligand.

[0362] In some contexts, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some respects, first-generation CARs are CARs that provide only CD3 chain induction signals upon antigen binding; in some respects, second-generation CARs are CARs that provide both such signals and co-stimulatory signals, such as CARs containing intracellular signaling domains from co-stimulatory receptors (e.g., CD28 or CD137); and in some respects, third-generation CARs are CARs containing multiple co-stimulatory domains from different co-stimulatory receptors.

[0363] For example, in some embodiments, the CAR comprises 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 a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR comprises 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 4-1BB or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor also comprises a spacer region containing a portion of an Ig molecule (e.g., a human Ig molecule), such as an Ig hinge, like an IgG4 hinge, or a hinge-only spacer region.

[0364] For example, in some embodiments, the CAR comprises an antibody, such as an antibody fragment (including scFv), a spacer region (e.g., a spacer region 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 a spacer region containing an Ig hinge), a transmembrane domain containing all or part of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, the CAR comprises an antibody or fragment (e.g., scFv), a spacer region (e.g., any spacer region containing an Ig hinge), a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3ζ-derived signaling domain.

[0365] Recombinant receptors (e.g., CARs) expressed by cells administered to a subject typically recognize or specifically bind to molecules expressed, associated with, and / or specific to the disease or condition being treated or its cells. Upon specific binding to this molecule (e.g., an antigen), the receptor typically delivers an immune-stimulating signal (e.g., an ITAM-transduced signal) to the cell, thereby promoting an immune response against the disease or condition. For example, in some embodiments, cells express CARs that specifically bind to antigens expressed by cells or tissues of the disease or condition or associated with said disease or condition.

[0366] A therapeutic cell composition of engineered CD4+ T cells and engineered CD8+ T cells, each expressing the same anti-CD19 chimeric antigen receptor (CAR), was administered to subjects with relapsed or refractory large B-cell lymphoma (LBCL). Eligible subjects received a therapeutic T-cell composition containing engineered cells expressing the anti-CD19 CAR. The administered therapeutic T-cell composition was produced by a method involving immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukocyte ablation samples from the individual subject. The ablated CD4+ and CD8+ T cells were activated separately and transduced independently with a viral vector (e.g., a lentiviral vector) encoding the anti-CD19 CAR, subsequently expanded separately, and the engineered cell populations were cryopreserved in low volumes. The CAR contained an anti-CD19 scFv derived from a mouse antibody (the variable region was derived from FMC63, V...). L -Connector-V H The viral vector contains an immunoglobulin-derived spacer region, a transmembrane domain derived from CD28, a co-stimulatory region derived from 4-1BB, and an intracellular signal transduction domain of CD3-ζ. The viral vector also contains a sequence encoding a truncated receptor, which serves as a surrogate marker for CAR expression; it is separated from the CAR sequence by a T2A ribosomal skipping sequence.

[0367] The cryopreserved cell composition was thawed prior to intravenous administration. The therapeutic dose of T cells was administered as a defined cell composition by administering a formulated CD4+ CAR+ cell population and a formulated CD8+ CAR+ cell population at a target ratio of approximately 1:1.

[0368] Subjects were administered a single dose of CAR-expressing T cells (each single dose was administered separately via infusion of T cells expressing CD4+ CAR and T cells expressing CD8+ CAR), as follows: containing 5 x 10 7 A single dose of dose level 1 (DL-1) of total CAR-expressing T cells, or containing 1 x 10 8 A single dose of dose level 2 (DL-2) of total CAR-expressing T cells.

[0369] Exemplary antigen receptors (e.g., CARs) also include the following CARs: FDA-approved products BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some embodiments of any of the provided implementations, the CAR is one of the following: BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), or YESCARTA™ (axicabtagene ciloleucel). In some embodiments of any of the provided implementations, the CAR is a 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_1):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852). In some embodiments of any of the provided implementations, the CAR is a CAR of TECARTUS™ (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). In some implementations of any of the provided implementations, the CAR is the CAR of KYMRIAH™ (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).In some implementations of any of the provided implementations, the CAR is the CAR of YESCARTA™ (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).

[0370] V. Reagent Kit This document also provides kits for detecting the ctDNA molecules of this disclosure. In some embodiments, the kits provided herein contain reagents (e.g., one or more oligonucleotides, primers, probes, or decoys of this disclosure) for detecting the ctDNA provided herein. In some embodiments, the kits contain reagents (e.g., one or more oligonucleotides, primers, probes, or decoys of this disclosure) for detecting ctDNA of the cfDNA provided herein from a sample.

[0371] VI. Definition As used herein, the terms “tumor-specific” or “tumor-associated” with respect to cfDNA refer to the difference in the DNA sequence of cfDNA in a subject with a tumor-forming cancer (e.g., lung cancer) compared to a reference DNA, such as when cfDNA is compared to control DNA (gDNA) from non-tumor cells, as described herein. Alternatively, “tumor-specific” may refer to a comparison of pre-treatment cfDNA with cfDNA collected during or after treatment.

[0372] As used herein, the term "biomarker" generally refers to any measurable substance collected from a subject as a sample whose presence, absence, and / or amount indicates a phenomenon. Non-limiting examples of such phenomena may include disease states, symptoms, or exposure to compounds or environmental conditions. In the various embodiments described herein, biomarkers may be used for diagnostic purposes (e.g., to diagnose disease states, health states, asymptomatic states, symptomatic states, etc.). The term "biomarker" may be used interchangeably with the term "marker."

[0373] As used herein, "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (e.g., a patient) receiving administration of one or more agents, cells, cell populations, or compositions is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including juvenile, adolescent, puberty, adult, and old age. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0374] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to the complete or partial improvement or relief of a disease or symptom or disorder, or its associated symptoms, adverse effects or outcomes, or phenotype. The desired effects of treatment include, but are not limited to: prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or relief of the disease state, and relief or improvement of prognosis. This term does not imply a complete cure of the disease or the complete elimination of any symptom or effect of all symptoms or outcomes.

[0375] As used herein, “delayed disease development” refers to postponing, hindering, slowing, delaying, stabilizing, inhibiting, and / or postponing the development of a disease (e.g., cancer). This delay can vary in length depending on the individual’s medical history and / or the individual being treated. In some implementations, a sufficient or significant delay can effectively cover prevention because the individual has not developed the disease. For example, the development of advanced cancer (e.g., metastasis) can be delayed.

[0376] As used herein, “prevention” includes providing preventative measures against the onset or recurrence of a disease to subjects who may be susceptible to the disease but have not yet been diagnosed with it. In some embodiments, the provided cells and compositions are used to delay the development of the disease or slow its progression.

[0377] As used herein, "inhibition" function or activity refers to a reduction in function or activity compared to a condition that is otherwise identical except for the condition or parameter of interest, or compared to another condition. For example, cells that inhibit tumor growth reduce the tumor growth rate compared to the tumor growth rate in the absence of cells.

[0378] In the context of application, the “effective amount” of an agent (such as a pharmaceutical preparation, cell, or composition) refers to the amount that effectively achieves the desired result (such as a therapeutic or preventative outcome) within the required dose / amount and time period.

[0379] A “therapeutic effective amount” of a pharmaceutical agent (such as a drug formulation or cells) refers to the amount that effectively achieves the desired therapeutic outcome (e.g., for treating a disease, condition, or disorder) and / or therapeutic pharmacokinetic or pharmacodynamic effect within the required dose and time period. Therapeutic effective amounts can vary depending on factors such as disease state, age, sex, weight, and the cell population administered. In some embodiments, the provided method involves administering cells and / or the composition at an effective amount (e.g., a therapeutically effective amount).

[0380] "Prophylactic effective dose" refers to the amount that effectively achieves the desired preventive outcome within the required dosage and time period. It is usually, but not always, lower than the therapeutic effective dose because the prophylactic dose is administered to the subject before or in an early stage of the disease. In cases of low tumor burden, the prophylactic effective dose may be higher than the therapeutic effective dose in some respects.

[0381] As used herein, the term “about” refers to the typical range of error for various values ​​that is readily known to those skilled in the art. The description of “about” for a numerical value or parameter herein includes (and describes) embodiments for that numerical value or parameter itself.

[0382] As used herein, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural referents. For example, “a” or “an” means “at least one / a kind” or “one / a kind or more / a kind.”

[0383] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope designation is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the claimed subject matter. Therefore, the description of scope should be considered as specifically disclosing all possible sub-scopes and the individual values ​​within those scopes. For example, where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of that range, as well as any other specified value or intermediate value within that specified range, is included within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and also within the claimed subject matter, but are subject to any explicitly excluded limits within the specified range. When a specified range contains one or two limits, the range excluding any one or both of these included limits is also included within the claimed subject matter. This applies regardless of the breadth of the scope.

[0384] As used herein, a statement that a cell or cell population is “positive” for a particular biomarker means the presence of that particular biomarker (typically a surface biomarker) on or within the cell. When referring to a surface biomarker, the term means the presence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the biomarker and detecting the antibody, wherein the level of staining detected by flow cytometry is significantly higher than that detected by the same procedure under otherwise identical conditions using an isotype-matched control or a fluorescence-subtracted-one (FMO) gated control, and / or the level is significantly similar to that of cells known to be positive for the biomarker, and / or the level is significantly higher than that of cells known to be negative for the biomarker.

[0385] As used herein, a statement that a cell or cell population is “negative” for a particular biomarker means that the particular biomarker (typically a surface biomarker) is not substantially detectably present on or within the cell. When referring to a surface biomarker, the term means that the presence of surface expression is not detected by flow cytometry, for example by staining with an antibody that specifically binds to the biomarker and detecting the antibody, wherein the staining detected by flow cytometry is not at a level significantly higher than that detected under the same procedure with an isotype-matched control or a fluorescence-subtracted-one (FMO) gated control under otherwise identical conditions, and / or at a level significantly lower than that of cells known to be positive for the biomarker, and / or at a level significantly similar to that of cells known to be negative for the biomarker.

[0386] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from the commonly understood meaning in the art. All publications cited in this application, including patent documents, scientific articles, and databases, are incorporated herein by reference in their entirety for all purposes, as if each individual publication were individually incorporated by reference. If any definition set forth herein conflicts with or is otherwise inconsistent with the definitions set forth herein in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall prevail.

[0387] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0388] VII. Examples Example 1 In the absence of tumor tissue, multiple studies have shown that plasma cell-free DNA (cfDNA) can be used for blood-based, non-invasive genotyping of various human tumors, including aggressive lymphomas. Whether these same cfDNA advantages also apply to follicular lymphoma (FL) has not been investigated, and whether they can inform non-invasive minimal residual disease (MRD) surveillance in the context of modern protocols inducing deep FL remission remains to be explored. Here, we address these questions by monitoring ctDNA and MRD in previously untreated FL patients receiving standard first-line (1L) therapy using phased variant enrichment and detection sequencing (PhasED-Seq, Kurtz et al. 2021, Nat Biotech).

[0389] method We analyzed 298 samples from 61 patients with flaccid tumors (FL), including archived formalin-fixed paraffin-embedded (FFPE) tumor tissue and serial blood samples before, during, and after 1L treatment. All patients were treatment-naïve, with the majority (75%) presenting with advanced FL. Induction regimens primarily included bendamustine / rituximab (66%) and R-CHOP (26%). At a median follow-up of 23 months from the start of 1L therapy, 80% of patients were alive and progression-free, while 11% experienced progression within 24 months (POD24).

[0390] MRD was analyzed using PhasED-Seq (Foresight Diagnostics). Phased variants (PVs) were genotyped using baseline plasma or alternatively from FFPE tumor tissue; matched leukocytes were used as a source of constitutive DNA to review germline variation and potential-undetermined clonal hematopoiesis (CHIP). MRD was then assessed longitudinally serially during 1L therapy using baseline PV genotypes from each baseline source. Blood time points included baseline (pretreatment), day 1 of cycle 2 (C2D1), C3D1, C4D1, C5D1, C6D1, end of induction (EOI), and subsequent serial time points. ctDNA levels were compared with known prognostic factors for FL, including radiographic response, POD24, and progression-free survival (PFS).

[0391] result PVs were successfully identified from tumor FFPE samples in all patients (61 / 61), enabling ctDNA MRD assessment via PhasED-Seq. The median number of PVs identified was 530 (IQR 285-1152). Prior to treatment, ctDNA was detectable in 94% of cases (57 / 61) using tumor-derived PVs and in 75% of cases using plasma-derived PVs. The median tumor fraction in baseline plasma was 0.28%, significantly lower than the pre-treatment ctDNA level in diffuse large B-cell lymphoma (DLBCL) (median = 6.1% in DLBCL, Roschewski, ASH 2022, FL vs. DLBCL, P < 0.0001). The median pre-treatment ctDNA level was significantly higher in advanced disease (Stage I / II, 0.04%; Stage III / IV, 0.53%, P = 0.006) and correlated with the FLIPI risk score (P = 0.04). Figure 3 ).

[0392] Next, we tested whether sufficient PVs could be identified directly from plasma without requiring tumor tissue for MRD monitoring. Despite low baseline ctDNA levels in FL, sufficient PVs were identified directly from plasma for disease monitoring in 75% of cases (45 / 61). Furthermore, regardless of the baseline sample used for PV identification (i.e., tumor versus pre-treatment plasma, Spearman rho = 0.92, P < 0.0001), Figure 4 ctDNA levels were highly correlated. Although tumor and plasma genotypic concordance was high, we observed significant tumor heterogeneity across compartments, with varying rates of concordance among specific mutated genes. Figure 6 ).

[0393] Next, we evaluated the prognostic performance of ctDNA MRD in predicting clinical outcomes. Pretreatment ctDNA levels had no significant prognostic value for time to progression. In contrast, in patients assessed for ctDNA MRD after two cycles of treatment (i.e., at C3D1), the detection of residual ctDNA was significantly associated with poorer time to progression (log-rank P = 0.03, HR = 8.9). Figure 6 Finally, we evaluated the feasibility of serial ctDNA monitoring during radiographic remission. In patients who subsequently experienced clinical disease relapse, we detected the re-emergence of detectable MRD more than two years prior to clinical disease progression. Figure 7 ).

[0394] in conclusion Circulating tumor DNA analysis using PhasED-Seq is feasible in FL, with baseline ctDNA levels significantly lower than those observed in DLBCL. Plasma DNA can often be used as a substitute for tissue samples when none are available. ctDNA detection during treatment is closely associated with eventual treatment response, and serial disease monitoring using ctDNA can predict clinical relapse.

Claims

1. A method for determining the minimal residual disease (MRD) status of a subject diagnosed with cancer, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ratio of ctDNA to cfDNA molecules in the sample; (d) The subject’s MRD status is determined by the ratio of ctDNA to cfDNA molecules in the sample; Where the ratio is higher than 1:10 6 If the ctDNA:cfDNA molecule is present, then the subject has MRD, and if the ratio is less than 1:10 6 If ctDNA:cfDNA molecules are present, then the subject does not have MRD.

2. A method for evaluating the treatment response of a subject who has received cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ratio of ctDNA to cfDNA molecules in the sample; (d) Assess the treatment response of the subject by the ratio of ctDNA to cfDNA molecules in the sample; Where the ratio is less than 1:10 6 If the ctDNA:cfDNA molecule indicates a response to the treatment, and if the ratio is greater than 1:10, then the subject has responded to the treatment. 6 If ctDNA:cfDNA molecules are present, then the subject does not respond to the treatment.

3. A method for evaluating the treatment response of a subject who has received cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ratio of ctDNA to cfDNA molecules in the sample; (d) Assess the treatment response of the subject by the ratio of ctDNA to cfDNA molecules in the sample; If the ratio is lower than the detection threshold ratio of ctDNA:cfDNA molecules, the subject responds to the treatment; if the ratio is higher than the detection threshold ratio of ctDNA:cfDNA molecules, the subject does not respond to the treatment.

4. A method for evaluating the treatment response of a subject who has received cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ratio of ctDNA to cfDNA molecules in the sample; (d) Assess the treatment response of the subject by the ratio of ctDNA to cfDNA molecules in the sample; If the ctDNA level is below the detection threshold, the subject responds to the treatment; if the ctDNA level is above the detection threshold, the subject does not respond to the treatment.

5. A method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ratio of ctDNA to cfDNA molecules in the sample; (d) Assess the risk of cancer recurrence or relapse in the subject by the ratio of ctDNA to cfDNA molecules in the sample; Where the ratio is higher than 1:10 6 If the ctDNA:cfDNA molecule indicates that the subject is at risk of cancer recurrence or relapse, and if the ratio is less than 1:10... 6 If ctDNA:cfDNA molecules are present, then the subject is not at risk of cancer recurrence or relapse.

6. A method of treating a subject with a second cancer treatment, wherein the subject has received a first cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ctDNA:cfDNA molecule ratio in the sample; and (d) If the ctDNA:cfDNA ratio is higher than 1:10 6 ctDNA:cfDNA molecules, then a second cancer treatment is administered.

7. A method for treating cancer in a subject in need, said method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ctDNA:cfDNA molecule ratio in the sample; and (d) If the ctDNA:cfDNA ratio is higher than 1:10 6 ctDNA:cfDNA molecules, then an effective amount of cancer treatment is administered.

8. A method for treating cancer in a subject in need, wherein the subject is assessed as a candidate for cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ctDNA:cfDNA molecule ratio in the sample; and (d) If the ctDNA:cfDNA ratio is higher than 1:10 6 If the ctDNA:cfDNA molecule ratio is less than 1:10, the subject is identified as a candidate for cancer treatment. 6 ctDNA:cfDNA molecules, then the subject is identified as a candidate for non-cancer treatment; and (e) Administer an effective amount of cancer treatment to the subject.

9. A medicament used in a method for treating cancer in a subject of need, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ctDNA:cfDNA molecule ratio in the sample; and (d) If the ctDNA:cfDNA ratio is higher than 1:10 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

10. Use of cell therapy in the manufacture of a medicament for treating cancer in a subject of need, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Measure the level of cfDNA in the sample and measure the level 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) Determine the ctDNA:cfDNA molecule ratio in the sample; and (d) If the ctDNA:cfDNA ratio is higher than 1:10 6 If ctDNA:cfDNA molecules are present, then an effective amount of the drug is administered to the subject.

11. A method for evaluating the treatment response of a subject who has received cancer treatment, wherein, after administration of said cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from said subject are measured, and the ctDNA:cfDNA molecule ratio in said sample is determined, wherein if the ctDNA:cfDNA ratio is less than 1:10... 6 If the ctDNA:cfDNA ratio is greater than 1:10, then the subject has responded to the cancer treatment. 6 If ctDNA:cfDNA molecules are present, then the subject does not respond to the cancer treatment.

12. A method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, wherein, after administration of said cancer treatment, the levels of cell-free DNA (cfDNA) and cell-free tumor DNA (ctDNA) in a sample obtained from said subject are measured, and the ctDNA:cfDNA molecule ratio in said sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If the ctDNA:cfDNA ratio is less than 1:10, the subject is at risk of cancer recurrence or relapse. 6 If ctDNA:cfDNA molecules are present, then the subject is not at risk of cancer recurrence or relapse.

13. A method for treating cancer in a subject in need, comprising administering an effective amount of cancer treatment to the subject, wherein prior to administering the cancer treatment, 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 to cfDNA molecules in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 ctDNA:cfDNA molecules, then the subject is identified as a candidate for cancer treatment.

14. A method for treating cancer in a subject with a high-risk disease, comprising administering an effective amount of cancer treatment to the subject, wherein prior to administering the cancer treatment, 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 to cfDNA molecules in the sample is determined, wherein if the ctDNA:cfDNA ratio is greater than 1:10... 6 If ctDNA:cfDNA molecules are used, the subject will be identified as having a high-risk disease.

15. The method according to any one of claims 1 to 14, wherein the cancer is a blood cancer.

16. The method according to any one of claims 1 to 14, wherein the cancer is a solid tumor.

17. The method according to any one of claims 1 to 14, wherein the cancer is a B-cell malignancy.

18. The method according to any one of claims 1 to 14, wherein the cancer is leukemia or lymphoma.

19. The method according to any one of claims 1 to 14, wherein the cancer is large B-cell lymphoma (LBCL) or diffuse large B-cell lymphoma (DLBCL).

20. The method according to any one of claims 1 to 14, wherein the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute 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), and the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

21. The method according to any one of claims 1 to 14, wherein the cancer is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine carcinoma, CNS cancer, brain tumor, bone cancer, or soft tissue sarcoma.

22. The method according to any one of claims 2 to 14, wherein the treatment or the drug is a first-line therapy.

23. The method according to any one of claims 2 to 14, wherein the treatment is a second-line therapy.

24. The method according to any one of claims 2 to 14, wherein the method includes further monitoring of the subject.

25. The method according to any one of claims 2 to 14, wherein the treatment includes radiographic imaging of the subject.

26. The method according to any one of claims 2 to 14, wherein the treatment comprises computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI) of the subject.

27. The method according to any one of claims 2 to 9 and 11 to 14, wherein the treatment or drug comprises cell therapy.

28. The method according to any one of claims 2 to 9 and 11 to 14, wherein the treatment comprises CAR-T cell therapy.

29. The method of claim 28, wherein the CAR-T cell therapy is an anti-CD19 cell therapy.

30. The method of claim 27, wherein the cell therapy comprises genetically engineered cells.

31. The method of claim 30, wherein the genetically engineered cell is a T cell.

32. The method of claim 31, wherein the genetically engineered T cell comprises a chimeric antigen receptor (CAR).

33. The method of claim 32, wherein 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.

34. The method of claim 32, wherein the CAR specifically binds to two antigens associated with the disease or condition and / or is expressed by cells associated with the disease or condition.

35. The method according to claim 33 or 34, wherein the antigen is selected from the group consisting of: 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer-testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, 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 dimer, EGFR. vIII, estrogen receptor, fetal AchR, folate receptor α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Ra2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), cancer-fetal antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptor, BAFF-R, VEGF-R2, nephroblastoma 1 (WT-1), and pathogen-specific antigens.

36. The method according to claim 33 or 34, wherein the antigen is CD19.

37. The method according to claim 33 or 34, wherein the antigen is CD20.

38. The method of claim 32, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to the antigen, and an intracellular signal transduction domain comprising ITAM.

39. The method of claim 38, wherein the intracellular signal transduction domain comprises an intracellular domain of the CD3-zeta (CD3ζ) chain.

40. The method of claim 32, wherein the CAR further comprises a co-stimulatory signal transduction region.

41. The method of claim 40, wherein the co-stimulation signal transduction region comprises a CD28 or 4-1BB signal transduction domain.

42. The method of claim 41, wherein the signal conduction domain is a 4-1BB domain.

43. The method of claim 31, wherein the T cell is CD4+.

44. The method of claim 31, wherein the T cell is CD4+ or CD8+.

45. The method of claim 31, wherein the T cells are primary T cells obtained from the subject.

46. ​​The method of claim 30, wherein the genetically engineered cells are autologous to the subject.

47. The method of claim 30, wherein the genetically engineered cells are allogeneic to the subject.

48. The method according to any one of claims 1 to 47, wherein the subject is a human.

49. The method of claim 48, wherein the subject suffers from stage I / II disease.

50. The method of claim 48, wherein the subject suffers from stage III / IV disease.

51. The method according to any one of claims 2 to 47, wherein the subject has minimal residual disease (MRD).

52. The method according to any one of claims 1 to 51, wherein the subject is refractory to one or more prior therapies for the cancer.

53. The method according to any one of claims 1 to 51, wherein the subject has an inadequate response to one or more prior therapies for the cancer.

54. The method according to any one of claims 1 to 51, wherein the subject achieves a sustained response to the treatment or the drug.

55. The method of claim 54, wherein the durable response is defined as no recurrence or remission of the cancer for up to 3 months, 6 months, or 12 months.

56. The method according to any one of claims 1 to 51, wherein the subject has a high survival rate.

57. The method according to any one of claims 1 to 51, wherein the baseline characteristics of the disease of the subject are determined.

58. The method of claim 57, wherein the baseline features include International Prognostic Index (IPI) score, serum lactate dehydrogenase (LDH), sum of diameter products (SPD), disease stage, or any combination thereof.

59. The method of claim 57, wherein the baseline feature is defined by the Lugano 2014 standard.

60. The method of claim 58, wherein the subjects are classified according to the International Prognostic Index (IPI) score.

61. The method of claim 58, wherein the subject is classified as low-risk, low-intermediate-risk, or high-intermediate-risk based on the IPI score.

62. The method of claim 58, wherein the subject has no risk factors or has one risk factor and is considered to be in the low-risk group for IPI.

63. The method of claim 58, wherein the subject has two risk factors and is considered to be in the low-to-intermediate IPI group.

64. The method of claim 58, wherein the subject has three risk factors and is considered to be in the high-to-intermediate IPI group.

65. The method of claim 58, wherein the subject has a high or low IPI score.

66. The method according to any one of claims 1 to 51, wherein a volumetric measurement of the tumor burden of the subject is measured.

67. The method of claim 66, wherein the volume measurement of the tumor burden of the subject is the sum of diameter products (SPD).

68. The method of claim 66, wherein the volume measurement of the tumor burden is measured using computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI) of the subject.

69. The method of claim 67, wherein the SPD threshold is or approximately 30 / cm², or approximately 40 / cm², or approximately 50 / cm², or approximately 60 / cm², or approximately 70 / cm².

70. The method according to any one of claims 1 to 51, wherein the level of inflammatory markers in the subject is measured.

71. The method of claim 70, wherein the level of the inflammatory marker is or about 300 units / L, or about 400 units / L, or about 500 units / L, or about 600 units / L.

72. The method of claim 70, wherein the inflammatory marker is lactate dehydrogenase (LDH).

73. The method according to any one of claims 1 to 10, wherein the mutation is a point mutation, a deletion, or a frameshift mutation.

74. The method according to any one of claims 1 to 10, wherein the mutation is a somatic mutation.

75. The method according to any one of claims 1 to 10, wherein the mutation is a phase variation (PV).

76. The method according to any one of claims 1 to 10, wherein the mutation is a single nucleotide variant (SNV).

77. The method according to any one of claims 1 to 10, wherein the mutation includes SNV, insertion / deletion, rearrangement, or a combination thereof.

78. The method according to claim 3, wherein the detection threshold ratio of ctDNA:cfDNA molecules is 1:10, 1:100, 1:1000, or 1:

10. 4 1:10 5 1:10 6 1:10 7 1:10 8 .

79. The method according to claim 3, wherein the detection threshold ratio of ctDNA:cfDNA molecules is 1:

10. 6 .

80. The method according to any one of claims 1 to 10, wherein the presence of one or more mutations in one or more genes of the cfDNA is determined by genotyping of the cfDNA.

81. The method according to any one of claims 1 to 10, wherein the presence of one or more mutations in one or more genes of the cfDNA is determined by sequencing the cfDNA.

82. The method of claim 81, wherein the sequencing comprises high-throughput sequencing, pyrosequencing, sequencing synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule sequencing synthesis (SMSS) (Helicos), massively parallel sequencing, cloned 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 personalization analysis via deep sequencing (CAPP-Seq), or duplex sequencing (Duplex-Seq).

83. The method according to any one of claims 1 to 82, wherein measuring the level of ctDNA further comprises determining the absolute ctDNA concentration, expressed as mutant haploid genomic equivalents (hGE / mL) per milliliter of plasma.

84. The method according to any one of claims 1 to 82, wherein measuring the level of ctDNA further comprises determining the average variant allele frequency (AF).

85. The method of claim 84, wherein the average variant allele frequency (AF) greater than a predetermined range indicates the detectable ctDNA level.

86. The method of claim 4, wherein the detection threshold is a percentage of ctDNA level equal to or lower than the total cfDNA level in the sample.

87. The method of claim 4, wherein the detection threshold is a concentration of ctDNA that is equal to or lower than the total cfDNA level in the sample.

88. The method of claim 86 or 87, wherein the detection threshold is within 25%, 20%, 15%, 11%, or 5% of the median or average level, amount, or concentration of cfDNA in the biological sample obtained from the subject after administration of the treatment, and / or within one standard deviation above the median or average level, amount, or concentration.

89. The method according to claim 4, wherein 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.

90. The method of any one of claims 1 to 89, wherein the method further comprises determining the correlation between baseline ctDNA concentration (hGE / mL) and baseline characteristics of the subject.

91. The method according to any one of claims 1 to 90, wherein the method detects ctDNA levels in the sample with 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%.

92. The method according to any one of claims 1 to 90, wherein the method detects ctDNA levels in the sample with 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%.

93. The method of claim 89, wherein the genotyping comprises sequencing one or more genes to identify ctDNA.

94. The method according to any one of claims 1 to 93, wherein the sample is obtained 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after administration of the cancer treatment.

95. The method of claim 94, wherein the sample is obtained on day 15 after administration of the cancer treatment.

96. The method according to any one of claims 1 to 93, wherein the sample is obtained about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after administration of the cancer treatment.

97. The method according to any one of claims 1 to 93, wherein the sample is obtained about 1, 2, 3 or 12 months after the administration of the cancer treatment.

98. The method according to any one of claims 1 to 93, wherein the ctDNA is not detected at least 2, 4 or 6 weeks after administration of the cancer treatment, or 3, 6, 12, 18, 24, 30 or 36 months, or 1, 2, 3, 4, 5 years or longer.

99. The method according to any one of claims 1 to 93, wherein the sample comprises a fluid, cell, or tissue sample.

100. The method according to any one of claims 1 to 93, wherein the sample is a blood, serum, or plasma sample.

101. A method for assessing the risk of cancer recurrence or relapse in a subject who has received cancer treatment, the method comprising: (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from the subject; (b) Identify cell-free tumor DNA (ctDNA) in the sample, wherein the ctDNA is identified by the presence of one or more cfDNA molecules containing phase variation (PV); (c) Determine the subject's minimal residual disease (MRD) status from ctDNA in the sample; (d) Assess the risk of cancer recurrence or relapse in the subject based on the subject's MRD status; The treatment described therein includes at least two cycles of induction; and If MRD is detected after two cycles of the induction period, the subject is at risk of cancer recurrence or relapse.

102. The method of claim 101, wherein the cancer is a blood cancer.

103. The method of claim 101, wherein the cancer is a B-cell malignancy.

104. The method of claim 101, wherein the cancer is leukemia or lymphoma.

105. The method of claim 101, wherein the cancer is follicular lymphoma.

106. The method according to any one of claims 101 to 105, wherein the subject is a human.

107. The method of claim 106, wherein the subject suffers from stage I / II disease.

108. The method of claim 106, wherein the subject suffers from stage III / IV disease.

109. The method according to any one of claims 101 to 108, wherein the subject was untreated prior to the administration of the treatment.

110. The method according to any one of claims 101 to 109, wherein the treatment is a first-line therapy.

111. The method according to any one of claims 101 to 110, wherein the treatment comprises bendamustine / rituximab or R-CHOP.

112. The method of claim 111, wherein the induction period comprises six cycles of bendamustine / rituximab or R-CHOP.

113. The method according to any one of claims 101 to 112, wherein the sample is obtained on the first day of the treatment cycle.

114. The method according to any one of claims 101 to 113, further comprising, after administration of the treatment, continuously acquiring a plurality of biological samples over a period of at least one year, at least two years, or at least three years.

115. The method according to any one of claims 101 to 114, wherein the sample is a blood sample, a serum sample, or a plasma sample.

116. The method according to any one of claims 101 to 115, wherein the tumor fraction of the biological sample is less than 0.28%.

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