Methods for removing embedding medium from embedded samples

Abstract

The present disclosure provides methods for detecting analytes such as RNA and / or DNA, extracting analytes such as RNA and / or DNA, improving library construction for nucleic acid sequencing, and reducing levels of embedding agents in analyte samples, such as RNA and / or DNA samples extracted from embedded samples, such as paraffin-embedded samples.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 290,537, filed December 16, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to methods for removing embedding media, such as paraffin, from embedded samples. The methods described herein may be used to detect analytes, such as RNA and / or DNA, extract analytes, such as RNA and / or DNA, and improve library construction for nucleic acid sequencing from embedded samples, as well as methods for diagnosing, assessing, and treating diseases, such as cancer. [Background technology]

[0003] Cancers exhibit phenotypic endpoints of multiple genetic lesions that confer cells with a wide range of biological properties required for tumorigenesis. Indeed, a hallmark genomic feature of many cancers is the presence of numerous complex chromosomal structural abnormalities, including translocations, intrachromosomal inversions, point mutations, deletions, gene copy number changes, altered gene expression levels, gene fusions, and germline mutations, among others. The presence of these hallmark genomic features can serve as biomarkers for cancer.

[0004] One method for detecting such biomarkers is the analysis of nucleic acids extracted from tumor cells in tissue samples, such as formalin-fixed, paraffin-embedded (FFPE) tissue. It is particularly desirable to extract both RNA and DNA from a single tissue sample. Methods for extracting RNA and / or DNA from paraffin-embedded tissue samples involve removing the paraffin prior to nucleic acid extraction. One method for removing paraffin from embedded samples involves dissolving the paraffin in a harmful organic solvent, such as xylene, or another miscible solvent. These solvent-based methods for paraffin removal are not suitable for robotic automation, e.g., for multiplexing nucleic acid sample preparation. Therefore, there is a need in the art for methods for removing embedding agents, such as paraffin, from embedded samples, particularly methods suitable for robotic automation and high-throughput sample preparation.

[0005] All references cited herein, including patents, patent applications, and publications, are incorporated herein by reference in their entirety. To the extent that a reference incorporated by reference conflicts with the present disclosure, the present disclosure shall control. Summary of the Invention

[0006] Provided herein are methods for detecting alterations in RNA and / or DNA, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally with the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which the paraffin-embedded sample is not deparaffinized. In some embodiments, the alteration in the RNA and / or DNA is selected from the group consisting of a) copy number alteration, b) point mutation, c) in-frame deletion of one or more codons, d) intragenic deletion, e) intragenic insertion, f) whole gene deletion, g) inversion, h) interchromosomal translocation, i) tandem duplication, j) gene fusion, k) genomic rearrangement involving intronic sequences, and / or l) gene amplification or duplication. In some embodiments, the alteration in the RNA and / or DNA is a copy number alteration.

[0007] Further provided herein are methods for extracting RNA and / or DNA, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin, causing the paraffin to separate from the sample; and c) extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, RNA and / or DNA extraction is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, RNA and / or DNA extraction is improved compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0008] Further provided herein are methods for improving library construction for nucleic acid sequencing, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) preparing a sequencing library for sequencing the RNA and / or DNA. In some embodiments, the preparation of the sequencing library is improved compared to methods in which the paraffin is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the preparation of the sequencing library is improved compared to methods in which the paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0009] Further provided herein are methods for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to a method in which the paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to a method in which the paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0010] Further provided herein are methods for improving separation of paraffin from a paraffin-embedded sample, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; and c) extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of paraffin results in less filter clogging compared to methods that dissolve paraffin in a miscible solvent, where the miscible solvent is optionally xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of paraffin results in less filter clogging compared to methods that remove paraffin by cleaving it from a paraffin-embedded sample. In some embodiments, the filter is a filter in a spin column.

[0011] In some embodiments of the preceding method, step b) does not include dissolving the paraffin in a miscible solvent, and optionally the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments of the preceding method, the phase transition is melting. In some embodiments of the preceding method, step b) includes heating the paraffin-embedded sample. In some embodiments of the preceding method, the paraffin-embedded sample is heated to about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments of the preceding method, the paraffin-embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments of the preceding method, step b) comprises centrifuging and filtering the paraffin-embedded sample. In some embodiments of the preceding method, the paraffin-embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments of the preceding method, the paraffin-embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments of the preceding method, the paraffin-embedded sample is centrifuged at 1,811 rcf. In some embodiments of the preceding method, step b) comprises heating and centrifuging the paraffin-embedded sample. In some embodiments of the preceding method, the paraffin-embedded sample is heated to about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments of the preceding method, the paraffin-embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments of the preceding method, the paraffin-embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments of the preceding method, the paraffin-embedded sample is centrifuged at 1,811 rcf or greater.In some embodiments of the preceding method, the paraffin-embedded sample is centrifuged at 1,811 rcf. In some embodiments of the preceding method, step b) comprises heating, centrifuging, and filtering the paraffin-embedded sample.

[0012] Further provided herein are methods for detecting alterations in RNA and / or DNA, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods that do not deparaffinize paraffin-embedded samples. In some embodiments, the alterations in RNA and / or DNA are selected from the group consisting of a) copy number alterations, b) point mutations, c) in-frame deletions of one or more codons, d) intragenic deletions, e) intragenic insertions, f) whole-gene deletions, g) inversions, h) interchromosomal translocations, i) tandem duplications, j) gene fusions, k) genomic rearrangements involving intronic sequences, and / or l) gene amplifications or duplications. In some embodiments, the alterations in RNA and / or DNA are copy number alterations.

[0013] Further provided herein are methods for extracting RNA and / or DNA, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; and c) extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, extraction of RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, extraction of RNA and / or DNA is improved compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0014] Further provided herein are methods for improving library construction for nucleic acid sequencing, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) preparing a sequencing library for sequencing the RNA and / or DNA. In some embodiments, the preparation of the sequencing library is improved compared to methods in which the paraffin is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the preparation of the sequencing library is improved compared to methods in which the paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0015] Further provided herein are methods for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, the method comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to a method in which the paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to a method in which the paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0016] Further provided herein are methods for improving separation of paraffin from a paraffin-embedded sample, the methods comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; and c) extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is removed by separating the paraffin from the paraffin-embedded sample. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of paraffin results in less filter clogging compared to methods that dissolve paraffin in a miscible solvent, where the miscible solvent is optionally xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of paraffin results in less filter clogging compared to methods that remove paraffin by cleaving it from a paraffin-embedded sample. In some embodiments, the filter is a filter in a spin column.

[0017] In some embodiments of the preceding method, step b) does not include dissolving the paraffin in a miscible solvent, and optionally the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments of the preceding method, the immiscible solvent is mineral oil. In some embodiments, the paraffin-embedded sample is contacted with about 300, 350, 400, 450, 500, or 550 μL of mineral oil. In some embodiments, the paraffin-embedded sample is contacted with mineral oil at a ratio of about 1:4, 1:2, 1:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, or 50:1 of mineral oil to paraffin-embedded sample. In some embodiments, step b) comprises incubating the paraffin-embedded sample in mineral oil at about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., or about 75° C. In some embodiments, the mineral oil contacts the paraffin-embedded sample for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the mineral oil contacts the paraffin-embedded sample while shaking at about 25 rpm, about 50 rpm, about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, about 1000 rpm, or about 1100 rpm.

[0018] In some embodiments of the preceding method, step b) comprises contacting the sample with an immiscible solvent and centrifuging and filtering the paraffin-embedded sample. In some embodiments, the paraffin-embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the paraffin-embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the paraffin-embedded sample is centrifuged at 1,811 rcf.

[0019] In some embodiments of the preceding method, separation of paraffin from the sample is automated. In some embodiments of the preceding method, step b) is automated. In some embodiments of the preceding method, the method is automated. In some embodiments of the preceding method, two or more paraffin-embedded samples are processed in parallel. In some embodiments, 12, 24, 48, or 96 paraffin-embedded samples are processed in parallel. In some embodiments of the preceding method, the method is performed using a liquid handling robot. In some embodiments, the liquid handling robot is an Agilent, BioTek, Hamilton, Tecan, or ThermoFisher Scientific liquid handling robot, optionally, the liquid handling robot is a Hamilton AutoLys STAR, Hamilton STAR, BioTek Dispenser, or KingFisher Flex. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR. In some embodiments of the preceding method, step c) comprises extracting RNA and DNA from the deparaffinized sample. In some embodiments of the preceding method, step a) includes: i) identifying a target region containing tumor cells of interest in the paraffin-embedded tissue; ii) extracting a paraffin-embedded sample from the paraffin-embedded tissue; iii) identifying the location of the paraffin-embedded sample in the paraffin-embedded tissue; and iv) extracting RNA and / or DNA from the sample if the location of the paraffin-embedded sample overlaps with the target region containing tumor cells of interest. In some embodiments, steps ii) and iii) are repeated if the location of the paraffin-embedded sample does not overlap with the target region containing tumor cells of interest. In some embodiments, step ii) includes extracting the paraffin-embedded sample using a needle. In some embodiments, the paraffin-embedded sample is extracted by puncturing the needle through the paraffin-embedded tissue. In some embodiments, the needle is a disposable needle. In some embodiments, the needle is a 13-gauge needle, a 14-gauge needle, a 15-gauge needle, a 16-gauge needle, a 17-gauge needle, an 18-gauge needle, a 19-gauge needle, a 20-gauge needle, or a 21-gauge needle.In some embodiments, the paraffin-embedded sample extracted from the paraffin-embedded tissue is about 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, or 2.3 mm in diameter. In some embodiments, step ii) comprises extracting the paraffin-embedded sample using a laser microdissection (LMD) or a razor blade. In some embodiments, step iii) comprises preparing slides of sections of the paraffin-embedded tissue. In some embodiments, the sections of the paraffin-embedded tissue are stained. In some embodiments, the sections of the paraffin-embedded tissue are stained with hematoxylin and eosin (H&E). In some embodiments, step iii) is performed by visual inspection. In some embodiments, step iii) is performed by a computer system. In some embodiments, step iii) is performed using an image analysis system.

[0020] In some embodiments of the preceding methods, the paraffin-embedded sample is derived from an individual known to have or suspected of having cancer. In some embodiments of the preceding methods, the paraffin-embedded sample is derived from a biopsy, optionally a tumor biopsy. In some embodiments of the preceding methods, the paraffin-embedded sample is a fixed paraffin-embedded sample. In some embodiments, the fixed paraffin-embedded sample is selected from the group consisting of a formalin-fixed sample, an ethanol-fixed sample, and a methanol-fixed sample. In some embodiments of the preceding methods, the paraffin-embedded sample is obtained from formalin-fixed, paraffin-embedded (FFPE) tissue. In some embodiments of the preceding methods, the paraffin-embedded sample is obtained from cryopreserved tissue. In some embodiments of the preceding methods, the paraffin-embedded sample is obtained from fresh-frozen tissue. In some embodiments, the fresh-frozen tissue is frozen in optimal cutting temperature (OCT) compound. In some embodiments of the preceding methods, RNA is extracted before DNA is extracted. In some embodiments, the method further comprises digesting the paraffin-embedded sample prior to step b). In some embodiments, the paraffin-embedded sample is digested using a proteinase. In some embodiments, the proteinase is proteinase K. In some embodiments, the paraffin-embedded sample is digested using about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, or about 35 μL of 20 mg / mL proteinase K. In some embodiments, the paraffin-embedded sample is incubated with the proteinase at about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, or about 62°C. In some embodiments, the paraffin-embedded sample is incubated with the proteinase for about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes. In some embodiments, the paraffin-embedded sample is incubated with the proteinase while shaking at about 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, or 1500 rpm.In some embodiments, the paraffin-embedded sample is partially or completely digested. In some embodiments, the method further comprises de-crosslinking the digested sample after step b). In some embodiments, de-crosslinking comprises heating the digested sample to 80-90°C. In some embodiments, the digested sample is heated for about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes. In some embodiments, step c) comprises collecting a sample lysate containing RNA from the digested paraffin-embedded sample and purifying RNA from the RNA-containing sample lysate. In some embodiments, the method further comprises completely digesting the digested paraffin-embedded sample. In some embodiments, a proteinase is used to achieve the complete digestion. In some embodiments, the proteinase is proteinase K. In some embodiments, the digested paraffin-embedded sample is digested using about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, or about 35 μL of 20 mg / mL proteinase K. In some embodiments, the digested paraffin-embedded sample is incubated with the proteinase at about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., or about 75° C. In some embodiments, the digested paraffin-embedded sample is incubated with the proteinase for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, or about 22 hours. In some embodiments, the digested paraffin-embedded sample is incubated with the proteinase while shaking at about 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, or 1500 rpm. In some embodiments, the method further comprises collecting a sample lysate comprising DNA from the fully digested paraffin-embedded sample.In some embodiments, the method further comprises purifying DNA from a DNA-containing sample lysate. In some embodiments, the DNA is extracted before extracting RNA. In some embodiments, the method further comprises thoroughly digesting the paraffin-embedded sample after step b). In some embodiments, the thorough digestion is achieved using a proteinase. In some embodiments, the proteinase is proteinase K. In some embodiments, the paraffin-embedded sample is digested using about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, or about 35 μL of 20 mg / mL proteinase K. In some embodiments, the paraffin-embedded sample is incubated with the proteinase at about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the paraffin-embedded sample is incubated with the proteinase for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, or about 22 hours. In some embodiments, the method further comprises extracting DNA from the fully digested paraffin-embedded sample. In some embodiments, the method further comprises extracting RNA from the fully digested paraffin-embedded sample.

[0021] In some embodiments of the preceding methods, the method further comprises measuring the amount of RNA and / or DNA extracted from the paraffin-embedded sample. In some embodiments, the amount of RNA and / or DNA extracted from the paraffin-embedded sample is about 5 ng, about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 60 ng, about 70 ng, about 80 ng, about 100 ng, about 50 μg, or about 50 mg. In some embodiments of the preceding methods, the method further comprises analyzing the RNA and / or DNA extracted from the paraffin-embedded sample by one or more methods selected from the group consisting of a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, sequencing, next-generation sequencing, screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), or mass spectrometry genotyping. In some embodiments of the preceding methods, the method further comprises analyzing the RNA and / or DNA extracted from the paraffin-embedded sample by next-generation sequencing. In some embodiments of the preceding methods, the method further includes e) optionally ligating one or more adapters to RNA and / or DNA extracted from the paraffin-embedded sample to generate ligated nucleic acids, d) optionally amplifying nucleic acids from the ligated nucleic acids, f) optionally capturing a plurality of nucleic acids corresponding to one or more genes of interest, g) sequencing the plurality of nucleic acids with a sequencing device to obtain a plurality of sequence reads corresponding to the one or more genes of interest, h) analyzing the plurality of sequence reads, and i) detecting one or more mutations in the genes of interest based on the analysis. In some embodiments, the plurality of nucleic acids corresponding to the one or more genes of interest are captured from the amplified nucleic acids by hybridization with bait molecules.In some embodiments, a plurality of nucleic acids corresponding to one or more genes of interest are enriched from the amplified nucleic acids, and optionally, a plurality of nucleic acids corresponding to one or more genes of interest are enriched from the amplified nucleic acids by biotin / streptavidin tagging. In some embodiments, prior to step e), the RNA and / or DNA extracted from the paraffin-embedded sample is fragmented, and optionally, the RNA and / or DNA extracted from the paraffin-embedded sample is fragmented by sonication. In some embodiments, the fragmented RNA and / or DNA extracted from the paraffin-embedded sample is end-repaired. In some embodiments, the end-repaired and fragmented RNA and / or DNA extracted from the sample is dA-tailed or dT-tailed.

[0022] In some embodiments of the preceding methods, the method further comprises analyzing DNA extracted from the paraffin-embedded sample to detect somatic mutations selected from the group consisting of: i) point mutations, ii) in-frame deletions of one or more codons, iii) intragenic deletions, iv) intragenic insertions, v) whole-gene deletions, vi) inversions, vii) interchromosomal translocations, viii) tandem duplications, ix) gene fusions, x) genomic rearrangements involving intronic sequences, and / or xi) gene amplifications or duplications. In some embodiments, the somatic mutation is in a gene, and the gene is ABL1, AKT1, AKT2, AKT3, ALK, APC, AR, BRAF, CCND1, CDK4, CDKN2A, CEBPA, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FLT3, HRAS, JAK2, KIT, KRAS, MAP2K1, MAP2K2, MET, MLL, MYC, NF1, NOTCH1, NPM1, NRAS, NTRK3, PDGFRA, PIK3CA, PIK3CG, PIK3R1, PTCH1, PTCH2, PTEN, RB1, RET, SMO, STK11, SUFU, or TP53.In some embodiments, the somatic mutation is in a gene, including but not limited to ABL2, ARAF, ARFRP1, ARID1A, ATM, ATR, AURKA, AURKB, BAP1, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL6, BRCA1, BRCA2, CBL, CARD11, CBL, CCND2, CCND3, CCNE1, CD79A, CD79B, CDH1, CDH2, CDH20, CDH5, CDK6, CDK8, CD KN2B, CDKN2C, CHEK1, CHEK2, CRKL, CRLF2, DNMT3A, DOT1L, EPHA3, EPHA5, EPHA6, EPHA7, EPHB1, EPHB4, EPHB6, ERBB3, ERBB4, ERG, ETV1, ETV4, ETV5, ETV6, EWSR1, EZH2, FANCA, FBXW7, FGFR4, FLT1, FLT4, FOXP4, GATA1, GNA11, GNAQ, GNAS, GPR124, GUCY1 A2, HOXA3, HSP90AA1, IDH1, IDH2, IGF1R, IGF2R, IKBKE, IKZF1, INHBA, IRS2, JAK1, JAK3, JUN, KDM6A, KDR, LRP1B, LRP6, LTK, MAP2K4, MCL1, MDM2, MDM4, MEN1, MITF, MLH1, MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYCL1, MYCN, NF2, NKX2-1, NTRK1, NTRK2 , PAK3, PAX5, PDGFRB, PKHD1, PLCG1, PRKDC, PTPN11, PTPRD, RAF1, RARA, RICTOR, RPTOR, RUNX1, SMAD2, SMAD3, SMAD4, SMARCA 4, SMARCB1, SOX10, SOX2, SRC, TBX22, TET2, TGFBR2, TMPRSS2, TNFAIP3, TNK, TNKS2, TOP1, TSC1, TSC2, USP9X, VHL, or WT1.

[0023] In some embodiments of the prior methods, the method further comprises analyzing RNA extracted from the paraffin-embedded sample to detect an alteration selected from the group consisting of: i) gene fusions, ii) exon skipping events, iii) splice variants, and / or iv) altered gene expression. In some embodiments of the prior methods, the method further comprises detecting loss of heterozygosity (LOH) of one or more genes of interest in the paraffin-embedded sample. In some embodiments, the method detects ST7 / RAY1, ARH1 / NOEY2, TSLC1, RB, PTEN, SMAD2, SMAD4, DCC, TP53, ATM, miR-15a, miR-16-1, NAT2, BRCA1, BRCA2, hOGG1, CDH1, IGF2, CDKN1C / P57, MEN1, PRKAR1A, H19, KRAS, BAP1, PTCH1, SMO, SUFU, NOTCH1, PPP6C, LATS1, CASP8, PTPN14, ARID1A, FBXW7, M6P / IGF2R, IFN-α, olfactory receptor genes, CBFA2T3, DUTT1, FHIT, APC, P16, F In some embodiments, the method further comprises detecting LOH of CMD, TSC2, miR-34, c-MPL, RUNX3, DIRAS3, NRAS, miR-9, FAM50B, PLAGL1, ER, FLT3, ZDBF2, GPR1, c-KIT, NAP1L5, GRB10, EGFR, PEG10, BRAF, MEST, JAK2, DAPK1, LIT1, WT1, NF-1, PR, c-CBL, DLK1, AKT1, SNURF, cytochrome P450 genes (CYP), ZNF587, SOCS1, TIMP2, RUNX1, AR, CEBPA, C19MC, EMP3, ZNF331, CDKN2A, PEG3, NNAT, GNAS, and / or GATA5. In some embodiments, the method further comprises detecting LOH of human leukocyte antigen (HLA) genes in the paraffin-embedded sample.In some embodiments, the method further includes ligating one or more adapters to one or more of the RNA and / or DNA extracted from the paraffin-embedded sample to generate ligated nucleic acids; amplifying the ligated nucleic acids; capturing a plurality of nucleic acids from the amplified nucleic acids corresponding to HLA genes using bait molecules; sequencing the captured nucleic acids to obtain a plurality of sequence reads corresponding to the HLA genes; fitting, by one or more processors, one or more values ​​associated with one or more of the plurality of sequence reads to a model; and detecting LOH of the HLA genes and relative binding propensities for HLA alleles of the HLA genes based on the model. In some embodiments, LOH of an HLA gene and a relative binding propensity for an HLA allele of the HLA gene are detected by: a) obtaining an observed allele frequency for the HLA allele, wherein the observed allele frequency corresponds to a frequency of a nucleic acid encoding at least a portion of the HLA allele detected among a plurality of sequence reads corresponding to the HLA gene; b) obtaining a relative binding propensity of the HLA allele to a bait molecule, wherein the relative binding propensity of the HLA allele corresponds to a propensity of a nucleic acid encoding at least a portion of the HLA allele to bind to the bait molecule in the presence of nucleic acids encoding a portion of one or more other HLA alleles; c) applying an objective function to measure the difference between the relative binding propensity of the HLA allele and the observed allele frequency; d) applying an optimization model to minimize the objective function; e) determining an adjusted allele frequency of the HLA allele based on the optimization model and the observed allele frequency; and f) determining that LOH has occurred if the adjusted allele frequency of the HLA allele is less than a predetermined threshold. In some embodiments, the paraffin-embedded sample is from an individual known to have or suspected of having cancer, and further comprises administering to the individual an effective amount of a treatment other than an immune checkpoint inhibitor (ICI) based at least in part on the detection of LOH of an HLA gene.In some embodiments, the method further comprises recommending a treatment other than an immune checkpoint inhibitor (ICI) based at least in part on the detection of LOH of the HLA genes. In some embodiments, the method further comprises detecting or obtaining knowledge of a high tumor mutation burden (TMB) in the paraffin-embedded sample. In some embodiments, the paraffin-embedded sample is from an individual known to have cancer or suspected to have cancer, and further comprises administering to the individual an effective amount of an immune checkpoint inhibitor (ICI) based at least in part on the detection of LOH of the HLA genes and a high TMB. In some embodiments, the paraffin-embedded sample is from an individual known to have cancer or suspected to have cancer, and further comprises recommending to the individual a treatment comprising an immune checkpoint inhibitor (ICI) based at least in part on the detection of LOH of the HLA genes and a high TMB. In some embodiments, the HLA genes are human HLA-A, HLA-B, or HLA-C genes. In some embodiments, the TMB is determined based on the number of non-driver somatic coding mutations per megabase of the sequenced genome.

[0024] In some embodiments of the preceding methods, the method further comprises detecting a loss-of-function mutation in a phosphatase and tensin homolog (PTEN) gene in the paraffin-embedded sample. In some embodiments, the loss-of-function mutation in the PTEN gene comprises one or more of an insertion, deletion, or substitution of one or more nucleotides, a genomic rearrangement, a modification in the promoter, a gene fusion, or a copy number modification. In some embodiments, the loss-of-function mutation in the PTEN gene is detected in the paraffin-embedded sample by one or more of a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, sequencing, next-generation sequencing, screening analysis, fluorescent in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), or mass spectrometry genotyping.

[0025] In some embodiments of the preceding methods, the method further comprises measuring the level of tumor mutation burden (TMB) in the paraffin-embedded sample. In some embodiments, a TMB of at least about 10 mutations / megabase (Mb) or at least about 20 muts / Mb is detected. In some embodiments, TMB is measured in RNA and / or DNA from the paraffin-embedded sample by whole-exome sequencing, whole-genome sequencing, or gene-targeted sequencing. In some embodiments, TMB is measured on about 0.80 Mb of sequenced DNA. In some embodiments, TMB is measured on about 0.83 Mb to about 1.14 Mb of sequenced DNA. In some embodiments, TMB is measured on about 1.1 Mb of sequenced DNA. In some embodiments, TMB is measured on up to about 1.1 Mb of sequenced DNA.

[0026] In some embodiments of the preceding methods, the method further comprises detecting homozygous single-exon losses in the paraffin-embedded sample. In some embodiments, the homozygous single-exon losses are detected in RNA and / or DNA from the paraffin-embedded sample by whole-exome sequencing, whole-genome sequencing, or gene-targeted sequencing.

[0027] Further provided herein are methods for detecting an analyte, the method comprising: a) providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; c) extracting the analyte from the de-embedded sample; and d) analyzing the analyte to detect the analyte. In some embodiments, detection of the analyte is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, detection of the analyte is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample. In some embodiments, detection of the analyte is improved compared to methods in which the embedded sample is not de-embedded.

[0028] Further provided herein are methods for extracting an analyte, the method comprising: a) providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; and c) extracting the analyte from the de-embedded sample. In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0029] Further provided herein are methods for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, the method comprising: a) providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; c) extracting the analyte from the de-embedded sample; and d) purifying the extracted analyte to provide the analyte sample. In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0030] Further provided herein are methods for improving separation of an embedding medium from an embedded sample, the methods comprising: a) providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; and c) extracting the analyte from the de-embedded sample. In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods in which the embedding medium is dissolved in a miscible solvent, where the miscible solvent is optionally xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods in which the embedding medium is removed by separating it from the embedded sample. In some embodiments, the filter is a filter in a spin column.

[0031] In some embodiments of the preceding method, step b) does not include dissolving the embedding medium in a miscible solvent, and optionally the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments of the preceding method, the phase transition is selected from the group consisting of melting, freezing, vaporization, condensation, sublimation, and deposition. In some embodiments of the preceding method, the phase transition is melting. In some embodiments of the preceding method, step b) includes heating, cooling, increasing pressure, or decreasing pressure of the embedded sample. In some embodiments of the preceding method, step b) includes heating the embedded sample. In some embodiments, the embedded sample is heated to about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments of the preceding method, step b) comprises centrifuging and filtering the embedded sample to separate the embedding from the sample. In some embodiments, the embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the embedded sample is centrifuged at 1,811 rcf. In some embodiments of the prior method, step b) comprises heating, centrifuging, and filtering the embedded sample to separate the embedding medium from the sample, thereby producing a de-embedded sample. In some embodiments of the prior method, step b) comprises heating and centrifuging the embedded sample to separate the embedding medium from the sample, thereby producing a de-embedded sample.

[0032] Further provided herein are methods for detecting an analyte, the method comprising: a) providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedding medium is removed by contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample; c) extracting the analyte from the de-embedded sample; and d) analyzing the analyte to detect the analyte. In some embodiments, detection of the analyte is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, detection of the analyte is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample. In some embodiments, detection of the analyte is improved compared to methods in which the embedded sample is not de-embedded.

[0033] Further provided herein are methods for extracting an analyte, the method comprising: a) providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedding medium is removed by contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample; and c) extracting the analyte from the de-embedded sample. In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally the miscible solvent being xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0034] Further provided herein are methods for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, the method comprising: a) providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedding medium is removed by contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample; c) extracting the analyte from the de-embedded sample; and d) purifying the extracted analyte to provide the analyte sample. In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0035] Further provided herein are methods for improving separation of an embedding medium from an embedded sample, the methods comprising: a) providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedding medium is removed by contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample; and c) extracting the analyte from the de-embedded sample. In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods in which the embedding medium is dissolved in a miscible solvent, where the miscible solvent is optionally xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the method is performed on a liquid handling robot with a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods in which the embedding medium is removed by separating it from the embedded sample. In some embodiments, the filter is a filter in a spin column.

[0036] In some embodiments of the preceding method, step b) does not include dissolving the embedding agent in a miscible solvent, and optionally the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments of the preceding method, the density of the immiscible solvent is lighter than water and heavier than the embedding agent when the embedding agent is in liquid form. In some embodiments, the density of the immiscible solvent is heavier than liquid paraffin. In some embodiments of the preceding method, the immiscible solvent is vegetable oil. In some embodiments of the preceding method, the immiscible solvent is mineral oil. In some embodiments, the embedded sample is contacted with about 300, 350, 400, 450, 500, or 550 μL of mineral oil. In some embodiments, the embedded sample is contacted with mineral oil at a ratio of mineral oil to embedded sample of about 1:4, 1:2, 1:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, or 50:1. In some embodiments, the mineral oil is contacted with the embedded sample at about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., or about 75° C. In some embodiments, the mineral oil is contacted with the embedded sample for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the mineral oil contacts the embedded sample while shaking at about 25 rpm, about 50 rpm, about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, about 1000 rpm, or about 1100 rpm.

[0037] In some embodiments of the preceding method, step b) comprises contacting the sample with an immiscible solvent and centrifuging and filtering the embedded sample. In some embodiments, the embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the embedded sample is centrifuged at 1,811 rcf.

[0038] In some embodiments of the preceding methods, the analyte is selected from the group consisting of a polypeptide, RNA, DNA, a small molecule, a lipid, a polysaccharide, an exosome, a mitochondria, and a nucleus. In some embodiments of the preceding methods, the embedding agent is selected from the group consisting of paraffin, a resin, celloidin, Paraplast®, gelatin, an ester wax, a wax, polyethylene glycol, and nitrocellulose. In some embodiments, the embedding agent is paraffin.

[0039] Further provided herein are methods for extracting RNA from a paraffin-embedded sample, the method comprising: a) incubating the sample with a protease; b) incubating the sample at 50°C to 75°C for 1 to 40 minutes; c) centrifuging the sample at high speed to separate the paraffin from a lysate containing RNA; and d) aspirating the lysate containing RNA. In some embodiments, the method further comprises cooling the sample to room temperature after step c) and before step d). In some embodiments, the method further comprises centrifuging the sample and filtering the lysate after cooling the sample to room temperature. In some embodiments, incubating the sample with the protease comprises incubation at 50°C to 60°C. In some embodiments, incubating the sample with the protease comprises incubation for 1 to 20 minutes. In some embodiments, centrifuging the sample at high speed comprises centrifuging the sample at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, RNA is isolated from the lysate. In some embodiments, the method for extracting RNA is used in a high-throughput method. In some embodiments, the method further comprises analyzing the RNA. In some embodiments, the incubation with the protease is performed with shaking at 500-2000 rpm. In some embodiments, the incubation at 50°C-80°C is performed without shaking. In some embodiments, the method further comprises centrifuging the sample at 250 RCF-750 RCF after step c). In some embodiments, the method further comprises preparing cDNA from the RNA.

[0040] Further provided herein are methods for extracting DNA from a sample, the methods comprising: a) incubating the sample with a protease; b) incubating the sample at 50°C to 80°C for 2 to 48 hours; c) centrifuging the sample at high speed to produce a lysate containing DNA; and d) aspirating the lysate containing DNA. In some embodiments, the sample is incubated at 50°C to 80°C for 2 to 48 hours with shaking at 500 to 2000 rpm. In some embodiments, immediately after step b), the sample is centrifuged at 1000 RCF to 4000 RCF for 2 to 20 minutes. In some embodiments, after centrifugation at 1000 RCF to 4000 RCF for 2 to 20 minutes, the sample is refrigerated for at least 40 minutes. In some embodiments, step c) is performed after the sample has been refrigerated. In some embodiments, centrifuging the sample at high speed comprises centrifuging the sample at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf.

[0041] Further provided herein is a method for extracting RNA and DNA from a paraffin-embedded sample, the method comprising: a) incubating the sample with a protease; b) incubating the sample at 50°C to 80°C for 1 to 40 minutes; c) centrifuging the sample at high speed to separate the paraffin from a first lysate containing the RNA; d) aspirating the first lysate containing the RNA; e) isolating the RNA from the first lysate; f) incubating the sample from step c) with a protease; g) incubating the sample from step f) at 50°C to 80°C for 2 to 48 hours; h) centrifugation of the sample at high speed; i) aspirating a second lysate containing DNA; and j) isolating the DNA from the second lysate. In some embodiments, centrifuging the sample at high speed comprises centrifuging the sample at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the incubation with the protease in step a and / or step f is performed with shaking at 500-2000 rpm. In some embodiments, incubating the sample with the protease comprises incubation at 50°C-60°C. In some embodiments, incubating the sample with the protease comprises incubation for 1-20 minutes. In some embodiments, the sample is incubated at 50°C-80°C for 1-40 minutes without shaking.

[0042] Further provided herein are methods for extracting RNA from paraffin-embedded samples, the methods comprising: a) adding mineral oil to the sample; b) incubating the sample at 50°C to 80°C for 1 to 40 minutes; c) cooling the sample to room temperature; d) incubating the sample with a protease; f) incubating the sample at 50°C to 80°C for 1 to 40 minutes; g) centrifuging the sample at high speed to separate the paraffin and mineral oil from the RNA-containing lysate; and h) aspirating the RNA-containing lysate. In some embodiments, the incubation at 50°C to 80°C for 1 to 20 minutes is performed with shaking at 500 RPM to 2000 RPM. In some embodiments, centrifuging the sample at high speed comprises centrifuging the sample at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, after centrifugation at high speed, the sample is incubated at room temperature. In some embodiments, the method further comprises centrifuging the sample after incubation at room temperature.

[0043] Further provided herein are methods for extracting DNA from paraffin-embedded samples, the methods comprising: a) adding mineral oil to the sample; b) incubating the sample with a protease; c) incubating the sample at 50°C to 80°C for 2 to 48 hours; d) centrifuging the sample at high speed to separate the paraffin from a lysate containing the DNA; and e) aspirating the lysate containing the DNA. In some embodiments, the mineral oil is removed prior to step b). In some embodiments, centrifuging the sample at high speed comprises centrifuging the sample at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the sample is incubated at 50°C to 80°C for 2 to 48 hours with shaking at 500 RPM to 2000 RPM. In some embodiments, the sample is incubated at 50° C. to 80° C. for 2 to 48 hours, and then immediately prior to step d), the sample is centrifuged at 2000 to 5000 RCF.

[0044] Further provided herein is a method for extracting RNA and DNA from a paraffin-embedded sample, the method comprising: a) adding mineral oil to the sample; b) incubating the sample at 50°C to 80°C for 1 to 40 minutes to melt the paraffin; c) cooling the sample to room temperature; d) incubating the sample with a protease; e) incubating the sample at 50°C to 80°C for 1 to 40 minutes; f) centrifuging the sample at high speed to separate the paraffin and mineral oil from a lysate containing RNA; g) aspirating the lysate containing RNA; h) isolating the RNA from the lysate; i) centrifuging the lysate from step g) at high speed to separate the lysate from the mineral oil; j) incubating the sample from step i) with a protease; k) incubating the sample from step j) at 50°C to 80°C for 2 to 48 hours; l) centrifuging the sample at high speed; and m) aspirating a second lysate containing DNA. In some embodiments, centrifuging the sample at high speed comprises centrifuging the sample at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the sample is incubated at 50°C to 80°C for 2 to 48 hours with shaking at 500 RPM to 2000 RPM.

[0045] In some embodiments of the preceding method, the protease is proteinase K. In some embodiments of the preceding method, the sample has a size of about 30 μm. 3 In some embodiments of the preceding method, the sample is about 0.3 μm in size. 3 ~approximately 5.5 μm 3In some embodiments of the preceding methods, the method further comprises analyzing the RNA and / or DNA extracted from the paraffin-embedded sample by one or more methods selected from the group consisting of a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, sequencing, next-generation sequencing, screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), or mass spectrometry genotyping. In some embodiments, analyzing the RNA and / or DNA comprises next-generation sequencing. In some embodiments of the preceding methods, the method further comprises preparing a sequencing library for sequencing the RNA and / or DNA. In some embodiments of the preceding methods, the method further comprises sequencing the DNA and / or RNA using hybrid capture-based sequencing. In some embodiments of the preceding methods, the sample is from an individual known to have or suspected of having cancer. [Brief explanation of the drawings]

[0046] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application file with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0047] [Figure 1] An overview of exemplary RNA-first (top) and DNA-first (bottom) methods for extracting RNA and DNA from paraffin-embedded samples is provided. [Figure 2] A representative image of a turbid nucleic acid sample extracted from paraffin-embedded tissue after centrifugation at 10,000 rcf for 2 minutes is shown. The arrow points to the pellet containing wax (paraffin) contamination. [Figure 3A]A schematic diagram showing the three arms of the experiment used to test the RNA-first extraction workflow is shown: Arm 2 is the standard protocol (left column), Arm 3 is the oil method (middle column), and Arm 4 is the hot lysate spin method (right column). [Figure 3B] A schematic diagram showing the three arms of the experiment used to test the DNA-first extraction workflow is shown: Arm 2 is the standard protocol (left column), Arm 3 is the oil method (middle column), and Arm 4 is the hot lysate spin method (right column). [Figure 4] An exemplary method for performing precision enrichment of pathology specimens from formalin-fixed, paraffin-embedded (FFPE) blocks is outlined. [Figure 5A-5B] An overview of an exemplary comprehensive genomic profiling (CGP) method is provided. [Figure 6] 1 illustrates an exemplary device "Device 1100" according to some embodiments. [Figure 7] 1 illustrates an exemplary system, System 1200, according to some embodiments. [Figure 8] FIG. 1 illustrates a block diagram of an exemplary process for analyzing RNA and / or DNA extracted from paraffin-embedded samples according to some embodiments. [Figure 9A-9B] Illustrated are AutoLys tubes after the warm lysate process. [Figure 9A] The inner AutoLys tube is shown on the left, with the arrow indicating the paraffin layer remaining within the inner tube. The outer tube is shown on the right, with the arrow indicating the clear eluate with minimal residual paraffin in the lysate. [Figure 9B] The inner AutoLys tube is shown on top, with the arrow indicating the paraffin layer remaining within the inner tube. The outer tube is shown on the bottom, with the arrow indicating the clear eluate with minimal residual paraffin in the lysate. [Figures 10A-10B]The results of the mineral oil extraction process are illustrated. [Figure 10A] The white arrow identifies the oil layer, and the black arrow identifies the lysate layer, which is clear and has minimal residual paraffin. [Figure 10B] The two columns on the left were extracted using the mineral oil method, while the two columns on the right were extracted using the "standard" method. The images show clear eluates with minimal residual paraffin for samples extracted using the mineral oil method, and cloudy eluates with more residual paraffin for samples extracted using the standard method. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure is based, at least in part, on the development of a method for extracting nucleic acids from tissue that is accompanied by a histological quality assurance / quality control step to evaluate the enrichment process. The method described herein allows for the testing of specimens where previous enrichment methods would otherwise have yielded insufficient levels of tissue for analysis of tumor content.

[0049] In some embodiments, the method comprises combining DNA and RNA at 30 μM 2 This method has the advantage that it can be used to extract from small samples, such as 1000 μL or less. Furthermore, in some embodiments, the method can be used to extract DNA and RNA from the same sample. Moreover, in some embodiments, the method relates to the removal of paraffin from the sample, which allows for more efficient downstream processing, particularly in high-throughput methods. For example, in some embodiments, removal of paraffin using the method can reduce clogging of robots used in high-throughput sample processing methods.

[0050] definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0051] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0052] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0053] The terms "about" or "approximately," as used herein, refer to a normal error range for the respective value that would be readily apparent to one of ordinary skill in the art, e.g., an acceptable degree of error or deviation for the measured quantity given the nature or precision of the measurement. Reference herein to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0054] As used herein, the term "embedding medium" refers to any agent that may be used to embed a sample, such as a tissue sample. Exemplary embedding mediums include paraffin, the resin celloidin, Paraplast®, gelatin, ester wax, wax, polyethylene glycol, and nitrocellulose. In certain embodiments, the embedding medium is paraffin.

[0055] As used herein, the term "embedded sample" refers to any sample embedded in an embedding medium. In some embodiments, the embedded sample is an embedded tissue sample, such as an embedded tissue sample extracted from an individual known to have or suspected of having cancer. In certain embodiments, the embedded sample is a paraffin-embedded sample.

[0056] As used herein, the term "analyte" refers to any molecule that may be extracted from an embedded sample. Exemplary analytes include RNA, DNA, polypeptides, small molecules, lipids, polysaccharides, exosomes, mitochondria, and nuclei.

[0057] As used herein, "configured to hybridize to" refers to hybridizing a nucleic acid molecule to a T m indicates that the nucleic acid molecule has a nucleotide sequence of sufficient length and sequence complementarity to the nucleotide sequence of the target nucleic acid so as to hybridize to the target nucleic acid in an aqueous solution of 1x SCC (150 mM sodium chloride and 15 mM trisodium citrate) and 0.1% SDS. For example, other hybridization conditions may be used when hybridizing a nucleic acid molecule to a target nucleic acid molecule in the context of the described methods.

[0058] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. In some embodiments, the individual is a human patient, e.g., a human patient with a cancer described herein.

[0059] An "effective amount" or "therapeutically effective amount" of an agent, e.g., an anti-cancer agent, or pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time required, to achieve a desired therapeutic or prophylactic result, e.g., delaying or minimizing one or more symptoms associated with cancer, e.g., in the treatment or management of cancer. In some embodiments, an effective amount or therapeutically effective amount of an agent refers to the amount of agent, at dosages and for periods of time required, alone or in combination with other therapeutic agents that provide a therapeutic or prophylactic benefit in the treatment or management of a disease, such as cancer. In some embodiments, an effective amount or therapeutically effective amount of an agent is one that enhances the therapeutic or prophylactic efficacy of another therapeutic agent or another treatment modality.

[0060] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of the individual being treated and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, delaying the progression of disease, alleviating symptoms, attenuating any direct or indirect pathological consequence of the disease, reducing the rate of disease progression, ameliorating or palliation of the disease state, and ameliorating or improved prognosis. In some embodiments, the terms "treatment," "treat," or "treating" include preventing a disease, such as cancer, for example, before an individual becomes susceptible to cancer or to regrowth or recurrence of the cancer. In some embodiments, the terms "treatment," "treat," or "treating" include inhibiting or reducing the severity of a disease, such as cancer.

[0061] "Likely to" or "high probability" refers to a high probability that an event, item, object, thing, or person will occur. Thus, in one example, an individual who is likely to respond to an anti-cancer therapy, e.g., treatment with the anti-cancer therapies provided herein, alone or in combination, has a higher probability of responding to treatment with the anti-cancer therapies, alone or in combination, compared to a reference individual or group of individuals. "Likely" refers to a lower probability that an event, item, object, thing, or person will occur, compared to a reference individual or group of individuals. Thus, an individual who is unlikely to respond to an anti-cancer therapy, e.g., treatment with the anti-cancer therapies provided herein, alone or in combination, has a lower probability of responding to treatment with the anti-cancer therapies, alone or in combination, compared to a reference individual or group of individuals.

[0062] I. Removal of embedding medium from embedded samples In some aspects, provided herein are methods for detecting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding agent; removing the embedding agent from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding agent and the embedding agent is separated from the sample, thereby removing the embedding agent; extracting the analyte from the de-embedded sample; and analyzing the analyte to detect the analyte. Also provided herein are methods for extracting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding agent; removing the embedding agent from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding agent and the embedding agent is separated from the sample, thereby removing the embedding agent; and extracting the analyte from the de-embedded sample. Also provided herein is a method for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; extracting the analyte from the de-embedded sample; and purifying the extracted analyte to provide the analyte sample. Also provided herein is a method for improving the separation of embedding medium from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium.Also provided herein is a method for detecting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedded sample is contacted with an immiscible solvent to separate the embedding medium from the sample, extracting the analyte from the de-embedded sample; and analyzing the analyte to detect the analyte. Also provided herein is a method for extracting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedded sample is contacted with an immiscible solvent to separate the embedding medium from the sample, and extracting the analyte from the de-embedded sample. Also provided herein is a method for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedded sample is contacted with an immiscible solvent to separate the embedding medium from the sample, extracting the analyte from the de-embedded sample, and purifying the extracted analyte to provide the analyte sample. Also provided herein is a method for improving the separation of embedding medium from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedded sample is contacted with an immiscible solvent to separate the embedding medium from the sample, and extracting the analyte from the de-embedded sample.Also provided herein is a method for detecting alterations in RNA and / or DNA, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin and causing the paraffin to separate from the sample; extracting RNA and / or DNA from the deparaffinized sample; and analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA. Also provided herein is a method for extracting RNA and / or DNA, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin and causing the paraffin to separate from the sample; and extracting RNA and / or DNA from the deparaffinized sample. Also provided herein is a method for improving library construction for nucleic acid sequencing, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and preparing a sequencing library for sequencing the RNA and / or DNA. Also provided herein is a method for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample.Also provided herein is a method for improving separation of paraffin from a paraffin-embedded sample, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample. Also provided herein is a method for detecting alterations in RNA and / or DNA, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA.

[0009] Also provided herein is a method for extracting RNA and / or DNA, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample. Also provided herein is a method for improving library construction for nucleic acid sequencing, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and preparing a sequencing library for sequencing the RNA and / or DNA.Also provided herein is a method for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, the method comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, where the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample. Also provided herein is a method for improving the separation of paraffin from a paraffin-embedded sample, the method comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, where the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample.

[0063] The present disclosure is based in part on the development of a method for separating an embedded sample from an embedding agent. For example, the methods described herein may be used to separate paraffin from a paraffin-embedded sample, such as formalin-fixed, paraffin-embedded (FFPE) tissue. Without being bound by any particular theory, after the embedding agent is removed from the embedded sample, poor separation of the embedding agent presents various problems that arise during subsequent analysis of analytes extracted from the sample. For example, poor separation of the embedding agent from the sample extracted from the de-embedded sample may be an impurity in the analyte sample extracted from the de-embedded sample. In the case of paraffin, this can result in a cloudy preparation of the analyte sample and clogging of liquid handling robots used to process the analyte sample. In the case of RNA and / or DNA samples extracted from de-paraffinized samples, poor removal of paraffin can result in failure of library construction for next-generation sequencing analysis.

[0064] Importantly, the methods described herein do not rely on the use of toxic solvents such as xylene and are therefore suitable for automatable high-throughput analysis, e.g., high-throughput preparation of libraries for sequencing nucleic acids.

[0065] Induction of a phase transition in the embedding medium In some embodiments, provided herein are methods that involve inducing a phase transition in an embedding medium to remove the embedding medium from the embedded sample, thereby generating a de-embedded sample. The embedding medium may be any one of the embedding mediums described herein. The methods described herein may be used to separate the embedding medium from the embedded sample. In some embodiments, the methods described herein further include extracting an analyte from the de-embedded sample. The analyte may be any one of the analytes described herein.

[0066] In some embodiments, provided herein are methods for detecting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding agent; removing the embedding agent from the embedded sample to generate a de-embedded sample, wherein a phase transition is induced in the embedding agent to separate the embedding agent from the sample, thereby removing the embedding agent; extracting the analyte from the de-embedded sample; and analyzing the analyte to detect the analyte. In some embodiments, detection of the analyte is improved compared to methods in which the embedding agent is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, UltraClear™). In some embodiments, detection of the analyte is improved compared to methods in which the embedding agent is removed by separating the embedding agent from the embedded sample. In some embodiments, detection of the analyte is improved compared to methods in which the embedded sample is not de-embedded.

[0067] In some embodiments, provided herein are methods for extracting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to generate a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; and extracting the analyte from the de-embedded sample. In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, UltraClear™). In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0068] In some embodiments, provided herein are methods for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to generate a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; extracting the analyte from the de-embedded sample; and purifying the extracted analyte to provide the analyte sample. In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is removed by dissociating the embedding medium from the embedded sample. The level of embedding medium in the analyte sample may be measured, for example, by measuring the turbidity of the analyte sample. In some embodiments, the turbidity of the analyte sample is reduced compared to methods in which the embedding medium is dissolved in a miscible solvent and / or compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0069] In some embodiments, provided herein are methods for improving separation of an embedding medium from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein a phase transition is induced in the embedding medium to separate the embedding medium from the sample, thereby removing the embedding medium; and extracting the analyte from the de-embedded sample. In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample. In some embodiments, the method is performed on a liquid handling robot that includes a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). As described above, the level of embedding medium in the analyte sample may be measured, for example, by measuring the turbidity of the analyte sample. In some embodiments, the turbidity of the analyte sample is reduced compared to methods in which the embedding medium is dissolved in a miscible solvent and / or compared to methods in which the embedding medium is removed by detaching the embedding medium from the embedded sample. In some embodiments, the method is performed on a liquid handling robot that includes a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods in which the embedding medium is removed by detaching the embedding medium from the embedded sample. In some embodiments, the filter is a filter in a spin column. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR and the spin column is an AutoLys tube.

[0070] In some embodiments of any of the methods described herein, step b) does not include dissolving the embedding medium in a miscible solvent, hi some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

[0071] In some embodiments, the phase transition is selected from the group consisting of melting, freezing, vaporization, condensation, sublimation, and deposition. In some embodiments, step b) comprises heating, cooling, increasing pressure, or decreasing pressure of the embedded sample.

[0072] In some embodiments, the phase transition is melting. In some embodiments, step b) comprises melting the embedding medium to remove it from the embedded sample, thereby generating a de-embedded sample. In some embodiments, step b) comprises melting the embedding medium, causing it to separate from the sample. In some embodiments, step b) comprises heating the embedded sample. In some embodiments, the embedded sample is heated to about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes.

[0073] In some embodiments, step b) comprises centrifuging and filtering the embedded sample to separate the embedding from the sample. In some embodiments, the embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the embedded sample is centrifuged at 1,811 rcf. In some embodiments, the centrifuging and filtering are performed in a liquid handling robot, e.g., a Hamilton AutoLys STAR.

[0074] In some embodiments, step b) comprises heating, centrifuging, and filtering the embedded sample to separate the embedding medium from the sample, thereby producing a de-embedded sample. In some embodiments, the heating, centrifuging, and filtering are performed as described above. In some embodiments, the heating, centrifuging, and filtering are performed in a liquid handling robot, e.g., a Hamilton AutoLys STAR.

[0075] Methods involving paraffin-embedded samples In some embodiments, the embedding agent is paraffin. Thus, in some embodiments, methods are provided herein that involve removing paraffin from paraffin-embedded samples. Exemplary methods that involve removing paraffin from paraffin-embedded samples using a method involving phase transition are described herein in the Examples.

[0076] In some embodiments, provided herein are methods for detecting alterations in RNA and / or DNA, the methods comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which the paraffin is not deparaffinized. In some embodiments, the alteration in the RNA and / or DNA is selected from the group consisting of copy number alterations, point mutations, in-frame deletions of one or more codons, intragenic deletions, intragenic insertions, deletions of the entire gene, inversions, interchromosomal translocations, tandem duplications, gene fusions, genomic rearrangements involving intronic sequences, and / or gene amplifications or duplications. In certain embodiments, the alteration in the RNA and / or DNA is a copy number alteration. Methods for detecting alterations in RNA and / or DNA are described in detail below.

[0077] In some embodiments, provided herein are methods for extracting RNA and / or DNA, the methods comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, extraction of RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, extraction of RNA and / or DNA is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. Methods for extracting RNA and / or DNA are described in more detail below.

[0078] Also provided herein in some embodiments is a method for improving library construction for nucleic acid sequencing, comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and preparing a sequencing library for sequencing the RNA and / or DNA. In some embodiments, the preparation of the sequencing library is improved compared to methods in which paraffin is dissolved in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the preparation of the sequencing library is improved compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0079] In some embodiments, provided herein are methods for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, the method comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to methods in which paraffin is dissolved in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample. The level of paraffin in an RNA and / or DNA sample may be measured, for example, by measuring the turbidity of the RNA and / or DNA sample. In some embodiments, the turbidity of the RNA and / or DNA sample is reduced compared to a method in which paraffin is dissolved in a miscible solvent and / or a method in which paraffin is removed by cleaving the paraffin from the embedded sample. In some embodiments, the turbidity of an RNA and / or DNA sample prepared by a method in which paraffin is dissolved in a miscible solvent and / or a method in which paraffin is removed by cleaving the paraffin from a paraffin-embedded sample is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the turbidity of an RNA and / or DNA sample prepared using a method in which paraffin is removed by inducing a phase transition in the paraffin and causing it to separate from the sample.

[0080] In some embodiments, provided herein are methods for improving separation of paraffin from a paraffin-embedded sample, the methods comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is dissolved in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. As described above, the level of paraffin in an RNA and / or DNA sample may be measured, for example, by measuring the turbidity of the RNA and / or DNA sample. In some embodiments, measurements of the turbidity of RNA and / or DNA samples indicate improved paraffin separation compared to methods of dissolving paraffin in a miscible solvent and / or methods of removing paraffin by dissociating the paraffin from a paraffin-embedded sample. In some embodiments, the turbidity of RNA and / or DNA samples prepared by methods of dissolving paraffin in a miscible solvent and / or methods of removing paraffin by dissociating the paraffin from a paraffin-embedded sample is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the turbidity of RNA and / or DNA samples prepared using methods of removing paraffin by inducing a phase transition in the paraffin, causing it to dissociate from the sample. In some embodiments, the method is performed in a liquid handling robot equipped with a filter, and the improved paraffin separation reduces filter clogging compared to methods of dissolving paraffin in a miscible solvent.In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the method is performed in a liquid handling robot equipped with a filter, and the improved paraffin separation reduces filter clogging compared to methods that remove paraffin by cleaving it from the paraffin-embedded sample. In some embodiments, the filter is a filter in a spin column. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR and the spin column is an AutoLys tube.

[0081] In some embodiments of any one of the preceding methods, wherein the embedded sample is a paraffin-embedded sample, step b) does not include dissolving the paraffin in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments of any one of the preceding methods, wherein the embedded sample is a paraffin-embedded sample, step b) does not include dissolving the paraffin in a toxic solvent.

[0082] In some embodiments, step b) comprises heating the paraffin-embedded sample. In some embodiments, the paraffin-embedded sample is heated to about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the paraffin-embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes.

[0083] In some embodiments, step b) includes centrifuging and filtering the paraffin-embedded sample. Generally, the sample is centrifuged and filtered to separate the paraffin from the embedded sample based on the relative densities of the paraffin and the sample and the presence of particles in the sample. In some embodiments, the paraffin-embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the paraffin-embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the paraffin-embedded sample is centrifuged at 1,811 rcf.

[0084] In some embodiments, step b) comprises heating and centrifuging the paraffin-embedded sample. In some embodiments, the paraffin-embedded sample is heated to about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. The method of claim 31 or 32, wherein the paraffin-embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. The method of any one of claims 31 to 33, wherein the paraffin-embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. 35. The method of any one of claims 31 to 34, wherein the paraffin-embedded sample is centrifuged at 1,811 rcf or more. 36. The method of any one of claims 31 to 35, wherein the paraffin-embedded sample is centrifuged at 1,811 rcf.

[0085] In some embodiments, step b) comprises heating, centrifuging, and filtering the paraffin-embedded sample. The heating, centrifugation, and filtering may be performed as described above.

[0086] Contact of embedded samples with immiscible solvents In some embodiments, provided herein are methods that involve contacting an embedded sample with an immiscible solvent to remove an embedding medium from the embedded sample, thereby generating a de-embedded sample. The embedding medium may be any one of the embedding mediums described herein. The methods described herein may be used to separate the embedding medium from the embedded sample. In some embodiments, the methods described herein further include extracting an analyte from the de-embedded sample. The analyte may be any one of the analytes described herein.

[0087] In some embodiments, provided herein are methods for extracting an analyte, the method comprising: providing an embedded sample containing the analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample; contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample, thereby removing the embedding medium; and extracting the analyte from the de-embedded sample. In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, UltraClear™). In some embodiments, extraction of the analyte is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0088] In some embodiments, provided herein are methods for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedding medium is removed by contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample; extracting the analyte from the de-embedded sample; and purifying the extracted analyte to provide the analyte sample. In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the level of embedding medium in the analyte sample is reduced compared to a method in which the embedding medium is removed by dissociating the embedding medium from the embedded sample. The level of embedding medium in the analyte sample may be measured, for example, by measuring the turbidity of the analyte sample. In some embodiments, the turbidity of the analyte sample is reduced compared to methods in which the embedding medium is dissolved in a miscible solvent and / or compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample.

[0089] In some embodiments, provided herein are methods for improving separation of an embedding medium from an embedded sample, the method comprising: providing an embedded sample containing an analyte, wherein the sample is embedded in an embedding medium; removing the embedding medium from the embedded sample to produce a de-embedded sample, wherein the embedding medium is removed by contacting the embedded sample with an immiscible solvent to separate the embedding medium from the sample; and extracting the analyte from the de-embedded sample. In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is removed by separating the embedding medium from the embedded sample. In some embodiments, the method is performed on a liquid handling robot that includes a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods that dissolve the embedding medium in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the method is performed on a liquid handling robot that includes a filter, and the improved separation of the embedding medium results in less filter clogging compared to methods that remove the embedding medium by separating it from the embedded sample. In some embodiments, the filter is a filter in a spin column. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR and the spin column is an AutoLys tube.

[0090] In some embodiments, step b) does not include dissolving the embedding medium in a miscible solvent, hi some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

[0091] Without being bound by theory, it is believed that any immiscible solvent that is less dense than water and more dense than the embedding medium when the embedding medium is in crystalline form can be used in the methods described herein. For example, the density of paraffin is approximately 0.8 g / cm. 3 and the density of mineral oil is approximately 0.87 g / cm 3 and the density of water is 1g / cm 3 Mineral oil is an example of an immiscible solvent suitable for separating an aqueous sample from paraffin. Without being bound by theory, it is believed that the immiscible solvent promotes phase separation between the embedding medium (e.g., paraffin) and the sample (e.g., lysate) by creating an intermediate layer. Thus, in some embodiments, the density of the immiscible solvent is lighter than water, but heavier than the embedding medium when the embedding medium is in liquid form. In some embodiments, the density of the immiscible solvent is heavier than liquid paraffin. In some embodiments, the immiscible solvent is a vegetable oil.

[0092] In some embodiments, the immiscible solvent is mineral oil. In some embodiments, the embedded sample is contacted with about 300, 350, 400, 450, 500, or 550 μL of mineral oil. In some embodiments, the embedded sample is contacted with mineral oil at a ratio of mineral oil to embedded sample of about 1:4, 1:2, 1:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, or 50:1. In some embodiments, the mineral oil is contacted with the embedded sample at about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., or about 75° C. The method of any one of claims 218-221, wherein the mineral oil is contacted with the embedded sample for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the mineral oil is contacted with the embedded sample while shaking at about 25 rpm, about 50 rpm, about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, about 1000 rpm, or about 1100 rpm. In certain embodiments, the immiscible solvent is mineral oil and the embedding agent is paraffin.

[0093] In some embodiments, step b) comprises contacting the sample with an immiscible solvent and centrifuging and filtering the embedded sample. In some embodiments, the embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the embedded sample is centrifuged at 1,811 rcf.

[0094] Methods involving paraffin-embedded samples In some embodiments, the embedding agent is paraffin. Thus, in some embodiments, methods are provided herein that involve removing paraffin from a paraffin-embedded sample. Exemplary methods that involve removing paraffin from a paraffin-embedded sample using a method that involves contacting the paraffin-embedded sample with an immiscible solvent are described herein in the Examples.

[0095] In some embodiments, provided herein are methods for detecting alterations in RNA and / or DNA, the method comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. In some embodiments, detection of alterations in RNA and / or DNA is improved compared to methods in which the paraffin-embedded sample is not deparaffinized. In some embodiments, the alteration in the RNA and / or DNA is selected from the group consisting of copy number alterations, point mutations, in-frame deletions of one or more codons, intragenic deletions, intragenic insertions, whole gene deletions, inversions, interchromosomal translocations, tandem duplications, gene fusions, genomic rearrangements involving intronic sequences, and / or gene amplifications or duplications. In some embodiments, the alteration in the RNA and / or DNA is a copy number alteration. Methods for detecting alterations in RNA and / or DNA are described in more detail below.

[0096] In some embodiments, provided herein are methods for extracting RNA and / or DNA, the methods comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, extraction of RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, extraction of RNA and / or DNA is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. Methods for extracting RNA and / or DNA are described in more detail below.

[0097] In some embodiments, provided herein are methods for improving library construction for nucleic acid sequencing, the methods comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and preparing a sequencing library for sequencing the RNA and / or DNA. In some embodiments, the preparation of the sequencing library is improved compared to methods in which paraffin is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the preparation of the sequencing library is improved compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample.

[0098] In some embodiments, provided herein are methods for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, the method comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to dissociate the paraffin from the sample; extracting RNA and / or DNA from the deparaffinized sample; and purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample. In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to methods in which paraffin is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, the level of paraffin in the RNA and / or DNA sample is reduced compared to methods in which paraffin is removed by dissociating the paraffin from the paraffin-embedded sample. The level of paraffin in an RNA and / or DNA sample may be measured, for example, by measuring the turbidity of the RNA and / or DNA sample. In some embodiments, the turbidity of the RNA and / or DNA sample is reduced compared to a method in which paraffin is dissolved in a miscible solvent and / or a method in which paraffin is removed by cleaving the paraffin from the embedded sample. In some embodiments, the turbidity of an RNA and / or DNA sample prepared by a method in which paraffin is dissolved in a miscible solvent and / or a method in which paraffin is removed by cleaving the paraffin from a paraffin-embedded sample is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the turbidity of an RNA and / or DNA sample prepared using a method in which paraffin is removed by inducing a phase transition in the paraffin and causing it to separate from the sample.

[0099] In some embodiments, provided herein are methods of improving separation of paraffin from a paraffin-embedded sample, the methods comprising: providing a paraffin-embedded sample; removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by contacting the paraffin-embedded sample with an immiscible solvent to separate the paraffin from the sample; and extracting RNA and / or DNA from the deparaffinized sample. In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). In some embodiments, separation of paraffin from a paraffin-embedded sample is improved compared to methods in which paraffin is removed by cleaving the paraffin from the paraffin-embedded sample. In some embodiments, the method is performed on a liquid handling robot equipped with a filter, and the improved paraffin separation reduces filter clogging compared to methods in which paraffin is dissolved in a miscible solvent (e.g., xylene, ethyl acetate, CitriSolv™, or UltraClear™). As described above, the level of paraffin in an RNA and / or DNA sample may be measured, for example, by measuring the turbidity of the RNA and / or DNA sample. In some embodiments, measuring the turbidity of the RNA and / or DNA sample indicates improved paraffin separation compared to methods in which paraffin is dissolved in a miscible solvent and / or compared to methods in which paraffin is removed by cleaving it from a paraffin-embedded sample. In some embodiments, the turbidity of an RNA and / or DNA sample prepared by a method of removing paraffin by dissolving the paraffin in a miscible solvent and / or by dissociating the paraffin from a paraffin-embedded sample is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the turbidity of an RNA and / or DNA sample prepared using a method of removing paraffin by inducing a phase transition in the paraffin and causing it to separate from the sample.In some embodiments, the method is performed on a liquid handling robot that includes a filter, and the improved paraffin separation results in less filter clogging compared to methods that dissolve paraffin in a miscible solvent. In some embodiments, the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™. In some embodiments, the method is performed on a liquid handling robot that includes a filter, and the improved paraffin separation results in less filter clogging compared to methods that remove paraffin by cleaving it from the paraffin-embedded sample. In some embodiments, the filter is a filter in a spin column. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR, and the spin column is an AutoLys tube.

[0100] In some embodiments, step b) does not include dissolving the paraffin in a miscible solvent, and optionally the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

[0101] As mentioned above, the density of paraffin is approximately 0.8 g / cm 3 and the density of mineral oil is approximately 0.87 g / cm 3 and the density of water is 1g / cm 3Thus, mineral oil is believed to be an example of a suitable immiscible solvent for separating an aqueous sample from paraffin. Without being bound by theory, it is believed that the immiscible solvent promotes phase separation between the embedding agent (e.g., paraffin) and the sample (e.g., lysate) by creating an intermediate layer. In some embodiments, the immiscible solvent is vegetable oil. In some embodiments, the immiscible solvent is mineral oil. In some embodiments, the paraffin-embedded sample is contacted with about 300, 350, 400, 450, 500, or 550 μL of mineral oil. In some embodiments, the paraffin-embedded sample is contacted with mineral oil at a ratio of about 1:4, 1:2, 1:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, or 50:1 of mineral oil to paraffin-embedded sample. In some embodiments, the mineral oil contacts the paraffin-embedded sample at about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., or about 75° C. In some embodiments, the mineral oil contacts the paraffin-embedded sample for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the mineral oil contacts the paraffin-embedded sample while shaking at about 25 rpm, about 50 rpm, about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, about 1000 rpm, or about 1100 rpm.

[0102] In some embodiments, step b) comprises contacting the sample with an immiscible solvent and centrifuging and filtering the paraffin-embedded sample. In some embodiments, the paraffin-embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf. In some embodiments, the paraffin-embedded sample is centrifuged at 1,811 rcf or greater. In some embodiments, the paraffin-embedded sample is centrifuged at 1,811 rcf.

[0103] In some embodiments, separation of paraffin from the sample is automated. In some embodiments, step b) is automated. In some embodiments, the method is automated. In some embodiments, two or more paraffin-embedded samples are processed in parallel. In some embodiments, 12, 24, 48, or 96 paraffin-embedded samples are processed in parallel.

[0104] In some embodiments, the method is performed using a liquid handling robot. In some embodiments, the liquid handling robot is an Agilent, BioTek, Hamilton, Tecan, or ThermoFisher Scientific liquid handling robot, optionally the liquid handling robot is a Hamilton AutoLys STAR, Hamilton STAR, BioTek Dispenser, or KingFisher Flex. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR.

[0105] In some embodiments, step c) comprises extracting RNA and DNA from the deparaffinized sample. Methods for extracting RNA and / or DNA are described in more detail below.

[0106] II.Embedded sample The embedding medium may be removed from an embedded sample using the methods described herein. The embedding medium may be any one of the embedding mediums described herein. In some embodiments, the embedding medium is selected from the group consisting of paraffin, resin, celloidin, Paraplast®, gelatin, ester wax, wax, polyethylene glycol, and nitrocellulose. In some embodiments, the embedding medium is Paraplast®. In some embodiments, the embedding medium is paraffin.

[0107] In some embodiments, the sample is a biological sample, such as a sample containing cells and / or tissue. In some embodiments, the sample is obtained from an individual. In some embodiments, the sample is a mammalian sample. In some embodiments, the sample is a human sample.

[0108] Paraffin-embedded samples In some embodiments, provided herein are methods for removing paraffin from a paraffin-embedded sample to produce a deparaffinized sample.

[0109] A variety of paraffin-embedded samples are known in the art and suitable for use in the methods described herein. In some embodiments, the paraffin-embedded sample is derived from an individual known to have or suspected of having cancer. In some embodiments, the individual is suspected of having any one of the cancers described herein. In some embodiments, the paraffin-embedded sample is derived from a biopsy, optionally a tumor biopsy.

[0110] In some embodiments, the paraffin-embedded sample is a fixed paraffin-embedded sample. In some embodiments, the fixed paraffin-embedded sample is selected from the group consisting of a formalin-fixed sample, an ethanol-fixed sample, and a methanol-fixed sample. In some embodiments, the paraffin-embedded sample is obtained from formalin-fixed, paraffin-embedded (FFPE) tissue.

[0111] In some embodiments, the paraffin-embedded sample is obtained from cryopreserved tissue. In some embodiments, the paraffin-embedded sample is obtained from fresh-frozen tissue. In some embodiments, the fresh-frozen tissue is frozen in optimal cutting temperature (OCT) compound.

[0112] In some embodiments, the paraffin-embedded sample is obtained from a primary sample obtained directly from the source of interest by any suitable means. For example, in some embodiments, the paraffin-embedded sample is obtained from tissue obtained by a method selected from biopsy (e.g., fine needle aspiration or tissue biopsy) and surgery. In one embodiment, the paraffin-embedded sample is obtained from tissue containing one or more cells associated with a tumor, such as tumor cells or tumor-infiltrating lymphocytes (TILs). In one embodiment, the paraffin-embedded sample is obtained from tissue containing one or more pre-malignant or malignant cells. In one embodiment, the paraffin-embedded sample is obtained from tissue obtained from a hematological malignancy (or pre-malignancy), such as a hematological malignancy (or pre-malignancy) described herein. In one embodiment, the paraffin-embedded sample is obtained from tissue obtained from a cancer, such as a cancer described herein. In some embodiments, the paraffin-embedded sample is obtained from a solid tumor, a soft tissue tumor, or a metastatic lesion. In other embodiments, the paraffin-embedded sample is obtained from tissue or tissue containing cells from a surgical margin. In some embodiments, the paraffin-embedded sample comprises tumor cells of interest. In some embodiments, the paraffin-embedded sample further comprises non-tumor cells. Provided herein are methods that include extracting a paraffin-embedded sample from tissue from an individual suspected of having cancer. In some embodiments, the tissue comprises tumor cells of interest.

[0113] In some embodiments, the individual is suspected of having any one of the cancers described herein. In some embodiments, the tumor cell of interest is a tumor cell associated with any one of the cancers described herein. In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia ("ALL"), acute myeloid leukemia ("AML"), adenocarcinoma, adenocarcinoma of the lung, adrenocortical cancer, adrenocortical carcinoma, anal cancer, appendix cancer, B-cell derived leukemia, B-cell derived lymphoma, B-cell lymphoma, bladder cancer, brain cancer, breast cancer (e.g., triple-negative breast cancer (TNBC) or non-triple-negative breast cancer), cancer of the fallopian tubes, cancer of the testes, carcinoma, cerebral cancer), cervical cancer, bile duct cancer, choriocarcinoma, chronic myeloid leukemia, central nervous system (CNS) tumors, CNS cancer, colon cancer, colorectal cancer (e.g., colon adenocarcinoma), diffuse intrinsic pontine glioma (DIPG), diffuse large B-cell lymphoma ("DLBCL"), embryonal rhabdomyosarcoma (ERMS), endometrial cancer, epithelial cancer, epithelial neoplasms, thymoma, esophageal cancer, Ewing's sarcoma, eye cancer (e.g., uveal melanoma), eyelid cancer, follicular lymphoma ("FL"), gallbladder cancer, gastric cancer, gastrointestinal cancer, glioblastoma, polycythemia vera, glioblastoma multiforme, glioma (e.g., low-grade glioma), gullet cancer, head and neck cancer, blood cancer, hepatocellular cancer, hepatocellular carcinoma carcinoma), Hodgkin's lymphoma (HL), heavy chain disease, rectal cancer, renal cancer, kidney cancer (e.g., renal clear cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, papillary renal carcinoma), large B-cell lymphoma, large intestine cancercancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer), lymphoma, breast cancer, melanoma (e.g., metastatic malignant melanoma), Hodgkin's disease, Waldenstrom's macroglobulinemia, Merkel cell carcinoma, mesothelioma, monocytic leukemia, multiple myeloma, myeloma, myogenic sarcoma, nasopharyngeal carcinoma, CNS tumors of neuroblastic origin (e.g., neuroblastoma (NB)), neuroma, astrocytoma, pilocytic astrocytoma, anaplastic astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, vestibular schwannoma, adenoma, metastatic brain tumor, spinal cord tumor, non-Hodgkin's lymphoma (NHL), mouth cancer, oral cavity cancer, osteosarcoma, ovarian cancer cancer), ovarian carcinoma, pancreatic adenocarcinoma, pancreatic cancer, peritoneal cancer, pheochromocytoma, primary mediastinal B-cell lymphoma, primary peritoneal cancer, prostate cancer (e.g., hormone-refractory prostate cancer), rectal cancer (rectum carcinoma), recurrent or refractory classical Hodgkin's lymphoma (cHL), salivary gland cancer (e.g., salivary gland tumors), skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma (e.g., anogenital squamous cell carcinoma, squamous cell carcinoma of the anus, squamous cell carcinoma of the cervix, squamous cell carcinoma of the esophagus, squamous cell carcinoma of the head and neck (SCHNC), squamous cell carcinoma of the lung, squamous cell carcinoma of the penis, squamous cell carcinoma of the vagina, or squamous cell carcinoma of the vulva), stomach cancer cancer, T-cell derived leukemia, T-cell lymphoma, testicular cancer, testicular tumor, thymic cancer, thyroid cancer (thyroid carcinoma), tongue cancer, conjunctival cancer, urinary bladder cancer, urothelial cell carcinoma, uterine cancer (e.g., endometrial cancer or uterine sarcoma such as uterine carcinosarcoma), endometrial cancer, uterus cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0114] In some embodiments, the cancer is a hematological cancer (e.g., a hematological malignancy), such as diffuse large B-cell lymphoma ("DLBCL"), Hodgkin's lymphoma ("HL"), non-Hodgkin's lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML"), acute lymphoblastic leukemia ("ALL"), multiple myeloma ("MM"), acute lymphoblastic leukemia ("ALL"). The hematological cancer may be B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute anaplastic leukemia, chronic myeloid leukemia ("CML"), chronic lymphocytic leukemia ("CLL"), or hairy cell leukemia. In some embodiments, the hematological cancer of the present disclosure is acute or chronic leukemia, e.g., lymphoblastic, myeloid, lymphocytic, or myelocytic leukemia. In some embodiments, the hematological cancer of the present disclosure is lymphoma (e.g., Hodgkin lymphoma, such as relapsed or refractory Hodgkin lymphoma (cHL), non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or precursor T-lymphoblastic lymphoma), lymphoepithelial carcinoma, or malignant histiocytosis.

[0115] In some embodiments, the cancer is a solid tumor (e.g., a solid malignant tumor), such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, or the like. cancer of the mouth, nose, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriomas, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, skin cancer, melanoma, neuroblastoma (NB), or retinoblastoma.

[0116] In certain embodiments, the cancer is cancer of the adrenal gland (such as neuroblastoma), bladder cancer (such as urothelial (transitional cell) carcinoma), brain tumor (such as anaplastic astrocytoma or glioblastoma), bone cancer (such as osteosarcoma), bone marrow cancer (such as B-cell acute leukemia (B-ALL) or multiple myeloma), breast cancer (such as invasive ductal carcinoma), head and neck cancer (such as adenocarcinoma, mucoepidermoid carcinoma, squamous cell carcinoma), lymph node cancer, lung cancer (e.g., mucoepidermoid carcinoma, sarcoma, small cell undifferentiated carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma, large cell neuroendocrine carcinoma, non-small cell lung carcinoma, non-small cell carcinoma not otherwise specified, or squamous cell carcinoma). epithelial cell carcinoma, etc.), cancers of the female reproductive tract (e.g., cancer of the fallopian tube, such as fallopian tube serous carcinoma; ovarian cancer, such as epithelial carcinoma, epithelial carcinoma not otherwise specified, high-grade serous carcinoma, low-grade serous carcinoma, and serous carcinoma; and uterine cancer, e.g., carcinosarcoma, endometrial adenocarcinoma, endometrial adenocarcinoma not otherwise specified, papillary serous endometrial adenocarcinoma, leiomyosarcoma, sarcoma, sarcoma not otherwise specified, or smooth muscle tumor of unknown malignant potential (STUMP)), gallbladder cancer (e.g., adenocarcinoma), cancer of the gastroesophageal junction (e.g., adenocarcinoma), lymph node cancer (anaplastic large cell lymphoma, B-cell lymphoma, B-cell lymphoma not otherwise specified, diffuse large cell lymphoma, cystic B-cell lymphoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma not otherwise specified, colon cancer (adenocarcinoma, etc.), colorectal cancer, skin cancer (melanoma or squamous cell carcinoma, etc.), small intestine cancer (adenocarcinoma), soft tissue cancer (Ewing's sarcoma, fibrosarcoma, histiocytosis, histiocytosis not otherwise specified, juvenile xanthogranuloma or non-Langerhans cell histiocytosis, inflammatory myofibroblastic tumor, leiomyosarcoma, neurofibroma, neuroblastoma, sarcoma not otherwise specified, sarcoma, undifferentiated sarcoma, or undifferentiated soft tissue carcinoma, etc.), pancreatic cancer (carcinoma, carcinoma not otherwise specified, ductal adenocarcinoma, or mucinous adenocarcinoma, etc.) cystadenocarcinoma, etc.), prostate (e.g., acinar adenocarcinoma), pericardial cancer (e.g., mesothelioma), peritoneal cancer (e.g., mesothelioma), salivary gland cancer (e.g., carcinoma or carcinoma not otherwise specified), stomach cancer (e.g., adenocarcinoma, adenocarcinoma not otherwise specified, or diffuse carcinoma), kidney cancer (e.g., renal cell carcinoma or renal cell carcinoma not otherwise specified), thyroid cancer (e.g., carcinoma, carcinoma not otherwise specified, or papillary carcinoma), or carcinoma of unknown primary origin (e.g., adenocarcinoma, carcinoma, carcinoma not otherwise specified, leiomyosarcoma, malignant neoplasm, malignant neoplasm not otherwise specified, melanoma, myoepithelial carcinoma, squamous cell carcinoma (SCC), or undifferentiated neuroendocrine carcinoma).

[0117] In some embodiments, the cancer is a cancer that is relapsed or refractory to one or more previous anti-cancer therapies.

[0118] In some embodiments, the cancer is any cancer type provided in Ross et al., Oncologist (2017) 22(12):1444-1450, which is incorporated herein by reference.

[0119] Methods involving precision enrichment of paraffin-embedded samples In some embodiments, the methods described herein may be performed using samples obtained from precision enrichment-based methods. Methods involving precision enrichment are described, for example, in U.S. Provisional Application No. 63 / 189,602, the entire contents of which are incorporated herein by reference. In some embodiments of the methods described herein, step a) includes: i) identifying a target region containing tumor cells of interest in a paraffin-embedded tissue; ii) extracting a paraffin-embedded sample from the paraffin-embedded tissue; iii) identifying the location of the paraffin-embedded sample in the paraffin-embedded tissue; and iv) extracting RNA and / or DNA from the sample if the location of the paraffin-embedded sample overlaps with the target region containing tumor cells of interest. In some embodiments, if the location of the paraffin-embedded sample does not overlap with the target region containing tumor cells of interest, steps ii) and iii) are repeated.

[0120] This aspect of the present disclosure is based, at least in part, on the development of a method for extracting nucleic acids from tissue containing tumor cells of interest, such as formalin-fixed, paraffin-embedded (FFPE) tissue. The tissue may be derived from a subject suspected of or known to have cancer. Without being bound by any particular theory, including a step of analyzing the tissue after sample extraction can provide information about whether the sample was successfully enriched for tumor cells of interest or whether additional samples should be extracted from the tissue. For example, a tissue slide may be prepared after sample extraction to determine the degree of overlap between the sample and the tumor cells of interest. Such a histological quality assurance / quality control step is believed to enable evaluation of the tumor content enrichment process. Without such a quality assurance / quality control step, samples extracted from tissues may fail during sequencing analysis due to low tumor purity. These samples were unusable and deemed unusable due to "potential enrichment failure." Therefore, the method described herein allows for the testing of specimens that would otherwise have had insufficient tissue levels for tumor content analysis using previous enrichment methods, reducing the occurrence of unusable samples due to potential enrichment failures.Successful enrichment of tumor content is particularly important for the evaluation of certain biomarkers that may indicate the presence of cancer and require relatively high levels of tumor content to measure the biomarker.Therefore, the method described herein may be used to detect the presence of biomarkers that may not otherwise be detectable.This is believed to improve the specificity and accuracy of subsequent sequencing analysis of biomarkers.The method described herein may be referred to as a "precision enrichment" method, as it involves the precision enrichment of tumor cells of interest, and therefore tumor content, and nucleic acids obtained from tumor cells.Methods involving precision enrichment may be used in combination with any of the above methods.

[0121] In some embodiments, the methods described herein comprise extracting a paraffin-embedded sample from a paraffin-embedded tissue using a needle. In some embodiments, the sample is extracted by puncturing the paraffin-embedded tissue with the needle. In some embodiments, the needle is a disposable needle. In some embodiments, the needle is a thin-walled needle. In some embodiments, the needle is a blunt-end needle. In some embodiments, the needle is a stainless steel needle. In some embodiments, the needle is a hypodermic needle. In some embodiments, the needle comprises a Luer-compatible hub. In some embodiments, the needle is a 13-gauge needle, a 14-gauge needle, a 15-gauge needle, a 16-gauge needle, a 17-gauge needle, an 18-gauge needle, a 19-gauge needle, a 20-gauge needle, or a 21-gauge needle. In some embodiments, the paraffin-embedded sample extracted from the paraffin-embedded tissue is about 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, or 2.3 mm in diameter.

[0122] In some embodiments, step b) comprises extracting the paraffin-embedded sample using laser microdissection (LMD). In some embodiments, step b) comprises extracting the paraffin-embedded sample using a razor blade.

[0123] In some embodiments, step c) of the methods described herein includes preparing a slide of a paraffin-embedded tissue section. Generally, preparing a slide of a paraffin-embedded tissue section will enable evaluation of whether a paraffin-embedded sample (e.g., a paraffin-embedded sample extracted with a needle as described above) has been successfully enriched for tumor cells of interest. For example, a slide of a paraffin-embedded tissue section may reveal that the location of the paraffin-embedded sample (e.g., the location of the needle puncture from which the sample was extracted) overlaps with the location of the tumor cells of interest. An exemplary image of a slide of a paraffin-embedded tissue section is provided in FIG. 4. As shown in FIG. 4, the location of the needle puncture is visible in the paraffin-embedded tissue section. In some embodiments, the paraffin-embedded tissue section is stained. In some embodiments, the paraffin-embedded tissue section is stained to distinguish tumor cells of interest from other cells in the paraffin-embedded tissue. In some embodiments, the paraffin-embedded tissue section is stained with hematoxylin and eosin (H&E). In some embodiments, sections of paraffin-embedded tissue are immunostained, e.g., using a detectably labeled antibody. In some embodiments, the detectably labeled antibody binds to a protein expressed in tumor cells of interest. In some embodiments, step c) is performed by visual inspection. For example, in some embodiments, step c) is performed by a pathologist, who visually inspects slides of paraffin-embedded tissue sections to determine whether the location of the paraffin-embedded sample overlaps with tumor cells of interest. In some embodiments, step c) is performed by a computer system. For example, in some embodiments, step c) is performed using a computer system, which evaluates whether the location of the paraffin-embedded sample overlaps with tumor cells of interest. In some embodiments, step c) is performed using an image analysis system.

[0124] In some embodiments, the level of enrichment of tumor cells of interest in the paraffin-embedded sample is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or more. In some embodiments, the level of enrichment of tumor cells of interest in the paraffin-embedded sample is at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold higher than the level of tumor cells of interest in the remainder of the paraffin-embedded sample. In some embodiments, the paraffin-embedded sample contains cells that comprise at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% or more tumor cells of interest.

[0125] In some embodiments, step ii) further comprises inspecting the paraffin-embedded sample and, optionally, removing excess tissue from the paraffin-embedded sample.

[0126] III. Extraction of Analytes from Deembedded Samples The methods herein involve the extraction of analytes from a de-embedded sample. In some embodiments, where the de-embedded sample is a biological sample, the analyte may be any macromolecule or small molecule that can be extracted from the biological sample. In some embodiments, the analyte is selected from the group consisting of polypeptides, RNA, DNA, small molecules, lipids, polysaccharides, exosomes, mitochondria, and nuclei. In some embodiments, the analyte is an organelle. In some embodiments, two or more analytes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 analytes, may be extracted from the de-embedded sample.

[0127] In some embodiments, the analyte is one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise RNA and / or DNA. In some embodiments, the one or more nucleic acids comprise genomic DNA, cDNA, or mRNA. In some embodiments, the analyte is RNA. In some embodiments, the analyte is DNA.

[0128] Extraction of RNA and / or DNA from deparaffinized samples RNA preferential extraction method In some embodiments, the methods described herein involve extracting RNA and / or DNA from deparaffinized samples. In certain embodiments, RNA is extracted before DNA is extracted. Exemplary methods for extracting RNA before DNA are detailed in the Examples and diagrammed in Figures 1 and 3A.

[0129] In some embodiments, the method for extracting RNA before DNA further comprises digesting the paraffin-embedded sample before step b). In some embodiments, the paraffin-embedded sample is digested using a proteinase. In some embodiments, the proteinase is proteinase K. In some embodiments, the paraffin-embedded sample is digested using about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, or about 35 μL of 20 mg / mL proteinase K. In some embodiments, the paraffin-embedded sample is incubated with the proteinase at about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, or about 62°C. In some embodiments, the paraffin-embedded sample is incubated with the proteinase for about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes. In some embodiments, the paraffin-embedded sample is incubated with the proteinase while shaking at about 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, or 1500 rpm. In some embodiments, the paraffin-embedded sample is partially digested or fully digested.

[0130] In some embodiments, the method of extracting RNA before DNA further comprises, after step b), de-crosslinking the digested sample. In some embodiments, de-crosslinking comprises heating the digested sample to 80-90° C. In some embodiments, to de-crosslink the digested sample, the digested sample is heated for about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes.

[0131] In some embodiments, step c) comprises collecting an RNA-containing sample lysate from the digested paraffin-embedded sample and purifying RNA from the RNA-containing sample lysate. Methods for purifying RNA from RNA-containing sample lysates are known in the art.

[0132] In some embodiments, the method for extracting RNA before DNA further comprises completely digesting the digested paraffin-embedded sample. In some embodiments, the complete digestion is achieved using a proteinase. In some embodiments, the proteinase is proteinase K. In some embodiments, the digested paraffin-embedded sample is digested using about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, or about 35 μL of 20 mg / mL proteinase K. In some embodiments, the digested paraffin-embedded sample is incubated with the proteinase at about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the digested paraffin-embedded sample is incubated with the proteinase for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, or about 22 hours. In some embodiments, the digested paraffin-embedded sample is incubated with the proteinase while shaking at about 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, or 1500 rpm.

[0133] In some embodiments, the method of extracting RNA before DNA further comprises collecting a sample lysate containing DNA from the fully digested paraffin-embedded sample. In some embodiments, the method further comprises purifying DNA from the sample lysate containing DNA. Methods for purifying DNA from sample lysates containing DNA are known in the art.

[0134] The methods described herein for extracting RNA before DNA are suitable for automation and / or high-throughput analysis. In some embodiments, the methods described herein for extracting RNA before DNA are performed using a liquid handling robot. In some embodiments, the liquid handling robot is an Agilent, BioTek, Hamilton, Tecan, or ThermoFisher Scientific liquid handling robot, and optionally, the liquid handling robot is a Hamilton AutoLys STAR, Hamilton STAR, BioTek Dispenser, or KingFisher Flex. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR. In some embodiments, extracting RNA before DNA is performed on two or more samples processed in parallel. In some embodiments, 2, 4, 8, 16, 24, 48, 96, or more samples are processed in parallel.

[0135] DNA preferential extraction method In some embodiments, the methods described herein involve extracting RNA and / or DNA from deparaffinized samples. In certain embodiments, DNA is extracted before RNA is extracted. Exemplary methods for extracting RNA before DNA are detailed in the Examples and diagrammed in Figures 1 and 3B.

[0136] In some embodiments, the method for extracting DNA before RNA further comprises thoroughly digesting the paraffin-embedded sample after step b). In some embodiments, the thorough digestion is achieved using a proteinase. In some embodiments, the proteinase is proteinase K. In some embodiments, the paraffin-embedded sample is digested using about 10 μL, about 15 μL, about 20 μL, about 25 μL, about 30 μL, or about 35 μL of 20 mg / mL proteinase K. The method of any one of claims 125-127, wherein the paraffin-embedded sample is incubated with the proteinase at about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the paraffin-embedded sample is incubated with the proteinase for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, or about 22 hours.

[0137] In some embodiments, the method of extracting DNA before RNA further comprises extracting DNA from the fully digested paraffin-embedded sample. In some embodiments, the method further comprises purifying the extracted DNA. Methods for purifying extracted DNA are known in the art.

[0138] In some embodiments, the method of extracting DNA before RNA further comprises extracting RNA from the fully digested paraffin-embedded sample. In some embodiments, the method comprises purifying the extracted RNA. Methods for purifying RNA from extracted RNA are known in the art.

[0139] The methods described herein for extracting DNA before RNA are suitable for automation and / or high-throughput analysis. In some embodiments, the methods described herein for extracting DNA before RNA are performed using a liquid handling robot. In some embodiments, the liquid handling robot is an Agilent, BioTek, Hamilton, Tecan, or ThermoFisher Scientific liquid handling robot, and optionally, the liquid handling robot is a Hamilton AutoLys STAR, Hamilton STAR, BioTek Dispenser, or KingFisher Flex. In some embodiments, the liquid handling robot is a Hamilton AutoLys STAR. In some embodiments, DNA extraction before RNA is performed on two or more samples processed in parallel. In some embodiments, 2, 4, 8, 16, 24, 48, 96, or more samples are processed in parallel.

[0140] In some embodiments, regardless of whether DNA is extracted before RNA or RNA is extracted before DNA, the method further includes measuring the amount of RNA and / or DNA extracted from the paraffin-embedded sample. In some embodiments, the amount of RNA and / or DNA extracted from the paraffin-embedded sample is about 5 ng, about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 60 ng, about 70 ng, about 80 ng, about 100 ng, about 50 μg, or about 50 mg.

[0141] IV. Analysis of Analytes Extracted from De-embedded Samples Analytes extracted from the de-embedded sample as described above may be further analyzed. Suitable methods for analyzing the analytes described herein are known in the art.

[0142] RNA and / or DNA analysis 10. The method of any one of the preceding claims, further comprising analysing DNA extracted from the embedded sample to detect somatic mutations selected from the group consisting of: i) point mutations, ii) in-frame deletions of one or more codons, iii) intragenic deletions, iv) intragenic insertions, v) whole gene deletions, vi) inversions, vii) interchromosomal translocations, viii) tandem duplications, ix) gene fusions, x) genomic rearrangements involving intronic sequences, and / or xi) gene amplifications or duplications.

[0143] In some embodiments, the somatic mutation is in a gene, and the gene is ABL1, AKT1, AKT2, AKT3, ALK, APC, AR, BRAF, CCND1, CDK4, CDKN2A, CEBPA, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FLT3, HRAS, JAK2, KIT, KRAS, MAP2K1, MAP2K2, MET, MLL, MYC, NF1, NOTCH1, NPM1, NRAS, NTRK3, PDGFRA, PIK3CA, PIK3CG, PIK3R1, PTCH1, PTCH2, PTEN, RB1, RET, SMO, STK11, SUFU, or TP53.In some embodiments, the somatic mutation is in a gene, including but not limited to ABL2, ARAF, ARFRP1, ARID1A, ATM, ATR, AURKA, AURKB, BAP1, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL6, BRCA1, BRCA2, CBL, CARD11, CBL, CCND2, CCND3, CCNE1, CD79A, CD79B, CDH1, CDH2, CDH20, CDH5, CDK6, CDK8, CD KN2B, CDKN2C, CHEK1, CHEK2, CRKL, CRLF2, DNMT3A, DOT1L, EPHA3, EPHA5, EPHA6, EPHA7, EPHB1, EPHB4, EPHB6, ERBB3, ERBB4, ERG, ETV1, ETV4, ETV5, ETV6, EWSR1, EZH2, FANCA, FBXW7, FGFR4, FLT1, FLT4, FOXP4, GATA1, GNA11, GNAQ, GNAS, GPR124, GUCY1 A2, HOXA3, HSP90AA1, IDH1, IDH2, IGF1R, IGF2R, IKBKE, IKZF1, INHBA, IRS2, JAK1, JAK3, JUN, KDM6A, KDR, LRP1B, LRP6, LTK, MAP2K4, MCL1, MDM2, MDM4, MEN1, MITF, MLH1, MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYCL1, MYCN, NF2, NKX2-1, NTRK1, NTRK2 , PAK3, PAX5, PDGFRB, PKHD1, PLCG1, PRKDC, PTPN11, PTPRD, RAF1, RARA, RICTOR, RPTOR, RUNX1, SMAD2, SMAD3, SMAD4, SMARCA 4, SMARCB1, SOX10, SOX2, SRC, TBX22, TET2, TGFBR2, TMPRSS2, TNFAIP3, TNK, TNKS2, TOP1, TSC1, TSC2, USP9X, VHL, or WT1.

[0144] In some embodiments, the method further comprises analyzing RNA extracted from the embedded sample to detect alterations selected from the group consisting of: i) gene fusions, ii) exon skipping events, iii) splice variants, and / or iv) altered gene expression.

[0145] In some embodiments, RNA and / or DNA extracted from de-embedded samples are analyzed by one or more methods selected from the group consisting of nucleic acid hybridization assays, amplification-based assays, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays, real-time PCR, sequencing, next-generation sequencing, screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), or mass spectrometry genotyping. In some embodiments, RNA and / or DNA extracted from de-embedded samples are analyzed by next-generation sequencing. Exemplary methods for next-generation sequencing are described, for example, in Frampton, GM et al. (2013) Nat. Biotech. 31:1023-1031. In some embodiments, RNA and / or DNA extracted from de-embedded samples are analyzed according to the method outlined in Figures 5A-5B.

[0146] In some embodiments, the method further includes e) optionally ligating one or more adaptors to RNA and / or DNA extracted from the de-embedded sample to generate ligated nucleic acids, d) optionally amplifying nucleic acids from the ligated nucleic acids, f) optionally capturing a plurality of nucleic acids corresponding to the gene of interest, g) sequencing the plurality of nucleic acids with a sequencing device to obtain a plurality of sequence reads corresponding to the gene of interest, h) analyzing the plurality of sequence reads, and i) detecting one or more mutations in the gene of interest based on the analysis. In some embodiments, the plurality of nucleic acids corresponding to the gene of interest are captured from the amplified nucleic acids by hybridization with a bait molecule.

[0147] In some embodiments, biomarker nucleic acid molecules of the present disclosure are detected using any suitable method known in the art, such as a nucleic acid hybridization assay, an amplification-based assay (e.g., polymerase chain reaction, PCR), a PCR-RFLP assay, real-time PCR, sequencing (e.g., Sanger sequencing or next-generation sequencing), a screening assay (e.g., using spectral karyotyping), fluorescence in situ hybridization (FISH), breakaway FISH, spectral karyotyping, multiplex FISH, comparative genomic hybridization, in situ hybridization, sequence-specific primer polymerase chain reaction (SSP-PCR), high performance liquid chromatography (HPLC), or mass spectrometry genotyping. Methods for analyzing samples to detect, for example, nucleic acid molecules are described in U.S. Pat. No. 9,340,830 and WO 2012 / 092426(A1), which are incorporated by reference in their entireties.

[0148] In situ hybridization In some embodiments, biomarker nucleic acid molecules of the present disclosure are detected in RNA and / or DNA extracted from de-embedded samples using in situ hybridization methods, such as fluorescent in situ hybridization (FISH) methods.

[0149] In some embodiments, FISH analysis is used to identify chromosomal rearrangements that result in the mutations described herein. In some embodiments, FISH analysis is used to identify RNA molecules, including the biomarker nucleic acids described herein. Methods for performing FISH are known in the art and can be used with almost any type of tissue. In FISH analysis, a detectably labeled, e.g., fluorescently labeled, nucleic acid probe is bound to a specific region of DNA, e.g., a chromosome, or RNA, e.g., mRNA, and then examined, e.g., by microscopy. See, e.g., U.S. Patent No. 5,776,688. First, the DNA or RNA molecule is immobilized on a slide, and then the labeled probe is hybridized to the DNA or RNA molecule. Visualization is then achieved, e.g., using enzyme-linked label-based detection methods known in the art. Generally, the resolution of FISH analysis is the detection of DNA of 60 to 100,000 nucleotides, e.g., from 60 base pairs (bp) up to about 100 kilobase pairs. The nucleic acid probe used in FISH analysis comprises a single-stranded nucleic acid. Such probes are typically at least about 50 nucleotides in length. In some embodiments, the probe comprises about 100 to about 500 nucleotides. Probes that hybridize to centromeric DNA and locus-specific DNA or RNA are commercially available, for example, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.), or Cytocell (Oxfordshire, UK). Alternatively, probes may be produced non-commercially from chromosomal or genomic DNA or other sources of nucleic acid using standard techniques. Examples of probes, labeling, and hybridization methods are known in the art.

[0150] Several variations of FISH methods are known in the art and are suitable for use in accordance with the methods of the present disclosure, including single-molecule RNA FISH, Fiber FISH, Q-FISH, Flow-FISH, MA-FISH, breakaway FISH, hybrid fusion-FISH, and multi-color fluorescent FISH, or mFISH. In some embodiments, "breakaway FISH" is used in the methods provided herein. Breakaway FISH utilizes at least one probe targeting the fusion junction or breakpoint and at least one probe targeting an individual gene of the fusion, e.g., one or more exons and introns of the gene. In normal cells (i.e., cells harboring a fusion nucleic acid molecule described herein), both probes are observed (or a second color is observed due to the proximity of the two genes of the gene fusion), whereas in cells harboring a fusion nucleic acid molecule described herein, only one gene probe is observed due to the presence of a rearrangement that resulted in the fusion nucleic acid molecule.

[0151] Array-based methods In some embodiments, biomarker nucleic acid molecules of the present disclosure are detected in RNA and / or DNA extracted from deembedded samples using array-based methods, such as array-based comparative genomic hybridization (CGH). In array-based CGH, a first sample of nucleic acid (e.g., from a sample such as a tumor) is labeled with a first label, while a second sample of nucleic acid (e.g., from a control such as healthy cells / tissue) is labeled with a second label. In some embodiments, equal amounts of the two samples are mixed and co-hybridized to a DNA microarray of thousands of uniformly spaced clonal DNA fragments or oligonucleotides, spotted in triplicate on the array. After hybridization, a digital imaging system is used to capture and quantitate the relative fluorescence intensity of each hybridized fluorophore. The ratio of the resulting fluorescence intensities is proportional to the ratio of the copy numbers of the DNA sequences in the two samples. In some embodiments, if a chromosomal deletion or duplication is present, a difference in the ratio of signals from the two labels is detected, providing a measure of copy number. Array-based CGH can also be carried out by single-color labeling.In single-color CGH, control (for example, control nucleic acid sample, such as that derived from healthy cells / tissue) is labeled, hybridized with one array, and absolute signal is read; test sample (for example, nucleic acid sample obtained from individual or tumor) is labeled, hybridized with second array (having identical contents), and absolute signal is read.Copy number difference is calculated based on the absolute signal from two arrays.

[0152] Amplification-based methods In some embodiments, biomarker nucleic acid molecules of the present disclosure are detected in RNA and / or DNA extracted from de-embedded samples using amplification-based methods. As is known in the art, in such amplification-based methods, a nucleic acid sample, e.g., a sample obtained from an individual or tumor, is used as a template in an amplification reaction (e.g., polymerase chain reaction (PCR)) using one or more oligonucleotides or primers, such as one or more of the oligonucleotides or primers provided herein. The presence of a biomarker nucleic acid molecule of the present disclosure in a sample can be determined based on the presence or absence of an amplification product. Quantitative amplification methods are also known in the art and can be used in accordance with the methods provided herein. Methods for measuring DNA copy number at microsatellite loci using quantitative PCR analysis are known in the art. The known nucleotide sequence of a gene is sufficient to allow one of skill in the art to routinely select primers for amplifying any portion of the gene. Fluorogenic quantitative PCR can also be used. In fluorogenic quantitative PCR, quantification is based on the amount of fluorescent signal, e.g., TaqMan and Sybr Green.

[0153] Other amplification methods suitable for use in accordance with the methods provided herein include, for example, ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, dot PCR, and linker-adapter PCR.

[0154] Sequencing In some embodiments, the biomarker nucleic acid molecules of the present disclosure are detected in RNA and / or DNA extracted from de-embedded samples using sequencing methods. Any sequencing method known in the art can be used to detect the biomarker nucleic acid molecules provided herein. Exemplary sequencing methods that may be used to detect the biomarker nucleic acid molecules provided herein include those based on techniques developed by Maxam and Gilbert or Sanger. Automated sequencing procedures, including, for example, sequencing by mass spectrometry, can also be used.

[0155] In some embodiments, biomarker nucleic acid molecules of the disclosure are detected in RNA and / or DNA extracted from de-embedded samples using hybrid capture-based sequencing (hybrid capture-based NGS), e.g., using adapter ligation-based libraries. See, e.g., Frampton, GM et al. (2013) Nat. Biotech. 31:1023-1031. In some embodiments, biomarker nucleic acid molecules of the disclosure are determined using next-generation sequencing (NGS). Next-generation sequencing can be performed on individual nucleic acid molecules or on a large number of nucleic acids in a highly parallel manner (e.g., 10 5Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of any clonally propagated proxies for individual nucleic acid molecules (more than 100 molecules can be sequenced simultaneously). Next-generation sequencing methods suitable for use in accordance with the inventions provided herein are known in the art and include, but are not limited to, massively parallel short-read sequencing, template-based sequencing, pyrosequencing, real-time sequencing involving imaging of the sequential incorporation of dye-labeled nucleotides during DNA synthesis, nanopore sequencing, sequencing by hybridization, nanotransistor array-based sequencing, polony sequencing, scanning tunneling microscope (STM)-based sequencing, or nanowire molecular sensor-based sequencing. See, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference. Exemplary NGS methods and platforms that may be used to detect the biomarker nucleic acid molecules provided herein include, but are not limited to, the HeliScope Gene Sequencing system from Helicos BioSciences (Cambridge, MA, USA), the PacBio RS system from Pacific BioSciences (Menlo Park, CA, USA), massively parallel short-read sequencing such as the Solexa sequencer and other methods and platforms from Illumina Inc. (San Diego, CA, USA), 454 sequencing from 454 LifeSciences (Branford, CT, USA), Ion Torrent sequencing from ThermoFisher (Waltham, MA, USA), or the SOLiD sequencer from Applied Biosystems (Foster City, CA, USA).Additional exemplary methods and platforms that may be used to detect the biomarker nucleic acid molecules provided herein include, but are not limited to, the Genome Sequencer (GS) FLX System, G.007 Polonator system, Solexa Genome Analyzer, HiSeq 2500, HiSeq 3000, HiSeq 4000, and NovaSeq 6000 platforms from Roche (Basel, CHE), and Illumina Inc. (San Diego, CA, USA).

[0156] In some embodiments, one or more nucleic acids extracted from the RNA and / or DNA extracted from the de-embedded sample are analyzed by next-generation sequencing. In some embodiments, the method further includes: e) optionally ligating one or more adapters to the RNA and / or DNA extracted from the embedded sample to generate ligated nucleic acids; d) optionally amplifying nucleic acids from the ligated nucleic acids; f) optionally capturing a plurality of nucleic acids corresponding to one or more genes of interest; g) sequencing the plurality of nucleic acids with a sequencing device to obtain a plurality of sequence reads corresponding to the one or more genes of interest; h) analyzing the plurality of sequence reads; and i) detecting one or more mutations in the genes of interest based on the analysis. In some embodiments, prior to step e), the one or more nucleic acids extracted from the sample are fragmented, and optionally, the one or more nucleic acids extracted from the sample are fragmented by sonication. In some embodiments, the fragmented one or more nucleic acids extracted from the sample are end-repaired. In some embodiments, the end-repaired and fragmented one or more nucleic acids extracted from the sample are dA-tailed or dT-tailed. In some embodiments, the one or more nucleic acids extracted from the sample are prepared for sequencing according to the method described in Frampton, GM et al. (2013) Nat. Biotech. 31:1023-1031.

[0157] Biomarker detection In some aspects, provided herein are reagents for detecting biomarker nucleic acid molecules of the present disclosure, or fragments thereof, as described herein, in nucleic acids (i.e., RNA and / or DNA) extracted from, for example, de-embedded samples.

[0158] Generally, RNA and / or DNA samples extracted from de-embedded samples may be analyzed by the methods described herein to determine the presence of nucleic acid biomarkers, such as cancer biomarkers. Methods for determining the presence of specific nucleic acid biomarkers are described, for example, in International Publication No. WO 2021 / 096888, U.S. Pat. No. 9,884,060, U.S. Pat. No. 9,297,011, U.S. Pat. No. 10,000,814, U.S. Pat. No. 8,673,972, U.S. Pat. No. 9,410,954, U.S. Pat. No. 9,907,798, U.S. Pat. No. 6,673,972 ... Patent Publication No. 2016 / 0009785(A1), U.S. Patent Publication No. 2020 / 0299775(A1), U.S. Patent No. 10,980,804, U.S. Patent No. 9,861,633, U.S. Patent Publication No. 2018 / 0363066(A1), U.S. Patent Publication No. 2017 / 0356053(A1), U.S. Patent Publication No. 2019 / 0085403(A1), U.S. Patent Publication No. 2019 / 0219586(A1), International Publication No. WO2020 / 243021, International Publication No. WO2021 / 042066, International Application No. PCT / US2021 / 020752, International Application No. PCT / US2021 / 019982, International Publication No. WO2018009939, U.S. Provisional Patent Application No. 63 / 129,286, U.S. Provisional Patent Application No. 63 / 132,085, U.S. Provisional Patent Application No. No. 63 / 143,619, U.S. Provisional Patent Application No. 63 / 171,423, U.S. Provisional Patent Application No. 63 / 122,431, U.S. Provisional Patent Application No. 63 / 215,281, U.S. Provisional Patent Application No. 63 / 148,116, U.S. Provisional Patent Application No. 63 / 189,025, U.S. Provisional Patent Application No. 63 / 188,719, and U.S. Provisional Patent Application No. 63 / 215,356.

[0159] Additionally, exemplary nucleic acid biomarkers that may be detected in RNA and / or DNA samples extracted from de-embedded samples include loss of heterozygosity (LOH), LOH of human leukocyte antigen (HLA) genes (HLA LOH), loss of function of phosphatase and tensin homolog (PTEN) gene (PTEN LOF), tumor mutation burden (TMB), and homozygous single exon loss, which are described in more detail below.

[0160] In some embodiments, the detection reagents provided herein comprise a nucleic acid molecule, e.g., a DNA, RNA, or mixed DNA / RNA molecule, comprising a nucleotide sequence complementary to a nucleotide sequence on a target nucleic acid molecule, e.g., a nucleic acid comprising a biomarker nucleic acid molecule described herein, or a fragment or portion thereof. Provided herein are baits suitable for detecting the biomarker nucleic acid molecules of the present disclosure. In some embodiments, the bait comprises a capture nucleic acid molecule configured to hybridize to a target nucleic acid molecule comprising a biomarker nucleic acid molecule provided herein, or a fragment or portion thereof. In some embodiments, the capture nucleic acid molecule is configured to hybridize to a biomarker nucleic acid molecule of the target nucleic acid molecule. Also provided herein are probes, e.g., nucleic acid molecules, suitable for detecting the biomarker nucleic acid molecules provided herein. In some embodiments, the probes provided herein comprise a nucleic acid sequence configured to hybridize to a target nucleic acid molecule comprising a biomarker nucleic acid molecule provided herein, or a fragment or portion thereof. In some embodiments, the probes comprise a nucleic acid sequence configured to hybridize to a biomarker nucleic acid molecule of the target nucleic acid molecule, or a fragment or portion thereof. In some embodiments, the probe comprises a nucleic acid sequence configured to hybridize to a fragment or portion of a biomarker nucleic acid molecule of the target nucleic acid molecule, in some embodiments, the fragment or portion comprises about 5 to about 25 nucleotides, about 5 to about 300 nucleotides, about 100 to about 300 nucleotides, about 130 to about 230 nucleotides, or about 150 to about 200 nucleotides.

[0161] Loss of heterozygosity (LOH) of one or more genes of interest, e.g., human leukocyte antigen (HLA) genes In some embodiments, provided herein are methods comprising detecting loss of heterozygosity (LOH) of one or more genes of interest in RNA and / or DNA extracted from a de-embedded sample as described herein. In some embodiments, provided herein are methods comprising detecting LOH of human leukocyte antigen (HLA) genes in RNA and / or DNA extracted from a de-embedded sample as described herein. Exemplary methods for detecting LOH of HLA genes are described in International Application No. PCT / US2021 / 019982, which is incorporated herein by reference in its entirety.

[0162] In other embodiments, the gene of interest is selected from the group consisting of ST7 / RAY1, ARH1 / NOEY2, TSLC1, RB, PTEN, SMAD2, SMAD4, DCC, TP53, ATM, miR-15a, miR-16-1, NAT2, BRCA1, BRCA2, hOGG1, CDH1, IGF2, CDKN1C / P57, MEN1, PRKAR1A, H19, KRAS, BAP1, PTCH1, SMO, SUFU, NOTCH1, PPP6C, LATS1, CASP8, PTPN14, ARID1A, FBXW7, M6P / IGF2R, IFN-α, olfactory receptor genes, CBFA2T3, DUTT1, FHIT, APC, and P1 6, FCMD, TSC2, miR-34, c-MPL, RUNX3, DIRAS3, NRAS, miR-9, FAM50B, PLAGL1, ER, FLT3, ZDBF2, GPR1, c-KIT, NAP1L5, GRB10, EGFR, PEG10, BRAF, MEST, JAK2, DAPK1, LIT1, WT1, NF-1, PR, c-CBL, DLK1, AKT1, SNURF, cytochrome P450 genes (CYP), ZNF587, SOCS1, TIMP2, RUNX1, AR, CEBPA, C19MC, EMP3, ZNF331, CDKN2A, PEG3, NNAT, GNAS, or GATA5.

[0163] In some embodiments, any one of the above methods further comprises detecting loss of heterozygosity (LOH) of human leukocyte antigen (HLA) genes in RNA and / or DNA extracted from the de-embedded sample as described herein. In some embodiments according to any of the embodiments described herein, the HLA genes encode major histocompatibility (MHC) class I molecules. In some embodiments, the method further comprises, after determining the adjusted allele frequencies, determining that the genes have undergone loss of heterozygosity (LOH) based at least in part on the adjusted allele frequencies.

[0164] In yet some other aspects, provided herein are methods for detecting loss of heterozygosity (LOH) of human leukocyte antigen (HLA) genes in RNA and / or DNA extracted from a de-embedded sample, as described herein. In some embodiments, the method includes: a) obtaining an observed allele frequency for an HLA allele, wherein the observed allele frequency corresponds to a frequency of a nucleic acid encoding at least a portion of the HLA allele detected among a plurality of sequence reads corresponding to the HLA gene, the plurality of sequence reads being obtained by sequencing a nucleic acid encoding the gene or a portion thereof captured by hybridization with a bait molecule; and b) obtaining a relative binding propensity of the HLA allele to the bait molecule, wherein the relative binding propensity of the HLA allele corresponds to a frequency of one or more The method includes: obtaining a nucleic acid encoding at least a portion of an HLA allele corresponding to the tendency of the nucleic acid encoding a portion of another HLA allele to bind to a bait molecule; c) applying an objective function to measure the difference between the relative binding tendency of the HLA allele and the observed allele frequency; d) applying an optimization model to minimize the objective function; e) determining an adjusted allele frequency of the HLA allele based on the optimization model and the observed allele frequency; and f) determining that LOH has occurred if the adjusted allele frequency of the HLA allele is less than a predetermined threshold. In some embodiments, the HLA gene is an HLA-A, HLA-B, or HLA-C gene. In some embodiments, a plurality of sequence reads are obtained by sequencing nucleic acids obtained from RNA and / or DNA extracted from a de-embedded sample. In some embodiments, the method is for detecting loss of heterozygosity (LOH) of a polymorphic gene of interest in RNA and / or DNA extracted from a de-embedded sample.In some embodiments, the method comprises: a) obtaining an observed allele frequency for an allele of a gene of interest, wherein the observed allele frequency corresponds to a frequency of a nucleic acid encoding at least a portion of the allele detected among a plurality of sequence reads corresponding to the gene, wherein the plurality of sequence reads were obtained by sequencing a nucleic acid encoding the gene or a portion thereof captured by hybridization with a bait molecule; b) obtaining a relative binding propensity of the allele to the bait molecule, wherein the relative binding propensity of the allele corresponds to a propensity of a nucleic acid encoding at least a portion of the allele to bind to the bait molecule in the presence of nucleic acids encoding portions of one or more other alleles; c) applying an objective function to measure the difference between the relative binding propensity of the allele and the observed allele frequency; d) applying an optimization model to minimize the objective function; e) determining an adjusted allele frequency of the allele based on the optimization model and the observed allele frequency; and f) determining that LOH has occurred if the adjusted allele frequency of the allele is less than a predetermined threshold.In some embodiments, the polymorphic gene is ST7 / RAY1, ARH1 / NOEY2, TSLC1, RB, PTEN, SMAD2, SMAD4, DCC, TP53, ATM, miR-15a, miR-16-1, NAT2, BRCA1, BRCA2, hOGG1, CDH1, IGF2, CDKN1C / P57, MEN1, PRKAR1A, H19, KRAS, BAP1, PTCH1, SMO, SUFU, NOTCH1, PPP6C, LATS1, CASP8, PTPN14, ARID1A, FBXW7, M6P / IGF2R, IFN-α, olfactory receptor genes, CBFA2T3, DUTT1, FHIT, APC, P 16, FCMD, TSC2, miR-34, c-MPL, RUNX3, DIRAS3, NRAS, miR-9, FAM50B, PLAGL1, ER, FLT3, ZDBF2, GPR1, c-KIT, NAP1L5, GRB10, EGFR, PEG10, BRAF, MEST, JAK2, DAPK1, LIT1, WT1, NF-1, PR, c-CBL, DLK1, AKT1, SNURF, cytochrome P450 genes (CYP), ZNF587, SOCS1, TIMP2, RUNX1, AR, CEBPA, C19MC, EMP3, ZNF331, CDKN2A, PEG3, NNAT, GNAS, or GATA5.

[0165] In yet another aspect, any of the methods of the present disclosure further comprises measuring TMB in RNA and / or DNA extracted from the de-embedded sample, e.g., as described herein. In some embodiments, the method comprises determining LOH and assessing TMB, e.g., in RNA and / or DNA extracted from the de-embedded sample. As demonstrated herein, HLA LOH and high TMB (and optionally intact HLA genes) can be predictive of increased overall survival, a higher probability of survival, and / or an increased likelihood of responding to ICI therapy, e.g., when compared to HLA LOH without high TMB. In some embodiments, high TMB refers to a TMB of 10 mutations / Mb or greater or 13 mutations / Mb or greater. In some embodiments, TMB is obtained from multiple sequence reads, e.g., multiple sequence reads obtained by sequencing nucleic acids of at least a portion of a genome (e.g., from an enriched or unenriched sample). In some embodiments, TMB is determined based on the number of non-driver somatic coding mutations per megabase of the sequenced genome.

[0166] In some embodiments, any of the methods of the disclosure involves obtaining knowledge of LOH of an HLA gene (e.g., in RNA and / or DNA extracted from a de-embedded sample) and obtaining knowledge of TMB (e.g., in RNA and / or DNA extracted from a de-embedded sample). In some embodiments, any of the methods of the disclosure involves detecting LOH of an HLA gene (e.g., in RNA and / or DNA extracted from a de-embedded sample) and obtaining knowledge of TMB (e.g., in RNA and / or DNA extracted from a de-embedded sample). In some embodiments, any of the methods of the disclosure involves obtaining knowledge of LOH of an HLA gene (e.g., in RNA and / or DNA extracted from a de-embedded sample) and detecting or determining TMB (e.g., in RNA and / or DNA extracted from a de-embedded sample). In some embodiments, any of the methods of the present disclosure includes detecting LOH of an HLA gene (e.g., in RNA and / or DNA extracted from a de-embedded sample) and detecting or determining TMB (e.g., in RNA and / or DNA extracted from a de-embedded sample). In some embodiments, the sample used to detect / determine LOH and TMB is the same. In some embodiments, the samples used to detect / determine LOH and TMB are different.

[0167] Phosphatase and tensin homolog (PTEN) Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently disrupted tumor suppressors in cancer. The lipid phosphatase activity of PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K) / AKT / mTOR pathway to suppress tumor cell growth and survival. Therefore, loss-of-function mutations in the PTEN gene can serve as biomarkers for cancer.

[0168] In some embodiments, provided herein are methods that include detecting loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene in RNA and / or DNA extracted from a de-embedded sample as described herein. In some embodiments, the loss-of-function mutations in the PTEN gene include one or more of an insertion, deletion, or substitution of one or more nucleotides, a genomic rearrangement, an alteration in the promoter, a gene fusion, or a copy number alteration.

[0169] Tumor mutation burden (TMB) In some embodiments, provided herein are methods that include measuring a level of tumor mutation burden (TMB) in RNA and / or DNA extracted from a de-embedded sample as described herein. In some embodiments, the methods provided herein include obtaining knowledge that the RNA and / or DNA extracted from the de-embedded sample has a tumor mutation burden of at least about 10 mut / Mb or at least about 20 mut / Mb. In some embodiments, obtaining knowledge that the RNA and / or DNA extracted from the de-embedded sample has a tumor mutation burden of at least about 10 mut / Mb or at least about 20 mut / Mb includes measuring the level of tumor mutation burden in the RNA and / or DNA extracted from the de-embedded sample, e.g., in the RNA and / or DNA extracted from the de-embedded sample, where the embedded sample was obtained from the individual. In some embodiments, the methods provided herein include detecting a tumor mutation burden of at least about 10 mut / Mb or at least about 20 mut / Mb in the RNA and / or DNA extracted from the de-embedded sample. In some embodiments, the method includes administering an effective amount of immunotherapy in response to knowledge that the RNA and / or DNA extracted from the de-embedded sample has a tumor mutational burden of at least about 10 mut / Mb or at least about 20 mut / Mb. In some embodiments, the method includes providing a report to an interested party.

[0170] In some embodiments, tumor mutational burden is assessed in RNA and / or DNA extracted from a de-embedded sample obtained from the individual. In some embodiments, the sample from the individual comprises a tumor biopsy. In some embodiments, the embedded sample from the individual comprises nucleic acid.

[0171] In some embodiments, tumor mutational burden is assessed using any suitable method known in the art. For example, tumor mutational burden may be measured using whole exome sequencing (WES), next-generation sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or sequencing of a panel of genes, for example, a panel including cancer-related genes. See, for example, Melendez et al., Transl Lung Cancer Res (2018) 7(6):661-667. In some embodiments, tumor mutational burden is measured using gene-targeted sequencing, for example, using a nucleic acid hybridization capture method, for example, in combination with sequencing. See, for example, Fancello et al., J Immunother Cancer (2019) 7:183.

[0172] In some embodiments, tumor mutational burden is measured according to the methods provided in WO2017 / 151524(A1), which is incorporated herein by reference in its entirety.

[0173] In some embodiments, tumor mutational burden is measured in RNA and / or DNA extracted from de-embedded samples by whole exome sequencing. In some embodiments, tumor mutational burden is measured in RNA and / or DNA extracted from de-embedded samples using next-generation sequencing. In some embodiments, tumor mutational burden is measured in RNA and / or DNA extracted from de-embedded samples using whole genome sequencing. In some embodiments, tumor mutational burden is measured in RNA and / or DNA extracted from de-embedded samples by gene-targeted sequencing. In some embodiments, tumor mutational burden is measured on about 0.8 Mb to about 1.1 Mb of sequenced DNA. In some embodiments, the tumor mutation burden is determined by the following: about 0.8 Mb, about 0.81 Mb, about 0.82 Mb, about 0.83 Mb, about 0.84 Mb, about 0.85 Mb, about 0.86 Mb, about 0.87 Mb, about 0.88 Mb, about 0.89 Mb, about 0.9 Mb, about 0.91 Mb, about 0.92 Mb, about 0.93 Mb, about 0.94 Mb, about 0.95 Mb In some embodiments, tumor mutation burden is measured on about 0.8 Mb of sequenced DNA.

[0174] In some embodiments, the RNA and / or DNA extracted from the de-embedded sample has a high tumor mutation burden, for example, at least about 10 mut / Mb. In some embodiments, the RNA and / or DNA extracted from the de-embedded sample has a tumor mutation burden of at least about 10 mut / Mb. In some embodiments, the RNA and / or DNA extracted from the de-embedded sample has a tumor mutation burden of at least about 20 mut / Mb. In some embodiments, the RNA and / or DNA extracted from the de-embedded sample is from about 10 mut / Mb to about 15 mut / Mb, from about 15 mut / Mb to about 20 mut / Mb, from about 20 mut / Mb to about 25 mut / Mb, from about 25 mut / Mb to about 30 mut / Mb, from about 30 mut / Mb to about 35 mut / Mb, from about 35 mut / Mb to about 40 mut / Mb, from about 40 mut / Mb to about 45 mut / Mb, from about 45 mut / Mb to about 50 mut / Mb, from about 50 mut / Mb to about 55 and having a tumor mutation burden of any of the following: about 55 mut / Mb to about 60 mut / Mb, about 60 mut / Mb to about 65 mut / Mb, about 65 mut / Mb to about 70 mut / Mb, about 70 mut / Mb to about 75 mut / Mb, about 75 mut / Mb to about 80 mut / Mb, about 80 mut / Mb to about 85 mut / Mb, about 85 mut / Mb to about 90 mut / Mb, about 90 mut / Mb to about 95 mut / Mb, or about 95 mut / Mb to about 100 mut / Mb.In some embodiments, the RNA and / or DNA extracted from the de-embedded sample is from about 100 mut / Mb to about 110 mut / Mb, from about 110 mut / Mb to about 120 mut / Mb, from about 120 mut / Mb to about 130 mut / Mb, from about 130 mut / Mb to about 140 mut / Mb, from about 140 mut / Mb to about 150 mut / Mb, from about 150 mut / Mb to about 160 mut / Mb, or from about 160 mut / Mb. b ~ approx. 170mut / Mb, approx. 170mut / Mb ~ approx. 180mut / Mb, approx. 180mut / Mb ~ approx. 190mut / Mb, approx. 190mut / Mb ~ approx. 200mut / Mb, approx. 210mut / M b ~ approx. 220mut / Mb, approx. 220mut / Mb ~ approx. 230mut / Mb, approx. 230mut / Mb ~ approx. 240mut / Mb, approx. 240mut / Mb ~ approx. 250mut / Mb, approx. 250mut / Mb ~260mut / Mb, approximately 260mut / Mb~270mut / Mb, approximately 270mut / Mb~280mut / Mb, approximately 280mut / Mb~290mut / Mb, approximately 290mut / Mb ~300mut / Mb, approximately 300mut / Mb~310mut / Mb, approximately 310mut / Mb~320mut / Mb, approximately 320mut / Mb~330mut / Mb, approximately 330mut / Mb~ The tumor has a mutational burden of about 340 mut / Mb, about 340 mut / Mb to about 350 mut / Mb, about 350 mut / Mb to about 360 mut / Mb, about 360 mut / Mb to about 370 mut / Mb, about 370 mut / Mb to about 380 mut / Mb, about 380 mut / Mb to about 390 mut / Mb, about 390 mut / Mb to about 400 mut / Mb, or greater than 400 mut / Mb.

[0175] In some embodiments, measuring the tumor mutational burden comprises assessing mutations in RNA and / or DNA extracted from a de-embedded sample derived from the cancer in the individual. In some embodiments, measuring the tumor mutational burden comprises assessing mutations in RNA and / or DNA extracted from a de-embedded sample derived from the cancer in the individual and a matched normal sample, e.g., a de-embedded sample from the individual derived from cancer-free tissue or other source.

[0176] Homozygous single-exon loss Generally, homozygous single-exon loss refers to the deletion of both copies of a given exon. In some embodiments, methods are provided herein that include detecting homozygous single-exon loss in RNA and / or DNA extracted from a de-embedded sample. In some embodiments, homozygous single-exon loss is detected in RNA and / or DNA extracted from a de-embedded sample by whole-exome sequencing, whole-genome sequencing, or gene-targeted sequencing.

[0177] Systems, Software, and Devices In some other aspects, a non-transitory computer-readable storage medium is provided herein. In some embodiments, the non-transitory computer-readable storage medium includes one or more programs executed by one or more processors of a device, the one or more programs including instructions that, when executed by the one or more processors, cause the device to perform a method according to any of the embodiments described herein.

[0178] FIG. 6 illustrates an example of a computing device according to one embodiment. The device 1100 may be a host computer connected to a network. The device 1100 may be a client computer or a server. As shown in FIG. 6, the device 1100 may be any suitable type of microprocessor-based device, such as a personal computer, a workstation, a server, or a handheld computing device (a portable electronic device, e.g., a phone or tablet). The device may include, for example, one or more of a processor 1110, an input device 1120, an output device 1130, storage 1140, a communication device 1160, a power supply 1170, an operating system 1180, and a system bus 1190. The input device 1120 and the output device 1130 may generally correspond to those described herein and may be connectable to or integrated with the computer.

[0179] The input device 1120 may be any suitable device that provides input, such as a touchscreen, a keyboard or keypad, a mouse, or a voice recognition device. The output device 1130 may be any suitable device that provides output, such as a touchscreen, a tactile device, or a speaker.

[0180] Storage 1140 may be any suitable device that provides storage (e.g., electrical, magnetic, or optical memory, including RAM (volatile and non-volatile), cache, hard drive, or removable storage disk). Communication device 1160 may include any suitable device that can send and receive signals over a network, such as a network interface chip or device. The components of a computer may be connected in any suitable manner, for example, via wired media (e.g., a physical bus, Ethernet, or any other wired transmission technology) or wirelessly (e.g., Bluetooth, Wi-Fi, or any other wireless technology). For example, in FIG. 6, the components are connected by system bus 1190.

[0181] The detection module 1150 can be stored as executable instructions in the storage 1140 and executed by the processor 1110, and can include, for example, processes that embody the functionality of the present disclosure (e.g., embodied in the devices described above).

[0182] The detection module 1150 may also be stored in and / or transferred to any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device (e.g., those described herein) and may fetch and execute instructions associated with the software from the instruction execution system, apparatus, or device. In the context of the present disclosure, a computer-readable storage medium may be any medium, such as storage 1140, that may contain or store processes for use by or in connection with an instruction execution system, apparatus, or device. Examples of computer-readable storage media may include memory units, such as hard drives, flash drives, and distribution modules, that operate as a single functional unit. Additionally, various processes described herein may be embodied as modules configured to operate in accordance with the above embodiments and techniques. Furthermore, while processes may be shown and / or described separately, those skilled in the art will understand that the above processes may be routines or modules within other processes.

[0183] The detection module 1150 may also propagate within any transmission medium for use by or in connection with an instruction execution system, apparatus, or device (e.g., those described above) and may fetch instructions associated with the software from and execute the instructions. In the context of the present disclosure, a transmission medium may be any medium that can communicate, propagate, or transmit transmission programming for use by or in connection with an instruction execution system, apparatus, or device. Transmission-readable media may include, but are not limited to, electronic, magnetic, optical, electromagnetic, or infrared wired or wireless propagation media.

[0184] Device 1100 may be connected to a network (e.g., network 1204 shown in FIG. 7 and / or described below), which may be any suitable type of interconnected communications system. The network may implement any suitable communications protocol and may be protected by any suitable security protocol. The network may include any suitable arrangement of network links capable of implementing the transmission and reception of network signals, such as a wireless network connection (T1 or T3 line), a cable network, DSL, or telephone lines.

[0185] Device 1100 may implement any operating system (e.g., operating system 1180) suitable for operating on a network. Detection module 1150 may be written in any suitable programming language, such as C, C++, Java, or Python. In various embodiments, application software embodying functionality of the present disclosure may be deployed in different configurations (e.g., in a client / server arrangement, or via a web browser, as a web-based application or web service). In some embodiments, operating system 1180 is executed by one or more processors, e.g., processor 1110.

[0186] The device 1100 may further include a power source 1170, which may be any suitable power source.

[0187] 7 illustrates an example of a computing system according to one embodiment. In system 1200, device 1100 (e.g., as described above and shown in FIG. 6) is connected to network 1204, which is also connected to device 1206. In some embodiments, device 1206 is a sequencing device. Exemplary sequencing devices may include, but are not limited to, Roche / 454's Genome Sequencer (GS) FLX System, Illumina / Solexa's Genome Analyzer (GA), Illumina's HiSeq 2500, HiSeq 3000, HiSeq 4000, and NovaSeq 6000 sequencing systems, Life / APG's Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator's G.007 system, Helicos BioSciences' HeliScope Gene sequencing system, or Pacific Biosciences' PacBio RS system. Device 1100 and device 1206 may communicate via network 1204, such as, for example, a local area network (LAN), a virtual private network (VPN), or the Internet, using a suitable communication interface. In some embodiments, network 1204 may be, for example, the Internet, an intranet, a virtual private network, a cloud network, a wired network, or a wireless network. Device 1100 and device 1206 may communicate partially or entirely via wireless or wired communications, such as Ethernet, IEEE 802.11b wireless, etc. Additionally, device 1100 and device 1206 may communicate via a second network, such as, for example, a mobile / cellular network, using a suitable communication interface. Communications between devices 1100 and 1206 may further include or communicate with various servers, such as a mail server, a mobile server, a media server, a telephone server, etc.In some embodiments, device 1100 and device 1206 may communicate directly (instead of, or in addition to, communication via network 1204), e.g., via wireless or wired communication, such as Ethernet, IEEE 802.11b wireless, etc. In some embodiments, device 1100 and device 1206 communicate via communication 1208, which may be a direct connection or may occur over a network (e.g., network 1204).

[0188] One or all of devices 1100 and 1206 generally include logic (e.g., http web server logic) or are programmed to format data accessed from local or remote databases or other sources of data and content to provide and / or receive information over network 1204 in accordance with various embodiments described herein.

[0189] FIG. 8 illustrates an exemplary process 1300 for detecting in an analyte sample extracted from an embedded sample, according to some embodiments. Process 1300 may be performed, for example, using one or more electronic devices implementing a software program. In some examples, process 1300 is performed using a client-server system, with the blocks of process 1300 being divided in any manner between a server and a client device. In other examples, the blocks of process 1300 are divided between a server and multiple client devices. Thus, while portions of process 1300 are described herein as being performed by a particular device in a client-server system, it should be understood that process 1300 is not so limited. In some embodiments, the steps performed may be performed across many systems, for example, in a cloud environment. In other examples, process 1300 is performed using only a client device or only multiple client devices. In process 1300, some blocks are optionally combined, the order of some blocks is optionally changed, and some blocks are optionally omitted. In some examples, additional steps may be performed in combination with process 1300. Accordingly, the operations illustrated (and described in more detail below) are exemplary in nature and, therefore, should not be considered limiting.

[0190] At block 1302, a plurality of sequence reads for one or more nucleic acids are obtained, where the one or more nucleic acids are derived from a de-embedded sample obtained from an individual. In some embodiments, the sample is obtained from an individual with cancer (e.g., a cancer described herein). In some embodiments, the sequence reads are obtained using a sequencing device (e.g., one described herein or otherwise known in the art). In some embodiments, the nucleic acids include one or more nucleic acids corresponding to a biomarker of the present disclosure, or portions thereof. Optionally, prior to obtaining the sequence reads, the sample is purified, concentrated (e.g., for nucleic acids corresponding to a biomarker of the present disclosure, or portions thereof), and / or PCR amplified. At block 1304, an exemplary system (e.g., one or more electronic devices) analyzes the plurality of sequence reads for the presence of one or more mutations in the biomarker or portions thereof. At block 1306, the system detects one or more mutations in the biomarker or portions thereof in the sample (e.g., based on analysis). V. Diagnostic and Therapeutic Methods and Kits

[0191] Diagnostic, evaluation, screening, monitoring, or prognostic methods In some aspects, provided herein are methods for diagnosing or assessing a biomarker in a cancer, such as a cancer provided herein, in an individual. In some embodiments, the method comprises obtaining knowledge of the presence of a nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample obtained from the individual. In some embodiments, the method comprises detecting a biomarker nucleic acid molecule provided herein in the RNA and / or DNA sample extracted from the embedded sample obtained from the individual. In some embodiments, the biomarker nucleic acid molecule is detected in the RNA and / or DNA sample extracted from the embedded sample obtained from the individual using any method known in the art, such as one or more of the methods for detecting biomarker nucleic acid molecules described herein. In some embodiments, the method further comprises providing a diagnosis or assessment of the biomarker nucleic acid molecule. In some embodiments, the diagnosis or assessment identifies the presence or absence of a biomarker nucleic acid molecule in the RNA and / or DNA sample extracted from the embedded sample. In some embodiments, the diagnosis or assessment identifies a cancer, such as a cancer provided herein, as likely to respond to an anti-cancer therapy, e.g., an anti-cancer therapy provided herein. In some embodiments, the presence of biomarker nucleic acid molecules in an RNA and / or DNA sample extracted from the embedded sample identifies the cancer as likely to respond to an anti-cancer therapy, such as an anti-cancer therapy provided herein. In some embodiments, the RNA and / or DNA sample extracted from the embedded sample is a sample described herein. In some embodiments, the embedded sample comprises cells derived from or is obtained from cells derived from a cancer. In some embodiments, the individual has cancer, is suspected of having cancer, is being tested for cancer, is being treated for cancer, or is being tested for susceptibility to cancer, and the cancer is, for example, a cancer described herein.

[0192] In some aspects, provided herein are methods of diagnosing or assessing cancer in an individual, e.g., a cancer provided herein. In some embodiments, the method of diagnosing or assessing cancer comprises detecting a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample obtained from the individual, e.g., an embedded sample containing cells derived from the cancer. In some embodiments, the method comprises detecting a biomarker nucleic acid molecule described herein in an RNA and / or DNA sample extracted from an embedded sample obtained from the individual using any method known in the art, such as one or more of the methods for detecting a biomarker nucleic acid molecule described herein. In some embodiments, detecting a biomarker nucleic acid molecule described herein, or a fragment thereof, in an RNA and / or DNA sample extracted from an embedded sample obtained from the individual identifies the cancer as likely to respond to an anti-cancer therapy, e.g., an anti-cancer therapy described herein. In some embodiments, the presence of a biomarker nucleic acid molecule described herein, or a fragment thereof, in an RNA and / or DNA sample extracted from an embedded sample obtained from the individual identifies the cancer as likely to respond to an anti-cancer therapy, e.g., an anti-cancer therapy provided herein. In some embodiments, the method further includes providing a diagnosis or assessment of the cancer or the fusion nucleic acid molecule. In some embodiments, the diagnosis or assessment identifies the cancer as likely to respond to an anti-cancer therapy, such as an anti-cancer therapy provided herein. In some embodiments, the diagnosis or assessment identifies the presence or absence of a biomarker nucleic acid molecule in an RNA and / or DNA sample extracted from the embedded sample.

[0193] In some aspects, provided herein are methods of predicting survival of an individual having cancer, e.g., a cancer provided herein. In some embodiments, the individual is undergoing treatment with an anti-cancer therapy, such as an anti-cancer therapy described herein. In some embodiments, the method comprises obtaining knowledge of the presence of a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample from the individual. In some embodiments, the method comprises detecting a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample from the individual. In some embodiments, in response to obtaining knowledge of a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from the embedded sample, the individual is predicted to have a longer survival time after treatment with an anti-cancer therapy, e.g., an anti-cancer therapy provided herein, compared to, e.g., an individual whose cancer does not exhibit the biomarker nucleic acid molecule. In some embodiments, in response to detecting a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from the embedded sample, the individual is predicted to have a longer survival time after treatment with an anti-cancer therapy, e.g., an anti-cancer therapy provided herein, compared to, e.g., an individual whose cancer does not exhibit the biomarker nucleic acid molecule. In some embodiments, the method further comprises providing a diagnosis or assessment. In some embodiments, the diagnosis or assessment identifies the presence or absence of a biomarker nucleic acid molecule in an RNA and / or DNA sample extracted from the embedded sample. In some embodiments, the diagnosis or assessment identifies an individual as predicted to have a longer survival time following treatment with an anticancer therapy, e.g., an anticancer therapy provided herein, compared to, for example, an individual whose cancer does not exhibit the biomarker nucleic acid molecule. In some embodiments, the RNA and / or DNA sample extracted from the embedded sample is a sample described herein. In some embodiments, the embedded sample comprises cells derived from the cancer.

[0194] In some aspects, provided herein are methods of screening individuals who have cancer, are suspected of having cancer, are being tested for cancer, are being treated for cancer, or are being tested for susceptibility to cancer, where the cancer is, for example, a cancer provided herein. In some embodiments, the individual is being treated with an anti-cancer therapy, such as an anti-cancer therapy described herein. In some embodiments, the method comprises obtaining knowledge of the presence of a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample from the individual. In some embodiments, the method comprises detecting a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample from the individual. In some embodiments, in response to obtaining knowledge of a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample, the individual is predicted to be at increased risk of cancer recurrence, aggressive cancer, anti-cancer therapy resistance, or poor prognosis, for example, compared to an individual whose cancer does not exhibit the biomarker nucleic acid molecule. In some embodiments, upon detection of a biomarker nucleic acid molecule provided herein in an RNA and / or DNA sample extracted from an embedded sample extracted from a tissue, the individual is predicted to be at increased risk of cancer recurrence, aggressive cancer, resistance to anti-cancer therapy, or a poor prognosis, e.g., compared to an individual whose cancer does not exhibit the biomarker nucleic acid molecule. In some embodiments, the method further comprises providing a diagnosis or assessment. In some embodiments, the diagnosis or assessment identifies the presence or absence of a biomarker nucleic acid molecule in the RNA and / or DNA sample extracted from the embedded sample extracted from the tissue. In some embodiments, the diagnosis or assessment identifies the individual as being at increased risk of cancer recurrence, aggressive cancer, resistance to anti-cancer therapy, or a poor prognosis, e.g., compared to an individual whose cancer does not exhibit the biomarker nucleic acid molecule. In some embodiments, the RNA and / or DNA sample extracted from the embedded sample is as described herein. In some embodiments, the embedded sample comprises cells derived from a cancer.

[0195] In some embodiments, the method further comprises selectively enriching one or more nucleic acids comprising biomarker nucleotide sequences, e.g., using reagents known in the art or provided herein, e.g., baits, probes, or oligonucleotides described herein, to generate an enriched sample.

[0196] Anti-cancer therapy Certain aspects of the present disclosure relate to anti-cancer therapies and methods for identifying individuals who may benefit from treatment with anti-cancer therapies, methods for selecting anti-cancer therapies for treating individuals, methods for identifying anti-cancer therapies as treatment options, methods for treating cancer or delaying the progression of cancer including administering anti-cancer therapies, uses for anti-cancer therapies (e.g., in methods for treating or delaying the progression of cancer in an individual, or in methods for the manufacture of medicaments for treating or delaying the progression of cancer), etc. These methods and uses are based, at least in part, on the detection of biomarkers from tumor cells of interest in embedded samples, as described above. Without being bound by theory, it is believed that these biomarkers can identify patients who may benefit from appropriate anti-cancer therapies, such as one or more of small molecule inhibitors, chemotherapeutic agents, cancer immunotherapies, antibodies, cell therapies, nucleic acids, surgery, radiation therapy, anti-angiogenic therapies, anti-DNA repair therapies, anti-inflammatory therapies, anti-neoplastic agents, growth inhibitory agents, cytotoxic agents, or any combination thereof.

[0197] In some embodiments, the anti-cancer therapy comprises a cyclin-dependent kinase (CDK) inhibitor. In some embodiments, the CDK inhibitor inhibits CDK4. In some embodiments, the CDK inhibitor inhibits cyclin D / CDK4. In some embodiments, the anti-cancer therapy / CDK inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of CDK4, (b) an antibody that inhibits one or more activities of CDK4 (e.g., by binding to CDK4 and inhibiting one or more of its activities, by binding to CDK4 and inhibiting its expression, and / or by binding to a cell expressing CDK4 and inhibiting one or more of its activities, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of CDK4 (e.g., antisense oligonucleotides, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the CDK inhibitor inhibits CDK4 and CDK6. In some embodiments, the CDK inhibitor is a small molecule inhibitor of CDK4 (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of CDK inhibitors include palbociclib, ribociclib, and abemaciclib, and pharmaceutically acceptable salts thereof.

[0198] In some embodiments, the anti-cancer therapy comprises a mouse double minute 2 homolog (MDM2) inhibitor. In some embodiments, the anti-cancer therapy / MDM2 inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of MDM2 (e.g., binds to p53), (b) an antibody that inhibits one or more activities of MDM2 (e.g., by binding to MDM2 and inhibiting one or more of its activities, by binding to MDM2 and inhibiting its expression, and / or by binding to cells expressing MDM2 and inhibiting one or more of its activities, e.g., by inducing antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP)), or (c) a nucleic acid that inhibits expression of MDM2 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the MDM2 inhibitor is a small molecule inhibitor of MDM2 (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of MDM2 inhibitors include nutlin-3a, RG7112, idasanutlin (RG7388), AMG-232, MI-63, MI-291, MI-391, MI-77301 (SAR405838), APG-115, DS-3032b, NVP-CGM097, and HDM-201 (siremadlin), and pharmaceutically acceptable salts thereof. In some embodiments, the MDM2 inhibitor inhibits or disrupts the interaction between MDM2 and p53.

[0199] In some embodiments, the anti-cancer therapy is an antimetabolite, a DNA damaging agent, or a platinum-containing therapeutic (e.g., 5-azacitadine, 5-fluorouracil, acadesine, busulfan, carboplatin, cisplatin, chlorambucil, CPT-11, cytarabine, daunorubicin, decitabine, doxorubicin, etoposide, fludarabine, gemcitabine, idarubicin, radiation, oxaliplatin, temozolomide, topotecan, trabectedin, GSK2830371, or rucaparib); a pro-apoptotic agent (e.g., For example, a BCL2 inhibitor or down-modulator, a SMAC mimetic, or a TRAIL agonist, e.g., ABT-263, ABT-737, oridonin, venetoclax, a combination of venetoclax with an anti-CD20 antibody, e.g., obinutuzumab or rituximab, 1396-11, ABT-10, SM-164, D269H / E195R, or rhTRAIL; a tyrosine kinase inhibitor (e.g., those described herein); an inhibitor of the RAS, RAF, MEK, or MAPK pathway (e.g., AZD6244, dabrafenib, L GX818, PD0325901, pimasertib, trametinib, or vemurafenib; inhibitors of PI3K, mTOR, or Akt (e.g., those described herein); CDK inhibitors (e.g., those described herein); PKC inhibitors (e.g., LXS196 or sotrastaurin); antibody-based therapies (e.g., anti-PD-1 or anti-PDL1 antibodies, e.g., atezolizumab, pembrolizumab, nivolumab, or spartalizumab; anti-CD20 antibodies, e.g., obinutuzumab or rituximab; or anti-DR5 antibodies, e.g., doxorubicin, davidib ... proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, or MG-132); HDAC inhibitors (e.g., SAHA or VPA); antibiotics (e.g., actinomycin D); zinc-containing therapeutics (e.g., zinc or ZMC1); HSP inhibitors (e.g., geldanamycin); ATPase inhibitors (e.g., alkazolid); mitotic inhibitors (e.g., paclitaxel or vincristine); metformin; methotrexate; tanshinone IIA; and / or P5091.

[0200] In some embodiments, the anti-cancer therapy comprises a tyrosine kinase inhibitor. In some embodiments, the anti-cancer therapy / tyrosine kinase inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of a tyrosine kinase, (b) an antibody that inhibits one or more activities of a tyrosine kinase (e.g., by binding to the tyrosine kinase and inhibiting one or more activities of the tyrosine kinase, by binding to the tyrosine kinase and inhibiting expression (e.g., cell surface expression) of the tyrosine kinase, and / or by binding to a cell expressing the tyrosine kinase and inhibiting one or more activities thereof, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of a tyrosine kinase (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the tyrosine kinase inhibitor is a small molecule inhibitor of a tyrosine kinase (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of tyrosine kinase inhibitors include imatinib, crenolanib, linifanib, ninetedanib, axitinib, dasatinib, imetelstat, midostaurin, pazopanib, sorafenib, sunitinib, motesanib, masitinib, vatalanib, cabozanitinib, tivozanib, OSI-930, Ki8751, telatinib, dovitinib, tyrphostin AG 1296, and amuvatinib, and pharmaceutically acceptable salts thereof.

[0201] In some embodiments, the anti-cancer therapy comprises a mitogen-activated protein kinase (MEK) inhibitor. In some embodiments, the MEK inhibitor inhibits one or more activities of MEK1 and / or MEK2. In some embodiments, the anti-cancer therapy / MEK inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of MEK, (b) an antibody that inhibits one or more activities of MEK (e.g., by binding to MEK and inhibiting one or more activities of MEK, by binding to MEK and inhibiting expression of MEK, and / or by binding to cells expressing MEK and inhibiting one or more activities thereof, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of MEK (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the MEK inhibitor is a small molecule inhibitor of MEK (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of MEK inhibitors include trametinib, cobimetinib, binimetinib, CI-1040, PD0325901, selumetinib, AZD8330, TAK-733, GDC-0623, refametinib, pimasertib, RO4987655, RO5126766, WX-544, and HL-085, and pharmaceutically acceptable salts thereof. In some embodiments, the anti-cancer therapy inhibits the activity of one or more of the Raf / MEK / ERK pathways, including inhibitors of Raf, MEK, and / or ERK.

[0202] In some embodiments, the anti-cancer therapy comprises a mammalian target of rapamycin (mTOR) inhibitor. In some embodiments, the anti-cancer therapy / mTOR inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of mTOR, (b) an antibody that inhibits one or more activities of mTOR (e.g., by binding to mTOR and inhibiting one or more activities of mTOR, by binding to mTOR and inhibiting expression of mTOR, and / or by binding to cells expressing mTOR and inhibiting one or more activities thereof, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of mTOR (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the mTOR inhibitor is a small molecule inhibitor of mTOR (e.g., a competitive inhibitor, e.g., an ATP-competitive inhibitor, or a non-competitive inhibitor, e.g., a rapamycin analog). Non-limiting examples of mTOR inhibitors include temsirolimus, everolimus, ridaforolimus, dactolisib, GSK2126458, XL765, AZD8055, AZD2014, MLN128, PP242, NVP-BEZ235, LY3023414, PQR309, PKI587, and OSI027, and pharmaceutically acceptable salts thereof. In some embodiments, the anti-cancer therapy inhibits one or more activities of the Akt / mTOR pathway, including inhibitors of Akt and / or mTOR.

[0203] In some embodiments, the anti-cancer therapy comprises a PI3K inhibitor or an Akt inhibitor. In some embodiments, the PI3K inhibitor inhibits one or more activities of PI3K. In some embodiments, the anti-cancer therapy / PI3K inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of PI3K; (b) an antibody that inhibits one or more activities of PI3K (e.g., by binding to PI3K and inhibiting one or more of its activities, by binding to PI3K and inhibiting its expression, and / or by binding to cells expressing PI3K and inhibiting one or more of its activities, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP); or (c) a nucleic acid that inhibits expression of PI3K (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the PI3K inhibitor is a small molecule inhibitor of PI3K (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of PI3K inhibitors include GSK2636771, buparlisib (BKM120), AZD8186, copanlisib (BAY80-6946), LY294002, PX-866, TGX115, TGX126, BEZ235, SF1126, idelalisib (GS-1101, CAL-101), pictilisib (GDC-094 ), GDC0032, IPI145, INK1117 (MLN1117), SAR260301, KIN-193 (AZD6482), duvelisib, GS-9820, GSK2636771, GDC-0980, AMG319, pazovanib, and alpelisib (BYL719, Piqray), and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor inhibits one or more activities of AKT (e.g., AKT1).In some embodiments, the anti-cancer therapy / AKT inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of AKT1, (b) an antibody that inhibits one or more activities of AKT1 (e.g., by binding to AKT1 and inhibiting one or more of its activities, by binding to AKT1 and inhibiting its expression, and / or by binding to a cell expressing AKT1 and inhibiting one or more of its activities, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits the expression of AKT1 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the AKT1 inhibitor is a small molecule inhibitor of AKT1 (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of AKT1 inhibitors include GSK690693, GSK2141795 (uprosertib), GSK2110183 (afuresertib), AZD5363, GDC-0068 (ipatasertib), AT7867, CCT128930, MK-2206, BAY 1125976, AKT1 and AKT2-IN-1, perifosine, and VIII, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT1 inhibitor is a pan-Akt inhibitor.

[0204] In some embodiments, the anti-cancer therapy is a hedgehog (Hh) inhibitor. In some embodiments, the anti-cancer therapy / Hh inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of Hh, (b) an antibody that inhibits one or more activities of Hh (e.g., by binding to Hh and inhibiting one or more of its activities, by binding to Hh and inhibiting its expression, and / or by binding to cells expressing Hh and inhibiting one or more of its activities, e.g., by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of Hh (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapy, etc.). In some embodiments, the Hh inhibitor is a small molecule inhibitor of Hh (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of Hh inhibitors include sonidegib, vismodegib, erismodegib, slidegib, BMS833923, PF-04449913, and LY2940680, and pharmaceutically acceptable salts thereof.

[0205] In some embodiments, the anti-cancer therapy comprises a heat shock protein (HSP) inhibitor, a MYC inhibitor, an HDAC inhibitor, immunotherapy, a neoantigen, a vaccine, or a cell therapy.

[0206] In some embodiments, the anti-cancer therapy comprises one or more of an immune checkpoint inhibitor, chemotherapy, a VEGF inhibitor, an integrin β3 inhibitor, a statin, an EGFR inhibitor, an mTOR inhibitor, a PI3K inhibitor, a MAPK inhibitor, or a CDK4 / 6 inhibitor.

[0207] In some embodiments, the anti-cancer therapy includes a kinase inhibitor. In some embodiments, the methods provided herein include administering a kinase inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the kinase inhibitor is crizotinib, alectinib, ceritinib, lorlatinib, brigutinib, ensartinib (X-396), repotrectinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP-37440, belizatinib (TSR-011), ASP3026, KRCA-0008, TQ-B3139, TPX-0131, or TAE684 (NVP-TAE684). In some embodiments, the kinase inhibitor is an ALK kinase inhibitor, e.g., as described in Examples 3-39 of WO2005016894, which is incorporated herein by reference.

[0208] In some embodiments, the anti-cancer therapy includes a heat shock protein (HSP) inhibitor. In some embodiments, the methods provided herein include administering an HSP inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the HSP inhibitor is a pan-HSP inhibitor such as KNK423. In some embodiments, the HSP inhibitor is an HSP70 inhibitor such as cmHsp70.1, quercetin, VER155008, or 17-AAD. In some embodiments, the HSP inhibitor is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is 17-AAD, Debio0932, ganetespib (STA-9090), retaspimycin hydrochloride (retaspimycin, IPI-504), AUY922, alvespimycin (KOS-1022, 17-DMAG), tanespimycin (KOS-953, 17-AAG), DS 2248, or AT13387 (onarespib). In some embodiments, the HSP inhibitor is an HSP27 inhibitor, such as apatorsen (OGX-427).

[0209] In some embodiments, the anti-cancer therapy comprises a MYC inhibitor. In some embodiments, the methods provided herein comprise administering a MYC inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the MYC inhibitor is MYCi361 (NUCC-0196361), MYCi975 (NUCC-0200975), Omomyc (a dominant-negative peptide), ZINC16293153 (Min9), 10058-F4, JKY-2-169, 7594-0035, or an inhibitor of MYC / MAX dimerization and / or MYC / MAX / DNA complex formation.

[0210] In some embodiments, the anti-cancer therapy comprises a histone deacetylase (HDAC) inhibitor. In some embodiments, the methods provided herein comprise administering an HDAC inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the HDAC inhibitor is belinostat (PXD101, Beleodac®), SAHA (vorinostat, suberoylanilide hydroxamine, Zolinza®), panobinostat (LBH589, LAQ-824), ACY1215 (rocilinostat), xinostat (JNJ-26481585), abexinostat (PCI-24781), pracinostat (SB939), gibinostat (ITF2357), resminostat (4SC-201), trichostatin A (TSA), MS-275 (echinostat), romidepsin (depsipeptide, FK228), MGCD0103 (mosetinostat), BML-210, CAY10603 , valproic acid, MC1568, CUDC-907, CI-994 (tacedinaline), Pivanex (AN-9), AR-42, chidamide (CS055, HBI-8000), CUDC-101, CHR-3996, MPT0E028, BRD8430, MRLB-223, apicidin, RGFP966, BG45, PCI-34051, C149 (NCC1 49), TMP269, Cpd2, T247, T326, LMK235, C1A, HPOB, Nextulastat A, Befexamac, CBHA, phenylbutyric acid, MC1568, SNDX275, Scriptaid, Merck60, PX089344, PX105684, PX117735, PX117792, PX117245, PX105844, compound 12 described in Li et al., Cold Spring Harb Perspect Med (2016) 6 (10): a026831, or PX117445.

[0211] In some embodiments, the anti-cancer therapy includes a VEGF inhibitor. In some embodiments, the methods provided herein include administering a VEGF inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the VEGF inhibitor is bevacizumab (Avastin®), BMS-690514, ramucirumab, pazopanib, sorafenib, sunitinib, golvatinib, vandetanib, cabozantinib, levantinib, axitinib, cediranib, tivozanib, lucitanib, semaxanib, nindentanib, regorafenib, or aflibercept.

[0212] In some embodiments, the anti-cancer therapy comprises an integrin β3 inhibitor. In some embodiments, the methods provided herein comprise administering to an individual an integrin β3 inhibitor, e.g., in combination with another anti-cancer therapy. In some embodiments, the integrin β3 inhibitor is anti-avb3 (clone LM609), cilengitide (EMD121974, NSC, 707544), siRNA, GLPG0187, MK-0429, CNTO95, TN-161, etaracizumab (MEDI-522), intetumumab (CNTO95) (anti-αV subunit antibody), abituzumab (EMD525797 / DI17E6) (anti-αV subunit antibody), JSM6427, SJ749, BCH-15046, SCH221153, or SC56631. In some embodiments, the anti-cancer therapy comprises an αIIbβ3 integrin inhibitor. In some embodiments, the methods provided herein include administering to an individual an αIIbβ3 integrin inhibitor, e.g., in combination with another anti-cancer therapy. In some embodiments, the αIIbβ3 integrin inhibitor is abciximab, eptifibatide (Integrilin®), or tirofiban (Aggrastat®).

[0213] In some embodiments, the anti-cancer therapy includes a statin or statin-based drug. In some embodiments, the methods provided herein include administering a statin or statin-based drug to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the statin or statin-based drug is simvastatin, atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, or cerivastatin.

[0214] In some embodiments, the anti-cancer therapy includes a MAPK inhibitor. In some embodiments, the methods provided herein include administering a MAPK inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the MAPK inhibitor is SB203580, SKF-86002, BIRB-796, SC-409, RJW-67657, BIRB-796, VX-745, RO3201195, SB-242235, or MW181.

[0215] In some embodiments, the anti-cancer therapy includes an EGFR inhibitor. In some embodiments, the methods provided herein include administering an EGFR inhibitor to an individual, for example, in combination with another anti-cancer therapy. In some embodiments, the EGFR inhibitor is cetuximab, panitumumab, lapatinib, gefitinib, vandetanib, dacomitinib, icotinib, osimertinib (AZD9291), afatanib, olmutinib, EGF816 (nazartinib), avitinib (AC0010), rociletinib (CO-1686), BMS-690514, YH5448, PF-06747775, ASP8273, PF299804, AP26113, or erlotinib. In some embodiments, the EGFR inhibitor is gefitinib or cetuximab.

[0216] In some embodiments, the anti-cancer therapy comprises a cancer immunotherapy, such as a checkpoint inhibitor, a cancer vaccine, a cell-based therapy, a T cell receptor (TCR)-based therapy, an adjuvant immunotherapy, a cytokine immunotherapy, and an oncolytic virus therapy. In some embodiments, the methods provided herein comprise administering to an individual a cancer immunotherapy, such as a checkpoint inhibitor, a cancer vaccine, a cell-based therapy, a T cell receptor (TCR)-based therapy, an adjuvant immunotherapy, a cytokine immunotherapy, and an oncolytic virus therapy, e.g., in combination with another anti-cancer therapy. In some embodiments, the cancer immunotherapy comprises a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a toxin, a cell-based agent, or a cell-binding agent. Examples of cancer immunotherapies are described in more detail herein and are not intended to be limiting. In some embodiments, the cancer immunotherapy activates one or more aspects of the immune system to attack cells (e.g., tumor cells) that express neoantigens, e.g., neoantigens expressed by the cancers of the present disclosure. The cancer immunotherapies of the present disclosure are contemplated for use as monotherapy or in a combination approach including any combination or number of two or more, subject to medical judgment. Any of the cancer immunotherapies (optionally as monotherapy or in combination with another cancer immunotherapy or other therapeutic agent described herein) can be used in any of the methods described herein.

[0217] In some embodiments, the cancer immunotherapy comprises a cancer vaccine. Various cancer vaccines using different approaches to promote immune responses against cancer have been tested (see, for example, Emens LA, Expert Opin Emerg Drugs 13(2):295-308 (2008) and US20190367613). Approaches have been designed to enhance the response of B cells, T cells, or professional antigen-presenting cells to tumors. Exemplary types of cancer vaccines include, but are not limited to, DNA-based vaccines, RNA-based vaccines, viral transduction vaccines, peptide-based vaccines, dendritic cell vaccines, oncolytic viruses, whole tumor cell vaccines, tumor antigen vaccines, and the like. In some embodiments, the cancer vaccine can be prophylactic or therapeutic. In some embodiments, the cancer vaccine is formulated as a peptide-based vaccine, a nucleic acid-based vaccine, an antibody-based vaccine, or a cell-based vaccine. For example, vaccine compositions can include naked cDNA in cationic lipid formulations, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), naked cDNA or peptides encapsulated in, for example, poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991; Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune-stimulating complexes (ISCOMs) (e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Nature 344:873-875, 1990), and the like. al, Clin. Exp. Immunol. 113:235-243, 1998), or multiple antigen peptide systems (MAP) (see, e.g., Tam, JP, Proc. Natl. Acad. Sci. USA Natl. Acad. Sci. USA 85:5409-5413, 1988; Tam, JP, J. Immunol. Methods 196:17-32, 1996).In some embodiments, the cancer vaccine is formulated as a peptide-based vaccine or a nucleic acid-based vaccine (wherein the nucleic acid encodes the polypeptide). In some embodiments, the cancer vaccine is formulated as an antibody-based vaccine. In some embodiments, the cancer vaccine is formulated as a cell-based vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, and in some embodiments, a personalized peptide vaccine. In some embodiments, the cancer vaccine is a multivalent long peptide, multiple peptides, peptide mixtures, hybrid peptides, or peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104:14-21), 2013). In some embodiments, such cancer vaccines enhance anti-cancer responses.

[0218] In some embodiments, the cancer vaccine comprises a polynucleotide encoding a neoantigen, e.g., a neoantigen expressed by a cancer of the present disclosure. In some embodiments, the cancer vaccine comprises DNA encoding a neoantigen, e.g., a neoantigen expressed by a cancer of the present disclosure. In some embodiments, the cancer vaccine comprises RNA encoding a neoantigen, e.g., a neoantigen expressed by a cancer of the present disclosure. In some embodiments, the cancer vaccine comprises a polynucleotide encoding a neoantigen, e.g., a neoantigen expressed by a cancer of the present disclosure. In some embodiments, the cancer vaccine further comprises one or more additional antigens, neoantigens, or other sequences that enhance antigen presentation and / or immune response. In some embodiments, the polynucleotide is complexed with one or more additional agents, such as a liposome or lipoplex. In some embodiments, the polynucleotide is taken up and translated by antigen-presenting cells (APCs), which then present the neoantigen via MHC class I on the APC cell surface.

[0219] In some embodiments, the cancer vaccine is selected from sipuleucel-T (Provenge®, Dendreon / Valeant Pharmaceuticals), which is approved for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant (hormone-refractory) prostate cancer, and talimogene laherparepvec (Imlygic®, BioVex / Amgen, formerly known as T-VEC), a genetically modified oncolytic virus therapy approved for the treatment of unresectable cutaneous, subcutaneous, and lymph node disease in melanoma. In some embodiments, the cancer vaccine is selected from the group consisting of pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), which is a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312), pelareorep (Reolysin®, Oncolytics), and others.Biotech) (a variant of a respiratory enteric orphan virus (reovirus) that does not replicate in cells in which RAS is not activated, affecting many cancers, including colorectal cancer (NCT01622543), prostate cancer (NCT01619813), head and neck squamous cell carcinoma (NCT01166542), pancreatic adenocarcinoma (NCT00998322), and non-small cell lung cancer (NSCLC) (NCT00861627)); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAdl) (ovarian cancer (NCT02028117), metastatic or These include adenoviruses engineered to express full-length CD80 and antibody fragments specific for the T-cell receptor CD3 protein in advanced epithelial tumors (e.g., colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036)), ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF in melanoma (NCT03003676), and peritoneal disease, colorectal cancer, or ovarian cancer (NCT02963831), and GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux). GmbH) (which are vaccinia viruses engineered to express β-galactosidase (β-gal) / β-glucoronidase or β-gal / human sodium iodide symporter (hNIS), respectively, studied in peritoneal carcinomatosis (NCT01443260), fallopian tube cancer, and ovarian cancer (NCT02759588)), or CG0070 (Cold Genesys) (which is an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818)), anti-gp100, STINGVAX, GVAX, DCVaxL, and oncolytic virotherapy such as DNX-2401. In some embodiments, the cancer vaccine is selected from JX-929 (SillaJen / formerly Jennerex)In some embodiments, the cancer vaccine is selected from the group consisting of TILT Biotherapeutics (a TK-deficient and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which can convert the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil), TGO1 and TG02 (Targovax / formerly Oncos) (peptide-based immunotherapeutics targeting difficult-to-treat RAS mutations), TILT-123 (TILT Biotherapeutics) (an engineered adenovirus called Ad5 / 3-E2F-δ24-hTNFα-IRES-hIL20), and VSV-GP (ViraTherapeutics) (a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express antigens designed to elicit antigen-specific CD8+ T cell responses). In some embodiments, the cancer vaccine comprises a vector-based tumor antigen vaccine. Vector-based tumor antigen vaccines can be used as a method to provide a steady supply of antigens to stimulate anti-tumor immune responses. In some embodiments, vectors encoding tumor antigens are injected into individuals (perhaps along with pro-inflammatory agents or other attractants such as GM-CSF) and are taken up by cells in vivo to produce the specific antigen, which then elicits the desired immune response. In some embodiments, vectors can be used to deliver more than one tumor antigen at a time to increase the immune response. Furthermore, recombinant viral, bacterial, or yeast vectors can themselves elicit an immune response, which may also enhance the overall immune response.

[0220] In some embodiments, the cancer vaccine comprises a DNA-based vaccine. In some embodiments, DNA-based vaccines can be used to stimulate anti-tumor responses. The ability to induce a protective immune response by directly injecting DNA encoding an antigenic protein has been demonstrated in numerous experimental systems. Vaccination by directly injecting DNA encoding an antigenic protein to induce a protective immune response often provokes both cellular and humoral responses. Furthermore, reproducible immune responses to DNA encoding various antigens have been reported in mice, which persist essentially throughout the animal's lifetime (see, e.g., Yankauckas et al. (1993) DNA Cell Biol., 12:771-776). In some embodiments, plasmid (or other vector) DNA containing a protein-encoding sequence operably linked to regulatory elements necessary for gene expression is administered to an individual (e.g., a human patient, a non-human mammal, etc.). In some embodiments, the individual's cells take up the administered DNA, and the coding sequence is expressed. In some embodiments, the antigen so produced becomes the target of an immune response.

[0221] In some embodiments, the cancer vaccine comprises an RNA-based vaccine. In some embodiments, RNA-based vaccines can be used to stimulate anti-tumor responses. In some embodiments, the RNA-based vaccine comprises a self-replicating RNA molecule. In some embodiments, the self-replicating RNA molecule can be an RNA replicon derived from an alphavirus. Self-replicating RNA (or "SAM") molecules are well known in the art and can be generated, for example, by using replication elements derived from alphaviruses to replace structural viral proteins with nucleotide sequences encoding proteins of interest. Self-replicating RNA molecules are typically positive-strand molecules that can be directly translated after delivery to cells; this translation provides an RNA-dependent RNA polymerase that generates both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded polypeptide or can be transcribed to provide additional transcripts of the same sense strand as the delivered RNA, which can be translated to provide in situ expression of the antigen.

[0222] In some embodiments, cancer immunotherapy comprises cell-based therapy. In some embodiments, cancer immunotherapy comprises T cell-based therapy. In some embodiments, cancer immunotherapy comprises adoptive therapy, e.g., adoptive T cell-based therapy. In some embodiments, the T cells are autologous or allogeneic to the recipient. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the intent that the cells will mediate direct or indirect specific immunity against (i.e., mount an immune response against) cancer cells. In some embodiments, the immune response results in inhibition of growth and / or proliferation of tumor cells and / or metastatic cells, and in related embodiments, death and / or resorption of tumor cells. Immune cells can be derived from a different organism / host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells). In some embodiments, immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or γδ T cells), NK cells, invariant NK cells, or NKT cells) can be genetically engineered to express an antigen receptor, such as an engineered TCR and / or chimeric antigen receptor (CAR). For example, host cells (e.g., autologous or allogeneic T cells) are modified to express a T cell receptor (TCR) with antigen specificity for a cancer antigen. In some embodiments, NK cells are engineered to express a TCR. NK cells may be further engineered to express a CAR. Multiple CARs and / or TCRs, e.g., for different antigens, can be added to a single cell type, such as a T cell or NK cell. In some embodiments, the cells comprise one or more nucleic acids / expression constructs / vectors introduced via genetic engineering that encode one or more antigen receptors, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous. That is, it is not normally present in the cell or sample obtained from the cell, such as one obtained from another organism or cell, and is not normally found, for example, in the cell being manipulated and / or the organism from which such cell is derived.In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature (e.g., a chimera). In some embodiments, the population of immune cells can be obtained from a subject in need of therapy or a subject suffering from a disease associated with decreased immune cell activity. Thus, the cells are autologous to the subject in need of therapy. In some embodiments, the population of immune cells can be obtained from a donor (e.g., a histocompatibility-matched donor). In some embodiments, the immune cell population can be harvested from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. In some embodiments, the immune cells can be isolated from a pool of subjects and / or donors (e.g., pooled umbilical cord blood). In some embodiments, if the population of immune cells is obtained from a donor different from the subject, the donor can be allogeneic, as long as the resulting cells are subject-compatible in that they can be introduced into the subject. In some embodiments, allogeneic donor cells may or may not be human leukocyte antigen (HLA)-compatible. In some embodiments, allogeneic cells can be treated to reduce immunogenicity in order to be subject-compatible.

[0223] In some embodiments, cell-based therapies include T cell-based therapies, including autologous cells, e.g., tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCRs or chimeric TCR molecules (known as "T bodies," which exhibit antibody-like tumor recognition capabilities). Several approaches for the isolation, derivation, manipulation or modification, activation, and expansion of functional anti-tumor effector cells have been described over the past 20 years and can be used according to any of the methods provided herein. In some embodiments, T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells, such as cells isolated directly from a subject and / or cells isolated and frozen from a subject. In some embodiments, the cells include T cells or other cell types, e.g., one or more subsets of the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, proliferation, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the cells may be allogeneic and / or autologous. In some embodiments, such as off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0224] In some embodiments, T cell-based therapy includes chimeric antigen receptor (CAR)-T cell-based therapy. This approach involves engineering a CAR. The CAR specifically binds to an antigen of interest and contains one or more intracellular signaling domains for T cell activation. The CAR is then expressed on the surface of engineered T cells (CAR-T) and administered to a patient, resulting in a T cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the CAR specifically binds to a neo-antigen.

[0225] In some embodiments, T cell-based therapies involve T cells expressing a recombinant T cell receptor (TCR). This approach involves identifying a TCR that specifically binds to an antigen of interest. This is then used to replace the endogenous or natural TCR on the surface of engineered T cells. When administered to a patient, this results in a T cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the recombinant TCR specifically binds to a neo-antigen.

[0226] In some embodiments, the T cell-based therapy includes tumor-infiltrating lymphocytes (TILs). For example, TILs can be isolated from a tumor or cancer of the present disclosure, then isolated and expanded in vitro. Some or all of these TILs can specifically recognize antigens expressed by the tumor or cancer of the present disclosure. In some embodiments, the TILs are exposed to one or more neoantigens, e.g., one neoantigen, after being isolated in vitro. The TILs are then administered to the patient (optionally in combination with one or more cytokines or other immune stimulants).

[0227] In some embodiments, the cell-based therapy comprises natural killer (NK) cell-based therapy. Natural killer (NK) cells are a subpopulation of lymphocytes that possess spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are important effectors of the early innate immune response against transformed and virus-infected cells. NK cells can be detected by specific surface markers, such as human CD16, CD56, and CD8. NK cells do not express T cell antigen receptors, the pan-T marker CD3, or surface immunoglobulin B cell receptors. In some embodiments, NK cells are obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art.

[0228] In some embodiments, the cell-based therapy comprises dendritic cell (DC)-based therapy, e.g., a dendritic cell vaccine. In some embodiments, the DC vaccine comprises antigen-presenting cells capable of inducing specific T cell immunity, harvested from a patient or donor. In some embodiments, the DC vaccine can then be exposed to peptide antigens in vitro, generating T cells for such in the patient. In some embodiments, the antigen-loaded dendritic cells are then injected back into the patient. In some embodiments, immunizations can be repeated multiple times as needed. Methods for harvesting, expanding, and administering dendritic cells are known in the art. See, e.g., WO 2019 / 178081. Dendritic cell vaccines (e.g., Sipuleucel-T, also known as APC8015 and PROVENGE®) are vaccines that involve the administration of dendritic cells that function as APCs to present one or more cancer-specific antigens to the patient's immune system. In some embodiments, the dendritic cells are autologous or allogeneic to the recipient.

[0229] In some embodiments, the cancer immunotherapy comprises a TCR-based therapy. In some embodiments, the cancer immunotherapy comprises administration of one or more TCRs or TCR-based therapeutics that specifically bind to an antigen expressed by the cancer of the present disclosure. In some embodiments, the TCR-based therapeutic may further comprise a moiety that binds to an immune cell (e.g., a T cell), such as an antibody or antibody fragment (e.g., an anti-CD3 antibody or antibody fragment) that specifically binds to a T cell surface protein or receptor.

[0230] In some embodiments, immunotherapy includes adjuvant immunotherapy, which includes the use of one or more agents that activate components of the innate immune system, such as HILTONOL® (imiquimod), which targets the TLR7 pathway.

[0231] In some embodiments, immunotherapy comprises cytokine immunotherapy. Cytokine immunotherapy involves the use of one or more cytokines to activate components of the immune system. Examples include, but are not limited to, aldesleukin (PROLEUKIN®, interleukin-2), interferon alpha-2a (ROFERON®-A), interferon alpha-2b (INTRON®-A), and PEG-interferon alpha-2b (PEGINTRON®).

[0232] In some embodiments, immunotherapy includes oncolytic virotherapy, which uses genetically modified viruses to replicate in and kill cancer cells, releasing antigens that stimulate an immune response. In some embodiments, replication-competent oncolytic viruses that express tumor antigens include any naturally occurring (e.g., from a "field source") or modified replication-competent oncolytic viruses. In some embodiments, in addition to expressing tumor antigens, oncolytic viruses may be modified to increase the selectivity of the virus for cancer cells. In some embodiments, replication-competent oncolytic viruses include, but are not limited to, Myoviridae, Siphoviridae, Podoviridae, Tesiviridae, Corticoviridae, Plasmaviridae, Liposthrixviridae, Fuselloviridae, Poxyiridae, Iridoviridae, Phycodnaviridae, Baculoviridae, Herpesviridae, Adnoviridae, Papovaviridae, Polydnaviridae, Inoviridae, Microviridae, Geminiviridae, Circoviridae, Parvoviridae, Hepadnaviruses Oncolytic viruses include those that are members of the following families: Retroviridae, Cytoviridae, Reoviridae, Birnaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Leviviridae, Picornaviridae, Sequiviridae, Comoviridae, Potyviridae, Caliciviridae, Astroviridae, Nodaviridae, Tetraviridae, Tombusviridae, Coronaviridae, Graviviridae, Togaviridae, and Barnaviridae. In some embodiments, replication-competent oncolytic viruses include adenoviruses, retroviruses, reoviruses, rhabdoviruses, Newcastle disease virus (NDV), polyomaviruses, vaccinia viruses (VacV), herpes simplex viruses, picornaviruses, coxsackieviruses, and parvoviruses. In some embodiments, replicating oncolytic vaccinia viruses expressing tumor antigens may be engineered to lack one or more functional genes to enhance the cancer selectivity of the virus.In some embodiments, the oncolytic vaccinia virus is engineered to lack thymidine kinase (TK) activity. In some embodiments, the oncolytic vaccinia virus may be engineered to lack vaccinia virus growth factor (VGF). In some embodiments, the oncolytic vaccinia virus may be engineered to lack both VGF and TK activity. In some embodiments, the oncolytic vaccinia virus may be engineered to lack one or more genes involved in evading the host interferon (IFN) response, such as E3L, K3L, B18R, or B8R. In some embodiments, the replicative oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain and lacks a functional TK gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain that lacks a functional B18R and / or B8R gene. In some embodiments, the replicating oncolytic vaccinia virus expressing a tumor antigen may be administered locally or systemically to a subject, for example, via intratumoral, intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intracranial, subcutaneous, or intranasal administration.

[0233] In some embodiments, the anti-cancer therapy comprises an immune checkpoint inhibitor. In some embodiments, the methods provided herein comprise administering an immune checkpoint inhibitor to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the methods provided herein comprise administering an effective amount of an immune checkpoint inhibitor to an individual. As is known in the art, checkpoint inhibitors target at least one immune checkpoint protein to alter the regulation of the immune response. Immune checkpoint proteins include, for example, CTLA4, PD-L1, PD-1, PD-L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CEACAM, LAIR1, CD80, CD86, CD276, VTCN1, MHC class I, MHC class II, GALS, adenosine, TGFR, CSF1R, MICA / B, arginase, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, OX40, and A2aR.In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to, PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-CD, CD273), CTLA-4 (CD152), HVEM, BTLA (CD272), killer cell immunoglobulin-like receptors (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM-1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRβ, A2AR, GITR (CD357), CD80 (B7-1), CD86 (B7- 2), CD276 (B7-H3), VTCNI (B7-H4), MHC class I, MHC class II, GALS, adenosine, TGFR, B7-H1, OX40 (CD134), CD94 (KLRD1), CD137 (4-1BB), CD137L (4-1BBL), CD40, IDO, CSF1R, CD40L, CD47, CD70 (CD27L), CD226, HHLA2, ICOS (CD278), ICOSL (CD275), LIGHT (TNFSF14, CD258), NKG2a, NKG2d, OX40L (CD134L), PVR (NECL5, CD155), SIRPa, MICA / B, and / or arginase. In some embodiments, immune checkpoint inhibitors (i.e., checkpoint inhibitors) decrease the activity of checkpoint proteins that negatively regulate immune cell function, e.g., to enhance T cell activation and / or anti-cancer immune responses. In other embodiments, checkpoint inhibitors increase the activity of checkpoint proteins that positively regulate immune cell function, e.g., to enhance T cell activation and / or anti-cancer immune responses. In some embodiments, the checkpoint inhibitor is an antibody.Examples of checkpoint inhibitors include, but are not limited to, PD-1 axis-binding antagonists, PD-L1 axis-binding antagonists (e.g., anti-PD-L1 antibodies, e.g., atezolizumab (MPDL3280A)), antagonists against co-inhibitory molecules (e.g., CTLA4 antagonists (e.g., anti-CTLA4 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or LAG-3 antagonists (e.g., anti-LAG-3 antibodies)), or any combination thereof. In some embodiments, immune checkpoint inhibitors include drugs such as small molecules, recombinant forms of ligands or receptors, or antibodies (e.g., human antibodies) (see, e.g., International Patent Publication No. WO 2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012, both of which are incorporated herein by reference). In some embodiments, known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies.

[0234] In some embodiments, the checkpoint inhibitor is a PD-L1 axis-binding antagonist (e.g., a PD-1-binding antagonist, a PD-L1-binding antagonist, or a PD-L2-binding antagonist). PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is set forth in UniProtKB / Swiss-Prot accession number Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1," "PDCD 1LG1," "CD274," "B7-H," and "PDL1." An exemplary human PD-L1 is set forth in UniProtKB / Swiss-Prot accession number Q9NZQ7.1. PD-L2 (programmed death-ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1 LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is set forth in UniProtKB / Swiss-Prot accession number Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0235] In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain embodiments, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another example, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding ligand. In certain embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In another example, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partner. In certain embodiments, the PD-L2 binding ligand partner is PD-1. The antagonist can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the PD-1 binding antagonist is a small molecule, nucleic acid, polypeptide (e.g., antibody), carbohydrate, lipid, metal, or toxin.

[0236] In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), such as those described below. In some instances, the anti-PD-1 antibody is one or more of MDX-1106 (nivolumab), MK-3475 (pembrolizumab, Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, or BGB-108. In other examples, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some instances, the PD-1 binding antagonist is AMP-224. Other examples of anti-PD-1 antibodies include MEDI-0680 (AMP-514; AstraZeneca), PDR001 (CAS Registry Number 1859072-53-9; Novartis), REGN2810 (LIBTAYO® or cemiplimab-rwlc; Regeneron), BGB-108 (BeiGene), BGB-A317 (BeiGene), BI 754091, JS-001 (Shanghai Junshi), STI-A1110 (Sorrento), INCSHR-1210 (Incyte), PF-06801591 (Pfizer), TSR-042 (also known as ANB011; Tesaro / AnaptysBio), AM0001 (ARMO Biosciences), ENUM244C8 (Enumeral Biomedical Holdings), or ENUM388D4 (Enumeral Biomedical Holdings).In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of tislelizumab (BGB-A317), BGB-108, STI-A1110, AM0001, BI754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), and rituximab (RT-PCR). ), MEDI-0680 (AMP-514), MGA-012 (INCMGA0012), nivolumab (BMS-936558, MDX1106, ONO-4538), spartalizumab (PDR00l), pembrolizumab (MK-3475, SCH900475, Keytruda®), PF-06801591, cemiplimab (REGN-2810, REGEN 2810), dostallimab (TSR-042, ANB011), FITC-YT-16 (PD-1 binding peptide), APL-501 or CBT-501 or genolimuzumab (GB-226), AB-122, AK105, AMG404, BCD-100, F520, HLX10, HX008, JTX-4014, LZM009, Sym021, PSB205, AMP- 224 (fusion protein targeting PD-1), CX-188 (PD-1 probody), AGEN-2034, GLS-010, budigalimab (ABBV-181), AK-103, BAT-1306, CS-1003, AM-0001, TILT-123, BH-2922, BH-2941, BH-2950, ​​ENUM-244C8, ENUM-388D4, HAB-21, H EISCOI11-003, IKT-202, MCLA-134, MT-17000, PEGMP-7, PRS-332, RXI-762, STI-1110, VXM-10, XmAb-23104, AK-112, HLX-20, SSI-361, AT-16201, SNA-01, AB122, PD1-PIK, PF-06936308, RG-7 769, CABPD-1 Abs, AK-123, MEDI-3387, MEDI-5771, 4H1128Z-E27, REMD-288, SG-001, BY-24.3, CB-201, IBI-319, ONCR-177, Max-1, CS-4100, JBI-426, CCC-0701, or CCX-4503, or derivatives thereof.

[0237] In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA or PD-L1 and TIM3. In some embodiments, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody can bind to human PD-L1, for example, the human PD-L1 set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the PD-L1 binding antagonist is a small molecule, nucleic acid, polypeptide (e.g., antibody), carbohydrate, lipid, metal, or toxin.

[0238] Optionally, the PD-L1 binding antagonist is an anti-PD-L1 antibody, such as those described below. Optionally, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or the binding between PD-L1 and B7-1. Optionally, the anti-PD-L1 antibody is a monoclonal antibody. Optionally, the anti-PD-L1 antibody is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. Optionally, the anti-PD-L1 antibody is a humanized antibody. Optionally, the anti-PD-L1 antibody is a human antibody. Optionally, the anti-PD-L1 antibody is selected from YW243.55.S70, MPDL3280A (atezolizumab), MDX-1 105, MEDI4736 (durvalumab), or MSB0010718C (avelumab).In some embodiments, the PD-L1 axis binding antagonist is atezolizumab, avelumab, durvalumab (Imfinzi), BGB-A333, SHR-1316 (HTI-1088), CK-301, BMS-936559, embafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS-936559), LY330 0054, STI-A1014, FAZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M-7824, HTI-1088(H TI-131, SHR-1316), MSB-2311, AK-106, AVA-004, BBI-801, CA-327, CBA-0710, CBT-502, FPT-155 , IKT-201, IKT-703, 10-103, JS-003, KD-033, KY-1003, MCLA-145, MT-5050, SNA-02, BCD-135, AP L-502 (CBT-402 or TQB2450), IMC-001, KD-045, INBRX-105, KN-046, IMC-2102, IMC-2101, KD-005 , IMM-2502, 89Zr-CX-072, 89Zr-DFO-6E11, KY-1055, MEDI-1109, MT-5594, SL-279252, DSP-106, Gensci-047, REMD-290, N-809, PRS-344, FS-222, GEN-1046, BH-29xx, or FS-118, or derivatives thereof.

[0239] In some embodiments, the checkpoint inhibitor is an antagonist of CTLA4. In some embodiments, the checkpoint inhibitor is a small molecule antagonist of CTLA4. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody. CTLA4 is part of the CD28-B7 immunoglobulin superfamily of immune checkpoint molecules and acts to negatively regulate T cell activation (particularly CD28-dependent T cell responses). CTLA4 competes for binding to ligands shared with CD28, such as CD80 (B7-1) and CD86 (B7-2), and binds these ligands with higher affinity than CD28. Blockade of CTLA4 activity (e.g., using an anti-CTLA4 antibody) is thought to enhance CD28-mediated costimulation (leading to increased T cell activation / priming), affect T cell development, and / or deplete Tregs (e.g., intratumoral Tregs). In some embodiments, the CTLA4 antagonist is a small molecule, nucleic acid, polypeptide (e.g., an antibody), carbohydrate, lipid, metal, or toxin. In some embodiments, the CTLA-4 inhibitor comprises ipilimumab (IBI310, BMS-734016, MDX010, MDX-CTLA4, MEDI4736), tremelimumab (CP-675, CP-675, 206), APL-509, AGEN1884, CS1002, AGEN1181, abatacept (Orencia, BMS-188667, RG2077), BCD-145, ONC-392, ADU-1604, REGN4659, ADG116, KN044, KN046, or a derivative thereof.

[0240] In some embodiments, the anti-PD-1 antibody or antibody fragment is MDX-1106 (nivolumab), MK-3475 (pembrolizumab, Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, BGB-108, BGB-A317, JS-001, STI-A1110, INCSHR-1210, PF-06801591, TSR-042, AM0001, ENUM244C8, or ENUM388D4. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 immunoadhesin. In some embodiments, the anti-PD-1 immunoadhesin is AMP-224. In some embodiments, the anti-PD-L1 antibody or antibody fragment is YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), LY3300054, STI-A1014, KN035, FAZ053, or CX-072.

[0241] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 inhibitor (e.g., an antibody, antibody conjugate, or antigen-binding fragment thereof). In some embodiments, the LAG-3 inhibitor comprises a small molecule, a nucleic acid, a polypeptide (e.g., an antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the LAG-3 inhibitor comprises a small molecule. In some embodiments, the LAG-3 inhibitor comprises a LAG-3 binding agent. In some embodiments, the LAG-3 inhibitor comprises an antibody, antibody conjugate, or antigen-binding fragment thereof. In some embodiments, the LAG-3 inhibitor comprises eftiragimode alfa (IMP321, IMP-321, EDDP-202, EOC-202), leratolimab (BMS-986016), GSK2831781 (IMP-731), LAG525 (IMP701), TSR-033, EVIP321 (soluble LAG-3 protein), BI754111, IMP761, REGN3767, MK-4280, MGD-013, XmAb22841, INCAGN-2385, ENUM-006, AVA-017, AM-0003, iOnctura anti-LAG-3 antibody, Arcus Biosciences LAG-3 antibody, Sym022, a derivative thereof, or an antibody that competes with any of the foregoing.

[0242] In some embodiments, the anti-cancer therapy comprises an immunomodulatory molecule or cytokine. In some embodiments, the methods provided herein comprise administering an immunomodulatory molecule or cytokine to an individual, e.g., in combination with another anti-cancer therapy. An immunomodulatory profile is necessary to elicit an efficient immune response and balance the subject's immunity. Examples of suitable immunomodulatory cytokines include, but are not limited to, interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1α, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), and functional fragments thereof. In some embodiments, any immunomodulatory chemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, or CX3C chemokine receptor, can be used in the context of the present disclosure. Examples of chemokines include, but are not limited to, MIP-3α (Lax), MIP-3β, Hcc-1, MPIF-1, MPIF-2, MCP-2, MCP-3, MCP-4, MCP-5, eotaxin, Tarc, Elc, I309, IL-8, GCP-2 Groα, Gro-β, Nap-2, Ena-78, Ip-10, MIG, I-Tac, SDF-1, or BCA-1 (Blc), as well as functional fragments thereof. In some embodiments, an immunomodulatory molecule is included in any of the treatments provided herein.

[0243] In some embodiments, the immune checkpoint inhibitor is monovalent and / or monospecific, hi some embodiments, the immune checkpoint inhibitor is multivalent and / or multispecific.

[0244] In some embodiments, the anti-cancer therapy comprises a nucleic acid molecule such as a dsRNA, siRNA, or shRNA. In some embodiments, the methods provided herein comprise administering to an individual a nucleic acid molecule such as a dsRNA, siRNA, or shRNA, e.g., in combination with another anti-cancer therapy. As is known in the art, dsRNA having a double-stranded structure is effective at inducing RNA interference (RNAi). In some embodiments, the anti-cancer therapy comprises a small interfering RNA molecule (siRNA). dsRNA and siRNA can be used to silence gene expression in mammalian cells (e.g., human cells). In some embodiments, the dsRNA of the present disclosure comprises any of about 5 to about 10 base pairs, about 10 to about 12 base pairs, about 12 to about 15 base pairs, about 15 to about 20 base pairs, about 20 to 23 base pairs, about 23 to about 25 base pairs, about 25 to about 27 base pairs, or about 27 to about 30 base pairs. As known in the art, siRNA is a short dsRNA that optionally contains an overhang. In some embodiments, the double-stranded region of an siRNA is about 18 to 25 nucleotides, for example, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. siRNA also includes short hairpin RNAs (shRNAs), for example, those having a stem of approximately 29 base pairs and a 2-nucleotide 3' overhang. Methods for designing, optimizing, producing, and using dsRNA, siRNA, or shRNA are known in the art.

[0245] In some embodiments, the anti-cancer therapy comprises chemotherapy. In some embodiments, the methods provided herein comprise administering chemotherapy to an individual, e.g., in combination with another anti-cancer therapy. Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide), alkylsulfonates (e.g., busulfan, improsulfan, piposulfan), aziridines (e.g., benzodopa, carboquone, mesuredopa, and uredopa), ethylenimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine), acetogenins (particularly bullatacin and blutamin), and the like. latacinone), camptothecin (including the synthetic analog topotecan), bryostatin, kallistatin, CC-1065 (including the synthetic analogs adozelesin, carzelesin, and bizelesin), cryptophycins (particularly cryptophycin 1 and cryptophycin 8), dolastatin, duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictin, spongistatin, nitrogen mustards (e.g., chlorambucil, clorambucil, nitrosoureas (carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine); antibiotics (e.g., enediyne antibiotics such as calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); , dynemicins (including, for example, dynemicin A), bisphosphonates (for example, clodronate), esperamicin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,Doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin), antimetabolites (e.g., methotrexate, and 5-fluorouracil (5-FU)), folic acid analogs (e.g., denopterin, pteropterin, trimetrexate), purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine), androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and tesulfamethasone), antiadrenal agents (e.g., mitotane and trilostane), folic acid supplements (e.g., folic acid), aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatlaxate, defoamin, demecolcine, diaziconazole, elformitin, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids (e.g., maytansine and ansamitocin), mitoguazone, mitoxantrone, mopidanmol, nifedipine, Traeline, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid, triazicon, 2,2',2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C");Cyclophosphamide, taxoids (e.g., paclitaxel and docetaxel gemcitabine), 6-thioguanine, mercaptopurine, platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin), vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-1 l), the topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids (e.g., retinoic acid), capecitabine, carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0246] Some non-limiting examples of chemotherapeutic agents that can be combined with the anti-cancer therapies of the present disclosure include carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Folex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar). PFS), and vinblastine (Velban).

[0247] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor. In some embodiments, the methods provided herein comprise administering a kinase inhibitor to an individual, e.g., in combination with another anti-cancer therapy. Examples of kinase inhibitors include those that target one or more receptor tyrosine kinases, such as BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-α, PDGFR-β, cKit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, or ALK; one or more cytoplasmic tyrosine kinases, such as c-SRC, c-YES, Abl, or JAK-2; one or more serine / threonine kinases, such as ATM, Aurora A and B, CDK, mTOR, PKCi, PLK, b-Raf, S6K, or STK11 / LKB1; or one or more lipid kinases, such as PI3K or SKI. Small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, PD166326, NSC743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sutent (SU1 1248), sorafenib (BAY43-9006), or leflunomide (SU101). Additional non-limiting examples of tyrosine kinase inhibitors include imatinib (Gleevec / Glivec) and gefitinib (Iressa).

[0248] In some embodiments, the anti-cancer therapy includes an anti-angiogenic agent. In some embodiments, the methods provided herein include administering an anti-angiogenic agent to an individual, e.g., in combination with another anti-cancer therapy. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors need to survive. Non-limiting examples of angiogenesis-mediating molecules or angiogenesis-inhibiting molecules that can be used in the methods of the present disclosure include soluble VEGF (e.g., VEGF isoforms (e.g., VEGF121 and VEGF165), VEGF receptors (e.g., VEGFR1, VEGFR2), and co-receptors (e.g., neuropilin-1 and neuropilin-2)), NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMPs and CDAI, Meth-1 and Meth-2, IFN-α, IFN-β and IFN-γ, CXCL10, IL-4, IL-12, and IL-18, prothrombin (kringle domain- 2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, and drugs (e.g., bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-α, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiogenic steroids, and heparin), cartilage-derived angiogenesis inhibitor, matrix metalloproteinase inhibitor, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin vβ3 inhibitor, linomide, or tasquinimod. In some embodiments, known therapeutic candidates that can be used according to the methods of the present disclosure include naturally occurring angiogenesis inhibitors, including, but not limited to, angiostatin, endostatin, or platelet factor-4. In another embodiment, therapeutic drug candidates that may be used according to the methods of the present disclosure include specific inhibitors of endothelial cell proliferation, such as, but not limited to, TNP-470, thalidomide, and interleukin-12.Still other antiangiogenic agents that can be used in accordance with the methods of the present disclosure include, but are not limited to, those that neutralize angiogenic molecules, such as antibodies against fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, or antibodies or other types of inhibitors of EGF, VEGF, or PDGF receptors. In some embodiments, antiangiogenic agents that can be used in accordance with the methods of the present disclosure include, but are not limited to, suramin and its analogs, and tecogalan. In other embodiments, antiangiogenic agents that can be used in accordance with the methods of the present disclosure include, but are not limited to, agents that neutralize receptors for angiogenic factors or agents that disrupt vascular basement membranes and extracellular matrices, including, but not limited to, metalloprotease inhibitors and antiangiogenic steroids. Another group of antiangiogenic compounds that can be used in accordance with the methods of the present disclosure includes anti-adhesion molecules, such as, but not limited to, antibodies against integrin αvβ3. Still other antiangiogenic compounds or compositions that can be used in accordance with the methods of the present disclosure include kinase inhibitors, thalidomide, itraconazole, carboxyamidotriazole, CM101, IFN-α, IL-12, SU5416, thrombospondin, cartilage-derived angiogenesis inhibitor, 2-methoxyestradiol, tetrathiomolybdate, thrombospondin, prolactin, and linomide. In one particular embodiment, an antiangiogenic compound that can be used in accordance with the methods of the present disclosure is an antibody against VEGF, such as Avastin® / bevacizumab (Genentech).

[0249] In some embodiments, the anti-cancer therapy comprises an anti-DNA repair therapy. In some embodiments, the methods provided herein comprise administering an anti-DNA repair therapy to an individual, for example, in combination with another anti-cancer therapy. In some embodiments, the anti-DNA repair therapy is a PARP inhibitor (e.g., talazoparib, rucaparib, olaparib), a RAD51 inhibitor (e.g., RI-1), or an inhibitor of a DNA damage response kinase (e.g., CHCK1 (e.g., AZD7762), ATM (e.g., KU-55933, KU-60019, NU7026, or VE-821), and ATR (e.g., NU7026)).

[0250] In some embodiments, the anti-cancer therapy includes a radiosensitizer. In some embodiments, the methods provided herein include administering a radiosensitizer to an individual, for example, in combination with another anti-cancer therapy. Exemplary radiosensitizers include hypoxia radiosensitizers (e.g., misonidazole, metronidazole) and trans-sodium crocetinate, a compound that helps increase oxygen diffusion to hypoxic tumor tissue. Radiosensitizers can also be DNA damage response inhibitors that interfere with base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), including homologous recombination (HR), non-homologous end joining (NHEJ), and direct repair mechanisms. Single-strand break (SSB) repair mechanisms include the BER, NER, or MMR pathways, while double-strand break (DSB) repair mechanisms are composed of the HR and NHEJ pathways. Radiation causes DNA breaks that are lethal if not repaired. SSBs are repaired through a combination of BER, NER, and MMR mechanisms, using intact DNA strands as templates. The primary pathway for SSB repair is BER, which utilizes a family of related enzymes called poly(ADP-ribose) polymerases (PARPs). Therefore, radiosensitizers can include DNA damage response inhibitors, such as PARP inhibitors.

[0251] In some embodiments, the anti-cancer therapy includes an anti-inflammatory agent. In some embodiments, the methods provided herein include administering an anti-inflammatory agent to an individual, e.g., in combination with another anti-cancer therapy. In some embodiments, the anti-inflammatory agent is an agent that blocks, inhibits, or reduces inflammation or signaling from an inflammatory signaling pathway. In some embodiments, the anti-inflammatory agent inhibits or reduces the activity of any one or more of the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, interferon (IFN), e.g., IFNα, IFNβ, IFNγ, IFN-γ-inducing factor (IFN-γ), transforming growth factor-β (IFN-β), ... TGF-β), transforming growth factor-α (TGF-α), tumor necrosis factor (e.g., TNF-α, TNF-β, TNF-RI, TNF-RII), CD23, CD30, CD40L, EGF, G-CSF, GDNF, PDGF-BB, RANTES / CCL5, IKK, NF-κB, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and / or their cognate receptors. In some embodiments, the anti-inflammatory agent is IL-1 or an IL-1 receptor antagonist, e.g., anakinra (Kineret®), rilonacept, or canakinumab. In some embodiments, the anti-inflammatory agent is an IL-6 or IL-6 receptor antagonist, e.g., an anti-IL-6 antibody or an anti-IL-6 receptor antibody (such as tocilizumab (ACTEMRA®), olokizumab, clazakizumab, sarilumab, sirukumab, siltuximab, or ALX-0061). In some embodiments, the anti-inflammatory agent is a TNF-α antagonist, e.g., an anti-TNFα antibody (such as infliximab (Remicade®), golimumab (Simponi®), adalimumab (Humira®), certolizumab pegol (Cimzia®), or etanercept). In some embodiments, the anti-inflammatory agent is a corticosteroid.Examples of corticosteroids include, but are not limited to, cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, Ala-Cort®, Hydrocort Acetate®, hydrocortone phosphate, Lanacort®, Solu-Cortef®), Decadron (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, Dexasone®, Diodex®, Hexadrol®, Maxidex®), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, Duralone®, Medralone®, Medrol®, M-Prednisol®, Solu-Medrol®), prednisolone (Delta-Cortef®, ORAPRED®, Pediapred®, Prezone®), and prednisone (Deltasone®, Liquid Pred®, Meticorten®, Orasone®), and bisphosphonates (e.g., pamidronate (Aredia®), and zoledronic acid (Zometac®)).

[0252] In some embodiments, the anti-cancer therapy comprises an anti-hormonal agent. In some embodiments, the methods provided herein comprise administering to an individual an anti-hormonal agent, e.g., in combination with another anti-cancer therapy. Anti-hormonal agents are agents that act to regulate or inhibit hormone action on tumors. Examples of antihormonal agents include antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON®, toremifene, aromatase inhibitors (which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands) (e.g., 4(5)-imidazole, aminoglutethimide, MEGACE® megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® (anastrozole). anti-androgens (e.g., flutamide, nilutamide, bicalutamide, leuprolide, goserelin), troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides (particularly oligonucleotides that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R)); vaccines such as gene therapy vaccines (e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine), PROLEUKIN® rIL-2, LURTOTECAN® topoisomerase 1 inhibitor, ABARELIX® rmRH, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0253] In some embodiments, the anti-cancer therapy includes an antimetabolite chemotherapeutic agent. In some embodiments, the methods provided herein include administering an antimetabolite chemotherapeutic agent to an individual, e.g., in combination with another anti-cancer therapy. Antimetabolite chemotherapeutic agents are drugs that are structurally similar to metabolites but cannot be productively used by the body. Many antimetabolite chemotherapeutic agents interfere with the production of RNA or DNA. Examples of antimetabolite chemotherapeutic agents include gemcitabine (GEMZAR®), 5-fluorouracil (5-FU), capecitabine (XELODA™), 6-mercaptopurine, methotrexate, 6-thioguanine, pemetrexed, raltitrexed, arabinosylcytosine, ARA-C, cytarabine (CYTOSAR-U®), dacarbazine (DTIC-DOMED), azocytosine, deoxycytosine, pyridomidene, fludarabine (FLUDARA®), cladrabine, and 2-deoxy-D-glucose. In some embodiments, the antimetabolite chemotherapeutic agent is gemcitabine. Gemcitabine hydrochloride is sold by Eli Lilly under the trademark GEMZAR®.

[0254] In some embodiments, the anti-cancer therapy comprises a platinum-based chemotherapeutic agent. In some embodiments, the methods provided herein comprise administering a platinum-based chemotherapeutic agent to an individual, e.g., in combination with another anti-cancer therapy. A platinum-based chemotherapeutic agent is a chemotherapeutic agent comprising an organic compound containing platinum as an integral part of the molecule. In some embodiments, the chemotherapeutic agent is a platinum agent. In some such embodiments, the platinum agent is selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin.

[0255] In some aspects, provided herein are therapeutic formulations comprising an anti-cancer therapy provided herein and a pharmaceutically acceptable carrier, excipient, or stabilizer. The formulations provided herein may contain more than one active compound, e.g., an anti-cancer therapy provided herein and one or more additional agents (e.g., anti-cancer agents).

[0256] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, for example, buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol; low molecular weight polypeptides (e.g., less than about 10 residues). the hydrophilic polymer, such as polyvinylpyrrolidone; an amino acid, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, a disaccharide, and other carbohydrate, including glucose, mannose, or dextrin; a chelating agent, such as EDTA; a sugar, such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion, such as sodium; a metal complex (e.g., a Zn-protein complex); a surfactant, such as a non-ionic surfactant; or a polymer, such as polyethylene glycol (PEG).

[0257] The active ingredient may be encapsulated in microcapsules. Such microcapsules may be prepared, for example, by coacervation techniques or by interfacial polymerization of, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or in macroemulsions. Such techniques are known in the art.

[0258] Sustained-release compositions may also be prepared. Suitable examples of sustained-release compositions include semipermeable matrices of solid hydrophobic polymers containing the anticancer therapy of the present disclosure. Such matrices may be in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0259] The formulations provided herein may also contain more than one active compound, e.g., those with complementary activities that do not adversely affect each other. The type and effective amount of such medicaments will depend, for example, on the amount and type of active compound present in the formulation and the clinical parameters of the subject.

[0260] For general information on formulations, see, for example, Gilman et al. (eds.) The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press, 1990; A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Pennsylvania, 1990; Avis et al. (eds.) Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New York, 1993; Lieberman et al. (eds.) Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New York, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York, 1990; and Walters (ed.) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol. 1 19, Marcel See Dekker, 2002.

[0261] The formulations to be used for in vivo administration are sterile, which is readily accomplished by filtration through sterile filtration membranes, or other methods known in the art.

[0262] In some embodiments, the anti-cancer therapy is administered as a monotherapy. In some embodiments, the anti-cancer therapy is administered in combination with one or more additional anti-cancer therapies or treatments. In some embodiments, the one or more additional anti-cancer therapies or treatments include one or more anti-cancer therapies described herein. In some embodiments, the methods of the present disclosure include administration of any combination of any of the anti-cancer therapies provided herein. In some embodiments, the additional anti-cancer therapy includes one or more of surgery, radiation therapy, chemotherapy, anti-angiogenic therapy, anti-DNA repair therapy, and anti-inflammatory therapy. In some embodiments, the additional anti-cancer therapy includes an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic therapy, radiation therapy, a cytotoxic agent, or a combination thereof. In some embodiments, the anti-cancer therapy may be administered in combination with chemotherapy or a chemotherapeutic agent. In some embodiments, the chemotherapy or chemotherapeutic agent is a platinum-based agent (including, but not limited to, cisplatin, carboplatin, oxaliplatin, and straplatin). In some embodiments, the anti-cancer therapy may be administered in combination with radiation therapy. In some embodiments, the anti-cancer therapy for use in any of the methods described herein (e.g., as a monotherapy or in combination with another therapy or treatment) is an anti-cancer therapy or treatment described by Pietrantonio et al., J Natl Cancer Inst (2017) 109(12) and / or Wang et al., Cancers (2020) 12(2):426, which are incorporated herein by reference.

[0263] kit Also provided herein are kits for improving nucleic acid sequence analysis and / or extraction according to any one of the methods described herein.

[0264] In some embodiments, the kits include reagents and instructions for carrying out the methods of the present disclosure.

[0265] Also provided herein are kits for detecting biomarker nucleic acid molecules of the present disclosure in RNA and / or DNA samples extracted from embedded samples, e.g., as described herein. In some embodiments, the kits provided herein include reagents for detecting the biomarker nucleic acid molecules provided herein (e.g., one or more oligonucleotides, primers, probes, or baits of the present disclosure). In some embodiments, the kits include reagents for detecting wild-type counterparts of the biomarker nucleic acid molecules provided herein (e.g., one or more oligonucleotides, primers, probes, or baits of the present disclosure). In some embodiments, the reagents include one or more oligonucleotides, primers, probes, or baits of the present disclosure that can hybridize to the biomarker nucleic acid molecules provided herein or the wild-type counterparts of the biomarker nucleic acid molecules provided herein. In some embodiments, the reagents include one or more oligonucleotides, primers, probes, or baits of the present disclosure that can distinguish the biomarker nucleic acid molecules provided herein from the wild-type counterparts of the biomarker nucleic acid molecules provided herein. In some embodiments, the kits are for use in accordance with any method of detecting biomarker nucleic acid molecules known in the art or described herein, such as sequencing, PCR, in situ hybridization methods, nucleic acid hybridization assays, amplification-based assays, PCR-RFLP assays, real-time PCR, sequencing, next-generation sequencing, screening assays, FISH, spectral karyotyping, MFISH, comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, HPLC, and mass spectrometry genotyping. In some embodiments, the kits provided herein further include instructions for detecting biomarker nucleic acid molecules of this disclosure, e.g., using one or more oligonucleotides, primers, probes, or baits of this disclosure.

[0266] Illustrative Embodiments The following exemplary embodiments are representative of some aspects of the present invention.

[0267] Embodiment 1. A method of detecting alterations in RNA and / or DNA comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to cause it to separate from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA.

[0268] Embodiment 2. The method of embodiment 1, wherein detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

[0269] Embodiment 3. The method of embodiment 1, wherein detection of alterations in RNA and / or DNA is improved compared to methods of removing paraffin by cleaving the paraffin from a paraffin-embedded sample.

[0270] Embodiment 4. The method of embodiment 1, wherein detection of alterations in RNA and / or DNA is improved compared to methods in which paraffin-embedded samples are not deparaffinized.

[0271] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the alteration in the RNA and / or DNA is selected from the group consisting of a) copy number alteration, b) point mutation, c) in-frame deletion of one or more codons, d) intragenic deletion, e) intragenic insertion, f) whole gene deletion, g) inversion, h) interchromosomal translocation, i) tandem duplication, j) gene fusion, k) genomic rearrangement involving intronic sequences, and / or l) gene amplification or duplication.

[0272] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the modification in the RNA and / or DNA is a copy number modification.

[0273] Embodiment 7. A method of extracting RNA and / or DNA comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin, causing the paraffin to separate from the sample; and c) extracting RNA and / or DNA from the deparaffinized sample.

[0274] Embodiment 8. The method of embodiment 7, wherein extraction of RNA and / or DNA is improved compared to methods in which paraffin is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

[0275] Embodiment 9. The method of embodiment 7, wherein extraction of RNA and / or DNA is improved compared to methods of removing paraffin by cleaving the paraffin from paraffin-embedded samples.

[0276] Embodiment 10. A method for improving library construction for nucleic acid sequencing, comprising: a) providing a paraffin-embedded sample; b) removing paraffin from the paraffin-embedded sample to produce a deparaffinized sample, wherein the paraffin is removed by inducing a phase transition in the paraffin to cause it to separate from the sample; c) extracting RNA and / or DNA from the deparaffinized sample; and d) preparing a se...

Claims

1. 1. A method for detecting an analyte, comprising: a) providing an embedded sample containing an analyte, the sample being embedded in an embedding agent; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, (i) inducing a phase transition in said embedding medium; (ii) contacting the embedded sample with an immiscible solvent; removing the embedding medium by separating the embedding medium from the sample; c) extracting the analyte from the de-embedded sample; d) analyzing said analyte to detect said analyte.

2. 2. The method of claim 1, wherein the detection of the analyte is improved compared to a method in which the embedding medium is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

3. The method of claim 1 , wherein the detection of the analyte is improved compared to a method in which the embedding medium is removed by separating the embedding medium from the embedded sample.

4. 10. The method of claim 1, wherein the detection of the analyte is improved compared to a method in which the embedded sample is not de-embedded.

5. 1. A method for extracting an analyte, comprising: a) providing an embedded sample containing an analyte, the sample being embedded in an embedding agent; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, (i) inducing a phase transition in said embedding medium; (ii) contacting the embedded sample with an immiscible solvent; removing the embedding medium by separating the embedding medium from the sample; c) extracting said analyte from said de-embedded sample.

6. 6. The method of claim 5, wherein the extraction of the analyte is improved compared to a method in which the embedding agent is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

7. 6. The method of claim 5, wherein the extraction of the analyte is improved compared to a method of removing the embedding medium by separating the embedding medium from the embedded sample.

8. 1. A method for reducing the level of embedding medium in an analyte sample extracted from an embedded sample, comprising: a) providing an embedded sample containing an analyte, the sample being embedded in an embedding agent; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, (i) inducing a phase transition in said embedding medium; (ii) contacting the embedded sample with an immiscible solvent; removing the embedding medium by separating the embedding medium from the sample; c) extracting the analyte from the de-embedded sample; d) purifying the extracted analyte to provide an analyte sample.

9. 9. The method of claim 8, wherein the level of embedding agent in the analyte sample is reduced compared to a method in which the embedding agent is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

10. 9. The method of claim 8, wherein the level of embedding medium in the analyte sample is reduced compared to a method of removing the embedding medium by separating the embedding medium from the embedded sample.

11. 1. A method for improving the separation of an embedding medium from an embedded sample, comprising: a) providing an embedded sample containing an analyte, the sample being embedded in an embedding agent; b) removing the embedding medium from the embedded sample to produce a de-embedded sample, (i) inducing a phase transition in said embedding medium; (ii) contacting the embedded sample with an immiscible solvent; removing the embedding medium by separating the embedding medium from the sample; c) extracting an analyte from said de-embedded sample.

12. 12. The method of claim 11, wherein separation of the embedding medium from the embedded sample is improved compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

13. 12. The method of claim 11, wherein separation of the embedding medium from the embedded sample is improved compared to methods that remove the embedding medium by detaching it from the embedded sample.

14. 12. The method of claim 11, wherein the method is performed on a liquid handling robot equipped with a filter, and the improved separation of the embedding medium results in less clogging of the filter compared to methods in which the embedding medium is dissolved in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

15. The method of claim 11, wherein the method is performed on a liquid handling robot equipped with a filter, and the improved separation of the embedding medium reduces clogging of the filter compared to methods of removing the embedding medium by separating it from the embedded sample.

16. 16. The method of claim 14 or 15, wherein the filter is a filter in a spin column.

17. 16. The method of any one of claims 1 to 15, wherein step b) does not comprise dissolving the embedding medium in a miscible solvent, optionally wherein the miscible solvent is xylene, ethyl acetate, CitriSolv™, or UltraClear™.

18. The method of any one of claims 1 to 15, wherein the phase transition is selected from the group consisting of melting, freezing, vaporization, condensation, sublimation, and deposition.

19. 20. The method of claim 18, wherein the phase transition is melting.

20. 16. The method of any one of claims 1 to 15, wherein step b) comprises heating, cooling, increasing pressure, or decreasing pressure of the embedded sample.

21. The method of any one of claims 1 to 15, wherein step b) comprises heating the embedded sample.

22. 22. The method of claim 21, comprising heating the embedded sample to 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C.

23. 22. The method of claim 21, wherein the embedded sample is heated for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes.

24. 16. The method of any one of claims 1 to 15, wherein step b) comprises centrifuging and filtering the embedded sample to separate the embedding from the sample.

25. 25. The method of claim 24, wherein the embedded sample is centrifuged at about 1,700 rcf, about 1,750 rcf, about 1,800 rcf, about 1,850 rcf, or about 1,900 rcf.

26. 25. The method of claim 24, wherein the embedded sample is centrifuged at 1,811 rcf or greater.

27. 25. The method of claim 24, wherein the embedded sample is centrifuged at 1,811 rcf.

28. 16. The method of any one of claims 1 to 15, wherein step b) comprises heating, centrifuging and filtering the embedded sample to separate the embedding medium from the sample, thereby producing a de-embedded sample.

29. 16. The method of any one of claims 1 to 15, wherein step b) comprises heating and centrifuging the embedded sample to separate the embedding medium from the sample, thereby producing a de-embedded sample.

30. 16. The method of any one of claims 1 to 15, wherein the density of the immiscible solvent is lighter than water and heavier than the embedding medium when the embedding medium is in liquid form.

31. 16. The method of any one of claims 1 to 15, wherein the immiscible solvent is a vegetable oil or a mineral oil.

32. 16. The method of any one of claims 1 to 15, wherein step b) comprises contacting the sample with an immiscible solvent and centrifuging and filtering the embedded sample.

33. 16. The method of any one of claims 1 to 15, wherein the embedding agent is selected from the group consisting of paraffin, resin, celloidin, Paraplast®, gelatin, ester wax, wax, polyethylene glycol, and nitrocellulose, preferably the embedding agent is paraffin.

34. A method for extracting RNA and / or DNA, comprising: a) providing a paraffin-embedded sample; b) removing paraffin from said paraffin-embedded sample to produce a deparaffinized sample, (i) inducing a phase transition in said paraffin; (ii) contacting the paraffin-embedded sample with an immiscible solvent, removing the paraffin by separating the paraffin from the sample; c) extracting RNA and / or DNA from the deparaffinized sample.

35. The immiscible solvent is mineral oil, and optionally: (i) the paraffin-embedded sample is contacted with about 300, 350, 400, 450, 500, or 550 μL of mineral oil; (ii) contacting the paraffin-embedded sample with an immiscible solvent and separating the paraffin from the sample comprises incubating the paraffin-embedded sample in mineral oil at 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C; (iii) the mineral oil is in contact with the paraffin-embedded sample for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes; and / or (iv) the mineral oil is contacted with the paraffin-embedded sample while being shaken at about 25 rpm, about 50 rpm, about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, about 1000 rpm, or about 1100 rpm; 35. The method of claim 34.

36. (i) step b) of removing the paraffin from the paraffin-embedded sample is automated; (ii) the method is automated; (iii) 12, 24, 48, or 96 paraffin-embedded samples are processed in parallel; (iv) the method is carried out using a liquid handling robot; and / or (v) the paraffin-embedded sample is obtained from formalin-fixed, paraffin-embedded (FFPE) tissue, cryopreserved tissue, or fresh-frozen tissue; 35. The method of claim 34.

37. The method of claim 34, wherein the paraffin-embedded sample is a fixed paraffin-embedded sample, and optionally, the fixed paraffin-embedded sample is selected from the group consisting of a formalin-fixed sample, an ethanol-fixed sample, and a methanol-fixed sample.

38. A method for detecting modifications in RNA and / or DNA, comprising: a) extracting RNA and / or DNA from the deparaffinized sample by the method of claim 34; b) analyzing the RNA and / or DNA to detect alterations in the RNA and / or DNA.

39. A method for reducing the level of paraffin in an RNA or DNA sample extracted from a paraffin-embedded sample, comprising: a) extracting RNA and / or DNA by the method of claim 34; d) purifying the extracted RNA and / or DNA to provide an RNA and / or DNA sample; A method comprising:

40. 40. The method of claim 39, wherein the RNA is isolated from a lysate.

41. 40. The method of claim 39, further comprising analyzing the RNA.

42. 40. The method of claim 39, further comprising preparing cDNA from the RNA.

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