Methods for identifying druggable targets and methods for treating cancer - Patents.com

Abstract

The present disclosure provides a method, set and kit for predicting and identifying druggable targets for treating cancer.The present disclosure further provides a method for treating cancer.The method and test set described herein provide improved detection and / or treatment of cancer.In some embodiments, the method comprises identifying one or more druggable targets for treating cancer in a subject.In some embodiments, the method comprises predicting and confirming druggable targets for treating cancer in a subject.

Description

[Technical field]

[0001] (cross reference) This application claims the benefit under 5 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 364,841, filed May 17, 2022. [Background technology]

[0002] (background) Early detection of cancer is an ongoing challenge that has been limited for decades by diagnostic methods that rely on solid tissue biopsy. Recently, liquid biopsy has emerged as a promising method for non-invasive diagnosis or monitoring of cancer. However, there remains a need in the art for improved methods that streamline the diagnosis and treatment selection process when using liquid biopsy samples. The present disclosure addresses this long-felt need by providing improved methods and sets of tests for diagnosing patients with cancer, predicting and confirming druggable targets, selecting appropriate treatments for cancer patients, and / or administering appropriate treatments to cancer patients in a streamlined and cost-reduced manner. Summary of the Invention [Means for solving the problem]

[0003] (overview) The methods and sets of tests described herein provide improved detection and / or treatment of cancer. In some embodiments, the methods include identifying one or more druggable targets for treating cancer in a subject. In some embodiments, the methods include predicting and validating a druggable target for treating cancer in a subject. In some embodiments, the methods include selecting a targeted cancer diagnostic test for a subject. In some embodiments, the methods include treating cancer in a subject. In some embodiments, the present disclosure provides a set of a first test and a second test for identifying one or more druggable targets for treating cancer in a subject. In some embodiments, the present disclosure provides a set of a first test and a second test for predicting and evaluating one or more druggable targets for treating cancer in a subject. The evaluating includes detecting the presence (or absence) of a genetic variant that is useful for determining the selection of a particular therapeutic agent or therapeutic agents.

[0004] In various aspects, the present disclosure provides a method for identifying one or more druggable targets for treating cancer in a subject, the method comprising: performing or performing a first test on a liquid biopsy sample of the subject to identify that the subject has cancer, and performing a second test, the second test evaluating genetic variants in the subject to identify the presence of one or more druggable targets. In some embodiments, the second test is performed on a liquid biopsy sample of the subject. The first test and the second test can be performed on separate liquid biopsy samples of the subject. Alternatively, the first test and the second test can be performed on the same liquid biopsy sample.

[0005] In various aspects, the present disclosure provides a method of treating cancer in a subject, comprising: performing or having performed a first test on a liquid biopsy sample of the subject to identify that the subject has cancer; performing or having performed a second test, the second test evaluating genetic variants in the subject to identify the presence of one or more druggable targets; and administering to the subject an effective amount of a drug that targets at least one of the identified druggable targets. In some embodiments, the second test is performed or has been performed on the liquid biopsy sample of the subject. The first test and the second test can be performed on separate liquid biopsy samples of the subject. Alternatively, the first test and the second test can be performed on the same liquid biopsy sample. In embodiments where two or more samples are tested, the samples can be collected at the same time or at different times. In some embodiments, a single sample can be divided into separate portions to perform separate tests on each portion.

[0006] In various embodiments, the present disclosure provides a method of selecting a targeted cancer diagnostic test for a subject, the method comprising: performing or having performed a first test on a liquid biopsy sample of the subject to identify the subject as having cancer, the first test generating a predictive profile for one or more druggable targets in the subject; and selecting a second test as the targeted cancer diagnostic test, the second test evaluating the presence or absence of one or more druggable targets predicted by the profile.

[0007] In various aspects, the present disclosure provides a method for predicting and confirming (or detecting) a druggable target for treating cancer in a subject, the method comprising: performing or performing a first test on a liquid biopsy sample of the subject to identify the subject as having cancer, the first test generating a predictive profile for one or more druggable targets (e.g., increased likelihood of having) in the subject; selecting a second test, the second test evaluating the presence or absence of the one or more druggable targets; and performing the second test, the second test confirming (e.g., detecting) the presence of at least one of the one or more druggable targets predicted by the profile generated by the first test. In some embodiments, the second test is performed on a liquid biopsy sample of the subject. The first test and the second test may be performed on separate liquid biopsy samples of the subject. Alternatively, the first test and the second test may be performed on the same liquid biopsy sample.

[0008] In various aspects, the present disclosure provides a method of treating cancer in a subject, comprising: performing or performing a first test on a liquid biopsy sample of the subject to identify the subject as having cancer, the first test generating a predictive profile for one or more druggable targets in the subject; selecting or selecting a second test, the second test evaluating the presence or absence of the one or more druggable targets; performing or performing the second test, the second test confirming the presence of at least one of the one or more druggable targets predicted by the profile generated by the first test; and administering to the subject an effective amount of a drug targeting the at least one confirmed druggable target. In some embodiments, the second test is performed or has been performed on the liquid biopsy sample of the subject. The first test and the second test can be performed on separate liquid biopsy samples of the subject. Alternatively, the first test and the second test can be performed on the same liquid biopsy sample.

[0009] In various embodiments, the disclosure provides a method of selecting a targeted cancer diagnostic test for a subject, the method comprising: performing or having performed a first test on a liquid biopsy sample of the subject to identify the subject as having cancer, the first test generating a first profile or a second profile, optionally generating a first epigenetic profile or a second epigenetic profile, the first profile being predictive for one or more druggable targets of a first panel in the subject and the second profile being predictive for one or more druggable targets of a second panel in the subject; and selecting the second test as the targeted cancer diagnostic test. In some embodiments, if the first test produces the first profile, the second test evaluates the presence or absence of one or more druggable targets of the first group predicted by the first profile; if the first test produces the second profile, the second test evaluates the presence or absence of one or more druggable targets of the second group predicted by the first profile. In various embodiments, observation of a particular profile suggests that one group is more predictive than another group, thereby providing guidance in selecting the more predictive group.

[0010] In various embodiments, the disclosure provides a method of predicting and validating druggable targets for treating cancer in a subject, the method comprising: performing or having performed a first test on a liquid biopsy sample of the subject to identify the subject as having cancer, the first test generating a first profile or a second profile, optionally generating a first epigenetic profile or a second epigenetic profile, the first profile being predictive for one or more druggable targets of a first group in the subject, and the second profile being predictive for one or more druggable targets of a second group in the subject. the first test is predictive of the druggable target of the first group; selecting or selecting a second test as the targeted cancer diagnostic test, where if the first test produces the first profile, the second test evaluates the presence or absence of one or more druggable targets of the first group; if the first test produces the second profile, the second test evaluates the presence or absence of one or more druggable targets of the second group; and performing the second test, where the second test confirms the presence of at least one of the one or more druggable targets. In some embodiments, the second test is performed on a liquid biopsy sample of the subject. The first test and the second test can be performed on separate liquid biopsy samples of the subject. Alternatively, the first test and the second test can be performed on the same liquid biopsy sample.

[0011] In various embodiments, the disclosure provides a method of treating cancer in a subject, comprising the steps of: performing or having performed a first test on a liquid biopsy sample of the subject to identify the subject as having cancer, the first test generating a first profile or a second profile, optionally generating a first epigenetic profile or a second epigenetic profile, the first profile being predictive for a first group of one or more druggable targets in the subject, and the second profile being predictive for a second group of one or more druggable targets in the subject; The method includes the steps of: selecting or selecting as a diagnostic test, where if the first test produces the first profile, the second test evaluates the presence or absence of one or more druggable targets of the first group; if the first test produces the second profile, the second test evaluates the presence or absence of one or more druggable targets of the second group; performing or performing the second test, where the second test confirms the presence of at least one of the one or more druggable targets; and administering to the subject an effective amount of a drug targeting the at least one confirmed druggable target. In some embodiments, the second test is performed or has been performed on a liquid biopsy sample of the subject. The first test and the second test may be performed on separate liquid biopsy samples of the subject. Alternatively, the first test and the second test may be performed on the same liquid biopsy sample.

[0012] In some embodiments, the first test comprises a methylation assay, a nucleic acid assay (e.g., a genetic screen protein assay, a protein post-translational modification assay, a fragmentomics assay, a metabolomics assay, an RNA assay (e.g., a microRNA (miRNA) assay), a microbiome assay, an assay of one or more immune cell populations, or a combination thereof. In some embodiments, the first test comprises a methylation assay, a nucleic acid assay (e.g., a genetic screen), a protein assay, a fragmentomics assay, or a combination thereof. In some embodiments, the first test comprises a methylation assay, a nucleic acid assay (e.g., a genetic screen), a protein assay, and a fragmentomics assay. In some embodiments, the first test comprises a methylation assay.

[0013] In some embodiments, the profile generated by the first test includes data on methylation status, data on chromatin condensation, data on histone modifications, data on fragmentation patterns, data on topology, other epigenetic data, data on nucleic acid sequence, data on nucleic acid expression, data on protein translation, data on protein sequence, data on protein post-translational modifications (e.g., glycosylation), data on the presence of metabolites, data on microbiome composition, data on immune status, or combinations thereof. In some embodiments, the profile includes data on methylation status, data on chromatin condensation, data on histone modifications, data on fragmentation patterns, data on topology, other epigenetic data, or combinations thereof.

[0014] In some embodiments, the first test comprises a plurality of assays. In some embodiments, the profile generated by the first test comprises data from a plurality of assays. In other embodiments, the profile comprises data from a single assay of the plurality of assays.

[0015] In some embodiments, the first test includes a single assay and the profile generated by the first test includes data from that single assay.

[0016] In some embodiments, a second test is selected and / or performed as a result of the profile generated by the first test. In some embodiments, the second test includes testing for one or more genetic variants. In some embodiments, the second test identifies the presence, evaluates the presence or absence, and / or confirms the presence of at least one of the one or more druggable targets. In some embodiments, the one or more druggable targets include the one or more genetic variants. In some embodiments, the one or more druggable targets include ribonucleic acid expression products of the one or more genetic variants. In some embodiments, the one or more druggable targets include peptides or proteins encoded by the one or more genetic variants. In some embodiments, the one or more druggable targets include nucleic acids, peptides, or proteins that share a signaling pathway with the one or more genetic variants.

[0017] In some embodiments, the liquid biopsy sample comprises a blood sample. In some embodiments, the blood sample comprises one or more components of whole blood. In some embodiments, the blood sample comprises serum. In some embodiments, the blood sample comprises plasma. In some embodiments, the liquid biopsy sample comprises cell-free DNA.

[0018] In various embodiments, the disclosure provides a set of a first test and a second test for identifying one or more druggable targets for treating cancer in a subject, the set including a first liquid biopsy test that identifies whether the subject has cancer; and a second liquid biopsy test that evaluates genetic variants in the subject to identify the presence of one or more druggable targets.

[0019] In various embodiments, the present disclosure provides a set of first and second tests for predicting and evaluating one or more druggable targets for treating cancer in a subject, the set including a first liquid biopsy test that generates a predictive profile for one or more druggable targets in the subject; and a second liquid biopsy test that evaluates the presence or absence of one or more druggable targets predicted by the profile.

[0020] In various embodiments, the present disclosure provides a set of first and second tests for predicting and evaluating one or more druggable targets for treating cancer in a subject, the set including: a first liquid biopsy test that generates a first profile or a second profile, and optionally a first epigenetic profile or a second epigenetic profile, where the first profile is predictive for a first group of one or more druggable targets in the subject and the second profile is predictive for a second group of one or more druggable targets in the subject; and a second liquid biopsy test. In some embodiments, when the first liquid biopsy test produces the first profile, the second liquid biopsy test evaluates the presence or absence of one or more druggable targets in the first group; when the first liquid biopsy test produces the second profile, the second liquid biopsy test evaluates the presence or absence of one or more druggable targets in the second group predicted by the first profile.

[0021] In some embodiments, the set includes a first liquid biopsy test, the first liquid biopsy test includes a methylation assay, a nucleic acid assay (e.g., genetic screening), a protein assay, a protein post-translational modification assay, a fragmentomics assay, a metabolomics assay, an RNA assay (e.g., microRNA (miRNA) assay), a microbiome assay, an assay of one or more immune cell populations, or a combination thereof. In some embodiments, the set includes a first liquid biopsy test, the first liquid biopsy test includes a methylation assay, a nucleic acid assay (e.g., genetic screening), a protein assay, a fragmentomics assay, or a combination thereof. In some embodiments, the set includes a first test, the first test includes a methylation assay, a nucleic acid assay (e.g., genetic screening), a protein assay, and a fragmentomics assay. In some embodiments, the set includes a first liquid biopsy test including a methylation assay.

[0022] In some embodiments, the profile generated by the first liquid biopsy test of the set includes data on methylation status, data on chromatin condensation, data on histone modifications, data on fragmentation patterns, data on topology, other epigenetic data, data on nucleic acid sequence, data on nucleic acid expression, data on protein translation, data on protein sequence, data on protein post-translational modifications (e.g., glycosylation), data on the presence of metabolites, data on microbiome composition, data on immune status, or combinations thereof. In some embodiments, the profile generated by the first liquid biopsy test of the set includes data on methylation status, data on chromatin condensation, data on histone modifications, data on fragmentation patterns, data on topology, other epigenetic data, or combinations thereof.

[0023] In some embodiments, the set includes a first liquid biopsy test that includes a plurality of assays. In some embodiments, a profile generated by a first liquid biopsy test of the set includes data from a plurality of assays. In other embodiments, the profile includes data from a single assay of the plurality of assays.

[0024] In some embodiments, the set includes a first liquid biopsy test that includes a single assay, and the profile generated by the first liquid biopsy test includes data from that single assay.

[0025] In some embodiments, the set includes a second test selected and / or performed as a result of a profile generated by a first test of the set. In some embodiments, the second test of the set includes a test for one or more genetic variants. In some embodiments, the second test of the set identifies the presence, evaluates the presence or absence, or confirms the presence of at least one of the one or more druggable targets. In some embodiments, the one or more druggable targets include the one or more genetic variants. In some embodiments, the one or more druggable targets include a ribonucleic acid expression product of the one or more genetic variants. In some embodiments, the one or more druggable targets include a peptide or protein encoded by the one or more genetic variants. In some embodiments, the one or more druggable targets include a nucleic acid, peptide, or protein that shares a signaling pathway with the one or more genetic variants.

[0026] In some embodiments, a first liquid biopsy test of the set is configured to be performed on a blood sample. In some embodiments, a second liquid biopsy test of the set is configured to be performed on a blood sample. In some embodiments, a first liquid biopsy test and a second liquid biopsy test of the set are configured to be performed on a blood sample.

[0027] In some embodiments, a first liquid biopsy test of the set is configured to be performed on cell-free DNA. In some embodiments, the second liquid biopsy test is configured to be performed on cell-free DNA. In some embodiments, the first liquid biopsy test and the second liquid biopsy test are configured to be performed on cell-free DNA.

[0028] By using the profile observed in the first test to select a particular set of druggable targets to be analyzed in the second test, cost savings can be obtained by reducing the amount of target sequence information that needs to be analyzed.

[0029] Studies determining correlations between epigenetic profiles and genetic variation can be used in selecting genomic target regions for genetic analysis to inform drug selection.

[0030] Other features, objects, and advantages of the present invention will be apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is provided for illustrative purposes only and not for limiting purposes. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] (Detailed Description) The present disclosure provides methods and sets of tests for improved detection and / or treatment of cancer, which methods or sets of tests are performed on liquid biopsy samples.

[0032] (definition) As used herein, a "druggable target" refers to a biological target that is known or predicted to be amenable to therapeutic modulation. Specific examples of druggable targets and drugs capable of modulating druggable targets are provided in the present disclosure.

[0033] As provided herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Further, singular terms include their plurals and plural terms include the singular, unless otherwise required by context.

[0034] The phrases "and / or," "at least one," and "one or more," as used herein, each mean one, all, or any subcombination of the elements in the list of elements. Thus, as a non-limiting example, "A, B, and / or C" includes any of the following: A alone; B alone; C alone; A and B without C; A and C without B; B and C without A; and A, B and C.

[0035] As used herein, "or a combination thereof" means any two or more of the elements in a list of elements.

[0036] Throughout this disclosure, the words "have" and "comprise," or variations thereof (e.g., "has," "having," "comprises," or "comprising") are understood to mean the inclusion of the recited element or elements, but not the exclusion of any other elements or elements. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict between an incorporated reference and this disclosure, the present disclosure controls.

[0037] (Liquid biopsy) A liquid biopsy is a non-invasive method that uses a non-solid biological sample. In some embodiments, the liquid biopsy sample can be, for example, blood, cerebrospinal fluid, urine, saliva, sputum, pleural fluid, and / or amniotic fluid. The liquid biopsy sample (e.g., a blood sample) can contain a range of cell types (e.g., immune cells and / or circulating tumor cells) and cell products (e.g., DNA, RNA, peptides, and / or proteins). In some embodiments, the cell products originate from one or more tumor sites in the body. Exemplary biopsy samples include circulating tumor cells (CTCs), circulating nucleic acids (including circulating cell-free tumor DNA (ctDNA) and cell-free DNA (cfDNA), and cell-free RNA (e.g., mRNA, long non-coding RNA, microRNA, and / or circular RNA)), extracellular vesicles, tumor-secreted vesicles (e.g., exosomes, oncosomes, and / or apoptotic bodies), tumor-educated platelets, proteins, and metabolites (e.g., branched-chain amino acids (BCAAs)). In some embodiments, these samples provide information about the characteristics of the primary tumor or metastasis, including the site of origin, genomic mutations, and copy number changes. In some embodiments, these samples provide information about one or more of the transcriptome, epigenome, proteome, and metabolome. Some further details regarding certain liquid biopsies and their use in diagnostic methods are known in the art.See, e.g., Heitzer et al., "Current and future perspectives of liquid biopsies in genomics-driven oncology," Nat Rev Genet, 20, 71-88 (2019); Kilgour et al., "Liquid biopsy-based biomarkers of treatment response and resistance," Cancer Cell, 37:4, 485-495 (2020); Ignatiadis et al., "Liquid biopsy enters the clinic-implementation issues and future challenges," Nat Rev Clin Onc, 18, 297-312 (2021); and Wan et al., "Liquid biopsies come of age:towards implementation of circulating tumour DNA," Nat Rev Cancer, 17, 223-238 (2017).

[0038] In some embodiments, the liquid biopsy test detects or diagnoses cancer. In some embodiments, the liquid biopsy test does not detect cancer. In some embodiments, the liquid biopsy test detects a stage of cancer. In some embodiments, the liquid biopsy test detects early stage cancer. In some embodiments, the liquid biopsy test detects recurrent cancer after curative intent treatment of a primary tumor. In some embodiments, the liquid biopsy test detects metastatic cancer.

[0039] In some embodiments, the liquid biopsy test guides the selection of a suitable treatment for the cancer. In some embodiments, the liquid biopsy test guides the selection of a suitable second test from a group of possible second tests to identify one or more suitable and / or inappropriate treatments for the cancer. In some embodiments, the suitable treatment is a treatment to which the cancer may be sensitive. In some embodiments, the suitable treatment is a treatment to which the cancer is unlikely to be resistant. In some embodiments, the suitable treatment targets one or more biomarkers detected by the test described herein. In some embodiments, the liquid biopsy test identifies that the cancer is resistant or likely to be resistant to one or more possible treatments, and thus identifies that one or more possible treatments are inappropriate.

[0040] In some embodiments, the liquid biopsy sample is a blood sample, a serum sample, or a plasma sample. In some embodiments, the liquid biopsy sample is a serum sample. In some embodiments, the liquid biopsy sample is a plasma sample. In some embodiments, the liquid biopsy sample is a cell-free sample. Certain methods for collecting blood samples for analysis of cell-free DNA are known in the art. See, for example, Aggarwal et al., "Strategies for the successful implementation of plasma-based NSCLC genotyping in clinical practice," Nat Rev Clin Oncol, 18, 56-62 (2020).

[0041] Assay As used herein, the term "assay" refers to a technique for determining one or more characteristics of one or more substances (e.g., nucleic acids, proteins, cells, tissues, and / or organs). An assay (e.g., a first assay or a second assay) may include a methylation assay, a nucleic acid assay (e.g., genetic screening), a protein assay, a protein post-translational modification assay, a fragmentomics assay, a metabolomics assay, an RNA assay (e.g., a microRNA (miRNA) assay), a microbiome assay, an assay of one or more immune cell populations, or a combination thereof. An assay may be used to detect the characteristics of one or more components of a liquid biopsy (e.g., cfDNA, extracellular vesicles, proteins, metabolites, and / or circulating tumor cells). A "test" or a "liquid biopsy test" may include one or more assays.

[0042] In some embodiments, the first liquid biopsy sample is assayed to generate a profile, which profile includes data regarding methylation status, data regarding chromatin condensation, data regarding histone modifications, data regarding fragmentation patterns, data regarding topology, other epigenetic data, data regarding nucleic acid sequence, data regarding nucleic acid expression, data regarding protein translation, data regarding protein sequence, data regarding protein post-translational modifications (e.g., glycosylation), data regarding the presence of metabolites, data regarding microbiome composition, data regarding immune status, or combinations thereof.

[0043] In some embodiments, the test comprises a nucleic acid assay. In some embodiments, the nucleic acid assay comprises a DNA assay. In some embodiments, the DNA assay evaluates one or more genetic variants in the subject. In some embodiments, the DNA assay generates a profile that includes data on one or more genetic variants of the subject. Any method of isolating and evaluating DNA can be used. See, e.g., WO2018083467A1; WO2017181202A2; WO2019241250A1; WO2019018757A1; WO2017151524A1; WO2017151502A1; WO2016149261A1; WO2020047378A1; Alekseyev et al., "A next-generation sequencing primer - how does it work and what can it do?", Academic Pathology, 5, 1-11 (2018); and McCombie et al., "Next-generation sequencing technologies", Cold Spring Harb Perspect Med (2019).

[0044] In some embodiments, the DNA assay may involve sequencing techniques known in the art. Exemplary sequencing technologies include targeted sequencing using PCR amplicons, hybrid capture sequencing, target capture sequencing, whole genome sequencing (WGS), shallow WGS, targeted sequencing of single nucleotide polymorphisms, BEAMing (beads, emulsion, amplified, and magnetic), Intplex, COLD-PCR (co-amplification at lower denaturation temperature PCR), multiplex PCR, SCODA (synchronous coefficient of drag alteration), NaME-PrO (nuclease-assisted minor-allele enrichment with probe-overlap), ARMS-PCR kits for companion diagnostics (CDx), cobas EGFR, therascreen EGFR, single-cell reduced representation bisulfite sequencing, and genomic DNA sequencing. sequencing), TAm-Seq (tagged amplicon deep sequencing), enhanced TAm-Seq, Safe-SeqS, exome sequencing, CAPP-Seq (cancer personalized profiling by deep sequencing), digital sequencing, TEC-seq (targeted error correction sequencing), Plasma-Seq, PARE (personalized analysis of rearranged ends), FAST-SeqS (fast aneuploidy screening test-sequencing system), mFAST-SeqS (modified fast aneuploidy screening test-sequencing system)These include: allele-specific PCR, whole exome sequencing (WES), UroSEEK, PapSEEK, MSK-ACCESS (Memorial Sloan Kettering-Analysis of Circulating Cell-free DNA to Evaluate Somatic Status), Archer Reveal ctDNA 28 assay, FoundationACT (incorporates measurement of hematologic tumor mutation burden (bTMB)), FoundationOne Liquid CDx, Guardant360 CDx, Inivata InVision, OncoDNA OncoSTRAT&GO, PGDx elio plasma resolve, Resolution Bioscience Resolution ctDx, multiplexed targeted digital sequencing (TARDIS), single molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation, Sanger sequencing, and droplet digital PCR (ddPCR). In some embodiments, the DNA assay is allele-specific.

[0045] In some embodiments, the DNA assay evaluates a cell-free DNA (cfDNA; also known as circulating DNA) sample. In some embodiments, the DNA assay generates a profile that includes data about the cfDNA. In some embodiments, the cfDNA is identified or quantified using methods known in the art, including DNA sequencing techniques. In some embodiments, the cfDNA sample comprises a subject's bodily fluid (e.g., blood, whole blood, plasma, serum, urine, cerebrospinal fluid, feces, saliva, sweat, tears, pleural fluid, pericardial fluid, or ascites). Certain exemplary cell-free nucleic acids include RNA, mitochondrial DNA, and genomic DNA. In some embodiments, the cfDNA is nucleosome-bound. In some embodiments, the cfDNA comprises DNA from healthy cells and / or cancerous cells. In some embodiments, the cfDNA may have one or more epigenetic modifications. Certain exemplary modifications include acetylation, 5-methylation, ubiquitination, phosphorylation, sumoylation, ribosylation, and citrullination.

[0046] In some embodiments, the DNA assay evaluates circulating tumor DNA (ctDNA; also known as circulating cell-free tumor DNA). In some embodiments, the DNA assay generates a profile that includes data on ctDNA. In some embodiments, ctDNA is identified or quantified using methods known in the art, including fragment length or methylation status. See, e.g., Mardis, "The emergence of cancer genomics in diagnosis and precision medicine," Nat Cancer, 2, 1263-1264 (2021). In some embodiments, ctDNA levels vary according to disease state, metabolic tumor volume, tumor histology, and / or radiological appearance of the tumor. See, e.g., Rolfo and Russo, "Liquid biopsy for early stage lung cancer moves ever closer," Nat Rev Clin Onc, 17, 523-524 (2020).

[0047] In some embodiments, the nucleic acid assay comprises an RNA assay. In some embodiments, the RNA assay generates an RNA profile comprising data on RNA molecules (e.g., mRNA, microRNA, piRNA, lncRNA, and / or snoRNA). In some embodiments, the RNA assay evaluates one or more genetic variants in the subject. In some embodiments, the RNA assay generates a profile comprising data on one or more genetic variants in the subject. Certain methods of isolating and evaluating RNA are known to those skilled in the art. Certain exemplary methods include RNA-seq, qRT-PCR, two-tailed qRT-PCR, microarray, high-coverage capture sequencing (CaptureSeq), TaqMan miRNA assay, exome capture transcriptome sequencing, electrochemical detection, nanosensors, nanomechanical detection, and Single Molecule Array (Simoa). See, e.g., Stark et al., "RNA sequencing: the teenage years," Nat Rev Genet, 20, 631-656 (2019); Dufva, "Introduction to microarray technology," DNA Microarrays for Biomed Res, 529, 1-22 (2009); Skrzypski, "Quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) in translational oncology: lung cancer perspective," 59:2, 147-154 (2008). In some embodiments, the RNA assay is RNA-seq.

[0048] In some embodiments, the test comprises an epigenetic assay. In some embodiments, the epigenetic assay generates a profile that includes data on epigenetic changes. Certain exemplary data generated by epigenetic assays include methylation status, chromatin condensation, histone modifications, fragmentation patterns, topology, nucleosome positioning, and terminal overhang raggedness. Certain methods for evaluating epigenetic modifications are known in the art. Certain exemplary methods include fractionation based on CpG methylation density using DNA capture with methyl-CpG binding domain proteins (e.g., MBD2) followed by sequencing and bioinformatics analysis and DNA fragmentation pattern analysis (fragmentomics). See, e.g., Li and Zhou et al., "Methylation extends the reach of liquid biopsy in cancer detection," Nat Rev Clin Oncol, 17, 655-656 (2020); and Barefoot et al., "Detection of cell types contributing to cancer from circulating, cell-free methylated DNA," Frontiers in Genetics, 12, 1-14 (2021).

[0049] In some embodiments, the epigenetic assay is selected from the group consisting of cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), MeDIP-seq, methyl-CpG binding domain sequencing (MBD-seq), methylated DNA capture sequencing (MethylCap-seq), hMe-Seal, Epi proColon, whole genome bisulfite sequencing (WGBS), WGBS / CMS-IP-seq, bisulfite amplicon sequencing (BSAS), reduced representation bisulfite sequencing (RRBS), methylated CpG tandem amplification and sequencing (MCTA-seq), methylation array, methylation specific PCR (MSP), Capture-seq, methylation sensitive restriction enzyme sequencing (MRE-seq or MRSE-seq), MRSE-qPCR, Hpall-tiny fragment enrichment by ligation-mediated PCR, and the like. PCR (HELP), methyl-sensitive break counting (MSCC), enzymatic methyl sequencing (EM-seq), TET-assisted pyridine borane sequencing (TAPS), Tet-assisted bisulfite sequencing (TAB-seq), APOBEC-linked epigenetic sequencing (ACE-seq), Tet-assisted 5-methylcytosine sequencing (TAmC-seq), oxidative bisulfite sequencing (oxBS-seq), nanopore sequencing, single molecule real-time (SMRT)-seq, 450K arrays, targeted next-generation bisulfite sequencing (tNGBS), cell-free nucleosome occupancy and methylation sequencing (cfNOME-seq), PanSeer, assay for transposase-accessible chromatin using high-throughput sequencing for transposase-accessible chromatin with high-throughputIn some embodiments, the epigenetic assay comprises one or more of: advanced genomic sequencing (ATAC-seq), single cell ATAC-seq, and single cell ATAC-seq. In some embodiments, the epigenetic assay is a methylation assay. In some embodiments, the methylation assay comprises MBD-seq or MethyCap-Seq. In some embodiments, the methylation assay comprises the GRAIL Galleri test (also called the Galleri Multi-Cancer Early Detection (MCED) test). See, e.g., WO2021174072A1; WO2021202423A1; WO2021041840A1; WO2020163410A1; WO2021250677A1; Liu et al., "Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA," Ann Oncol, 31:6, 745-759 (2020); and Klein et al., "Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set," Ann Oncol, 32:9, 1167-1177 (2021). In some embodiments, the methylation assay comprises a MethylMiner kit.

[0050] In some embodiments, the epigenetic assay generates a profile that includes data regarding fragmentomics. Certain methods for assessing DNA fragmentation patterns are known in the art. Exemplary methods include one or more of DNA evaluation of fragments for early interception (DELFI), large-scale co-fragmentation patterns (FREE-C), fragment coverage near transcription-start sites (TSS), cfDNA-accessibility score near the transcription factor-binding sites (TFBS), orientation-aware cfDNA fragmentation (OCF), windowed protection score (WPS), cfDNA-fragmentation hotspots, inference of DNA methylation from cfDNA-fragmentation patterns, cfDNA preferred end positions, end motif frequency and motif-diversity score (MDS), jagged ends, and extrachromosomal patterns. See, e.g., Liu, "At the dawn: cell-free DNA fragmentomics and gene regulation," Br J Cancer, 126, 379-390 (2022); and Lo et al., "Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies," Science, 372:6538, eaaw361 (2021).

[0051] In some embodiments, the test comprises a protein assay. In some embodiments, the protein assay generates a profile that includes data on peptide expression, data on protein expression, and / or data on the proteome. In some embodiments, the protein assay generates a profile that includes data on protein post-translational modifications. Specific methods for evaluating individual peptides and proteins or proteomes are known to those skilled in the art. Exemplary methods include one or more of enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), immunohistochemistry (IHC), liquid-bead immunoassay, immunoblotting, antibody array, antigen array, reverse-phase protein array (RPPA), proximity extension assay (PEA), bead-based array, liquid chromatography-mass spectrometry (LC-MS / MS), multidimensional protein identification technology (MudPIT), surface-enhanced laser desorption / ionization (SELDI)-MS, slow off-rate modified aptamers (SOMA) scanning assay, AptoDetect-Lung, and OVERA. See, e.g., Ding et al., "Proteomics technologies for cancer liquid biopsies," Mol Cancer, 21:53, (2022).

[0052] In some embodiments, the test includes a metabolic assay. In some embodiments, the metabolic assay generates a profile including metabolic and / or metabolomic data. Particular methods for assaying the metabolome use nuclear magnetic resonance spectroscopy (NMR-spec) or are mass spectrometry-based. See, e.g., McCartney et al., "Metabolomics in breast cancer: a decade in review," Cancer Treat Rev, 67, 88-96 (2018); and Spratlin et al., "Clinical applications of metabolomics in oncology: a review," Clin Cancer Res, 15:2, 431-440 (2009). In some embodiments, the metabolic assay includes one or more of nuclear magnetic resonance spectroscopy (NMR-spec), gas chromatography-mass spectrometry (GC-MS), LC-MS, and Fourier transform ion cyclotron resonance (Fourier transform / MS).

[0053] In some embodiments, the test comprises a microbiome assay. In some embodiments, the microbiome assay generates a microbial profile. In some embodiments, the microbiome can be profiled by sequencing methods known in the art. See, for example, Adlung et al., "Microbiome genomics for cancer prediction," Nat Cancer, 1, 379-381 (2020); Dzutsev and Trinchieri, "Microbial DNA signature in plasma enables cancer diagnosis," Nat Rev Clin Oncol, 17, 453-454 (2020); and Riquelme et al., "Tumor microbiome diversity and composition influence pancreatic cancer outcomes," Cell, 178:4, 795-806 (2019). In some embodiments, the microbiome assay comprises one or more of RNA-seq, WGS, and 16S ribosomal RNA sequencing.

[0054] In some embodiments, the test includes an assay to evaluate circulating tumor cells (CTCs). In some embodiments, circulating tumor cells are enriched from the biological sample using size exclusion or protein expression markers using methods known in the art. See, for example, Zhang et al., "Integrative diagnosis of cancer by combining CTCs and associated peripheral blood cells in liquid biopsy," Clin Transl Oncol, 21:7, 828-835 (2019) and Hofman et al., "Liquid biopsy in the era of immune-oncology: is it ready for prime-time use for cancer patients?", Ann Oncol, 30:9, 1448-1459 (2019). Specific methods for detecting circulating tumor cells include CellSearch, CellSieve, EPIC CTC Platform, Vortex, ISET, negative depletion, magnetic separation, Maintrac, RT-qPCR, microfluidic devices, and GO chips. In some embodiments, the circulating tumor cells express one or more of epithelial cell adhesion molecule (EpCAM), programmed cell death receptor ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), cytokeratin (CK), and RAD50. In some embodiments, the circulating tumor cells are identified by the absence of CD45. Once isolated, in some embodiments, the circulating tumor cells are assayed for nucleic acid biomarkers, epigenetic biomarkers, peptide biomarkers, protein biomarkers, and / or metabolic biomarkers.

[0055] In some embodiments, the test includes an assay that evaluates immune cell populations. In some embodiments, the immune cell assay generates an immune cell profile. Certain methods for evaluating immune cells from liquid biopsies are known in the art. Certain exemplary methods include one or more of qRT-PCR, RNA-seq, single-cell RNA-seq, NanoString's nCounter, flow cytometry, phosphoflow, cytometry by time-of-flight (CyTOF), microengraving, and barcode microchip assays. See, e.g., Lyons et al., "Immune cell profiling in cancer: molecular approaches to cell-specific identification," Npj Precision Onc, 1:26, 1-8 (2017).

[0056] In some embodiments, the test includes an assay to evaluate extracellular vesicles. In some embodiments, the extracellular vesicles are tumor-derived exosomes. Tumor-derived exosomes are known to circulate in the bloodstream, and tumor-derived exosomes can be isolated by certain methods known to those skilled in the art. See, for example, WO2019068269A1; Yoshioka et al., "Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen," Nat Commun, 5, 3591 (2014); Logozzi et al., "Exosomes: a source for new and old biomarkers in cancer," Cancers, 12:9, 2566 (2020); and Makler and Asghar, "Exosomal biomarkers for cancer diagnosis and patient monitoring," Expert Rev Mol Diagn, 20:4, 387-400 (2020). In some embodiments, the exosomes are enriched by ultrafiltration, ultracentrifugation, sucrose gradient ultracentrifugation, size exclusion chromatography, immunocapture, immunoaffinity, immunoprecipitation, polymer precipitation, extracellular vesicle arrays, ExoScreen, immunocapture-based enzyme-linked immunosorbent assay (IC-ELISA), nanosensors, or microfluidics-based assays.

[0057] In some embodiments, the test evaluates both epigenetic changes (e.g., methylation status) and nucleic acid, and the nucleic acid test includes both DNA assay and RNA assay. See, for example, WO2017181146A1. In some embodiments, the test includes the Guardant Reveal test. In some embodiments, the test includes the Shield test. For example, Kim et al., “Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early stage colorectal cancer (CRC),” Cancer Res., 79(Suppl. 13), 916 (2019); Liles et al., “Uptake of a colorectal cancer screening blood test is higher than of a fecal test offered in clinic:a randomized trial,” Cancer Treat Res. Comm., 10, 27–31 (2017); Westesson et al., “Integrated genomic and epigenomic cell-free DNA (cfDNA) analysis for the detection of early-stage colorectal cancer,” Cancer Res., 80 (Supplement 16), 2316 (2020); Mack et al., “Residual circulating tumor DNA (ctDNA) after two months of therapy to predict progression-free and overall survival in patients treated on S1403 with afatinib + / - cetuximab”, J Clin Oncol, 38:15 Suppl, 9532-9532 (2020).

[0058] In some embodiments, the test evaluates both nucleic acids and proteins. In some embodiments, the test comprises a Cancer SEEK test. See, e.g., WO2020150656A1; Cohen et al., "Detection and localization of surgically resectable cancer with a multi-analyte blood test," Science, 359:6378, 926-930 (2018). In some embodiments, the test is a multiomics assay. In some embodiments, the multiomics assay comprises the Freenome multiomics platform. See, e.g., WO2021202351A1; WO2021222220A2; Ulz et al., "Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection," Nat Commun, 10:4666 (2019); Wan et al., "Machine learning enables detection of early-stage colorectal cancer by whole-genome sequencing of plasma cell-free DNA," BMC Cancer, 19:832 (2019); and Ignjatovic et al., "Mass spectrometry-based plasma proteomics: considerations from sample collection to achieving translational data," J. Proteome Res, 18:12, 4085-4097 (2019). In some embodiments, the test comprises LUNAR-2.See, e.g., Kim et al., “Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early-stage colorectal cancer (CRC),” Cancer Res, 79:13 Suppl, Abstract 916 (2019).

[0059] In some embodiments, the assay or assays are available as a kit. In some embodiments, the assay or assays are available as a kit approved for clinical use.

[0060] (First and second tests) In some embodiments, a first liquid biopsy test is performed and a profile generated by the first test determines whether to perform a second test, which in some embodiments is a second liquid biopsy test.

[0061] In some embodiments, the first test detects cancer and the second test is performed to identify one or more druggable targets for treating the cancer (e.g., to select one or more appropriate treatments). In some embodiments, the subject is treated with one or more drugs selected according to the second test.

[0062] In some embodiments, the second test evaluates the presence or absence of one or more genetic variants that identify one or more druggable targets. In some embodiments, the second test identifies an effective dosage for a drug that targets the one or more druggable targets. In some embodiments, the second test eliminates one or more possible treatments as inappropriate for the subject, for example, by identifying a high likelihood of resistance to a given drug (e.g., based on nucleic acid data).

[0063] In some embodiments, the first test does not detect cancer and the second test is not performed. Without being limited by theory, such embodiments provide a more cost-effective and targeted alternative to traditional methods that combine cancer screening with exhaustive and expensive testing (including for subjects who do not have cancer and therefore do not require such testing).

[0064] In some embodiments, a first liquid biopsy test and a second test are performed, and a profile or result from the first liquid biopsy test determines the selection of the second test. In some embodiments, the second test is a second liquid biopsy test.

[0065] In some embodiments, the profile from the first test narrows the set of possible second tests to a particular second test. In some embodiments, the profile from the first test narrows the set of possible druggable targets in the subject to a targeted set of possible druggable targets (e.g., to be evaluated by the second test). In some embodiments, the profile from the first test narrows the set of possible genetic variants in the subject to a targeted set of possible genetic variants (e.g., to be evaluated by the second test). For example, in some embodiments, the profile from the first test narrows down the set of possible genetic variants to be evaluated by the second test, for example, from at least 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 possible variants to 10 or less possible variants, preferably 5 or less (e.g., 5, 4, 3, 2, or 1) possible variants. Without being limited by theory, such embodiments provide a more cost-effective and targeted alternative to the traditional method of performing exhaustive and expensive tests to determine or eliminate treatment options. Rather, the embodiments described herein help to focus testing on the most likely candidates, thereby facilitating a route to treatment that is more efficient and less costly than traditional methods.

[0066] In some embodiments, the first test generates more than one profile, each profile being predictive for one or more druggable targets in the subject. In some embodiments, the first test may generate 2, 3, 4, 5, 6, 7, 8, 9, 10 or more profiles. In some embodiments, the one or more druggable targets predicted by each profile may overlap but are not identical. In some embodiments, the one or more druggable targets predicted by each profile are unique for each profile. In some embodiments, a second test (optionally a second liquid biopsy test) is selected according to the profile generated by the first test. In some embodiments, the second test is selected from a group of possible second tests, each possible second test evaluating the presence or absence of one or more druggable targets. In some embodiments, the one or more druggable targets evaluated by each possible second test may overlap but are not identical. In some embodiments, the one or more druggable targets evaluated by each possible second test are unique.Without being limited by theory, such an embodiment provides a more cost-effective and targeted alternative than the conventional method of performing exhaustive and expensive tests to determine or eliminate treatment options.Rather, the embodiments described herein help to focus tests on the most likely candidates, thereby facilitating a more efficient and less expensive route to treatment compared to conventional methods.

[0067] In some embodiments, the first test generates a first profile or a second profile, the first profile being predictive for one or more druggable targets of a first group in the subject, and the second profile being predictive for one or more druggable targets of a second group in the subject. In some embodiments, the first profile generated by the first test predicts one or more druggable targets in a first pathway, and the second profile generated by the first test predicts one or more druggable targets in a second pathway. In some embodiments, the first test generates the first profile, and the second test evaluates the presence or absence of the one or more druggable targets in the first pathway. In some embodiments, the first test generates the second profile, and the second test evaluates the presence or absence of the one or more druggable targets in the second pathway. In some embodiments, the first pathway and the second pathway have one or more overlapping druggable targets, but do not have the same group of possible druggable targets. In some embodiments, the first pathway and the second pathway do not have any overlapping possible druggable targets.In some embodiments, at least one of the one or more druggable targets is a gene variant.In some embodiments, at least one of the one or more druggable targets is a peptide or protein.

[0068] In some embodiments, one or more calling criteria for the second test are adjusted based on the profile generated by the first test. In some embodiments, the profile includes data from multiple assays (i.e., any combination of assays described herein). For example, in some embodiments, the profile generated by the first test includes nucleic acid data (e.g., data predicting the likelihood of one or more gene variants) and data from another assay (e.g., methylation status data, microbiome data, protein sequence data or post-translational modification data, fragmentation patterns, metabolomics data), and the conventional detectable threshold for predicting gene variants is lowered to take into account the data from the other assay. In other words, in some embodiments, the data of the profile generated by the first test is weighted to select possible gene variants or possible druggable targets in the subject, and thus to select the second test from a group of possible second tests. Without being limited by theory, such embodiments improve conventional methods by applying adjusted (e.g., lowered) thresholds or criteria that facilitate accurate diagnosis and treatment of the subject.

[0069] In some embodiments, the first test generates one or more profiles that identify cancer in the subject. In some embodiments, the first test generates one or more profiles that indicate the absence of cancer in the subject. In some embodiments, the one or more profiles generated by the first test predict the tissue of origin of the cancer in the subject. In some embodiments, the one or more profiles generated by the first test identify the tissue of origin of the cancer in the subject. In some embodiments, the one or more profiles generated by the first test predict one or more druggable targets in the subject. In some embodiments, the one or more profiles generated by the first test guide the selection of an appropriate second test from a panel of possible second tests to confirm (e.g., detect) or eliminate one or more druggable targets predicted by the one or more profiles generated by the first test.

[0070] In some embodiments, the second test generates one or more profiles that identify one or more druggable targets in the subject. In some embodiments, the second test generates one or more profiles that evaluate the presence or absence of one or more druggable targets predicted by the first test. In some embodiments, the second test generates one or more profiles that confirm the presence of one or more druggable targets predicted by the first test. In some embodiments, the one or more profiles generated by the second test guide the selection of an appropriate treatment for the cancer. In some embodiments, the one or more profiles generated by the second test identify one or more appropriate and / or inappropriate treatments for the cancer. In some embodiments, the appropriate treatment is a treatment to which the cancer is likely to be sensitive. In some embodiments, the appropriate treatment is a treatment to which the cancer is unlikely to be resistant. In some embodiments, the second test identifies an effective dosage for the appropriate treatment. In some embodiments, the profile or profiles generated by the second test identify the cancer as resistant or likely to be resistant to one or more potential treatments, and, as a result, identify those one or more potential treatments as inappropriate.

[0071] In some embodiments, the first test comprises a DNA assay. In some embodiments, the first test comprises an RNA assay. In some embodiments, the first test comprises an epigenetic assay. In some embodiments, the first test comprises a methylation assay. In some embodiments, the first test comprises a fragmentomics assay. In some embodiments, the first test comprises a protein assay. In some embodiments, the first test comprises a metabolic assay. In some embodiments, the first test comprises a microbiome assay. In some embodiments, the first test evaluates circulating tumor cells. In some embodiments, the first test evaluates immune cells. In some embodiments, the first test evaluates extracellular vesicles. In some embodiments, the first test comprises a DNA assay, a methylation assay, a fragmentomics assay, and a protein assay.

[0072] In some embodiments, the second test comprises a DNA assay. In some embodiments, the second test comprises an RNA assay. In some embodiments, the second test comprises an epigenetic assay. In some embodiments, the second test comprises a methylation assay. In some embodiments, the second test comprises a fragmentomics assay. In some embodiments, the second test comprises a protein assay. In some embodiments, the second test comprises a metabolic assay. In some embodiments, the second test comprises a microbiome assay. In some embodiments, the second test evaluates circulating tumor cells. In some embodiments, the second test evaluates immune cells. In some embodiments, the second test evaluates extracellular vesicles. In some embodiments, the second test comprises a DNA assay, a methylation assay, a fragmentomics assay, and a protein assay.

[0073] In some embodiments, the first test identifies the presence or absence of cancer in a subject, the first test includes an epigenetic assay, and the second test includes a nucleic acid assay (e.g., a DNA assay and / or an RNA assay). In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0074] In some embodiments, the first test identifies the presence or absence of cancer in a subject, the first test comprises an epigenetic assay, and the second test comprises a DNA assay and a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0075] In some embodiments, the first test identifies the presence or absence of cancer in a subject, the first test comprises an epigenetic assay, and the second test comprises an RNA assay and a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0076] In some embodiments, the first test identifies the presence or absence of cancer in a subject, the first test comprises an epigenetic assay, and the second test comprises a DNA assay, an RNA assay, and a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0077] In some embodiments, the first test identifies the presence or absence of cancer in the subject, and the first test comprises an epigenetic assay and the second test comprises a methylation assay, a fragmentomics assay, a nucleic acid assay (e.g., a DNA assay and / or an RNA assay), and a protein assay.

[0078] In some embodiments, the first test predicts one or more druggable targets, the first test comprises an epigenetic assay, and the second test comprises a DNA assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0079] In some embodiments, the first test predicts one or more druggable targets, the first test comprises an epigenetic assay, and the second test comprises a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0080] In some embodiments, the first test predicts one or more druggable targets, the first test includes a DNA assay and an epigenetic assay, and the second test includes a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0081] In some embodiments, the first test predicts one or more druggable targets, the first test includes a DNA assay and an epigenetic assay, and the second test includes a nucleic acid assay (e.g., a DNA assay and / or an RNA assay). In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0082] In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay and a fragmentomics assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay and a fragmentomics assay, and the second test comprises a nucleic acid assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay and a fragmentomics assay, and the second test comprises a protein assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay and a fragmentomics assay, and the second test comprises a nucleic acid assay and a protein assay.

[0083] In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay, a fragmentomics assay, and a nucleic acid assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay, a fragmentomics assay, and a nucleic acid assay, and the second test comprises a further nucleic acid assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay, a fragmentomics assay, and a nucleic acid assay, and the second test comprises a protein assay.

[0084] In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay, a fragmentomics assay, a nucleic acid assay, and a protein assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay, a fragmentomics assay, a nucleic acid assay, and a protein assay, and the second test comprises a further nucleic acid assay. In some embodiments, the first test predicts one or more druggable targets, and the first test comprises a methylation assay, a fragmentomics assay, a nucleic acid assay, and a protein assay, and the second test comprises a further protein assay.

[0085] (set) In some embodiments, the first test and the second test are provided in a set.In some embodiments, both the first test and the second test of the set are carried out.In some embodiments, only the first test of the set is carried out.In some embodiments, the first test indicates that the subject does not have cancer, and the second test of the set (for example, to identify one or more druggable targets for treating cancer) is not carried out.

[0086] In some embodiments, the second test of the set evaluates the presence or absence of one or more genetic variants that identify one or more druggable targets. In some embodiments, the second test of the set confirms the presence of one or more genetic variants that identify one or more druggable targets predicted by the first test. In some embodiments, the second test of the set identifies an effective dosage for a drug that targets the one or more druggable targets.

[0087] In some embodiments, the profile from the first test of the set narrows the group of possible second tests to a specific second test. In some embodiments, the profile from the first test of the set narrows the group of possible druggable targets in the subject to a targeted group of possible druggable targets. In some embodiments, the profile from the first test of the set narrows the group of possible genetic variants in the subject to a targeted group of possible genetic variants.

[0088] In some embodiments, the first test of the set generates more than one profile, and each profile is predictive for one or more druggable targets in the subject. In some embodiments, the first test of the set can generate 2, 3, 4, 5, 6, 7, 8, 9, 10 or more profiles. In some embodiments, the second test of the set (optionally, the second liquid biopsy test) is selected according to the profile generated by the first test of the set. In some embodiments, the second test of the set is selected from a group of possible second tests, and each second test evaluates the presence or absence of one or more druggable targets.

[0089] In some embodiments, the first test of the set generates one or more profiles that identify cancer in the subject. In some embodiments, the first test of the set generates one or more profiles that indicate the absence of cancer in the subject. In some embodiments, the one or more profiles generated by the first test of the set predict the tissue of origin of the cancer in the subject. In some embodiments, the one or more profiles generated by the first test of the set identify the tissue of origin of the cancer in the subject. In some embodiments, the one or more profiles generated by the first test of the set predict one or more druggable targets in the subject. In some embodiments, the one or more profiles generated by the first test of the set guide the selection of an appropriate second test from a panel of possible second tests to confirm (e.g., detect) or eliminate one or more druggable targets predicted by the one or more profiles generated by the first test.

[0090] In some embodiments, one or more criteria for a second test of the set are adjusted based on a weighted profile generated by a first test of the set, in some embodiments, the profile includes data from multiple assays (i.e., any combination of the assays described herein).

[0091] In some embodiments, the second test of the set generates one or more profiles that identify one or more druggable targets in the subject. In some embodiments, the second test of the set generates one or more profiles that evaluate the presence or absence of one or more druggable targets predicted by the first test. In some embodiments, the one or more profiles generated by the second test of the set guide the selection of an appropriate treatment for the cancer. In some embodiments, the one or more profiles generated by the second test of the set identify one or more appropriate and / or inappropriate treatments for the cancer. In some embodiments, the appropriate treatment is a treatment to which the cancer is likely to be sensitive. In some embodiments, the appropriate treatment is a treatment to which the cancer is unlikely to be resistant. In some embodiments, the second test of the set identifies an effective dosage for the appropriate treatment. In some embodiments, the one or more profiles generated by the second test of the set identify that the cancer is resistant or likely to be resistant to one or more possible treatments, and as a result, identify that the one or more possible treatments are inappropriate.

[0092] In some embodiments, the first test of the set comprises a DNA assay. In some embodiments, the first test of the set comprises an RNA assay. In some embodiments, the first test of the set comprises an epigenetic assay. In some embodiments, the first test of the set comprises a methylation assay. In some embodiments, the first test of the set comprises a fragmentomics assay. In some embodiments, the first test of the set comprises a protein assay. In some embodiments, the first test of the set comprises a metabolic assay. In some embodiments, the first test of the set comprises a microbiome assay. In some embodiments, the first test of the set evaluates circulating tumor cells. In some embodiments, the first test of the set evaluates immune cells. In some embodiments, the first test of the set evaluates extracellular vesicles. In some embodiments, the first test of the set comprises a DNA assay, a methylation assay, a fragmentomics assay, and a protein assay.

[0093] In some embodiments, the second test of the set comprises a DNA assay. In some embodiments, the second test of the set comprises an RNA assay. In some embodiments, the second test of the set comprises an epigenetic assay. In some embodiments, the second test of the set comprises a methylation assay. In some embodiments, the second test of the set comprises a fragmentomics assay. In some embodiments, the second test of the set comprises a protein assay. In some embodiments, the second test of the set comprises a metabolic assay. In some embodiments, the second test of the set comprises a microbiome assay. In some embodiments, the second test of the set evaluates circulating tumor cells. In some embodiments, the second test of the set evaluates immune cells. In some embodiments, the second test of the set evaluates extracellular vesicles. In some embodiments, the second test of the set comprises a DNA assay, a methylation assay, a fragmentomics assay, and a protein assay.

[0094] In some embodiments, a first test of the set identifies the presence or absence of cancer in a subject, the first test of the set includes an epigenetic assay, and the second test of the set includes a nucleic acid assay (e.g., a DNA assay and / or an RNA assay). In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0095] In some embodiments, a first test of the set identifies the presence or absence of cancer in a subject, the first test of the set includes an epigenetic assay, and the second test of the set includes a DNA assay and a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0096] In some embodiments, a first test of the set identifies the presence or absence of cancer in a subject, the first test of the set includes an epigenetic assay, and the second test of the set includes an RNA assay and a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0097] In some embodiments, a first test of the set identifies the presence or absence of cancer in a subject, the first test of the set includes an epigenetic assay, and the second test of the set includes a DNA assay, an RNA assay, and a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0098] In some embodiments, a first test of the set identifies the presence or absence of cancer in a subject, the first test of the set includes an epigenetic assay, and a second test of the set includes a methylation assay, a fragmentomics assay, a nucleic acid assay (e.g., a DNA assay and / or an RNA assay), and a protein assay.

[0099] In some embodiments, a first test of the set predicts one or more druggable targets, the first test of the set includes an epigenetic assay, and the second test of the set includes a DNA assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0100] In some embodiments, a first test of the set predicts one or more druggable targets, the first test of the set includes an epigenetic assay, and the second test of the set includes a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0101] In some embodiments, a first test of the set predicts one or more druggable targets, the first test of the set includes a DNA assay and an epigenetic assay, and the second test of the set includes a protein assay. In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0102] In some embodiments, a first test of the set predicts one or more druggable targets, the first test of the set includes a DNA assay and an epigenetic assay, and the second test of the set includes a nucleic acid assay (e.g., a DNA assay and / or an RNA assay). In some embodiments, the epigenetic assay generates both a methylation profile and a fragmentomics profile.

[0103] In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay and a fragmentomics assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay and a fragmentomics assay, and the second test of the set comprises a nucleic acid assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay and a fragmentomics assay, and the second test of the set comprises a protein assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay and a fragmentomics assay, and the second test of the set comprises a nucleic acid assay and a protein assay.

[0104] In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay, a fragmentomics assay, and a nucleic acid assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay, a fragmentomics assay, and a nucleic acid assay, and the second test of the set comprises a further nucleic acid assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set comprises a methylation assay, a fragmentomics assay, and a nucleic acid assay, and the second test of the set comprises a protein assay.

[0105] In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set includes a methylation assay, a fragmentomics assay, a nucleic acid assay, and a protein assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set includes a methylation assay, a fragmentomics assay, a nucleic acid assay, and a protein assay, and the second test of the set includes a further nucleic acid assay. In some embodiments, the first test of the set predicts one or more druggable targets, and the first test of the set includes a methylation assay, a fragmentomics assay, a nucleic acid assay, and a protein assay, and the second test of the set includes a further protein assay.

[0106] (Biomarkers and Genetic Variants) The first test of the methods, kits, and sets described herein may predict or identify a biomarker in a subject (e.g., a biomarker that is predictive of treatment efficacy or resistance). The first profile of the methods, kits, and sets described herein may predict or identify a biomarker in a subject (e.g., a biomarker that is predictive of treatment efficacy or resistance). The second test of the methods, kits, and sets described herein may identify or evaluate the presence or absence of one or more biomarkers in a subject (e.g., a biomarker that is predictive of treatment efficacy or resistance). Biomarkers may include, but are not limited to, druggable targets. Furthermore, biomarkers may include, but are not limited to, nucleic acids (e.g., gene variants in the subject or the microbiome of the subject), epigenetic markers, peptides, proteins, lipids, or immune cell profiles, for example, as further described herein.

[0107] As used herein, "genetic variant" refers to any change in a gene or gene product (e.g., RNA, peptide, and / or protein), and "genetic variant" refers to, for example, the presence of a mutation or mutations in the gene or gene product, a change in the copy number of the gene or gene product, or a translocation of the gene or gene product. The genetic variant may affect the integrity, sequence, structure, amount, or activity of the gene or gene product compared to the wild-type gene. Certain exemplary genetic variants include genetic variants associated with the nucleic acid sequence of all or a portion of the genome (e.g., nucleotide polymorphism, indel, sequence rearrangement, mutation frequency, and / or chromosomal translocation), genetic variants associated with the copy number of one or more specific nucleotide sequences in the genome (e.g., copy number, ploidy, and / or allele frequency fraction of a single chromosome or the whole genome), and genetic variants associated with the expression profile of the genome of the organism (e.g., gene expression level, isotype expression level, and / or gene expression ratio). In some embodiments, the genetic variant comprises a gene fusion. In some embodiments, the genetic variant is a druggable target. In some embodiments, the genetic variant is a biomarker for a druggable target.

[0108] In some embodiments, the DNA profile includes genetic variants indicative of biomarkers. Certain cancer-associated biomarkers are known to those of skill in the art. Certain exemplary biomarkers include ALK, AKT1, APC, AR (androgen receptor), ASXL1, ATM, BRAF, BRCA1, BRACA2, c-Kit, c-MET, CDK4, CDK12, CDKN2A, CTNNB1, DNMT3A, EGFR, ERα, ERBB2, ESR1, FBXW7, FGFR1, FGFR2, FGFR3, GNAS, HER2, HRAS, IDH1, IDH2, JAK2, KRAS (including variants G12C, G12D, and G12R), MED1, MEK, MET, MLH1, MSH2, MSH6, NTRK, NRAS, PDGFR, PIK3CA, PMS2, POLE, POLD, PPP2R1A, PTEN, Rb1, ROS1, RET, ROS1, TET2, TP53, VEGF, and VEGFR. In some embodiments, the biomarker comprises microsatellite instability (MSI) status.

[0109] In some embodiments, the DNA profile includes genetic variants indicative of signaling pathways associated with tumorigenesis (e.g., cell cycle, Hippo, Myc, Notch, oxidative stress response / Nrf2, RAS / MAPK, Akt / PI3K / mTORC1, TGFβ, p53, and / or β-catenin / Wnt signaling pathways). See, e.g., Sanchez-Vega et al., "Oncogenic signaling pathways in The Cancer Genome Atlas," Cell, 173:2, 321-337.e10 (2018).

[0110] In some embodiments, the RNA profile comprises RNA biomarker. In some embodiments, the RNA biomarker comprises RNA molecule. Particular exemplary RNA molecules include mRNA, long non-coding RNA (lncRNA), microRNA, small nucleolar RNA (snoRNA), circular RNA (circRNA), and Piwi-binding RNA (piRNA). In some embodiments, the mRNA biomarker is the translation product of any DNA gene variant biomarker described herein. Particular non-coding RNA biomarkers are known in the art. Certain exemplary RNA biomarkers include AC021218.2, AFAP1-AS1, ANRIL, BALR-1, BALR-2, BALR-6, ​​BANCR, CCAT-2, CRNDE.h, FALEC, FAM83H-AS-1, GAPLINC, GAS-5, H19, HOTAIR, HOX-AS-S, HYMA-1, LIMT, LINC00310, LINC00858, LINC00958, LincRNA-P21, Linc-ROR, LINK-00477, lnc RNA-LRB1, Lnc-PCDH9-13:1, lncRNA-P21, LOC_152578, LOC100506688, LOC149086, MALAT-1, MIAT, NEAT-1, NR_026817, NR_029373, N R_034119, OTX2-AS1, P34822, PCA3, PCAT18, PVT-1, RP11-138J23.1, RP11-160H22.5, RP11-317J10.2, RP11-435O5.2, RP11-445H22.4. SNHG-6, SPRY4-IT1, TUBA-4B, TUG-1, UCA-1, XLOC_000303, XLOC_006844, XLOC-014172, XLOC-109948, ZFAS1, piR-651, piR-823, miR-10b, miR-17-5p, miR-18a, miR-18b-5p, miR-19a-3p, miR-20a-5p, miR-21, miR-29a, miR-34a, miR-93-5p, miR-98-5p, miR-101-3p, miR-103-3p, miR-106a, miR-107, miR-122, miR-125, miR-125b, miR-126-3p, miR-126-5p, miR-130a-3p, miR-141, miR-144-3p, miR-144-5p, miR-145, miR-155, miR-190a-5p, miR-192, miR-193b, miR-194, miR-200a, miR-200b, miR-210, miR-221, miR-222, miR-224, miR-301, miR-301a-3p, miR-301b-3p, miR-454-3p, miR-664b-5p, miR-1246, miR-1275, miR-4485-5p, miR-5793, miR-6749-5p, miR-200c, miR-375, let-7f-5p, let-7d-5p, SNORA25, SNORD33, SNORD66, SNORA74A, and SNORD76 are included. For example, see WO2018055093A1; Xi et al., "RNA biomarkers: frontier of precision medicine for cancer", NoncodingRNA, 3:1, 9 (2017); and Sarfi et al., "Long noncodingRNAs: biomarker-based assessment", J Cell Phys, 234:10, 16971-16986 (2019).

[0111] In some embodiments, the epigenetic profile includes information about specific epigenetic changes (e.g., methylation, 5-methylcytosine (5mC), hydroxymethylation, 5-hydroxymethylcytosine, N6-methyladenine, chromatin accessibility, transcription factor binding site accessibility, histone occupancy, terminal overhang irregularity, and / or DNA fragment length). Methylation status and other epigenetic modifications are known to correlate with the presence of several disease states (e.g., cancer), and specific methylation patterns have been determined to be associated with specific cancer states. See, e.g., WO2022002423A1; Jones, "DNA methylation and cancer," Oncogene, 21:35, 5358-5360 (2002); and Paska and Hudler, "Aberrant methylation patters in cancer: a clinical view," Biochemia Med, 25:2, 161-176 (2015). Methylation patterns can also be observed in cell-free DNA, see Warton and Samimi, "Methylation of cell-free circulating DNA in the diagnosis of cancer," Front Mol Biosci, 2:13 (2015).

[0112] In some embodiments, the epigenetic profile comprises methylation data that indicates cancer biomarkers. In some embodiments, the methylation biomarkers can comprise one or more genes that are hypermethylated. Certain genes known in the art are silenced by promoter hypermethylation in cancer. Specific exemplary hypermethylated genes include Rb, p16INK4a, BRCA1, VHL, CDH1, MLH1, IGSF4, HOXA, PCDH, SEMA3F, SEPT9, WIFI, DACT2, SOSTDC1, BRMS1, SLC6A1, F13A1, BARHL1, CSMD2, and chromosome region 2q14.2. See, for example, WO2022013880A1. In some embodiments, the methylation biomarkers can comprise one or more genes that are hypomethylated. Certain exemplary genes known in the art to be hypomethylated include the Jagged1 gene, the Notch gene, hTERT, or Iroquois homeobox 1 (IRX1). In some embodiments, the methylation biomarkers may include non-coding RNA molecules (e.g., microRNAs). Certain non-coding RNAs that have differential epigenetic regulation compared to wild-type epigenetic regulation are biomarkers for cancer diagnosis. See, for example, Wang et al., "Mutual regulation of microRNAs and DNA methylation in human cancers," Epigenetics, 12:3, 187-197 (2017). Certain exemplary hypermethylated microRNA biomarkers include miR-137, miR-124-2, miR-124-3, miR-9-3, miR-203a, miR-148a, miR-34b / c, miR-34a, miR-203, miR-124, and miR-212. In some embodiments, the microRNA biomarker comprises a hypomethylated gene (e.g., miR135b).

[0113] In some embodiments, the peptide profile, protein profile, or proteomic profile comprises a biomarker. In some embodiments, the biomarker comprises a peptide or protein biomarker. In some embodiments, the peptide or protein biomarker is a cancer-associated peptide biomarker or a cancer-associated protein biomarker. Specific exemplary peptide or protein biomarkers include AFP (alpha-fetoprotein), breast cancer resistance protein (BCRP), carbohydrate antigen 15-3 (CA15-3), CA19-9, CA27-29, CA-125, carbonic anhydrase IX (CA IX), carcinoembryonic antigen (CEA), developmental endothelial locus-1 (Del-1), fibronectin, gastrokine 1 (GKN1), glycoprotein leucine-rich alpha-2 glycoprotein 1 (LRG1), glypican-1 (GPC1), soluble HER2 (sHER2), human chorionic gonadotropin beta subunit (hCG-β), human growth factor (HGF), interleukin-8 (IL-8), leucine-rich alpha-2-glycoprotein 1 (LRG1), leptin, melanoma inhibitory activity (MEA), and the like. These include myeloma immune activity (MIA), mucin 16, NY-ESO-1, osteopontin (OPN), soluble PD-L1, prolactin, prostate-specific antigen (PSA), S100B, survivin, thrombospondin-2 (THBS2), tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), apolipoprotein Cl, apolipoprotein(a), neural cell adhesion molecule LI-like protein, carbonic anhydrase 1, olfactomedin-4, neudecin, desmoplakin, and tissue inhibitor of metalloproteinase 1 (TIMP1).

[0114] In some embodiments, the peptide or protein biomarker comprises a post-translational modification to a peptide or protein, hi some embodiments, the protein post-translational modification comprises phosphorylation, glycosylation, lipidation, nitrosylation, ubiquitination, methylation, hydroxylation, and / or acetylation.

[0115] In some embodiments, the metabolic or metabolomic profile comprises information on metabolite biomarkers (e.g., cancer metabolites). In some embodiments, the metabolic biomarkers comprise one or more of linoleic acid, glutamine, threonine, isoleucine, phospholipids, total choline-containing compounds (tCho), phosphocholine, glucose, glycerophosphorylcholine, lactate, alanine, citrate, spermine, D-2-hydroxyglutarate, L-2-hydroxyglutarate, succinate, and fumarate. In some embodiments, the metabolic or metabolomic profile comprises information on one or more of glycolytic capacity, glucose metabolism, glutamine metabolism, glutaminolysis function, and lipidomics.

[0116] In some embodiments, the microbial profile includes information about one or more microorganisms. Specific exemplary microorganisms include bacteria, viruses, and fungi. In some embodiments, the microbiome profile includes one or more bacterial biomarkers. The bacterial biomarkers may be one or more of Fusobacterium spp., Alphapapillomavirus spp., Proteobacteria, Pseudoxanthomonas, Actinobacteria, Saccharopolyspora, and Streptomyces. In some embodiments, the microbiome profile includes a measure of the alpha diversity of the bacterial community.

[0117] In some embodiments, the microbial profile includes viral nucleic acids. Specific exemplary viral nucleic acids include Hepatitis B virus (HBV), human papilloma virus (HPV), human herpes virus, and Epstein-Barr virus (EBV).

[0118] In some embodiments, the immune cell profile includes information regarding one or more of the following populations: B cells, T cells, neutral killer (NK) cells, neutrophils, eosinophils, basophils, monocytes, dendritic cells, macrophages, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). In some embodiments, the T cell profile includes information regarding one or more of the following populations: cytolytic T lymphocytes (CTLs), regulatory T cells (Tregs), CD3+ T cells, CD4+ T cells, CD8+ T cells, and CD95+ T helper cells. The immune cell profile may include information regarding the T cell receptor repertoire.

[0119] Any DNA profile, RNA profile, epigenetic profile, peptide profile, protein or proteomic profile, or metabolic or metabolomic profile described herein can include data from tumor-derived exosomes.

[0120] (Druggable targets and drugs) In some embodiments, any biomarker described herein may show one or more druggable targets.Specific exemplary druggable targets include gene variants; the RNA expression product of one or more gene variants; the peptide or protein encoded by one or more gene variants; the nucleic acid, peptide or protein that shares signal pathway with the one or more gene variants; the metabolic product produced by one or more gene variants; the metabolic pathway that is affected by the one or more gene variants; and microbial targets.

[0121] Certain druggable targets, including genetic variants, are known in the art. Certain exemplary targetable genetic variants include epidermal growth factor receptor (EGFR; e.g., L858R, T790M), KRAS, NRAS, BRAF (e.g., V600E, V600K), phosphoinositide 3 kinase p110α (PIK3CA), phosphatase and tensin homolog (PTEN), human epidermal growth factor receptor 2 (HER2), c-Met, anaplastic lymphoma kinase (ALK), ROS1, and RET. In some embodiments, the druggable target includes a signal transduction pathway. Certain exemplary signal transduction pathways include RAS / MAPK, PI3K / AKT / mTORC1, Notch, JAK / STAT, and VEGF / VEGFR.

[0122] In some embodiments, the druggable target is targeted by a small molecule drug. In some embodiments, the druggable target is targeted by a biological drug (also called a biopharmaceutical or biologic). In some embodiments, the biological drug comprises a vaccine, whole blood, blood components, allergenic, somatic cells, gene therapy, tissue, recombinant therapeutic protein, or a combination thereof. In some embodiments, the biological drug comprises a sugar, a peptide, a protein, a nucleic acid, or a combination thereof. In some embodiments, the biological drug comprises a living cell or living tissue. In some embodiments, the biological drug is isolated from a living source (e.g., human, animal, plant, fungus, and / or microorganism).

[0123] Certain targeted drug therapies are known in the art. Certain exemplary targeted drug therapies include drugs that target: EGFR, ALK1, ROS1, tyrosine kinase receptor (TRK), RET, fibroblast growth factor receptor (FGFR), HER2, RAS, BRAF, tyrosine kinase, PI3K, target of rapamycin (mTOR), Akt, Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2), insulin-like growth factor 1 receptor (IGF1R), immune checkpoints, neurotrophic factors. Tyrosine receptor kinase (NTRK) fusions, human growth factor (HGF)-c-Met, c-Kit, platelet-derived growth factor receptor (PDGFR), MEK / MAPK, cyclin-dependent kinase (CDK)4 / 6, isocitrate dehydrogenase (IDH1 or IDH2), BRCA1 / 2 and ATM, estrogen receptor (ERα), aromatase, vascular endothelial growth factor receptor (VEGFR), poly(ADP-ribose) polymerase (PARP), and pyruvate kinase M2 (PKM2).

[0124] Particular exemplary EGFR targeting therapy includes cetuximab, panitumumab, gefitinib, osimertinib, erlotinib, afatinib, and rociletinib.Particular exemplary ALK1 targeting therapy includes crizotinib, alectinib, ceritinib, lorlatinib, and brigutinib.Particular exemplary ROS1 targeting therapy includes crizotinib and entrectinib.Particular exemplary TRK targeting therapy includes entrectinib and larotrectinib.Particular exemplary RET targeting therapy includes selpercatinib, pralsetinib, cabozantinib, and vandetanib. Specific exemplary FGFR targeting therapy includes erdafitinib, pemigatinib, infigratinib, rogaratinib, AZD4547, and dovitinib.Specific exemplary HER2 targeting therapy includes trastuzumab, T-DM1, lapatinib, neratinib, tucatinib, pertuzumab, and antibody drug conjugates (e.g., trastuzumab-emtansine and trastuzumab-deruxtecan).Specific exemplary RAS targeting therapy includes sotrasib, adagrasib, ARS3248, cetuximab, panitumumab, and JNJ-74699157. Specific exemplary BRAF targeting therapy includes encorafenib, cetuximab, binimetinib, vemurafenib, dabrafenib, trametinib, and cobimetinib. Specific exemplary tyrosine kinase inhibitor (TKI) therapy includes lapatinib, tucatinib, imatinib, sunitinib, regorafenib, crenolanib, avapritinib, erlotinib, and gefitinib. Specific exemplary PI3K targeting therapy includes alpelisib, buparisib, and taselisib. Specific exemplary mTOR targeting therapy includes everolimus, temsirolimus, and sirolimus. Specific exemplary Akt targeting therapy includes capivasertib and ipatasertib.Particular exemplary immune checkpoint inhibitors include pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab.Particular exemplary NTRK fusion targeting therapy includes entrectinib and larotrectinib.Particular exemplary HGF-c-Met targeting therapy includes crizotinib, capmatinib, savolitinib, tepotinib, cabozantinib, foretinib, tivantinib, emibetuzumab, onartuzumab, ficlatuzumab, and rilotumumab.Particular exemplary c-Kit targeting therapy includes imatinib, sunitinib, regorafenib, sorafenib, dasatinib, and nilotinib. Specific exemplary PDGFR targeted therapy includes imatinib, dasatinib, sunitinib, regorafenib, crenolanib, and avapritinib. Specific exemplary MEK / MAPK targeted therapy includes trametinib, cobimetinib, selumetinib, and binimetinib. Specific exemplary CDK4 / 6 targeted therapy includes palbociclib, ribociclib, and abemaciclib. Specific exemplary IDH1 targeted therapy includes ivosidenib. Specific exemplary IDH2 targeted therapy includes enasidenib. Specific exemplary ERα targeted therapy includes fulvestrant, tamoxifen, and raloxifene. Specific exemplary aromatase targeted therapy includes anastrozole and exemestane. Particular exemplary VEGFR targeting therapy includes bevacizumab, ramucirumab, sorafenib, sunitinib, axitinib, tivozanib, pazopanib, regorafenib and cediranib.Particular exemplary PARP targeting therapy includes olaparib, niraparib, rucaparib, talazoparib and veliparib.Particular exemplary microsatellite instability high (MSI high) targeting therapy includes checkpoint inhibitors, including the checkpoint inhibitors described herein.

[0125] In some embodiments, the methods, kits, and sets described herein are used to diagnose cancer in pediatric, adolescent, or adult subjects. In some embodiments, the methods and sets described herein are used to diagnose any type of cancer, predict druggable targets for the cancer, validate druggable targets for the cancer, select treatments for the cancer, and / or treat the cancer. Certain exemplary cancers include lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumors, heart cancer, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, endometrial cancer, esophageal cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, gallbladder cancer, gastric cancer (stomach cancer), and ovarian cancer (GI cancer). cancer), gastrointestinal cancer, ovarian cancer, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma (HCC), pancreatic islet cell tumor, pancreatic neuroendocrine tumor, renal (renal cell) cancer, leukemia, lung cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, melanoma, mesothelioma, multiple myeloma (MM), pancreatic cancer, paraganglioma, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, sarcoma, rhabdomyosarcoma, Kaposi's sarcoma, Ewing's sarcoma, osteosarcoma, skin cancer, small intestine cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is colorectal cancer.

Claims

1. A method for obtaining a first test result and a second test result as indicators for identifying one or more drug-worthy targets for treating cancer in a subject, (a) a step of performing or having performed a first test on the liquid biopsy sample of the subject, wherein the result of the first test identifies the subject as having cancer; and (b) A step of performing a second test, wherein the results of the second test are used to evaluate gene variants in the subject to identify the presence of one or more drug-potential targets. Methods that include...

2. A drug for use in a method for treating cancer in a subject, wherein the method is: A step of administering an effective amount of the drug to the subject, wherein the drug targets at least one of the identified drug-developable targets, and the drug-developable target is identified according to the method of claim 1. Drugs, including

3. The method according to Claim 1, further comprising the step of selecting a cancer diagnostic test targeted for a subject, The first test of step (a) generates a predictive profile for one or more drug-potential targets in the subject, The second test of step (b) is selected as the targeted cancer diagnostic test, and the second test evaluates the presence or absence of the one or more drug-available targets predicted by the profile. method.

4. The method according to Claim 1, further comprising the step of predicting and confirming the drug-worthy target for treating cancer in a subject, The first test of step (a) generates a predictive profile for one or more drug-potential targets in the subject, Step (b) further comprises selecting the second test, the second test evaluating the presence or absence of the one or more drug-worthy targets predicted by the profile. method.

5. The first test of step (a) generates a predictive profile for one or more drug-worthy targets in the subject, Step (b) further includes selecting or having selected the second test, the second test evaluating the presence or absence of the one or more drug-potentially discoverable targets. A drug for use in the method of claim 2.

6. The method according to claim 3, The first test of step (a) generates a first profile or a second profile, and optionally generates a first epigenetic profile or a second epigenetic profile, wherein the first profile is predictive for one or more drug-worthy targets of a first group in the subject, and the second profile is predictive for one or more drug-worthy targets of a second group in the subject. Step (b) further includes selecting the second test as the targeted cancer diagnostic test, (i) If the first test generates the first profile, the second test evaluates the presence or absence of one or more drug-worthy targets of the first group predicted by the first profile; (ii) If the first test generates the second profile, the second test evaluates the presence or absence of one or more drug-worthy targets of the second group predicted by the first profile. method.

7. The method according to claim 6, further comprising the step of predicting and confirming drug-worthy targets for treating cancer in a subject, (c) A step of performing the second test, wherein the second test confirms the presence of at least one of the one or more drug-potentially discoverable targets. Methods that further include the above.

8. The method further comprises the step of treating cancer in a subject, The first test of step (a) generates a first profile or a second profile, and optionally generates a first epigenetic profile or a second epigenetic profile, wherein the first profile is predictive for one or more drug-worthy targets of a first group in the subject, and the second profile is predictive for one or more drug-worthy targets of a second group in the subject. Step (b) further includes selecting or having selected the second test as the targeted cancer diagnostic test. (i) If the first test generates the first profile, the second test evaluates the presence or absence of one or more drug-worthy targets in the first group; (ii) If the first test generates the second profile, the second test evaluates the presence or absence of one or more drug-worthy targets in the second group. A drug for use in the method according to claim 5.

9. A drug for use according to the method or method of any one of claims 1 to 8, wherein the second test is performed on or has been performed on the liquid biopsy sample of the subject, and the first test and the second test are performed on separate liquid biopsy samples of the subject, if necessary.

10. The first test comprises a methylation assay, a nucleic acid assay (e.g., gene screening), a protein assay, a post-translational modification assay, a fragment mix assay, a metabolomics assay, an RNA assay (e.g., a microRNA (miRNA) assay), a microbiome assay, an assay of one or more immune cell populations, or a combination thereof. If necessary, the first test may include a methylation assay, a nucleic acid assay (e.g., gene screening), a protein assay, a fragment mix assay, or a combination thereof. If necessary, the first test may include a methylation assay. A drug for use according to the method or method of any one of claims 1 to 8.

11. The profile generated by the first test includes data relating to methylation status, data relating to chromatin condensation, data relating to histone modification, data relating to fragmentation pattern, data relating to topology, other epigenetic data, data relating to nucleic acid sequence, data relating to nucleic acid expression, data relating to protein translation, data relating to protein sequence, data relating to post-translational modification (e.g., glycosylation), data relating to the presence of metabolites, data relating to microbiome composition, data relating to immune status, or a combination thereof. If necessary, the profile generated by the first test may include data on methylation status, data on chromatin condensation, data on histone modification, data on fragmentation patterns, data on topology, other epigenetic data, or a combination thereof. A drug for use according to the method or method of any one of claims 1 to 8.

12. (a) The first test comprises a plurality of assays, and / or (b) The profile generated by the first test includes data from multiple assays, A drug for use according to the method or method of any one of claims 1 to 8.

13. The second test comprises a test for one or more gene variants, If necessary, one or more drug-potential targets (a) one or more gene variants, or (b) Ribonucleic acid expression products of one or more gene variants, (c) A peptide or protein encoded by one or more of the gene variants, (d) A nucleic acid, peptide, or protein that shares a signaling pathway with one or more of the gene variants. A drug for use according to the method or method described in any one of claims 1 to 8, including the method described in any one of claims 1 to 8.

14. A drug for use according to the method or method of any one of claims 1 to 8, wherein the liquid biopsy sample comprises a blood sample.

15. A drug for use according to the method or method of any one of claims 1 to 8, wherein the liquid biopsy sample contains cell-free DNA.