ESR1 mutations, ESR1 co-occurring mutations, and uses thereof

Abstract

Provided herein are methods related to measuring clonal fraction of one or more estrogen receptor 1 (ESRI) mutations in a liquid biopsy sample from an individual having cancer, as well as uses, systems, and computer readable storage media related thereto. In some embodiments, the methods comprise measuring a variant allele frequency of one or more ESRI mutations in a liquid biopsy sample obtained from the individual, measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

Description

ESRI MUTATIONS, ESRI CO-OCCURRING MUTATIONS, AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit of U.S. Provisional Application No. 63 / 606,027, filed on December 4, 2023, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (197102017440seqlist.xml; Size: 9,676 bytes; and Date of Creation: November 25, 2024) are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0003] Provided herein are methods related to measuring clonal fraction of one or more estrogen receptor 1 (ESRE) mutations in a liquid biopsy sample from an individual having cancer, as well as uses, systems, and computer readable storage media related thereto.BACKGROUND

[0004] Endocrine therapy (ET) has been highly successful in targeted treatment for cancers such as estrogen receptor-positive (ER+) breast cancer, including selective ER modulators or degraders that inhibit estradiol-ER binding and aromatase inhibitors that reduce estradiol production. However, ESRI mutations (ESRI mut) are an established biomarker of ET resistance in patients (pts) with hormone receptor positive (HR+) MBC. Moreover, ES7? / mut acquired in response to standard-of-care ET now confer access to novel ET recently approved by health authorities. ESRI mutations have been described in breast cancer and associated with resistance to ET (see, e.g., Hartmaier, R.J. et al. (2018) Ann Oncol. 29(4):872-880;Nagy, Z. and Jeselsohn, R. (2023) Front Oncol. 12:1037531; and Jeselsohn, R .et al. (2014) Clin Cancer Res. 2Q(iy. 151 16T).

[0005] Currently, the binary presence or absence of ESRI mutations informs therapeutic recommendations for treatment of cancers, including HR+ breast cancers. However, a more comprehensive and nuanced analysis of ESRI mutations in a more quantitative manner could provide more accurate ways to monitor ET resistance and provide treatment options. Thus,there is a need in the art for improved methods for selecting cancer therapies and monitoring ET resistance based on ESRI mutational status of a cancer.

[0006] All references cited herein, including patents, patent applications and publications, are hereby incorporated by reference in their entirety. To the extent that any reference incorporated by reference conflicts with the instant disclosure, the instant disclosure shall control.SUMMARY OF THE INVENTION

[0007] In some aspects, provided herein is a method of measuring clonal fraction of one or more estrogen receptor 1 (ESRE) mutations in a liquid biopsy sample from an individual having cancer, the method comprising measuring a variant allele frequency (VAF) of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction (TF) of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

[0008] In some aspects, provided herein is a method of selecting a therapy for an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is greater than or equal to a threshold ratio identifies the individual as one who may benefit from a treatment comprising an anti-cancer agent other than a selective estrogen receptor modulator (SERM) or an aromatase inhibitor.

[0009] In some aspects, provided herein is a method of identifying one or more treatment options for an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report comprising one or more treatment options identified for the individual based, at least in part, on the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction, wherein the one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor, and wherein the calculated ratio is greater than or equal to a threshold ratio.

[0010] In some aspects, provided herein is a method of treating or delaying progression of cancer. In some embodiments, the method comprises acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual; and responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments, the method comprises administering to an individual having cancer an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor, wherein the anti-cancer agent is administered responsive to acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual. In some embodiments, the method comprises measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or anaromatase inhibitor. In some embodiments, the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is greater than or equal to a threshold ratio.

[0011] In some aspects, provided herein is a method of monitoring resistance to endocrine therapy in an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is greater than or equal to a threshold ratio identifies the individual as one who may be resistant to endocrine therapy.

[0012] In some aspects, provided herein is a method of selecting a therapy for an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is less than a threshold ratio identifies the individual as one who may benefit from an endocrine therapy.

[0013] In some aspects, provided herein is a method of identifying one or more treatment options for an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRImutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report comprising one or more treatment options identified for the individual based, at least in part, on the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction, wherein the one or more treatment options comprise an endocrine therapy, and wherein the calculated ratio is less than a threshold ratio.

[0014] In some aspects, provided herein is a method of predicting responsiveness to endocrine therapy in an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is less than a threshold ratio identifies the individual as one who may be responsive to endocrine therapy.

[0015] In some aspects, provided herein is a method of treating or delaying progression of cancer. In some embodiments, the method comprises acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual; and responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an endocrine therapy. In some embodiments, the method comprises administering to an individual having cancer an effective amount of a treatment that comprises an endocrine therapy, wherein the endocrine therapy is administered responsive to acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual. In some embodiments, the method comprises measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumornucleic acid molecules in the liquid biopsy sample; and administering to the individual an effective amount of a treatment that comprises an endocrine therapy. In some embodiments, the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is less than a threshold ratio.

[0016] In some aspects, provided herein is a method of selecting a therapy for an individual having cancer, the method comprising measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and identifying the individual as one who may benefit from a treatment comprising an anti-cancer agent other than a SERM or an aromatase inhibitor when the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is greater than or equal to a threshold ratio; or identifying the individual as one who may benefit from a treatment comprising an endocrine therapy when the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is less than the threshold ratio.

[0017] In some aspects, provided herein is a method of monitoring resistance to endocrine therapy in an individual with cancer, the method comprising measuring a first variant allele frequency of the one or more ESRI mutations in a first liquid biopsy sample obtained from the individual at a first timepoint, wherein the first liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the first variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the first liquid biopsy sample; measuring a first tumor fraction of tumor nucleic acid molecules in the first liquid biopsy sample; calculating a ratio of the first variant allele frequency of the one or more ESRI mutations to the first tumor fraction of tumor nucleic acid molecules in the first liquid biopsy sample, thereby measuring a first clonal fraction of the one or more ESRI mutations in the first liquid biopsy sample; measuring a second variant allele frequency of the one or more ESRI mutations in a second liquid biopsy sample obtained from the individual at a second timepoint after the first timepoint, wherein the second liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the second variant allele frequency of the one or more ESRI mutations is measured from tumornucleic acid molecules or nucleic acid molecules derived therefrom in the second liquid biopsy sample; measuring a second tumor fraction of tumor nucleic acid molecules in the second liquid biopsy sample; calculating a ratio of the second variant allele frequency of the one or more ESRI mutations to the second tumor fraction of tumor nucleic acid molecules in the second liquid biopsy sample, thereby measuring a second clonal fraction of the one or more ESRI mutations in the second liquid biopsy sample; and providing an assessment of resistance to endocrine therapy in the individual based, at least in part, on the first and second clonal fractions of the one or more ESRI mutations; wherein the second clonal fraction is greater than the first clonal fraction. In some embodiments, the second timepoint is after administration of an endocrine therapy to the individual. In some embodiments, the method further comprises after providing the assessment of resistance to endocrine therapy, administering to the individual an effective amount of a treatment that comprises an anticancer agent other than a SERM or an aromatase inhibitor; wherein optionally the method further comprises halting administration of an endocrine therapy to the individual based, at least in part, on the first and second ratios of the clonal fraction of the one or more ESRI mutations.

[0018] In some aspects, provided herein is a method of measuring a clonal fraction of one or more ESRI mutations in a liquid biopsy sample, the method comprising providing a plurality of nucleic acid molecules obtained from a liquid biopsy sample from an individual having cancer, wherein the plurality of nucleic acid molecules comprises tumor nucleic acid molecules; optionally, ligating one or more adapters onto one or more tumor nucleic acid molecules from the plurality of nucleic acid molecules; optionally, amplifying the one or more ligated tumor nucleic acid molecules from the plurality of nucleic acid molecules; optionally, capturing amplified tumor nucleic acid molecules from the amplified tumor nucleic acid molecules; sequencing, by a sequencer, the captured tumor nucleic acid molecules to obtain a plurality of sequence reads that represent the captured tumor nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to one or more ESRI mutations; analyzing the plurality of sequence reads for presence of the one or more ESRI mutations; based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in theliquid biopsy sample. In some embodiments, the sequencer comprises a next-generation sequencer.

[0019] In some aspects, provided herein is a method of measuring a clonal fraction of one or more ESRI mutations in a liquid biopsy sample, the method comprising providing a liquid biopsy sample from an individual having cancer, wherein the sample comprises a plurality of nucleic acid molecules comprising tumor nucleic acid molecules; preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; amplifying said library; selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to one or more ESRI mutations in said library to produce an enriched sample; sequencing the enriched sample, thereby producing a plurality of sequence reads; analyzing the plurality of sequence reads for presence of the one or more ESRI mutations; based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

[0020] In some embodiments according to any of the embodiments described herein, the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more ESRI mutations, and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules. In some embodiments, the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique.

[0021] In some embodiments according to any of the embodiments described herein, the methods further comprise selectively enriching for one or more tumor nucleic acid molecules in the liquid biopsy sample comprising nucleotide sequences corresponding to the one or more ESRI mutations; wherein the selectively enriching produces an enriched sample. In some embodiments, the selectively enriching comprises: (a) combining one or more baitmolecules with the liquid biopsy sample, thereby hybridizing the one or more bait molecules to one or more tumor nucleic acid molecules in the sample comprising nucleotide sequences corresponding to the one or more ESRI mutations and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the one or more ESRI mutations. In some embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides. In some embodiments, one or more bait molecules are conjugated to an affinity reagent or to a detection reagent. In some embodiments, the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker. In some embodiments, the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule. In some embodiments, the selectively enriching comprises amplifying the one or more tumor nucleic acid molecules comprising nucleotide sequences corresponding to the one or more ESRI mutations using a polymerase chain reaction (PCR) to produce an enriched sample. In some embodiments, the methods further comprise sequencing the enriched sample. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; optionally wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS).

[0022] In some embodiments according to any of the embodiments described herein, the one or more ESRI mutations comprise one or more base substitutions or short insertion / deletions. In some embodiments, the one or more ESRI mutations comprise one or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H,V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1. In some embodiments, the one or more ESRI mutations comprise one or more gene fusions, rearrangements, or ESRI gene amplifications. In some embodiments, the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations. In some embodiments, the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations that comprises two or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1. In some embodiments, the one or more ESRI mutations comprise a first ESRI mutation present in a first cell or set of cells of the cancer and a second ESRI mutation present in a different cell or set of cells of the cancer.

[0023] In some embodiments according to any of the embodiments described herein, the tumor nucleic acid molecules in the liquid biopsy sample are circulating tumor DNA (ctDNA) molecules. In some embodiments, the tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and non-tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the liquid biopsy sample further comprises non-tumor nucleic acid molecules. In some embodiments, the tumor fraction is greater than or equal to about 1%. In some embodiments, acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual comprises measuring a variant allele frequency of the one or more ESRI mutations in the liquid biopsy sample, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.In some embodiments, the methods further comprise obtaining the liquid biopsy sample from the individual. In some embodiments, the liquid biopsy sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the tumor nucleic acid molecules comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the liquid biopsy sample comprises circulating tumor cells (CTCs).

[0024] In some embodiments according to any of the embodiments described herein, the methods further comprise detecting one or more oncogenic or tumor suppressor mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the one or more oncogenic or tumor suppressor mutations are detected in tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample. In some embodiments, one or more oncogenic or tumor suppressor mutations are detected in the same liquid biopsy sample from which the variant allele frequency of the one or more ESRI mutations is measured. In some embodiments, the methods further comprise measuring frequency of one or more oncogenic or tumor suppressor mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the frequency of the one or more oncogenic or tumor suppressor mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample. In some embodiments, the frequency of the one or more oncogenic or tumor suppressor mutations is measured in the same liquid biopsy sample from which the variant allele frequency of the one or more ESRI mutations is measured. In some embodiments, the methods further comprise calculating a ratio of the frequency of the one or more oncogenic or tumor suppressor mutations to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample. In some embodiments, the tumor fraction used to measure the clonal fraction of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample is the same tumor fraction used to measure the clonal fraction of the one or more ESRI mutations in the liquid biopsy sample. In some embodiments, the one or more oncogenic or tumor suppressor mutations comprise a mutation in one or more of PIK3CA, RBI, PTEN, NF1, ARID1A, AKT1, FGFR2, FGFR3, KRAS, ERBB2, EGFR, and BRAF. In some embodiments, the frequency of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations. In some embodiments, the one or more oncogenicor tumor suppressor mutations and the one or more ESRI mutations are present in different cells or sets of cells of the cancer.

[0025] In some embodiments according to any of the embodiments described herein, the threshold ratio is a ratio of a threshold variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the threshold ratio is the ratio of a threshold variant allele frequency of the one or more ESRI mutations that is greater than zero to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample that is greater than or equal to 1%. In some embodiments, the threshold ratio is a ratio of a threshold frequency of one or more oncogenic or tumor suppressor mutations in tumor nucleic acid molecules in the liquid biopsy sample to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the anti-cancer agent other than a SERM or aromatase inhibitor is a targeted therapy targeting one of the one or more oncogenic or tumor suppressor mutations.

[0026] In some embodiments according to any of the embodiments described herein, the individual has received one or more prior treatments with an endocrine therapy. In some embodiments, the methods further comprise, prior to measuring or acquiring knowledge of the variant allele frequency of the one or more ESRI mutations, administering an effective amount of a treatment that comprises an endocrine therapy to the individual.

[0027] In some embodiments according to any of the embodiments described herein, the endocrine therapy comprises a SERM or an aromatase inhibitor. In some embodiments, the SERM is tamoxifen, raloxifene, EM652, GW7604, keoxifene, toremifene, bazedoxifene, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, ospemifene, or a pharmaceutically acceptable salt thereof. In some embodiments, the aromatase inhibitor is aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), 4-Androstene- 3,6, 17-trione (“6-OXO”), or a pharmaceutically acceptable salt thereof.

[0028] In some embodiments according to any of the embodiments described herein, the anticancer agent comprises one or more of a small molecule inhibitor, an antibody, a cellular therapy, a nucleic acid, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), a treatment for cancer comprising an ESRI mutation, an anti-cancer agent being tested in a clinical trial, a treatment for cancer comprising an ESRI mutation being tested in a clinical trial, or any combination thereof. In some embodiments, the anti-cancer agent comprises a selective estrogen receptor covalent antagonist (SERCA). In someembodiments, the anti-cancer agent is H3B-5942 or H3B-6545. In some embodiments, the anti-cancer agent comprises a selective estrogen receptor degrader (SERD). In some embodiments, the anti-cancer agent is fulvestrant, elacestrant, amcenestrant, camizestrant, giredestrant, rintodestrant, imlunestrant, ZB-716, Zn-c5, LSZ102, LY3484356, or D-0502, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-cancer agent comprises a PROTAC. In some embodiments, the PROTAC is ARV-471. In some embodiments, the anti-cancer agent comprises a CDK4 / 6 inhibitor. In some embodiments, the CDK4 / 6 inhibitor is palbociclib, abemaciclib, ribociclib, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-cancer agent comprises a PI3K inhibitor. In some embodiments, the PI3K inhibitor is GSK2636771, buparlisib, AZD8186, copanlisib, LY294002, PX-866, TGX115, TGX126, BEZ235, SF1126, idelalisib, pictilisib, GDC0032, IPI145, INK1117, SAR260301, KIN-193, duvelisib, GS-9820, GSK2636771, GDC-0980, AMG319, paxalisib, or alpelisib, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-cancer agent comprises an mTOR inhibitor. In some embodiments, the mTOR inhibitor is temsirolimus, everolimus, ridaforolimus, dactolisib, GSK2126458, XL765, AZD8055, AZD2014, MLN128, PP242, NVP-BEZ235, LY3023414, PQR309, PKI587, or OSI027, or a pharmaceutically acceptable salt thereof. In some embodiments, the nucleic acid inhibits the expression of an ESRI nucleic acid molecule, or an ESRI polypeptide encoded by the ESRI nucleic acid molecule. In some embodiments, the nucleic acid is a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In some embodiments, the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophagebased therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy.

[0029] In some embodiments according to any of the embodiments described herein, the cancer is breast cancer. In some embodiments, the breast cancer is hormone receptor positive (HR+) breast cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is advanced or metastatic. In some embodiments, the treatment or the one or more treatment options further comprise an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti- DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growthinhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)- based therapy. In some embodiments, the nucleic acid comprises a double- stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In some embodiments, the methods further comprise generating a report, wherein the report: (a) indicates the presence of the one or more ESRI mutations in the sample from the individual; and / or (b) indicates a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the presence of the one or more ESRI mutations in the sample from the individual, wherein the treatment or the one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments, the methods further comprise generating a molecular profile for the individual, based, at least in part, on detecting or acquiring knowledge of the one or more ESRI mutations in the sample from the individual. In some embodiments, the molecular profile for the individual further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test. In some embodiments, the molecular profile for the individual further indicates presence of the one or more ESRI mutations in the liquid biopsy sample. In some embodiments, the report or the molecular profile for the individual further indicates the variant allele frequency or the clonal fraction of the one or more ESRI mutations in the liquid biopsy sample. In some embodiments, the methods further comprise selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments, the methods further comprise selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an endocrine therapy. In some embodiments, the methods further comprise generating a report, wherein the report comprises the molecular profile for the individual. In some embodiments,the report further comprises information on a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the treatment or one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments, the report further comprises information on a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the treatment or one or more treatment options comprise an endocrine therapy. In some embodiments, the methods further comprise providing the report to the individual, a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office.

[0030] In some embodiments according to any of the embodiments described herein, the individual is a human.

[0031] In some aspects, provided herein is a system for measuring clonal fraction of one or more ESRI mutations in a liquid biopsy sample from an individual having cancer, e.g., according to any one of the embodiments disclosed herein. In some embodiments, the system comprises a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to perform the method according to any one of the embodiments disclosed herein. In some embodiments, the system comprises a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from a liquid biopsy sample obtained from an individual having cancer, wherein the liquid biopsy sample comprises tumor nucleic acid molecules; analyze the plurality of sequence reads for one or more ESRI mutations present in tumor nucleic acid molecules from the liquid biopsy sample; measure, based on the analyzing, a variant allele frequency of the one or more ESRI mutations in the liquid biopsy sample; measure a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculate a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample. In some embodiments, the one or more program instructionswhen executed by the one or more processors are further configured to analyze the plurality of sequence reads for one or more oncogenic or tumor suppressor mutations present in tumor nucleic acid molecules from the liquid biopsy sample; and measure, based on the analyzing, frequency of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample. In some embodiments, the one or more program instructions when executed by the one or more processors are further configured to calculate a ratio of the frequency of the one or more oncogenic or tumor suppressor mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

[0032] In some aspects, provided herein is a non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for measuring clonal fraction of one or more ESRI mutations in a liquid biopsy sample from an individual having cancer, e.g., according to any one of the embodiments disclosed herein. In some embodiments, the method comprises obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from a liquid biopsy sample obtained from an individual having cancer, wherein the liquid biopsy sample comprises tumor nucleic acid molecules; analyzing, using the one or more processors, the plurality of sequence reads for one or more ESRI mutations present in tumor nucleic acid molecules from the liquid biopsy sample; measuring, using the one or more processors, a variant allele frequency of the one or more ESRI mutations in the liquid biopsy sample; measuring, using the one or more processors, a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating, using the one or more processors, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample. In some embodiments, the method further comprises analyzing, using the one or more processors, the plurality of sequence reads for one or more oncogenic or tumor suppressor mutations present in tumor nucleic acid molecules from the liquid biopsy sample; and measuring, using the one or more processors, frequency of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample. In some embodiments, the method further comprises calculating, using the one or more processors, a ratio of the frequency of the one or more oncogenic or tumor suppressor mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

[0033] In some embodiments according to any of the embodiments described herein, the one or more ESRI mutations comprise one or more base substitutions or short insertion / deletions. In some embodiments, the one or more ESRI mutations comprise one or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1. In some embodiments, the one or more ESRI mutations comprise one or more gene fusions, rearrangements, or ESRI gene amplifications. In some embodiments, the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations. In some embodiments, the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations that comprises two or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1. In some embodiments, the tumor nucleic acid molecules in the liquid biopsy sample are circulating tumor DNA (ctDNA) molecules. In some embodiments, the tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and nontumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the liquid biopsy sample further comprises non-tumor nucleic acid molecules. In some embodiments, the tumor fraction is greater than or equal to about 1%. In some embodiments, the liquid biopsy sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the tumor nucleic acid molecules comprise mRNA, DNA, circulatingtumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the liquid biopsy sample comprises circulating tumor cells (CTCs). In some embodiments, the one or more oncogenic or tumor suppressor mutations comprise a mutation in one or more of PIK3CA, RBI, PTEN, NF1, ARID1A, AKT1, FGFR2, FGFR3, KRAS, ERBB2, EGFR, and BRAF. In some embodiments, the frequency of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations. In some embodiments, the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene- targeted sequencing, or next-generation sequencing.

[0034] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGS. 1 & 2 show the ESRI mutational landscape observed in liquid and tissue biopsies from patients with metastatic breast cancer (MBC). Shown are ESRI mutations such as substitutions, indels, and fusions (as indicated) that were detected in liquid and tissue biopsies (FIG. 1). Rarer ESRI mutations are shown in FIG. 2. Graphs show raw count (n) observed for each mutation or fusion, as indicated.

[0036] FIG. 3 shows variant allele frequency of ESRI mutations such as substitutions, indels, and fusions (as indicated) detected in liquid biopsies with highly polyclonal ESRI resistance. Shown are percent tumor fraction and variant allele frequency for 77 liquid biopsies with 4 or more ESRI mutations or fusions.

[0037] FIG. 4 shows ESRI co-occurring mutations in liquid biopsies.

[0038] FIG. 5 shows ESRI co-occurring mutations in tissue biopsies.

[0039] FIG. 6 depicts an exemplary device, in accordance with some embodiments.

[0040] FIG. 7 depicts an exemplary system, in accordance with some embodiments.

[0041] FIG. 8 depicts a block diagram of an exemplary process for measuring clonal fraction of one or more ESRI mutations, in accordance with some embodiments.DETAILED DESCRIPTION

[0042] The present disclosure relates generally to measuring clonal fraction of one or more ESRI mutations in a sample from an individual having cancer, e.g., a liquid biopsy sample. Further provided herein are related methods for selecting a therapy, identifying treatment options, treating or delaying progression of cancer, monitoring resistance to endocrine therapy, and predicting responsiveness to endocrine therapy. The present disclosure describes the analysis of ESRI genomic alterations and co-occuring oncogenic / tumor suppressor mutations. A wide spectrum of ESRI mutations, including missense mutations, indels, etc. were observed and found to coexist with complementary or competing resistance mechanisms, particularly when ESRI was a minor allele, which could impact benefit from novel ET approved for patients with ESRI mutations.

[0043] Without wishing to be bound to theory, it is thought that a more quantitative approach to assessing ESRI mutations (instead of assessing simple binary presence or absence of ESRI mutation) could be used to provide improved treatment recommendations or techniques to monitor ET resistance. For example, measuring an overall variant allele frequency of multiple ESRI mutations present in a liquid biopsy may provide more accurate information on a cancer and how much is ES7? / mut that factors in clonality or sub-clonality of the mutations as well as overall ESEJmut positivity across a primary tumor and metastatic site(s), as opposed to analysis of a single tissue biopsy from a primary tumor or metastatic lesion, or binary presence / absence of an ESEJmut. Moreover, relating overall variant allele frequency to tumor fraction can be more informative to overall ESRI mutational status and / or potential for endocrine therapy resistance. For example, a liquid biopsy sample with a higher variant allele frequency of ESRI mutation than another sample with the same tumor fraction can indicate that more of the shedding tumor has ESRI mutation(s), as opposed to ESRI mutation(s) being sub-clonal. As another example, a certain variant allele frequency of ESRI mutation in a liquid biopsy sample with a higher tumor fraction could indicate that the tumor has significant presence of ESRI wild-type cells, whereas the same variant allele frequency of ESRI mutation in a liquid biopsy sample with a lower tumor fraction could indicate that the tumor is predominantly or entirely ESEJmut. Thus, the methods of the present disclosure are thought to allow for a determination of how much of a cancer is ESRI mat, rather than simply detecting presence or absence of a particular ESRI mutation in a tumor or liquid biopsy sample.I. General Techniques

[0044] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).II. Definitions

[0045] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.

[0046] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value orparameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0047] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0048] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.

[0049] The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive as referred to herein.

[0050] As used herein, the term “ESR1” refers to a gene encoding an estrogen receptor 1 polypeptide. The human ESRI gene is located on chromosome 6q25.1-q25.2. ESRI is also known as ER, ESR, Era, ESRA, ESTRR, and NR3A1. In some embodiments, an ESRI gene is a human ESRI gene. An exemplary ESRI amino acid sequence is provided below. In some embodiments, references to a specific ESRI mutation by amino acid position refer to amino acid numbering according to SEQ ID NO:1.MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEG AAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQ VPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGY HYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRML KHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAP NLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRV LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV (SEQ ID NO:1)

[0051] “Polynucleotide,” “nucleic acid,” or “nucleic acid molecule” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA orRNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double- stranded DNA, DNA including single- and double- stranded regions, single- and double- stranded RNA, and RNA including single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or include single- and double- stranded regions. In addition, the term “polynucleotide” as used herein refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs.

[0052] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl-, 2'-fluoro-, or 2'- azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), "(0)NR2("amidate"), P(0)R, P(0)OR', CO orCH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1 -20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. A polynucleotide can contain one or more different types of modifications as described herein and / or multiple modifications of the same type. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0053] “Oligonucleotide,” as used herein, generally refers to short, single stranded, polynucleotides that are, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0054] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0055] “Antibody fragments” comprise a portion of an intact antibody comprising the antigen-binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0056] The term “detection” includes any means of detecting, including direct and indirect detection. The term “biomarker” as used herein refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and / or clinical features (e.g., responsiveness to therapy including an immunotherapy, such as a checkpoint inhibitor). In some embodiments, a biomarker is a collection of genes and / or a collective number of mutations / alterations (e.g., somatic mutations) in a collection of genes, for example, a biomarker may comprise an ESRI alteration (e.g., an ESRI base substitution, missense mutation, truncation mutation, shortinsertion / deletion, rearrangement, or gene fusion) and / or an oncogenic or tumor suppressor mutation (e.g., a base substitution, missense mutation, truncation mutation, short insertion / deletion, rearrangement, or gene fusion in an oncogene or tumor suppressor gene). Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide alterations (e.g., polynucleotide copy number alterations, e.g., DNA copy number alterations), polypeptides, polypeptide and polynucleotide modifications (e.g., post- translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0057] “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.

[0058] The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid.

[0059] The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, for instance, by histopathological criteria, orby molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0060] The term “aiding diagnosis” is used herein to refer to methods that assist in making a clinical determination regarding the presence, or nature, of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method of aiding diagnosis of a disease or condition (e.g., cancer) can comprise measuring certain somatic mutations in a biological sample from an individual.

[0061] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. In some embodiments, the sample is a liquid biopsy sample. Liquid biopsy samples include but are not limited to samples comprising blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some instances, the sample is a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the liquid biopsy sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, cell-free RNA, and / or circulating tumor cells (CTCs).

[0062] A “tumor cell” as used herein, refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein.

[0063] The term “segmentation” (or “sequence segmentation”), as used herein, refers to a process for partitioning of sequence read data into a number of non-overlapping segments that cover all sequence read data points, such that each segment of a plurality of segments is as homogeneous as possible and all sequence reads associated with a given segment have the same copy number. In some instances, segmentation may be performed by processing aligned sequence read data (or other sequencing-related data, e.g., coverage data, allele frequency data, etc., derived from the sequence read data) using any of a variety of methods known to those of skill in the art (see., e.g., Braun and Miller (1998), “Statistical methods for DNA sequence segmentation”, Statistical Science 13(2): 142-162). Examples of segmentation methods include, but are not limited to, circular binary segmentation (CBS) methods, maximum likelihood methods, hidden Markov chain methods, walking Markovmethods, Bayesian methods, long-range correlation methods, change point methods, or any combination thereof.

[0064] A “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refer to a sample, cell, tissue, standard, or level that is used for comparison purposes.

[0065] By ‘ ‘correlate” or “correlating” is meant comparing, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocol and / or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0066] ‘ ‘Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down or complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down, or complete stopping) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e. reduction, slowing down, or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extension in the length of survival, including overall survival and progression free survival; and / or (7) decreased mortality at a given point of time following treatment.

[0067] An “effective response” of a patient or a patient's “responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and / or progression-free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.

[0068] An “effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent, and in some embodiments stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent, and in some embodiments stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), response rates (e.g., CR and PR), duration of response, and / or quality of life.

[0069] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0070] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0071] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating” , and the like) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0072] As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, e.g., a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non- human primates) for which treatment is desired. In particular embodiments, the patient, individual or subject herein is a human.

[0073] As used herein, “administering” (and grammatical variations thereof such as “administration” or “administer”, and the like) refers to a method of giving a dosage of an agent or a pharmaceutical composition (e.g., a pharmaceutical composition including the agent) to a subject (e.g., a patient). Administering can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include, for example, intramuscular, intravenous, 1intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules, including, but not limited to, single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0074] The terms “concurrently” or “in combination” are used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen wherein the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0075] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0076] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a reagent for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0077] The terms “allele frequency” and “allele fraction” are used interchangeably herein and refer to the fraction of sequence reads corresponding to a particular allele relative to the total number of sequence reads for a genomic locus. The terms “variant allele frequency” and “variant allele fraction” are used interchangeably herein and refer to the fraction of sequence reads corresponding to a particular variant allele relative to the total number of sequence reads for a genomic locus, e.g., ESRI.

[0078] The phrase “based on” when used herein means that the information about one or more biomarkers is used to inform a treatment decision, information provided on a package insert, or marketing / promotional guidance, etc.III. Methods, Systems, and Devices

[0079] In some aspects, provided herein are methods for measuring clonal fraction of one or more ESRI mutations in a liquid biopsy sample, e.g., from an individual having a cancer. In other aspects, provided herein are methods for selecting a therapy for an individual havingcancer. In other aspects, provided herein are methods for identifying one or more treatment options for an individual having cancer. In other aspects, provided herein are methods for treating or delaying progression of cancer in an individual. In other aspects, provided herein are methods for monitoring resistance to endocrine therapy in an individual having cancer. In other aspects, provided herein are methods for predicting responsiveness to endocrine therapy in an individual having cancer. In some embodiments, the individual is a human.

[0080] In some embodiments of any of the methods provided herein, the methods comprise measuring a variant allele frequency of one or more ESRI mutations in a liquid biopsy sample (e.g., that comprises tumor nucleic acid molecules and optionally non-tumor nucleic acid molecules); measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

[0081] In other aspects, provided herein are systems and non-transitory computer readable storage media. In some embodiments, the systems and non-transitory computer readable storage media provided herein are for (e.g., are configured for) performing a method according to any one of the embodiments disclosed herein. In some embodiments, the systems and non-transitory computer readable storage media provided herein are for (e.g., are configured for) measuring clonal fraction of one or more ESRI mutations in a liquid biopsy sample, e.g., from an individual having a cancer. In some embodiments, the systems and non-transitory computer readable storage media provided herein are for (e.g., are configured for) identifying one or more treatment options for an individual having cancer. In some embodiments, the systems and non-transitory computer readable storage media provided herein are for (e.g., are configured for) treating or delaying progression of cancer in an individual. In some embodiments, the systems and non-transitory computer readable storage media provided herein are for (e.g., are configured for) monitoring resistance to endocrine therapy in an individual having cancer. In some embodiments, the systems and non- transitory computer readable storage media provided herein are for (e.g., are configured for) predicting responsiveness to endocrine therapy in an individual having cancer.Clonal Fraction

[0082] Certain aspects of the present disclosure relate to calculation of a clonal fraction of mutations in a gene, e.g., of one or more ESRI mutations, or of one or more oncogenic or tumor suppressor mutations. As used herein, the clonal fraction of a mutated gene e.g., an ESRI gene, or an oncogene / tumor suppressor gene) refers to a ratio of the variant allelefrequency of one or more mutations in the gene (e.g., one or more ESRI mutations, or one or more oncogenic or tumor suppressor mutations) in a liquid biopsy sample to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. For example, the clonal fraction of one or more ESRI mutations can refer to a ratio of the variant allele frequency of one or more ESRI mutations in a liquid biopsy sample to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. The clonal fraction of one or more oncogenic or tumor suppressor mutations (e.g., in a particular oncogene or tumor suppressor gene) can refer to a ratio of the variant allele frequency of one or more oncogenic or tumor suppressor mutations (e.g., mutations in a particular oncogene or tumor suppressor gene) in a liquid biopsy sample to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

[0083] In some embodiments, the variant allele frequency of one or more ESRI mutations in a liquid biopsy sample is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample. In some embodiments, the variant allele frequency of one or more oncogenic or tumor suppressor mutations in a liquid biopsy sample is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample. In some embodiments, the variant allele frequency of mutation(s) in a gene (e.g., of one or more ESRI mutations, or of one or more oncogenic or tumor suppressor mutations) is based on a total frequency (e.g., variant allele frequency) representing a plurality of mutations in the gene (e.g., a plurality of ESRI or oncogenic / tumor suppressor mutations). For example, the variant allele frequency of ESRI mutations in a liquid biopsy sample can be based on a total or sum of the variant allele frequencies of a plurality of ESRI mutations in the sample.

[0084] In some embodiments, different mutations in a gene (e.g., ESRI mutations) can be present in multiple cells or sets of cells (e.g., clones or sub-clones) of a cancer. That is, ESRI mutations from which a variant allele frequency is measured can comprise a first ESRI mutation present in a first cell or set of cells of the cancer and a second ESRI mutation present in a different cell or set of cells of the cancer. As demonstrated herein, 12% of liquid biopsies analyzed from an MBC cohort had 3 or more ESRI mutations present, and up to 10 different ESRI mutations were observed in a single liquid biopsy sample. These different mutations can be present as sub-clones in a single tumor, or be present in different primary / metastatic sites, and / or may originate independently from one another.Advantageously, the methods of the present disclosure allow for the sum total of such varied ESRI mutations to be accounted for and compared against a total tumor fraction of a liquidbiopsy sample, e.g., to provide an overall quantification of ES7? / mut presence or load in a cancer or an individual having cancer. In some embodiments, one or more oncogenic or tumor suppressor mutations and one or more ESRI mutations are present in different cells or sets of cells of the cancer.

[0085] In some embodiments, the methods of the present disclosure comprise measuring a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample. Methods for measuring tumor fraction are known in the art; see, e.g., Husain, H. el al. (2022) JCO Precis Oncol. Oct:6:e2200261. In some embodiments, a liquid biopsy sample of the present disclosure further comprises non-tumor nucleic acid molecules. In some embodiments, tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and non-tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, tumor fraction is greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, greater than or equal to about 9%, or greater than or equal to about 10%.

[0086] In some embodiments, the methods of the present disclosure comprise comparing a clonal fraction e.g., clonal fraction of one or more ESRI mutations as described herein), e.g., a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample, with a threshold ratio. In some embodiments, the threshold ratio is a ratio of a threshold variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the threshold ratio is the ratio of a threshold variant allele frequency of the one or more ESRI mutations that is greater than zero to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample that is greater than or equal to 1%. In some embodiments, the threshold ratio is a ratio of a threshold frequency of one or more oncogenic or tumor suppressor mutations in tumor nucleic acid molecules in the liquid biopsy sample to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

[0087] In some embodiments, a clonal fraction of one or more ESRI mutations as described herein that is greater than or equal to the threshold ratio may indicate a greater presence of ESRJmut in a cancer and may lead to treatment or a recommendation for treatment with an anti-cancer agent other than a SERM or an aromatase inhibitor, e.g., as described herein. A clonal fraction of one or more ESRI mutations as described herein that is less than the threshold ratio may indicate a lesser or no presence of ES7? / mut in a cancer and may lead totreatment or a recommendation for treatment with an endocrine therapy, e.g., as described herein.

[0088] Certain aspects of the present disclosure relate to ESRI mutations, including without limitation ESRI base substitutions, missense mutations, truncation mutations, short insertion / deletions, rearrangements, or gene fusions. In some embodiments, the one or more ESRI mutations comprise one or more base substitutions or short insertion / deletions.Exemplary ESRI mutations have been described herein and are intended to illustrate, without limiting, the ESRI mutations of the present disclosure. In some embodiments, the one or more ESRI mutations comprise one or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1. In some embodiments, the one or more ESRI mutations comprise one or more ESRI gene fusions, ESRI rearrangements, or ESRI gene amplifications. In some embodiments, the variant allele frequency of one or more ESRI mutations can be based on a total frequency representing a plurality of ESRI mutations that comprises two or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1, and optionally further comprising one or more ESRI gene fusions, ESRI rearrangements, and / or ESRI gene amplifications.

[0089] As used herein “ESRE' refers to a gene encoding an ESRI mRNA or ESRI polypeptide. ESRI is also known as ER, ESR, Era, ESRA, ESTRR, and NR3A1. In someembodiments, an ESRI gene is a human ESRI gene. An exemplary ESRI gene is represented by NCBI Gene ID No. 2099. In some embodiments, an ESRI gene is located at chromosomal coordinates chr6:151, 656, 691-152, 129, 619 forward strand. An exemplary ESRI mRNA sequence is represented by NCBI Ref. Seq. NM_000125.4, provided below as SEQ ID NO: 2:AGCTGGCGGAGGGCGTTCGTCCTGGGACTGCACTTGCTCCCGTCGGGTCGCCCGG CTTCACCGGACCCGC AGGCTCCCGGGGCAGGGCCGGGGCCAGAGCTCGCGTGTCGGCGGGACATGCGCT GCGTCGCCTCTAACCT CGGGCTGTGCTCTTTTTCCAGGTGGCCCGCCGGTTTCTGAGCCTTCTGCCCTGCGG GGACACGGTCTGCA CCCTGCCCGCGGCCACGGACCATGACCATGACCCTCCACACCAAAGCATCTGGG ATGGCCCTACTGCATC AGATCCAAGGGAACGAGCTGGAGCCCCTGAACCGTCCGCAGCTCAAGATCCCCC TGGAGCGGCCCCTGGG CGAGGTGTACCTGGACAGCAGCAAGCCCGCCGTGTACAACTACCCCGAGGGCGC CGCCTACGAGTTCAAC GCCGCGGCCGCCGCCAACGCGCAGGTCTACGGTCAGACCGGCCTCCCCTACGGC CCCGGGTCTGAGGCTGCGGCGTTCGGCTCCAACGGCCTGGGGGGTTTCCCCCCACTCAACAGCGTGTCTCC GAGCCCGCTGATGCT ACTGCACCCGCCGCCGCAGCTGTCGCCTTTCCTGCAGCCCCACGGCCAGCAGGTG CCCTACTACCTGGAG AACGAGCCCAGCGGCTACACGGTGCGCGAGGCCGGCCCGCCGGCATTCTACAGG CCAAATTCAGATAATC GACGCCAGGGTGGCAGAGAAAGATTGGCCAGTACCAATGACAAGGGAAGTATG GCTATGGAATCTGCCAA GGAGACTCGCTACTGTGCAGTGTGCAATGACTATGCTTCAGGCTACCATTATGGA GTCTGGTCCTGTGAG GGCTGCAAGGCCTTCTTCAAGAGAAGTATTCAAGGACATAACGACTATATGTGTC CAGCCACCAACCAGT GCACCATTGATAAAAACAGGAGGAAGAGCTGCCAGGCCTGCCGGCTCCGTAAAT GCTACGAAGTGGGAAT GATGAAAGGTGGGATACGAAAAGACCGAAGAGGAGGGAGAATGTTGAAACACA AGCGCCAGAGAGATGAT GGGGAGGGCAGGGGTGAAGTGGGGTCTGCTGGAGACATGAGAGCTGCCAACCTT TGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGA TGGTCAGTGCCTTGTT GGATGCTGAGCCCCCGATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGT GAAGCTTCGATGATG GGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCG AAGAGGGTGCCAGGCT TTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGA GATCCTGATGATTGG TCTCGTCTGGCGCTCCATGGAGCACCCAGGGAAGCTACTGTTTGCTCCTAACTTG CTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACGGTCTGAGAGCTCCCTGGCTCCCACACGGTTCAGATAATCCCTGCTGCATTTTACCCTCATCATGCACCACTTTAGCCAAATTCTGTCTCCTGCATACACTCCGGCATGCATCCAACACCAATGGCTTTCTAGATGAGTGGCCATTCATTTGCTTGCTCAGTTCTTAGTGGCACATCTTCTGTCTTCTGTTGGGAACAGCCAAAGGGATTCCAAGGCTAAATCTTTGTAACAGCTCTCTTTCCCCCTTGCTATGTTACTAAGCGTGAGGATTCCCGTAGCTCTTCACAGCTGAACTCAGTCTATGGGTTGGGGCTCAGATAACTCTGTGCATTTAAGCTACTTGTAGAGACCCAGGCCTGGAGAGTAGACATTTTGCCTCTGATAAGCACTTTTTAAATGGCTCTAAGAATAAGCCACAGCAAAGAATTTAAAGTGGCTCCTTTAATTGGTGACTTGGAGAAAGCTAGGTCAAGGGTTTATTATAGCACCCTCTTGTATTCCTATGGCAATGCATCCTTTTATGAAAGTGGTACACCTTAAAGCTTTTATATGACTGTAGCAGAGTATCTGGTGATTGTCAATTCATTCCCCCTATAGGAATACAAGGGGCACACAGGGAAGGCAGATCCCCTAGTTGGCAAGACTATTTTAACTTGATACACTGCAGATTCAGATGTGCTGAAAGCTCTGCCTCTGGCTTTCCGGTCATGGGTTCCAGTTAATTCATGCCTCCCATGGACCTATGGAGAGCAGCAAGTTGATCTTAGTTAAGTCTCCCTATATGAGGGATAAGTTCCTGATTTTTGTTTTTATTTTTGTGTTACAAAAGAAAGCCCTCCCTCCCTGAACTTGCAGTAAGGTCAGCTTCAGGACCTGTTCCAGTGGGCACTGTACTTGGATCTTCCCGGCGTGTGTGTGCCTTACACAGGGGTGAACTGTTCACTGTGGTGATGCATGATGAGGGTAAATGGTAGTTGAAAGGAGCAGGGGCCCTGGTGTTGCATTTAGCCCTGGGGCATGGAGCTGAACAGTACTTGTGCAGGATTGTTGTGGCTACTAGAGAACAAGAGGGAAAGTAGGGCAGAAACTGGATACAGTTCTGAGGCACAGCCAGACTTGCTCAGGGTGGCCCTGCCACAGGCTGCAGCTACCTAGGAACATTCCTTGCAGACCCCGCATTGCCCTTTGGGGGTGCCCTGGGATCCCTGGGGTAGTCCAGCTCTTCTTCATTTCCCAGCGTGGCCCTGGTTGGAAGAAGCAGCTGTCACAGCTGCTGTAGACAGCTGTGTTCCTACAATTGGCCCAGCACCCTGGGGCACGGGAGAAGGGTGGGGACCGTTGCTGTCACTACTCAGGCTGACTGGGGCCTGGTCAGATTACGTATGCCCTTGGTGGTTTAGAGATAATCCAAAATCAGGGTTTGGTTTGGGGAAGAAAATCCTCCCCCTTCCTCCCCCGCCCCGTTCCCTACCGCCTCCACTCCTGCCAGCTCATTTCCTTCAATTTCCTTTGACCTATAGGCTAAAAAAGAAAGGCTCATTCCAGCCACAGGGCAGCCTTCCCTGGGCCTTTGCTTCTCTAGCACAATTATGGGTTACTTCCTTTTTCTTAACAAAAAAGAATGTTTGATTTCCTCTGGGTGACCTTATTGTCTGTAATTGAAACCCTATTGAGAGGTGATGTCTGTGTTAGCCAATGACCCAGGTGAGCTGCTCGGGCTTCTCTTGGTATGTCTTGTTTGGAAAAGTGGATTTCATTCATTTCTGATTGTCCAGTTAAGTGATCACCAAAGGACTGAGAATCTGGGAGGGCAAAAAAAAAAAAAAAGTTTTTATGTGCACTTAAATTTGGGGACAATTTTATGTATCTGTGTTAAGGATATGTTTAAGAACATAATTCTTTTGTTGCTGTTTGTTTAAGAAGCACCTTAGTTTGTTTAAGAAGCACCTTATATAGTATAATATATATTTTTTTGAAATTACATTGCTTGTTTATCAGACAATTGAATGTAGTAATTCTGTTCTGGATTTAATTTGACTGGGTTAACATGCAAAAACCAAGGAAAAATATTTAGTTTTTTTTTTTTTTTTTGTATACTTTTCAAGCTACCTTGTCATGTATACAGTCATTTATGCCTAAAGCCTGGTGATTATTCATTTAAATGAAGATCACATTTCATATCAACTTTTGTATCCACAGTAGACAAAATAGCACTAATCCAGATGCCTATTGTTGGATACTGAATGACAGACAATCTTATGTAGCAAAGATTATGCCTGAAAAGGAAAATTATTCAGGGCAGCTAATTTTGCTTTTACCAAAATATCAGTAGTAATATTTTTGGACAGTAGCTAATGGGTCAGTGGGTTCTTTTTAATGTTTATACTTAGATTTTCTTTTAAAAAAATTAAAATAAAACAAAAAAAAATTTCTAGGACTAGACGATGTAATACCAGCTAAAGCCAAACAATTATACAGTGGAAGGTTTTACATTATTCATCCAATGTGTTTCTATTCATGTTAAGATACTACTACATTTGAAGTGGGCAGAGAACATCAGATGATTGAAATGTTCGCCCAGGGGTCTCCAGCAACTTTGGAAATCTCTTTGTATTTTTACTTGAAGTGCCACTAATGGACAGCAGATATTTTCTGGCTGATGTTGGTATTGGGTGTAGGAACATGATTTAAAAAAAAACTCTTGCCTCTGCTTTCCCCCACTCTGAGGCAAGTTAAAATGTAAAAGATGTGATTTATCTGGGGGGCTCAGGTATGGTGGGGAAGTGGATTCAGGAATCTGGGGAATGGCAAATATATTAAGAAGAGTATTGAAAGTATTTGGAGGAAAATGGTTAATTCTGGGTGTGCACCAGGGTTCAGTAGAGTCCACTTCTGCCCTGGAGACCACAAATCAACTAGCTCCATTTACAGCCATTTCTAAAATGGCAGCTTCAGTTCTAGAGAAGAAAGAACAACATCAGCAGTAAAGTCCATGGAATAGCTAGTGGTCT GTGTTTCTTTTCGCCA TTGCCTAGCTTGCCGTAATGATTCTATAATGCCATCATGCAGCAATTATGAGAGG CTAGGTCATCCAAAGAGAAGACCCTATCAATGTAGGTTGCAAAATCTAACCCCTAAGGAAGTGCAGTCT TTGATTTGATTTCCCT AGTAACCTTGCAGATATGTTTAACCAAGCCATAGCCCATGCCTTTTGAGGGCTGA ACAAATAAGGGACTTACTGATAATTTACTTTTGATCACATTAAGGTGTTCTCACCTTGAAATCTTATACAC TGAAATGGCCATTGATTTAGGCCACTGGCTTAGAGTACTCCTTCCCCTGCATGACACTGATTACAAATA CTTTCCTATTCATACTTTCCAATTATGAGATGGACTGTGGGTACTGGGAGTGATCACTAACACCATAGTA ATGTCTAATATTCACAGGCAGATCTGCTTGGGGAAGCTAGTTATGTGAAAGGCAAATAGAGTCATACAG TAGCTCAAAAGGCAACCATAATTCTCTTTGGTGCAGGTCTTGGGAGCGTGATCTAGATTACACTGCACCAT TCCCAAGTTAATCCC CTGAAAACTTACTCTCAACTGGAGCAAATGAACTTTGGTCCCAAATATCCATCTT TTCAGTAGCGTTAATTATGCTCTGTTTCCAACTGCATTTCCTTTCCAATTGAATTAAAGTGTGGCCTCGTT TTTAGTCATTTAAAATTGTTTTCTAAGTAATTGCTGCCTCTATTATGGCACTTCAATTTTGCACTGTCTTT TGAGATTCAAGAAAAATTTCTATTCTTTTTTTTGCATCCAATTGTGCCTGAACTTTTAAAATATGTAAA TGCTGCCATGTTCCAAACCCATCGTCAGTGTGTGTGTTTAGAGCTGTGCACCCTAGAAACAACATATTG TCCCATGAGCAGGTGCCTGAGACACAGACCCCTTTGCATTCACAGAGAGGTCATTGGTTATAGAGACTTG AATTAATAAGTGACATTATGCCAGTTTCTGTTCTCTCACAGGTGATAAACAATGCTTTTTGTGCACTACAT ACTCTTCAGTGTAGAGCTCTTGTTTTATGGGAAAAGGCTCAAATGCCAAATTGTGTTTGATGGATTAAT ATGCCCTTTTGCCGA TGCATACTATTACTGATGTGACTCGGTTTTGTCGCAGCTTTGCTTTGTTTAATGAA ACACACTTGTAAACCTCTTTTGCACTTTGAAAAAGAATCCAGCGGGATGCTCGAGCACCTGTAAACAAT TTTCTCAACCTATTTGATGTTCAAATAAAGAATTAAACTAAA(SEQ ID NO: 2)

[0090] An exemplary ESRI amino acid sequence is provided below. In some embodiments, references to a specific ESRI mutation by amino acid position refer to amino acid numbering according to SEQ ID NO:1.MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEG AAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQ VPYYEENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGY HYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRML KHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAP NLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRV LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV (SEQ ID NO:1)

[0091] ESRI mutations may be assessed using any suitable method known in the art, e.g., for detecting specific polynucleotides in a liquid biopsy sample. Exemplary and non-limiting methods for detecting ESRI alterations include comprehensive genomic profiling (CGP), comparative genomic hybridization (CGH), sequencing, microarray based-methods, amplification-based methods, or any combination thereof. In some embodiments, an ESRI mutation is detected using a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. Certain methods for detecting ESRI mutations are described in further detail below as non-limiting examples.

[0092] In some embodiments, the methods of the present disclosure further comprise detecting one or more oncogenic or tumor suppressor mutations, e.g., in a liquid biopsy sample. In some embodiments, the one or more oncogenic or tumor suppressor mutations are detected in tumor nucleic acid molecules or nucleic acid molecules derived therefrom in a liquid biopsy sample. In some embodiments, the one or more oncogenic or tumor suppressor mutations are detected in the same liquid biopsy sample from which the variant allele frequency of the one or more ESRI mutations is measured.

[0093] In some embodiments, the methods of the present disclosure further comprise measuring frequency (e.g., variant allele frequency) of one or more oncogenic or tumor suppressor mutations, e.g., in a liquid biopsy sample. In some embodiments, the frequency (e.g., variant allele frequency) of the one or more oncogenic or tumor suppressor mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample. The variant allele frequency for a particular oncogenic or tumor suppressor mutation can be measured as disclosed herein for one or more ESRI mutations. In some embodiments, the frequency (e.g., variant allele frequency) of one or more oncogenic or tumor suppressor mutations is measured in the same liquid biopsy sample from which thevariant allele frequency of the one or more ESRI mutations is measured. In some embodiments, frequency (e.g., variant allele frequency) of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations, e.g., from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample.

[0094] In some embodiments, the methods of the present disclosure further comprise calculating a ratio of the frequency (e.g., variant allele frequency) of the one or more oncogenic or tumor suppressor mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample (e.g., a clonal fraction of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample). In some embodiments, the tumor fraction used to measure the clonal fraction of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample is the same tumor fraction used to measure the clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

[0095] In some embodiments, the one or more oncogenic or tumor suppressor mutations comprise a mutation in one or more of PIK3CA, RBI, PTEN, NF1, ARID1A, AKT1, FGFR2, FGFR3, KRAS, ERBB2, EGFR, and BRAF. Exemplary oncogenic or tumor suppressor mutations can include, without limitation, mutations in one or more of ABE1, BRAF, CDKN1A, EPHA3, FGFR4, IKZF1, MCE1, NKX2-1, PMS2, RNF43, TET2, ACVR1B, BRCA1, CDKN1B, EPHB1, FH, INPP4B, MDM2, NOTCH1, POED1, ROS1, TGFBR2, AKT1, BRCA2, CDKN2A, EPHB4, FLCN, IRF2, MDM4, NOTCH2, POEE, RPTOR, TIPARP, AKT2, BRD4, CDKN2B, ERBB2, FLT1, IRF4, MED12, NOTCH3, PPARG, SDHA, TNFAIP3, AKT3, BRIP1, CDKN2C, ERBB3, FET3, IRS2, MEF2B, NPM1, PPP2R1A, SDHB, TNFRSF14, AEK, BTG1, CEBPA, ERBB4, FOXE2, JAK1, MEN1, NRAS, PPP2R2A, SDHC, TP53, ALOXI2B, BTG2, CHEK1, ERCC4, FUBP1, JAK2, MERTK, NT5C2, PRDM1, SDHD, TSC1, AMER1, BTK, CHEK2, ERG, GABRA6, JAK3, MET, NTRK1, PRKAR1A, SETD2, TSC2, APC, Cllorf30, CIC, ERRFI1, GATA3, JUN, MITF, NTRK2, PRKCI, SF3B1, TYRO3, AR, CAER, CREBBP, GATA4, KDM5A, MKNK1, NTRK3, PTCHI, SGK1, U2AF1, ARAF, CARD11, CRKE, EZH2, GATA6, KDM5C, MEH1, P2RY8, PTEN, SMAD2, VEGFA, ARFRP1, CASP8, CSF1R, FAM46C, GID4, (C17orf39), KDM6A, MPE, PAEB2, PTPN11, SMAD4, VHL, ARID 1 A, CBFB, CSF3R, FANCA, GNA11, KDR, MRE11A, PARK2, PTPRO, SMARCA4, WHSCI, ASXE1, CBE, CTCF, FANCC, GNA13, KEAP1, MSH2, PARP1, QKI, SMARCB1, WHSC1E1, ATM, CCND1, CTNNA1, FANCG, GNAQ, KEL, MSH3, PARP2, RACE SMO, WT1, ATR, CCND2, CTNNB1, FANCE, GNAS, KIT, MSH6, PARP3, RAD2], SNCAIP, XPO1, ATRX, CCND3, CUE3, FAS, GRM3, KEHE6, MST1R, PAX5, RAD5I,S0CS1, XRCC2, AURKA, CCNE1, CUL4A, FBXW7, GSK3B, KMT2A, (MEL), MTAP, PBRM1, RAD51B, S0X2, ZNF217, AURKB, CD22, CXCR4, FGF10, H3F3A, KMT2D, (MLL2), MTOR, PDCD1, RAD51C, S0X9, ZNF703, AXIN1, CD274, CYP17A1, FGF12, HDAC1, KRAS, MUTYH, PDCD1LG2, RAD51D, SPEN, AXL, CD70, DAXX, FGF14, HGF, LTK, MYC, PDGFRA, RAD52, SPOP, BAP1, CD79A, DDR1, FGF19, HNF1A, LYN, MYCL, PDGFRB, RAD54L, SRC, BARD1, CD79B, DDR2, FGF23, HRAS, MAF, MYCN, PDK1, RAFI, STAG2, BCL2, CDC73, DIS3, FGF3, HSD3B1, MAP2K1, MYD88, PIK3C2B, RARA, STAT3, BCL2L1, CDH1, DNMT3A, FGF4, ID3, MAP2K2, NBN, PIK3C2G, RBI, STK11, BCL2L2, CDK12, D0T1L, FGF6, IDH1, MAP2K4, NF1, PIK3CA, RBM10, SUFU, BCL6, CDK4, EED, FGFR1, IDH2, MAP3K1, NF2, PIK3CB, REL, SYK, BCOR, CDK6, EGFR, FGFR2, IGF1R, MAP3K13, NFE2L2, PIK3R1, RET, TBX3, BC0RL1, CDK8, EP300, FGFR3, IKBKE, MAPK1, NFKBIA, PIM1, RICTOR, TEK, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, MYB, NUTM1, RSP02, SDC4, SLC34A2, TERC, TERT, and TMPRSS2.

[0096] In some embodiments, one or more mutations (e.g.. ESRI or oncogenic / tumor suppressor mutations) are detected in tumor nucleic acid molecules or nucleic acid molecules derived therefrom. For example, the one or more mutations can be detected directly in tumor nucleic acid molecules, or the one or more mutations can be detected in nucleic acid molecules that have been derived therefrom, such as nucleic acid molecules derived from steps disclosed herein including without limitation nucleic acid ligation, amplification, capture, sequencing, selective enrichment, and / or nucleic acid library preparation.

[0097] In some embodiments, ESRI mutations in a cancer (e.g., in a liquid biopsy sample) are assessed using an amplification-based method, such as a PCR method, e.g. quantitative PCR (qPCR) or digital droplet PCR (ddPCR), and the like. In some embodiments, primers specific to a particular ESRI mutations are used to specifically amplified altered ESRI polynucleotide(s). In some embodiments, general primers are used to amplify the ESRI locus, or a portion or coding sequence thereof, and the resulting amplicons are sequenced, e.g., in order to detect presence or absence of one or more ESRI mutation(s). In some embodiments, ESRI mutations in a cancer (e.g., in a liquid biopsy sample) are assessed using a qPCR or ddPCR method.

[0098] In some embodiments, ESRI mutations in a cancer (e.g., in a liquid biopsy sample) are assessed using a sequencing method. Any method of sequencing known in the art may be used to detect one or more ESRI mutations. Exemplary sequencing methods that may be used include those based on techniques developed by Maxam and Gilbert or Sanger. Automated sequencing procedures may also be used, e.g., including sequencing by mass spectrometry. Insome embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the massively parallel sequencing technique comprises next- generation sequencing (NGS). In some embodiments, the sequencing comprises hybrid capture-based sequencing (hybrid capturebased NGS), e.g., using adaptor ligation-based libraries. See, e.g., Frampton, G.M. et al. (2013) Nat. Biotech. 31:1023-1031.

[0099] Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 105molecules may be sequenced simultaneously). Next generation sequencing methods suitable for use according to the methods provided herein are known in the art and include, without limitation, massively parallel short-read sequencing, template-based sequencing, pyrosequencing, real-time sequencing comprising imaging the continuous incorporation of dye-labeling nucleotides during DNA synthesis, nanopore sequencing, sequencing by hybridization, nano-transistor array based sequencing, polony sequencing, scanning tunneling microscopy (STM)-based sequencing, or nanowire-molecule sensor based sequencing. See, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is hereby incorporated by reference. Exemplary NGS methods and platforms that may be used to detect ESRI mutations in a cancer (e.g., in a liquid biopsy sample) include, without limitation, 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 ESRI mutations in a cancer (e.g., in a liquid biopsy sample) include, without limitation, the Genome Sequencer (GS) FLX System from Roche (Basel, CHE), the G.007 polonator system, the Solexa Genome Analyzer, HiSeq 2500, HiSeq3000, HiSeq 4000, and NovaSeq 6000 platforms from Illumina Inc. (San Diego, CA, USA).

[0100] In some embodiments, methods for detecting ESRI mutations in a cancer (e.g., in a liquid biopsy sample) comprise providing a sample from an individual (e.g., an individual having cancer), wherein the sample comprises one or more nucleic acids. In someembodiments, an ESRI mutation is detected directly from one or more nucleic acids from the sample. In some embodiments, an ESRI mutation is detected from an amplicon, sequence read, or other nucleic acid otherwise derived from one or more nucleic acids from the sample.

[0101] In some embodiments, the methods further comprise preparing a nucleic acid sequencing library from the one or more nucleic acids in the sample. Methods for the preparation of nucleic acid sequencing libraries, e.g., suitable for any of the sequencing methods described herein (e.g., NGS and / or hybrid-capture NGS), are known in the art. In some embodiments, the sequencing library is prepared as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In an exemplary method, nucleic acids, e.g., double stranded DNA (dsDNA), are fragmented, for example, using sonication. In some embodiments, nucleic acids are fragmented to a length of about 200 base pairs. In some embodiments, the fragmented nucleic acids are purified, e.g., using any suitable method, such as using AMPure XP Beads (Agencourt) and / or solid phase reversible immobilization (SPRI) methods. In some embodiments, sequencing library construction using the purified nucleic acids is carried out using any suitable method, e.g., using commercially available library preparation kits, such as an NEBNext kit (e.g., available from New England Biolabs). In some embodiments, library preparation is performed using a “with-bead” protocol. See, e.g., Fisher et al., Genome Biol (2011) 12:R1. In some embodiments, the library preparation method is selected based on the sequencing method used, e.g., an NEBNext kit is suitable for use with NGS sequencing platforms from Illumina Inc. In some embodiments, a sequencing library indexed, e.g., with barcodes such as six base pair barcodes, is amplified, e.g., using any suitable method, such as PCR. In some embodiments, amplified nucleic acids are purified using any suitable method, such as SPRI purification. In some embodiments, the methods further comprise quantifying the amplified and / or purified nucleic acids, e.g., by qPCR. In some embodiments, the methods further comprise sizing the amplified and / or purified nucleic acids using any suitable method, such as using a LabChip GX system, e.g., available from Caliper Life Sciences. In some embodiments, size selection is not performed.

[0102] In some embodiments, the methods further comprise selectively enriching for one or more nucleic acids (e.g., one or more nucleic acids corresponding to an ESRI gene / coding sequence or a portion thereof) to produce an enriched sample. In some embodiments, the selectively enriching is performed on a sequencing library, e.g., a sequencing library prepared according to the methods described herein. In some embodiments, the selectively enriching is performed as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In an exemplary process, the methods comprise performing solution hybridization using 5’-biotinylated DNA oligonucleotide baits, which may be prepared or synthesized using any suitable method known in the art, e.g., as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, the methods comprise denaturing the sequencing library. In some embodiments, denaturing is performed at a temperature of about 95 °C, e.g., for about 5 minutes. In some embodiments, the methods further comprise incubating the denatured sequencing library at a temperature of about 68 °C, e.g., for about 5 minutes. In some embodiments, the methods further comprise mixing the sequencing library with baits, and optionally Cot, salmon sperm, and / or adaptor- specific blocker DNA in hybridization buffer. In some embodiments, the mixture is incubated for about 24 hours. In some embodiments, the methods further comprise capturing sequencing library-bait duplexes using any suitable method, such as using paramagnetic MyOne streptavidin beads (available from Invitrogen). In some embodiments, the methods further comprise washing to remove off- target library. In some embodiments, the methods further comprise amplifying the captured sequencing library, e.g., using PCR. In some embodiments, the methods further comprise purifying the amplification products using any suitable method, such as SPRI purification. In some embodiments, the methods further comprise quantifying the amplified and / or purified nucleic acids, e.g., by qPCR or any other suitable method. In some embodiments, the methods further comprise sizing the amplified and / or purified nucleic acids using any suitable method, such as using a LabChip GX system, e.g., available from Caliper Life Sciences. In some embodiments, the methods further comprise sequencing using any suitable method or system known in the art, e.g., as described herein. In some embodiments, sequencing is performed using a next-generation sequencer, such as an Illumina HiSeq 2000 system. In some embodiments, sequencing is performed using paired-end sequencing. In some embodiments, the sequencing is performed as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031.

[0103] In some embodiments, the methods further comprise analyzing sequence data obtained from the sequencing, e.g., a plurality of sequence reads, for one or more ESRI mutations. In some embodiments, the analysis is performed as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031, and / or Sun et al., PLoS Comput Biol. 2018 Feb 7;14(2):el005965. In some embodiments, analyzing sequence data, e.g., a plurality of sequence reads, for ESRI mutations comprises one or more, or all, of the steps as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031.

[0104] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (i) obtaining a sample from an individual e.g., anindividual having, suspected of having, or determined to have cancer), (ii) extracting nucleic acid molecules (e.g., a mixture of tumor or cancer nucleic acid molecules and optionally nontumor or non-cancer nucleic acid molecules) from the sample, (iii) ligating one or more adapters to the nucleic acid molecules extracted from the sample (e.g., one or more amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences), (iv) amplifying the nucleic acid molecules (e.g., using a polymerase chain reaction (PCR) amplification technique, a non- PCR amplification technique, or an isothermal amplification technique), (v) capturing nucleic acid molecules from the amplified nucleic acid molecules (e.g., by hybridization to one or more bait molecules, wherein the bait molecules each comprise one or more nucleic acid molecules (e.g., capture nucleic acid molecules) that each comprise a region that is complementary to a region of a captured nucleic acid molecule), (vi) sequencing the nucleic acid molecules extracted from the sample (or library proxies derived therefrom) using, e.g., a next-generation (massively parallel) sequencing technique, a whole genome sequencing (WGS) technique, a whole exome sequencing technique, a targeted sequencing technique, a direct sequencing technique, or a Sanger sequencing technique) using, e.g., a next-generation (massively parallel) sequencer, and (vii) generating, displaying, transmitting, and / or delivering a report (e.g., an electronic, web-based, or paper report) to the individual (or patient), a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office. In some instances, the report comprises output from the methods described herein. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal. In some instances, the report is transmitted via a computer network or peer-to-peer connection.

[0105] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a plurality of nucleic acid molecules obtained from a sample from an individual (e.g., an individual having cancer), wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an ESRI gene / coding sequence or portion thereof; (b) ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; (d) capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; (e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of theplurality of sequence reads correspond to an ESRI gene / coding sequence or portion thereof; (f) analyzing the plurality of sequence reads to detect one or more ESRI mutations, e.g., as described above; (g) based on the analysis, measuring a variant allele frequency of the one or more ESRI mutations; (h) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and (i) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the methods further comprise receiving, at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, the analyzing the plurality of sequence reads comprises identifying, using the one or more processors, the presence or absence of sequence reads corresponding to an ESRI gene / coding sequence or portion thereof. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.

[0106] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a sample from an individual (e.g., an individual having, suspected of having or determined to have cancer), wherein the sample comprises a plurality of nucleic acid molecules; (b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; (c) amplifying said library; (d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to an ESRI gene / coding sequence or portion thereof in said library to produce an enriched sample; (e) sequencing the enriched sample, thereby producing a plurality of sequence reads; (f) analyzing the plurality of sequence reads for presence of the one or more ESRI mutations, e.g., as described above; (g) based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations; (h) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and (i) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

[0107] In some embodiments of any of the methods provided herein, the plurality of nucleic acid molecules (e.g., from a liquid biopsy) or a liquid biopsy sample comprises a mixture of tumor nucleic acid molecules and non-tumor nucleic acid molecules. In some embodiments, the tumor nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample; and the non-tumor nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample or a cell-free DNA (cfDNA) fraction of the liquid biopsy sample.

[0108] In some embodiments of any of the methods, the one or more adapters comprise amplification primers, flow cell adaptor sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the one or more adapters comprise one or more sample index sequences. As is known in the art, sample indexes allow the sequencing of multiple samples on the same instrument flow cell or chip (i.e., multiplexing). Sample indexes are typically between about 8 and about 10 bases in length, and comprise a nucleotide sequence specific to a sample that is used to assign sequence reads to the correct sample during data analysis. In some embodiments, the one or more adapters comprise one or more unique molecule identifiers (UMIs). As is known in the art, UMIs comprise short nucleotide sequences that include a unique barcode that is incorporated into each molecule in a given sample library. UMIs are useful for identifying PCR duplicates created during library amplification steps, and / or for reducing the rate of false-positive variant calls and increasing variant detection, since variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0109] In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the captured nucleic acid molecules are captured from the amplified nucleic acid molecules by hybridization to one or more bait molecules. In some embodiments, the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS). In some embodiments, the sequencer comprises a next generation sequencer.

[0110] In some embodiments of any of the methods provided herein, the methods further comprise selectively enriching for one or more nucleic acids in the sample comprising nucleotide sequences corresponding to ESRI. In some embodiments, the selectively enriching produces an enriched sample. In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the sample, thereby hybridizing the one or morebait molecules to one or more nucleic acids in the sample comprising nucleotide sequences corresponding to ESRI and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the selectively enriching comprises amplifying the one or more nucleic acids comprising nucleotide sequences corresponding to ESRI using a polymerase chain reaction (PCR) to produce an enriched sample. In some embodiments, the methods further comprise sequencing the enriched sample.

[0111] In some embodiments of any of the methods for detection of ESRI mutation(s) provided herein, the methods further comprise analyzing sequence data (e.g., obtained from sequencing as described above), for the presence or absence of one or more alterations (e.g., a base substitution, a short insertion / deletion (indel), a copy number alteration, or a genomic rearrangement) in one or more genes (e.g., ESRI, or a panel of known / suspected oncogenes and / or tumor suppressors, or any combination thereof). In some embodiments, the presence or absence of one or more gene alterations of the disclosure is detected using any suitable method known in the art, e.g., as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, base substitution alterations are detected using Bayesian methodology, which allows detection of novel somatic mutations at low mutant allele frequency (MAF) and increased sensitivity for mutations at hotspot sites through the incorporation of tissue- specific prior expectations. See, e.g., Kim et al., Cancer Discov (2011) 1:44-53 and Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, insertion / deletion (indel) alterations are detected using any suitable method, such as de novo local assembly, e.g., using the de Bruijn approach, see, e.g., Compeau et al., Nat Biotechnol (2011) 29:987-991 and Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, gene fusion and genomic rearrangement alterations are detected using any suitable method, such as by analyzing chimeric read pairs (read pairs for which reads map to separate chromosomes, or at a distance of over 10 Mbp), see, e.g., Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, rearrangements are annotated for predicted function (e.g., creation of fusion gene or tumor suppressor inactivation).

[0112] In some embodiments of any of the methods for detection of ESRI mutations provided herein (see, e.g., sections i-iv herein), the methods further comprise generating a molecular profile for the individual or the sample, based, at least in part, on detecting the ESRI mutation(s) and / or a clonal fraction thereof. In some embodiments, the molecular profile for the individual or sample further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNAmethylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile further comprises results from a nucleic acid sequencing-based test. In some instances, a molecular profile may comprise information on the presence of genes (or variant sequences thereof), copy number variations, epigenetic traits, proteins (or modifications thereof), and / or other biomarkers in an individual’s genome and / or proteome, as well as information on the individual’s corresponding phenotypic traits and the interaction between genetic or genomic traits, phenotypic traits, and environmental factors.

[0113] In some embodiments of any of the methods provided herein, the methods further comprise selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an anticancer agent other than a SERM or an aromatase inhibitor, or an endocrine therapy, e.g., as described herein. In some embodiments of any of the methods provided herein, the methods further comprise generating a report indicating the presence or absence of an ESRI mutation and / or a clonal fraction thereof in the sample. In some embodiments of any of the methods for provided herein, the methods further comprise generating, by one or more processors, a report indicating the presence or absence of an ESRI mutation in the sample, or a clonal fraction thereof. In some embodiments, the report comprises the generated molecular profile. In some embodiments, the methods further comprise providing or transmitting the report, e.g., as described below. In some embodiments, the report is transmitted via a computer network or a peer-to-peer connection. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal.

[0114] In some embodiments of any of the methods provided herein, the methods for measuring a clonal fraction of one or more ESRI mutations may be implemented as part of a genomic profiling process that comprises identification of the presence of variant sequences at one or more gene loci in a sample derived from an individual as part of detecting, monitoring, predicting a risk factor, or selecting a treatment for a particular disease, e.g., cancer. In some instances, the variant panel selected for genomic profiling may comprise the detection of variant sequences at a selected set of gene loci. In some instances, the variant panel selected for genomic profiling may comprise detection of variant sequences at a number of gene loci through comprehensive genomic profiling (CGP), a next- generation sequencing (NGS) approach used to assess hundreds of genes (including relevant cancer biomarkers) in a single assay. Inclusion of the disclosed methods for determining the presence or absence of an ESRI mutation as part of a genomic profiling process can improvethe validity of, e.g., disease detection calls by, for example, independently confirming the presence of the ESRI mutation in a given patient sample.

[0115] The disclosed methods may be used with any of a variety of samples, e.g., as described in further detail below. For example, in some instances, the sample may comprise a liquid biopsy sample. In some instances, the sample may be a liquid biopsy sample and may comprise blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some instances, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample may be a liquid biopsy sample and may comprise cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some instances, the nucleic acid molecules extracted from a sample may comprise a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid molecules. In some instances, the tumor or cancer nucleic acid molecules may be derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample while the non-tumor or non-cancer nucleic acid molecules may be derived from a non-tumor or non-cancer, cell-free DNA (cfDNA) fraction of the liquid biopsy sample. In some embodiments of any of the methods provided herein, the method further comprises determining the circulating tumor DNA (ctDNA) fraction of a liquid biopsy sample.

[0116] In some embodiments of the methods provided herein, nucleic acid molecules (e.g., corresponding to one or more genes, such as ESRF) are captured (e.g., from amplified nucleic acids) by hybridization with a bait molecule. In some embodiments, a bait molecule comprises a capture nucleic acid molecule configured to hybridize to a target nucleic acid molecule, or a fragment or portion thereof. In some embodiments, the capture nucleic acid molecule is configured to hybridize to a fragment of a target (e.g., a fragment of one or more genes, such as ESRF). In some embodiments, the fragment comprises (or is) between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the fragment comprises (or is) about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, or about 300 nucleotides in length. In some embodiments, the capture nucleic acid molecule comprises (or is) between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises (or is) about 100 nucleotides, about 125 nucleotides, about 150nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, or about 300 nucleotides in length. In some embodiments, the capture nucleic acid molecule is configured to hybridize to a nucleotide sequence in an intron or an exon of a gene, e.g., an ESRI gene. In some embodiments, the capture nucleic acid molecule is a DNA, RNA, or a DNA / RNA molecule. In some embodiments, the capture nucleic acid molecule comprises any of between about 50 and about 1000 nucleotides, between about 50 and about 500 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises any of between about 50 nucleotides and about 100 nucleotides, about 100 nucleotides and about 150 nucleotides, about 150 nucleotides and about 200 nucleotides, about 200 nucleotides and about 250 nucleotides, about 250 nucleotides and about 300 nucleotides, about 300 nucleotides and about 350 nucleotides, about 350 nucleotides and about 400 nucleotides, about 400 nucleotides and about 450 nucleotides, about 450 nucleotides and about 500 nucleotides, about 500 nucleotides and about 550 nucleotides, about 550 nucleotides and about 600 nucleotides, about 600 nucleotides and about 650 nucleotides, about 650 nucleotides and about 700 nucleotides, about 700 nucleotides and about 750 nucleotides, about 750 nucleotides and about 800 nucleotides, about 800 nucleotides and about 850 nucleotides, about 850 nucleotides and about 900 nucleotides, about 900 nucleotides and about 950 nucleotides, or about 950 nucleotides and about 1000 nucleotides. In some embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises about 150 nucleotides. In some embodiments, the capture nucleic acid molecule is about 150 nucleotides. In some embodiments, the capture nucleic acid molecule comprises about 170 nucleotides. In some embodiments, the capture nucleic acid molecule is about 170 nucleotides.

[0117] In some embodiments, a bait provided herein includes a label, a tag or detection reagent. In some embodiments, the label, tag or detection reagent is a radiolabel, a fluorescent label, an enzymatic label, a sequence tag, biotin, or another ligand. In some embodiments, a bait provided herein includes a detection reagent such as a fluorescent marker. In some embodiments, a bait provided herein includes (e.g., is conjugated to) an affinity tag or reagent, e.g., that allows capture and isolation of a hybrid formed by a bait and a nucleic acidmolecule hybridized to the bait. In some embodiments, the affinity tag or reagent is an antibody, an antibody fragment, biotin, or any other suitable affinity tag or reagent known in the art. In some embodiments, a bait is suitable for solution phase hybridization.

[0118] Baits can be produced and used according to methods known in the art, e.g., as described in WO2012092426 Al and / or or in Frampton et al (2013) Nat Biotechnol, 31:1023- 1031, incorporated herein by reference. For example, biotinylated baits (e.g., RNA baits) can be produced by obtaining a pool of synthetic long oligonucleotides, originally synthesized on a microarray, and amplifying the oligonucleotides to produce the bait sequences. In some embodiments, the baits are produced by adding an RNA polymerase promoter sequence at one end of the bait sequences, and synthesizing RNA sequences using RNA polymerase. In one embodiment, libraries of synthetic oligodeoxynucleotides can be obtained from commercial suppliers, such as Agilent Technologies, Inc., and amplified using known nucleic acid amplification methods.

[0119] In some embodiments, a bait provided herein is between about 100 nucleotides and about 300 nucleotides in length. In some embodiments, a bait provided herein is between about 130 nucleotides and about 230 nucleotides in length. In some embodiments, a bait provided herein is between about 150 nucleotides and about 200 nucleotides in length. In some embodiments, a bait provided herein comprises a target- specific bait sequence and universal tails on each end. In some embodiments, the target- specific sequence is between about 40 nucleotides and about 300 nucleotides in length. In some embodiments, the targetspecific sequence is between about 100 nucleotides and about 200 nucleotides in length. In some embodiments, the target- specific sequence is between about 120 nucleotides and about 170 nucleotides in length. In some embodiments, the target- specific sequence is about 150 nucleotides or about 170 nucleotides in length. In some embodiments, a bait provided herein comprises an oligonucleotide comprising about 200 nucleotides, of which about 150 nucleotides or about 170 nucleotides are target-specific, and the other 50 nucleotides or 30 nucleotides (e.g., 25 or 15 nucleotides on each end of the bait) are universal arbitrary tails, e.g., suitable for PCR amplification.

[0120] The baits described herein can be used for selection of exons and short target sequences. In some embodiments, a bait of the disclosure distinguishes a target nucleic acid molecule, e.g., a genomic or transcribed nucleic acid molecule, e.g., a cDNA or RNA from a reference nucleotide sequence.

[0121] Provided herein are oligonucleotides, e.g., useful as primers, suitable for the detection of an ESRI mutation, e.g., according to any methods of detection known in the art and / ordescribed herein.

[0122] In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence configured to hybridize to a target nucleic acid molecule (e.g., corresponding to a gene, such as an ESRI gene), or a fragment or portion thereof. In some embodiments, the oligonucleotide comprises a nucleotide sequence configured to hybridize to an ESRI gene or a fragment thereof. In some embodiments, the oligonucleotide, e.g., the primer, comprises a nucleotide sequence configured to hybridize to a nucleotide sequence in an intron or an exon of a gene (e.g., an ESRI gene), or a fragment thereof. In some embodiments, the oligonucleotide comprises a nucleotide sequence corresponding to a gene, such as an ESRI gene. In some embodiments, the oligonucleotide comprises a nucleotide sequence corresponding to a fragment or a portion of a gene, such as an ESRI gene. In some embodiments, the fragment or portion comprises between about 10 and about 30 nucleotides, between about 12 and about 20 nucleotides, or between about 12 and about 17 nucleotides. In some embodiments, the oligonucleotide comprises a nucleotide sequence complementary to the sequence of a gene, such as an ESRI gene. In some embodiments, the oligonucleotide comprises a nucleotide sequence complementary to a fragment or a portion of the sequence of a gene, such as an ESRI gene. In some embodiments, the fragment or portion comprises between about 10 and about 30 nucleotides, between about 12 and about 20 nucleotides, or between about 12 and about 17 nucleotides.

[0123] In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence that is sufficiently complementary to its target nucleotide sequence such that the oligonucleotide specifically hybridizes to a nucleic acid molecule comprising the target nucleotide sequence, e.g., under high stringency conditions. In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence that is sufficiently complementary to its target nucleotide sequence such that the oligonucleotide specifically hybridizes to a nucleic acid molecule comprising the target nucleotide sequence under conditions that allow a polymerization reaction (e.g., PCR) to occur.

[0124] In some embodiments, an oligonucleotide, e.g., a primer, provided herein may be useful for initiating DNA synthesis via PCR (polymerase chain reaction) or a sequencing method. In some embodiments, the oligonucleotide may be used to amplify a target nucleic acid molecule (e.g., a gene such as an ESRI gene, or a portion thereof), e.g., using PCR. In some embodiments, the oligonucleotide may be used to sequence a target nucleic acid molecule (e.g., a gene such as an ESRI gene, or a portion thereof). In some embodiments, pairs of oligonucleotides, e.g., pairs of primers, are provided herein, which are configured tohybridize to a target nucleic acid molecule (e.g., a gene such as an ESRI gene, or a portion thereof), or a fragment thereof. In some embodiments, a pair of oligonucleotides of the disclosure may be used for directing amplification of a target nucleic acid molecule (e.g., a gene such as an ESRI gene, or a portion thereof), or fragment thereof, e.g., using a PCR reaction.

[0125] In some embodiments, an oligonucleotide, e.g., a primer, provided herein is a single stranded nucleic acid molecule, e.g., for use in sequencing or amplification methods. In some embodiments, an oligonucleotide provided herein is a double stranded nucleic acid molecule. In some embodiments, a double stranded oligonucleotide is treated, e.g., denatured, to separate its two strands prior to use, e.g., in sequencing or amplification methods. Oligonucleotides provided herein comprise a nucleotide sequence of sufficient length to hybridize to their target, and to prime the synthesis of extension products, e.g., during PCR or sequencing.

[0126] In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 8 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 10 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 12 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 15 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 30 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 30 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 25 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 15 deoxyribonucleotides or ribonucleotides. In someembodiments, an oligonucleotide provided herein comprises between about 12 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 17 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, the length and nucleotide sequence of an oligonucleotide provided herein is determined according to methods known in the art, e.g., based on factors such as the specific application (e.g., PCR, sequencing library preparation, sequencing), reaction conditions (e.g., buffers, temperature), and the nucleotide composition of the nucleotide sequence of the oligonucleotide or of its target complementary sequence.

[0127] In some embodiments, the sample is obtained from an individual having a cancer, such as a cancer described herein. In some embodiments, the methods provided herein comprise obtaining one or more samples from the individual (e.g., the individual having a cancer). In some embodiments, the one or more samples are obtained or derived from a cancer (e.g., a cancer in an individual).

[0128] In some embodiments, any of the methods of the present disclosure comprise acquiring knowledge of or detecting one or more ESRI mutations in one or more samples (e.g., as described above) obtained from an individual (e.g., an individual having a cancer).Cancers and Methods Related Thereto

[0129] Certain aspects of the present disclosure relate to methods for identifying an individual having a cancer who may benefit from a treatment comprising an anti-cancer therapy; selecting a treatment for an individual having a cancer; identifying one or more treatment options for an individual having a cancer; predicting survival of an individual having a cancer; treating or delaying progression of cancer; monitoring, evaluating or screening an individual having a cancer; monitoring progression or recurrence of a cancer in an individual; identifying a candidate treatment for a cancer in an individual in need thereof; monitoring resistance to endocrine therapy; or predicting responsiveness to endocrine therapy.

[0130] In some embodiments of any of the methods provided herein, the methods comprise measuring clonal fraction of one or more ESRI mutations and / or one or more oncogenic or tumor suppressor mutations, e.g., as described herein. In some embodiments, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction e.g., an ESRI clonal fraction) that is greater than or equal to a threshold ratio identifies the individual as one who may benefit from a treatment comprising an anti-cancer agent other than aselective estrogen receptor modulator (SERM) or an aromatase inhibitor, thereby predicting or monitoring resistance to endocrine therapy. In some embodiments, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., an ESRI clonal fraction) that is less than a threshold ratio identifies the individual as one who may be responsive to endocrine therapy, thereby predicting sensitivity or responsiveness to endocrine therapy.

[0131] In some embodiments, the methods comprise measuring a first clonal fraction of one or more ESRI mutations in a first liquid biopsy sample obtained from an individual at a first timepoint (e.g., based on a ratio of variant allele frequency of the one or more ESRI mutations to tumor fraction in the first liquid biopsy sample) and measuring a second clonal fraction of one or more ESRI mutations in a second liquid biopsy sample obtained from the individual at a second, later timepoint e.g., based on a ratio of variant allele frequency of the one or more ESRI mutations to tumor fraction in the second liquid biopsy sample). This can be used, for example, to monitor for or predict resistance to endocrine therapy over time. For example, in some embodiments, the second timepoint can be during or after treatment with an endocrine therapy. In some embodiments, a greater clonal fraction of one or more ESRI mutations in the second timepoint vs. the first timepoint indicates increasing resistance to endocrine therapy. In some embodiments, a lesser clonal fraction of one or more ESRI mutations in the second timepoint vs. the first timepoint indicates increasing sensitivity to endocrine therapy.

[0132] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based at least in part on a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations). In some embodiments, the report indicates the clonal fraction of one or more ESRI mutations and / or identity of particular ESRI mutation(s). In some embodiments, e.g., when the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., the clonal fraction of one or more ESRI mutations) is greater than or equal to a threshold ratio, the one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments, e.g., when the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., the clonal fraction of one or more ESRI mutations) is less than a threshold ratio, the one or more treatment options comprise an endocrine therapy.

[0133] In some embodiments of any of the methods provided herein, responsive to a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) in a liquid biopsy sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) being greater than or equal to a threshold ratio: (i) the individual is classified as a candidate to receive a treatment comprising an anti-cancer agent other than a SERM or an aromatase inhibitor; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments of any of the methods provided herein, responsive to a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) in a liquid biopsy sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) being less than a threshold ratio: (i) the individual is classified as a candidate to receive a treatment comprising an endocrine therapy; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an endocrine therapy.

[0134] In some embodiments, responsive to acquisition of knowledge of a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) in a liquid biopsy sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer), the methods comprise administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor, e.g., when the ratio is greater than or equal to a threshold ratio. In other embodiments, responsive to acquisition of knowledge of a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) in a liquid biopsy sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer), the methods comprise administering to the individual an effective amount of a treatment that comprises an endocrine therapy, e.g., when the ratio is less than a threshold ratio.

[0135] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based, at least in part, on a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) in a liquid biopsy sample from the individual. In some embodiments, e.g., when the ratio isgreater than or equal to a threshold ratio, the one or more treatment options comprise an anticancer agent other than a SERM or an aromatase inhibitor. In some embodiments, e.g., when the ratio is less than a threshold ratio, the one or more treatment options comprise an endocrine therapy.

[0136] In some embodiments, acquiring knowledge of a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) comprises measuring the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations) in the sample(s).

[0137] In some embodiments, the individual has been previously treated, or is being treated, with an endocrine therapy. In some embodiments, the individual has received one or more prior treatments with an endocrine therapy. In some embodiments, the methods of the present disclosure comprise (e.g., prior to acquiring knowledge of a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction (e.g., a clonal fraction of one or more ESRI mutations)) administering an effective amount of a treatment that comprises an endocrine therapy to the individual.

[0138] In some embodiments of any of the methods provided herein, the cancer is breast cancer. In some embodiments, the breast cancer is hormone receptor positive (HR+) breast cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is advanced or metastatic.

[0139] In some embodiments, the methods of the present disclosure comprise recommending a treatment, providing or identifying one or more treatment options, and / or administering an effective amount of a treatment. The treatment or treatment options can be based, at least in part, on the clonal fraction of one or more ESRI mutations, e.g., in comparison to a threshold ratio. Generally, a higher clonal fraction of one or more ESRI mutations may prompt treatment with an anti-cancer agent other than a SERM or an aromatase inhibitor, whereas a lower clonal fraction of one or more ESRI mutations may prompt treatment with an endocrine therapy.

[0140] Certain aspects of the present disclosure relate to treatment with an endocrine therapy. In some embodiments, the endocrine therapy comprises a SERM or an aromatase inhibitor. Examples of SERMs include, without limitation, tamoxifen (NOLVADEX®, SOLTAMOX®, GENOX), raloxifene (EVISTA®, OPTRUMA), EM652, GW7604, keoxifene, toremifene (FARESTON®), bazedoxifene, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, ospemifene, or pharmaceutically acceptable salts thereof.Examples of aromatase inhibitors include, without limitation, aminoglutethimide, testolactone (TESLAC®), anastrozole (ARIMIDEX®), letrozole (FEMARA®), exemestane (AROMASIN®), vorozole (RIVIZOR), formestane (LENT ARON®), fadrozole (AFEMA), 4-hydroxyandrostenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), 4-Androstene-3, 6, 17- trione (“6-OXO”), or pharmaceutically acceptable salts thereof.

[0141] Certain aspects of the present disclosure relate to treatment with an anti-cancer agent other than a SERM or aromatase inhibitor. In some embodiments, the anti-cancer agent is approved or tested for use in E.S7? / mut cancer. In some embodiments, the anti-cancer agent comprises one or more of a small molecule inhibitor, an antibody, a cellular therapy, a nucleic acid, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), a treatment for cancer comprising an ESRI mutation, an anti-cancer agent being tested in a clinical trial, a treatment for cancer comprising an ESRI mutation being tested in a clinical trial, or any combination thereof. In some embodiments, the anti-cancer agent comprises a selective estrogen receptor covalent antagonist (SERCA), including without limitation H3B-5942, H3B-6545, or a pharmaceutically acceptable salt thereof (see, e.g., Wang, Y. et al. (2023) Acta Pharmaceutica Sinica B 13( 12):4963-4982). In some embodiments, the anti-cancer agent comprises a selective estrogen receptor degrader (SERD), including without limitation fulvestrant (FASLODEX®), elacestrant (ORSERDU™), amcenestrant, camizestrant, giredestrant, rintodestrant, imlunestrant, ZB-716, Zn-c5, LSZ102, LY3484356, or D-0502, or a pharmaceutically acceptable salt thereof see, e.g., Patel, R. et al. (2023) NPJ Breast Cancer 9(l):20). In some embodiments, the anti-cancer agent comprises a PROTAC, including without limitation ARV-471. In some embodiments, the anti-cancer agent comprises a CDK4 / 6 inhibitor, including without limitation palbociclib, abemaciclib, ribociclib, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-cancer agent comprises a PI3K inhibitor, including without limitation GSK2636771, buparlisib, AZD8186, copanlisib, LY294002, PX-866, TGX115, TGX126, BEZ235, SF1126, idelalisib, pictilisib, GDC0032, IPI145, INK1117, SAR260301, KIN-193, duvelisib, GS- 9820, GSK2636771, GDC-0980, AMG319, paxalisib, or alpelisib, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-cancer agent comprises an mTOR inhibitor, including without limitation temsirolimus, everolimus, ridaforolimus, dactolisib, GSK2126458, XL765, AZD8055, AZD2014, MLN128, PP242, NVP-BEZ235, LY3023414, PQR309, PKI587, or OSI027, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-cancer agent is a nucleic acid that inhibits the expression of an ESRInucleic acid molecule e.g., bearing an ESRI mutation), or an ESRI polypeptide encoded by the ESRI nucleic acid molecule, including without limitation a double- stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In some embodiments, the anti-cancer agent is a cellular therapy that is an adoptive therapy, a T cellbased therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy. In some embodiments, the anti-cancer agent can be a targeted therapy that targets, or is otherwise approved for use in treating a cancer comprising a mutation in, one or more oncogenic or tumor suppressor mutations, e.g., detected in a liquid biopsy sample as disclosed herein. In some embodiments, the anti-cancer agent other than a SERM or aromatase inhibitor is a targeted therapy targeting one of the one or more oncogenic or tumor suppressor mutations.

[0142] In some embodiments, an anti-cancer therapy of the disclosure is a small molecule inhibitor; an antibody; a cellular therapy; a nucleic acid; a virus-based therapy; an antibodydrug conjugate; a recombinant protein; a fusion protein; a natural compound; a peptide; a PROteolysis-TArgeting Chimera (PROTAC); a targeted therapy; or any combination thereof, e.g., a described in further detail below. In some embodiments, the anti-cancer therapy is an immunotherapy, such as any immunotherapy known in the art or described herein (e.g., a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, or oncolytic virus therapy). In some embodiments, the anti-cancer therapy is an immune checkpoint inhibitor, such as any immune checkpoint inhibitor described herein or known in the art.

[0143] In some embodiments, the anti-cancer therapy comprises an immunotherapy (i.e., a cancer immunotherapy), such as a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy, as well as any combination thereof. In some embodiments, the cancer immunotherapy comprises a small molecule, nucleic acid, polypeptide, carbohydrate, toxin, cell-based agent, or cell-binding agent. Examples of cancer immunotherapies are described in greater detail herein but are not intended to be limiting. In some embodiments, the cancer immunotherapy activates one or more aspects of the immune system to attack a cell (e.g., a tumor cell) that expresses a neoantigen. The cancer immunotherapies of the present disclosure are contemplated for use as monotherapies, or in combination approaches comprising two or more in any combination or number, subject to medical judgement. Anyof the cancer immunotherapies (optionally as monotherapies or in combination with another cancer immunotherapy or other therapeutic agent described herein) may find use in any of the methods described herein.

[0144] In some embodiments, the cancer immunotherapy comprises a cancer vaccine. A range of cancer vaccines have been tested that employ different approaches to promoting an immune response against a cancer (see, e.g., Emens L A, 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 against tumors. Exemplary types of cancer vaccines include, but are not limited to, DNA-based vaccines, RNA-based vaccines, virus transduced vaccines, peptide-based vaccines, dendritic cell vaccines, oncolytic viruses, whole tumor cell vaccines, tumor antigen vaccines, etc. 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, a vaccine composition 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, e.g., in poly(DL-lactide- co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et ah, Molec. Immunol. 28:287-294, 1991: Alonso et al, Vaccine 12:299- 306, 1994; Jones et al, Vaccine 13:675-681, 1995); peptide composition contained in immune stimulating complexes (ISCOMS) (e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin. Exp. Immunol. 113:235-243, 1998); or multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196: 17-32, 1996). In some embodiments, a cancer vaccine is formulated as a peptide-based vaccine, or nucleic acid based vaccine in which the nucleic acid encodes the polypeptides. In some embodiments, a cancer vaccine is formulated as an antibody-based vaccine. In some embodiments, a cancer vaccine is formulated as a cell based vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104: 14-21, 2013). In some embodiments, such cancer vaccines augment an anti-cancer response.

[0145] In some embodiments, the cancer vaccine comprises a polynucleotide that encodes a neoantigen, e.g., neoantigen(s) expressed by a cancer of the disclosure, such as a cancer in an individual. In some embodiments, the cancer vaccine comprises DNA that encodes theneoantigen(s). In some embodiments, the cancer vaccine comprises RNA that encodes the neoantigen(s). In some embodiments, the cancer vaccine comprises a polynucleotide that encodes the neoantigen(s). In some embodiments, the cancer vaccine further comprises one or more additional antigens, neoantigens, or other sequences that promote antigen presentation and / or an 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(s) are taken up and translated by antigen presenting cells (APCs), which then present the neoantigen(s) via MHC class I on the APC cell surface.

[0146] In some embodiments, the cancer vaccine is selected from sipuleucel-T (e.g., Provenge®, Dendreon / V aleant Pharmaceuticals), which has been approved for treatment of asymptomatic, or minimally symptomatic metastatic castrate -resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (e.g., Imlygic®, BioVex / Amgen, previously known as T-VEC), a genetically modified oncolytic viral therapy approved for treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma. In some embodiments, the cancer vaccine is selected from an oncolytic viral therapy such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF, for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep (e.g., Reolysin®, Oncolytics Biotech), a variant of respiratory enteric orphan virus (reovirus) which does not replicate in cells that are not RAS-activated, in numerous cancers, including colorectal cancer (NCT01622543), prostate cancer (NCT01619813), head and neck squamous cell cancer (NCT01166542), pancreatic adenocarcinoma (NCT00998322), and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAdl), an adenovirus engineered to express a full length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117), metastatic or advanced epithelial tumors such as in 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); GE-ONC1 (GEV-lh68 / GEV-lhl53, Genelux GmbH), vaccinia viruses engineered to express beta-galactosidase (beta-gal) / beta-glucoronidase or beta-gal / human sodium iodide symporter (hNIS), respectively, were studied in peritoneal carcinomatosis (NCTO 1443260), fallopian tube cancer, ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818);anti-gplOO; STINGVAX; GV AX; DCVaxL; and DNX-2401. In some embodiments, the cancer vaccine is selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is able to convert the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapy agents targeted for difficult-to-treat RAS mutations; and TILT- 123 (TILT Biotherapeutics), an engineered adenovirus designated: Ad5 / 3-E2F-delta24-hTNFa-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 raise an antigen-specific CD8+ T cell response. In some embodiments, the cancer vaccine comprises a vector-based tumor antigen vaccine. Vectorbased tumor antigen vaccines can be used as a way to provide a steady supply of antigens to stimulate an anti-tumor immune response. In some embodiments, vectors encoding for tumor antigens are injected into an individual (possibly with pro-inflammatory or other attractants such as GM-CSF), taken up by cells in vivo to make the specific antigens, which then provoke the desired immune response. In some embodiments, vectors may be used to deliver more than one tumor antigen at a time, to increase the immune response. In addition, recombinant virus, bacteria or yeast vectors can trigger their own immune responses, which may also enhance the overall immune response.

[0147] In some embodiments, the cancer vaccine comprises a DNA-based vaccine. In some embodiments, DNA-based vaccines can be employed to stimulate an anti-tumor response. The ability of directly injected DNA that encodes an antigenic protein, to elicit a protective immune response has been demonstrated in numerous experimental systems. Vaccination through directly injecting DNA that encodes an antigenic protein, to elicit a protective immune response often produces both cell-mediated and humoral responses. Moreover, reproducible immune responses to DNA encoding various antigens have been reported in mice that last essentially for the lifetime of the animal (see, e.g., Yankauckas et al.(1993) DNA Cell Biol., 12: 771-776). In some embodiments, plasmid (or other vector) DNA that includes a sequence encoding a protein operably linked to regulatory elements required for gene expression is administered to individuals (e.g. human patients, non-human mammals, etc.). In some embodiments, the cells of the individual take up the administered DNA and the coding sequence is expressed. In some embodiments, the antigen so produced becomes a target against which an immune response is directed.

[0148] In some embodiments, the cancer vaccine comprises an RNA-based vaccine. In some embodiments, RNA-based vaccines can be employed to stimulate an anti-tumor response. In some embodiments, RNA-based vaccines comprise a self-replicating RNA molecule. In some embodiments, the self-replicating RNA molecule may be an alphavirus-derived RNA replicon. Self-replicating RNA (or "SAM") molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A selfreplicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces 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, may be translated themselves to provide in situ expression of an encoded polypeptide, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.

[0149] In some embodiments, the cancer immunotherapy comprises a cell-based therapy. In some embodiments, the cancer immunotherapy comprises a T cell-based therapy. In some embodiments, the cancer immunotherapy comprises an adoptive therapy, e.g., an adoptive T cell-based therapy. In some embodiments, the T cells are autologous or allogeneic to the recipient. In some embodiments, the T cells are CD 8+ T cells. In some embodiments, the T cells are CD4+ T cells. Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) cancer cells. In some embodiments, the immune response results in inhibition of tumor and / or metastatic cell growth and / or proliferation, and in related embodiments, results in neoplastic cell death and / or resorption. The immune cells can be derived from a different organism / host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells). In some embodiments, the immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, or NKT cells) can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, the host cells (e.g., autologous or allogeneic T-cells) are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen. In some embodiments, NK cells are engineered to express a TCR. The NK cells may befurther engineered to express a CAR. Multiple CARs and / or TCRs, such as to different antigens, may be added to a single cell type, such as T cells or NK cells. 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 genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g. chimeric). In some embodiments, a population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. In some embodiments, a population of immune cells can be obtained from a donor, such as a histocompatibility-matched donor. In some embodiments, the immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells reside in said subject or donor. In some embodiments, the immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood. In some embodiments, when the population of immune cells is obtained from a donor distinct from the subject, the donor may be allogeneic, provided the cells obtained 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, to be rendered subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

[0150] In some embodiments, the cell-based therapy comprises a T cell-based therapy, such as 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 virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non-tumor- specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibodylike tumor recognition capacity known as "T- bodies". Several approaches for the isolation, derivation, engineering or modification, activation, and expansion of functional anti-tumor effector cells have been described in the last two decades and may be used according to any of the methods provided herein. In some embodiments, the T cells are derived from theblood, 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 those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen-specificity, type of antigen receptor, 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 for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0151] In some embodiments, the T cell-based therapy comprises a chimeric antigen receptor (CAR)-T cell-based therapy. This approach involves engineering a CAR that specifically binds to an antigen of interest and comprises 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, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the CAR specifically binds a neoantigen, such as a neoantigen expressed in a cancer of a disclosure, e.g., in an individual.

[0152] In some embodiments, the T cell-based therapy comprises T cells expressing a recombinant T cell receptor (TCR). This approach involves identifying a TCR that specifically binds to an antigen of interest, which is then used to replace the endogenous or native TCR on the surface of engineered T cells that are administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the recombinant TCR specifically binds a neoantigen expressed in a cancer of a disclosure, e.g., in an individual.

[0153] In some embodiments, the T cell-based therapy comprises 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 may specifically recognize an antigen expressed by the tumor or cancer of the present disclosure. In some embodiments, the TILs are exposed to one or more neoantigens, e.g., expressed in a cancer of a disclosure, in vitro after isolation. TILs are then administered to the patient (optionally in combination with one or more cytokines or other immune-stimulating substances).

[0154] In some embodiments, the cell-based therapy comprises a natural killer (NK) cellbased therapy. Natural killer (NK) cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are critical effectors of the early innate immune response toward transformed and virus-infected cells. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. 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 derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art.

[0155] In some embodiments, the cell-based therapy comprises a dendritic cell (DC)-based therapy, e.g., a dendritic cell vaccine. In some embodiments, the DC vaccine comprises antigen-presenting cells that are able to induce specific T cell immunity, which are harvested from the patient or from a donor. In some embodiments, the DC vaccine can then be exposed in vitro to a peptide antigen, for which T cells are to be generated in the patient. In some embodiments, dendritic cells loaded with the antigen are then injected back into the patient. In some embodiments, immunization may be repeated multiple times if desired. Methods for harvesting, expanding, and administering dendritic cells are known in the art; see, e.g., W02019178081. Dendritic cell vaccines (such as Sipuleucel-T, also known as APC8015 and PROVENGE®) are vaccines that involve administration of dendritic cells that act 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.

[0156] 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 an antigen expressed by a cancer of the present disclosure, e.g., a neoantigen expressed in a cancer of a disclosure, e.g., in an individual. The TCR-based therapeutic may further include a moiety that binds an immune cell (e.g., a T cell), such as an antibody or antibody fragment that specifically binds a T cell surface protein or receptor (e.g., an anti-CD3 antibody or antibody fragment).

[0157] In some embodiments, the immunotherapy comprises adjuvant immunotherapy. Adjuvant immunotherapy comprises the use of one or more agents that activate components of the innate immune system, e.g., HILTONOL® (imiquimod), which targets the TLR7 pathway.

[0158] In some embodiments, the immunotherapy comprises cytokine immunotherapy. Cytokine immunotherapy comprises the use of one or more cytokines that activate components of the immune system. Examples include, but are not limited to, aldesleukin (e.g., PROLEUKIN®; interleukin-2), interferon alfa-2a (e.g., ROFERON®-A), interferon alfa-2b (e.g., INTRON®-A), and peginterferon alfa-2b (e.g., PEGINTRON®).

[0159] In some embodiments, the immunotherapy comprises oncolytic virus therapy. Oncolytic virus therapy uses genetically modified viruses to replicate in and kill cancer cells, leading to the release of antigens that stimulate an immune response. In some embodiments, replication-competent oncolytic viruses expressing a tumor antigen comprise any naturally occurring (e.g., from a “field source”) or modified replication-competent oncolytic virus. In some embodiments, the oncolytic virus, in addition to expressing a tumor antigen, may be modified to increase selectivity of the virus for cancer cells. In some embodiments, replication-competent oncolytic viruses include, but are not limited to, oncolytic viruses that are a member in the family of myoviridae, siphoviridae, podpviridae, teciviridae, corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae, poxyiridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae, adnoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hcpadnaviridae, retroviridae, cyctoviridae, reoviridae, birnaviridae, paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, Leviviridae, picornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae, glaviviridae, togaviridae, and bamaviridae. In some embodiments, replication-competent oncolytic viruses include adenovirus, retrovirus, reovirus, rhabdovirus, Newcastle Disease virus (NDV), polyoma virus, vaccinia virus (VacV), herpes simplex virus, picomavirus, coxsackie virus and parvovirus. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be engineered to lack one or more functional genes in order to increase the cancer selectivity of the virus. In some embodiments, an 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, an oncolytic vaccinia virus may be engineered to lack both VGF and TK activity. In some embodiments, an oncolytic vaccinia virus may be engineered to lack one or more genes involved in evading host interferon (IFN) response such as E3L, K3L, B18R, or B8R. In some embodiments, a 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 aWestern Reserve, Copenhagen, Lister or Wyeth strain lacking a functional B18R and / or B8R gene. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be locally or systemically administered to a subject, e.g. via intratumoral, intraperitoneal, intravenous, intra-arterial, intramuscular, intradermal, intracranial, subcutaneous, or intranasal administration.

[0160] In some embodiments, the anti-cancer therapy comprises an immune checkpoint inhibitor. In some embodiments, the methods provided herein comprise administering to an individual an effective amount of an immune checkpoint inhibitor. As is known in the art, a checkpoint inhibitor targets at least one immune checkpoint protein to alter the regulation of an immune response. Immune checkpoint proteins include, e.g., 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, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, 0X40, and A2aR. In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to: PD-1 (CD279), PD-L1 (B7- Hl, CD274), PD-L2 (B7-CD, CD273), CTLA-4 (CD152), HVEM, BTLA (CD272), a killercell immunoglobulin-like receptor (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM-1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRbeta, 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, 0X40 (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, an immune checkpoint inhibitor (i.e., a checkpoint inhibitor) decreases the activity of a checkpoint protein that negatively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In other embodiments, a checkpoint inhibitor increases the activity of a checkpoint protein that positively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In some embodiments, the checkpoint inhibitor is an antibody. Examples of checkpoint inhibitors include, without limitation, a PD- 1 axis binding antagonist, a PD-L1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., atezolizumab (MPDL3280A)), an antagonist directed against a co-inhibitory molecule (e.g., a CTLA4 antagonist (e.g., an anti-CTLA4 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof. In some embodiments, the immune checkpoint inhibitors comprise drugs such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (see, e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). In some embodiments, known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.

[0161] In some embodiments, the checkpoint inhibitor is a PD-L1 axis 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 shown in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1,” "PDCD1 LG1," "CD274," "B7- H," and "PDL1." An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot Accession No.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 shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

[0162] In some embodiments, the checkpoint inhibitor is a PD-1 binding antagonist / inhibitor. In some embodiments, the PD-1 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific embodiment, the PD-1 ligand binding partners are PD-L1 and / or PD-L2. In some embodiments, the checkpoint inhibitor is a PD-L1 binding antagonist / inhibitor. In some embodiments, a PD-L1 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-L1 to its binding ligands. In a specific embodiment, PD-L1 binding partners are PD-1 and / or B7-1. In some embodiments, the checkpoint inhibitor is a PD-L2 binding antagonist / inhibitor. In some embodiments, the PD-L2 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-L2 to its ligand binding partners. In a specific embodiment, the PD-L2 binding ligand partner is PD- 1. The antagonist or inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1, PD-L1, or PD-L1 binding antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0163] In some instances, the PD-1 binding antagonist or inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), for example, asdescribed below. In some instances, the anti-PD-1 antibody is one or more of MDX-1 106 (nivolumab), MK-3475 (pembrolizumab, e.g., Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, BGB-108, BGB-A317, JS-001, STI-Al l 10, INCSHR-1210, PF-06801591, TSR-042, AM0001, ENUM 244C8, ENUM 388D4, cemiplimab, or dostarlimab. In other instances, the PD-1 binding antagonist or inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an 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 or inhibitor is AMP-224. Other examples of anti-PD-1 antibodies include, but are not limited to, MEDI- 0680 (AMP-514; AstraZeneca), PDR001 (CAS Registry No. 1859072-53-9; Novartis), REGN2810 (e.g., LIBTAYO® or cemiplimab-rwlc; Regeneron), BGB-108 (BeiGene), BGB- A317 (BeiGene), BI 754091, JS-001 (Shanghai Junshi), STI-Al l 10 (Sorrento), INCSHR- 1210 (Incyte), PF-06801591 (Pfizer), TSR-042 (also known as ANB011;Tesaro / AnaptysBio), AM0001 (ARMO Biosciences), ENUM 244C8 (Enumeral Biomedical Holdings), or ENUM 388D4 (Enumeral Biomedical Holdings). In some embodiments, the PD-1 axis binding antagonist or inhibitor comprises tislelizumab (BGB-A317), BGB-108, STI-Al l 10, AM0001, BI 754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP-514), MGA-012 (INCMGA 0012), nivolumab (BMS-936558, MDX1106, ONO- 4538), spartalizumab (PDR001), pembrolizumab (MK-3475, SCH 900475, e.g., Keytruda®), PF-06801591, cemiplimab (REGN-2810, REGEN2810), dostarlimab (TSR-042, ANB011), FITC-YT-16 (PD-1 binding peptide), APL-501 or CBT-501 or genolimzumab (GB-226), AB- 122, AK105, AMG 404, 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 EISCOI 11-003, 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-7769, CAB PD-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, or an antibody that competes with any of the preceding.

[0164] In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-1. In some embodiments, the PD-L1 binding antagonist or inhibitor is asmall molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-L1 and VISTA or PD-L1 and TIM3. In some embodiments, the PD-L1 binding antagonist or inhibitor is CA-170 (also known as AUPM- 170). In some embodiments, the PD-L1 binding antagonist or inhibitor is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody can bind to a human PD-L1, for example a human PD-L1 as described above herein and / or as shown in UniProtKB / Swiss- Prot Accession No.Q9NZQ7.1, or a variant thereof. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0165] In some instances, the PD-L1 binding antagonist or inhibitor is an anti-PD-Ll antibody, for example, as described below. In some instances, the anti-PD-Ll antibody is capable of inhibiting the binding between PD-L1 and PD-1, and / or between PD-L1 and B7-1. In some instances, the anti-PD-Ll antibody is a monoclonal antibody. In some instances, the anti-PD-Ll antibody is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some instances, the anti-PD-Ll antibody is a humanized antibody. In some instances, the anti-PD-Ll antibody is a human antibody. In some instances, the anti- PD-Ll antibody is selected from YW243.55.S70, MPDL3280A (atezolizumab), MDX-1 105, MEDI4736 (durvalumab), MSB0010718C (avelumab), LY3300054, STI-A1014, KN035, FAZ053, or CX-072. In some embodiments, the PD-L1 axis binding antagonist or inhibitor comprises atezolizumab, avelumab, durvalumab (imfinzi), BGB-A333, SHR-1316 (HTI- 1088), CK-301, BMS-936559, envafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS- 936559), LY3300054, STLA1014, FAZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M-7824, HTI-1088 (HTI-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, APL-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-6El l, 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 a derivative thereof, or an antibody that competes with any of the preceding.

[0166] In some embodiments, the checkpoint inhibitor is an antagonist or inhibitor of CTLA4. In some embodiments, the checkpoint inhibitor is a small molecule antagonist or inhibitor 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 that acts to negatively regulate T cell activation, particularly CD28-dependent T cell responses. CTLA4 competes for binding to common ligands with CD28, such as CD80 (B7-1) and CD86 (B7-2), and binds to these ligands with higher affinity than CD28. Blocking 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 (such as intratumoral Tregs). In some embodiments, the CTLA4 antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the CTLA-4 antagonist or inhibitor comprises ipilimumab (IB 1310, 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, or an antibody that competes with any of the preceding.

[0167] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 antagonist or inhibitor (e.g., an antibody, an antibody conjugate, or an antigenbinding fragment thereof). In some embodiments, the LAG-3 antagonist or 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 antagonist or inhibitor comprises a small molecule. In some embodiments, the LAG-3 antagonist or inhibitor comprises a LAG-3 binding agent. In some embodiments, the LAG-3 antagonist or inhibitor comprises an antibody, an antibody conjugate, or an antigen-binding fragment thereof. In some embodiments, the LAG-3 antagonist or inhibitor comprises eftilagimod alpha (IMP321, IMP-321, EDDP-202, EOC-202), relatlimab (BMS-986016), GSK2831781 (IMP-731), LAG525 (IMP701), TSR-033, EVIP321 (soluble LAG-3 protein), BI 754111, 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 preceding.

[0168] In some embodiments, the immune checkpoint inhibitor is monovalent and / or monospecific. In some embodiments, the immune checkpoint inhibitor is multivalent and / or multispecific.

[0169] In some embodiments, an anti-cancer therapy of the disclosure (e.g., an immunotherapy) is administered in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancerimmunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti- neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof.

[0170] In some embodiments, an anti-cancer therapy of the disclosure comprises a cyclin- dependent kinase (CDK) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the CDK inhibitor inhibits CDK4. In some embodiments, the CDK inhibitor inhibits Cyclin D / CDK4. In some embodiments, the 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 and inhibiting one or more activities of CDK4, binding to and inhibiting expression of CDK4, and / or binding to and inhibiting one or more activities of a cell expressing CDK4, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of CDK4 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). 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, as well as pharmaceutically acceptable salts thereof.

[0171] In some embodiments, an anti-cancer therapy of the disclosure comprises a murine double minute 2 homolog (MDM2) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the MDM2 inhibitor is (a) a small molecule that inhibits one or more activities of MDM2 (e.g., binding to p53), (b) an antibody that inhibits one or more activities of MDM2 (e.g., by binding to and inhibiting one or more activities of MDM2, binding to and inhibiting expression of MDM2, and / or binding to and inhibiting one or more activities of a cell expressing MDM2, such as 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 therapeutic, and the like). In some embodiments, the MDM2 inhibitor is a small molecule inhibitor of MDM2 (e.g., a competitive or noncompetitive 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), as well as pharmaceutically acceptable salts thereof. In some embodiments, the MDM2 inhibitor inhibits or disrupts interaction between MDM2 and p53.

[0172] In some embodiments, an anti-cancer therapy of the disclosure comprises (alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor) one or more of an antimetabolite, DNA-damaging agent, or 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., a BCL2 inhibitor or downregulator, SMAC mimetic, or TRAIL agonist such as ABT-263, ABT-737, oridonin, venetoclax, combination of venetoclax and an anti-CD20 antibody such as obinutuzumab or rituximab, 1396-11, ABT- 10, SM-164, D269H / E195R, or rhTRAIL); a tyrosine kinase inhibitor; an inhibitor of RAS, RAF, MEK, or the MAPK pathway (e.g., AZD6244, dabrafenib, LGX818, PD0325901, pimasertib, trametinib, or vemurafenib); an inhibitor of PI3K, mTOR, or Akt; a CDK inhibitor; a PKC inhibitor (e.g., LXS196 or sotrastaurin); an antibody-based therapeutic (e.g., an anti-PD-1 or anti-PDLl antibody such as atezolizumab, pembrolizumab, nivolumab, or spartalizumab; an anti-CD20 antibody such as obinutuzumab or rituximab; or an anti-DR5 antibody such as drozitumab); a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, or MG- 132); an HD AC inhibitor (e.g., SAHA or VPA); an antibiotic (e.g., actinomycin D); a zinc-containing therapeutic (e.g., zinc or ZMC1); an HSP inhibitor (e.g., geldanamycin); an ATPase inhibitor (e.g., archazolid); a mitotic inhibitor (e.g., paclitaxel or vincristine); metformin; methotrexate; tanshinone IIA; and / or P5091.

[0173] In some embodiments, the anti-cancer therapy comprises an immunoregulatory molecule or a cytokine, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. An immunoregulatory profile is required to trigger an efficient immune response and balance the immunity in a subject. Examples of suitable immunoregulatory cytokines include, but are not limited to, interferons (e.g., IFNa, IFNP and ILNy), 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., TNEa and TNPP), erythropoietin (EPO), LLT-3 ligand, glplO, TCA-3, MCP-1, MIL, MIP-la, MIP-ip, Rantes, macrophage colony stimulating factor (M-CSL), granulocyte colony stimulating factor (G-CSL), or granulocyte-macrophage colony stimulating factor (GM-CSL), as well as 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-3a (Lax), MIP-3P, Hcc-1, MPIF-1, MPIF- 2, MCP-2, MCP-3, MCP-4, MCP-5, Eotaxin, Tare, Elc, 1309, IL-8, GCP-2 Groa, Gro-P, Nap-2, Ena-78, Ip-10, MIG, I-Tac, SDF-1, or BCA-1 (Bic), as well as functional fragments thereof. In some embodiments, the immunoregulatory molecule is included with any of the treatments provided herein.

[0174] In some embodiments, an anti-cancer therapy of the disclosure comprises a tyrosine kinase inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the 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 and inhibiting one or more activities of the tyrosine kinase, binding to and inhibiting expression, such as cell surface expression, of the tyrosine kinase, and / or binding to and inhibiting one or more activities of a cell expressing the tyrosine kinase, such as 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 therapeutic, and the like). In some embodiments, the tyrosine kinase inhibitor is a small molecule inhibitor of a tyrosine kinase (e.g., a competitive or noncompetitive inhibitor). Non-limiting examples of tyrosine kinase inhibitors include imatinib, crenolanib, linifanib, ninetedanib, axitinib, dasatinib, imetelstat, midostaurin, pazopanib, sorafenib, sunitinb, motesanib, masitinib, vatalanib, cabozanitinib, tivozanib, OSI-930, Ki8751, telatinib, dovitinib, tyrphostin AG 1296, and amuvatinib, as well as pharmaceutically acceptable salts thereof.

[0175] In some embodiments, an anti-cancer therapy of the disclosure comprises a mitogen- activated protein kinase (MEK) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint 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 and inhibiting one or more activities of MEK, binding to and inhibiting expression of MEK, and / or binding to and inhibiting one or more activities of a cell expressing MEK, such as 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 therapeutic, and thelike). 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, as well as pharmaceutically acceptable salts thereof. In some embodiments, the anti-cancer therapy inhibits one or more activities of the Raf / MEK / ERK pathway, including inhibitors of Raf, MEK, and / or ERK.

[0176] In some embodiments, an anti-cancer therapy of the disclosure comprises a mammalian target of rapamycin (mTOR) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the 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 and inhibiting one or more activities of mTOR, binding to and inhibiting expression of mTOR, and / or binding to and inhibiting one or more activities of a cell expressing mTOR, such as 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 therapeutic, and the like). In some embodiments, the mTOR inhibitor is a small molecule inhibitor of mTOR (e.g., a competitive inhibitor, such as an ATP-competitive inhibitor, or a non-competitive inhibitor, such as 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, as well as 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.

[0177] In some embodiments, an anti-cancer therapy of the disclosure comprises a PI3K inhibitor or Akt inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint 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 and inhibiting one or more activities of PI3K, binding to and inhibiting expression of PI3K, and / or binding to and inhibiting one or more activities of a cell expressing PI3K, such as by inducing antibodydependent 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 therapeutic, and the like). In some embodiments, the PI3K inhibitor is a small molecule inhibitor of PI3K (e.g., a competitive or non-competitive inhibitor). Nonlimiting 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, paxalisib, and alpelisib (BYL719, Piqray), as well as pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor inhibits one or more activities of AKT (e.g., AKT1). In some embodiments, the 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 and inhibiting one or more activities of AKT1, binding to and inhibiting expression of AKT1, and / or binding to and inhibiting one or more activities of a cell expressing AKT1, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of AKT1 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). 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, as well as pharmaceutically acceptable salts thereof. In some embodiments, the AKT1 inhibitor is a pan-Akt inhibitor.

[0178] In some embodiments, an anti-cancer therapy of the disclosure comprises a hedgehog (Hh) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the 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 and inhibiting one or more activities of Hh, binding to and inhibiting expression of Hh, and / or binding to and inhibiting one or more activities of a cell expressing Hh, such as 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 therapeutic, and the like). 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, saridegib, BMS833923, PF-04449913, and LY2940680, as well as pharmaceutically acceptable salts thereof.

[0179] In some embodiments, an anti-cancer therapy of the disclosure comprises a heat shock protein (HSP) inhibitor, a MYC inhibitor, an HD AC inhibitor, an immunotherapy, a neoantigen, a vaccine, or a cellular therapy, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor.

[0180] In some embodiments, the anti-cancer therapy comprises one or more of a chemotherapy, a VEGF inhibitor, an Integrin P3 inhibitor, a statin, an EGFR inhibitor, an mTOR inhibitor, a PI3K inhibitor, a MAPK inhibitor, or a CDK4 / 6 inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor.

[0181] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the kinase inhibitor is crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, ensartinib (X-396), repotrectinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP- 37440, belizatinib (TSR-011), ASP3026, KRCA-0008, TQ-B3139, TPX-0131, or TAE684 (NVP-TAE684). In some embodiments, the kinase inhibitor is an ALK kinase inhibitor, e.g., as described herein and / or in examples 3-39 of W02005016894.

[0182] In some embodiments, the anti-cancer therapy comprises a heat shock protein (HSP) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. 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 a 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 (onalespib). In some embodiments, the HSP inhibitor is an HSP27 inhibitor, such as Apatorsen (OGX-427).

[0183] In some embodiments, the anti-cancer therapy comprises a MYC inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the MYC inhibitor is MYCi361 (NUCC-0196361), MYCi975 (NUCC- 0200975), Omomyc (dominant negative peptide), ZINC16293153 (Min9), 10058-F4, JKY-2- 169, 7594-0035, or inhibitors of MYC / MAX dimerization and / or MYC / MAX / DNA complex formation.

[0184] In some embodiments, the anti-cancer therapy comprises a histone deacetylase (HD AC) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the HDAC inhibitor is belinostat (PXD101, e.g.,Beleodaq®), SAHA (vorinostat, suberoylanilide hydroxamine, e.g., Zolinza®), panobinostat (LBH589, LAQ-824), ACY1215 (Rocilinostat), quisinostat (JNJ-26481585), abexinostat (PCI-24781), pracinostat (SB939), givinostat (ITF2357), resminostat (4SC-201), trichostatin A (TSA), MS-275 (etinostat), Romidepsin (depsipeptide, FK228), MGCD0103 (mocetinostat), 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 (NCC149), TMP269, Cpd2, T247, T326, LMK235, CIA, HPOB, Nexturastat A, Befexamac, CBHA, Phenylbutyrate, MC1568, SNDX275, Scriptaid, Merck60, PX089344, PX105684, PX117735, PX117792, PX117245, PX105844, compound 12 as described by Li et al., Cold Spring Harb Perspect Med (2016) 6(10):a026831, or PX117445.

[0185] In some embodiments, the anti-cancer therapy comprises a VEGF inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the VEGF inhibitor is Bevacizumab (e.g., Avastin®), BMS-690514, ramucirumab, pazopanib, sorafenib, sunitinib, golvatinib, vandetanib, cabozantinib, levantinib, axitinib, cediranib, tivozanib, lucitanib, semaxanib, nindentanib, regorafinib, or aflibercept.

[0186] In some embodiments, the anti-cancer therapy comprises an integrin P3 inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the integrin P3 inhibitor is anti-avb3 (clone LM609), cilengitide (EMD121974, NSC, 707544), an siRNA, GLPG0187, MK-0429, CNTO95, TN-161, etaracizumab (MEDL522), intetumumab (CNTO95) (anti-alphaV subunit antibody), abituzumab (EMD 525797 / DI17E6) (anti-alphaV subunit antibody), JSM6427, SJ749, BCH- 15046, SCH221153, or SC56631. In some embodiments, the anti-cancer therapy comprises an allbp3 integrin inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the allbp3 integrin inhibitor is abciximab, eptifibatide (e.g., Integrilin®), or tirofiban (e.g., Aggrastat®).

[0187] In some embodiments, the anti-cancer therapy comprises an mTOR inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the mTOR inhibitor is temsirolimus (CCI-779), KU-006379, PP242, Torinl, Torin2, ICSN3250, Rapalink-1, CC-223, sirolimus (rapamycin), everolimus (RAD001), dactosilib (NVP-BEZ235), GSK2126458, WAY-001, WAY-600, WYE-687, WYE-354, SF1126, XL765, INK128 (MLN012), AZD8055, OSI027, AZD2014, or AP- 23573.

[0188] In some embodiments, the anti-cancer therapy comprises a statin or a statin-based agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the statin or statin-based agent is simvastatin, atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, or cerivastatin.

[0189] In some embodiments, the anti-cancer therapy comprises a MAPK inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. 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.

[0190] In some embodiments, the anti-cancer therapy comprises an EGFR inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. 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, necitumumab (e.g., Portrazza®), or erlotinib. In some embodiments, the EGFR inhibitor is gefitinib or cetuximab.

[0191] In some embodiments, the anti-cancer therapy comprises a nucleic acid molecule, such as a dsRNA, an siRNA, or an shRNA, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. As is known in the art, dsRNAs having a duplex structure are effective at inducing RNA interference (RNAi). In some embodiments, the anti-cancer therapy comprises a small interfering RNA molecule (siRNA). dsRNAs and siRNAs can be used to silence gene expression in mammalian cells (e.g., human cells). In some embodiments, a dsRNA of the disclosure comprises any of between about 5 and about 10 base pairs, between about 10 and about 12 base pairs, between about 12 and about 15 base pairs, between about 15 and about 20 base pairs, between about 20 and 23 base pairs, between about 23 and about 25 base pairs, between about 25 and about 27 base pairs, or between about 27 and about 30 base pairs. As is known in the art, siRNAs are small dsRNAs that optionally include overhangs. In some embodiments, the duplex region of an siRNA is between about 18 and 25 nucleotides, e.g., any of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. siRNAs may also include short hairpin RNAs (shRNAs), e.g., with approximately 29-base-pair stems and 2-nucleotide 3’ overhangs. Methods for designing, optimizing, producing, and using dsRNAs, siRNAs, or shRNAs, are known in the art.

[0192] In some embodiments, the anti-cancer therapy comprises a chemotherapy, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa andcyclo sphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L- norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6- mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as 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 1); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0193] Some non-limiting examples of chemotherapeutic drugs which can be combined with anti-cancer therapies of the present disclosure are 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, Amethop terin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0194] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor.Examples of kinase inhibitors include those that target one or more receptor tyrosine kinases, e.g., BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-a, PDGFR- 0, cKit, Flt-4, Flt3, FGFR1, FGFR2, FGFR3, FGFR4, CSF1R, c-Met, ROS1, RON, c-Ret, or ALK; one or more cytoplasmic tyrosine kinases, e.g., c-SRC, c-YES, Abl, or JAK-2; one or more serine / threonine kinases, e.g., ATM, Aurora A & B, CDKs, mTOR, PKCi, PLKs, b-Raf, c- Raf, S6K, or STK11 / LKB 1 ; or one or more lipid kinases, e.g., PI3K or SKI. Small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sunitinib (SU1 1248), sorafenib (BAY 43-9006), or leflunomide (SU101). Additional non-limiting examples of tyrosine kinase inhibitors include imatinib (Gleevec / Glivec) and gefitinib (Iressa).

[0195] In some embodiments, the anti-cancer therapy comprises an anti-angiogenic agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors require to survive. Non-limiting examples of angiogenesismediating molecules or angiogenesis inhibitors which may be used in the methods of the present disclosure include soluble VEGF (for example: 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, TIMP and CD Al, Meth-1 and Meth-2, IFNa, IFN-P and IFN-y, 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 such as bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-a platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids and heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactina v P3 inhibitors, linomide, or tasquinimod. In some embodiments, known therapeutic candidates that may be used according to the methods of the disclosure include naturally occurring angiogenic inhibitors, including without limitation, angiostatin, endostatin, or platelet factor-4. In another embodiment, therapeutic candidates that may be used according to the methods of the disclosure include, without limitation, specific inhibitors of endothelial cell growth, such as TNP-470, thalidomide, and interleukin- 12. Still other anti- angiogenic agents that may be used according to the methods of the disclosure include those that neutralize angiogenic molecules, including without limitation, antibodies to fibroblast growth factor, antibodies to vascular endothelial growth factor, antibodies to platelet derived growth factor, or antibodies or other types of inhibitors of the receptors of EGF, VEGF or PDGF. In some embodiments, anti-angiogenic agents that may be used according to the methods of the disclosure include, without limitation, suramin and its analogs, and tecogalan. In other embodiments, anti- angiogenic agents that may be used according to the methods of the disclosure include, without limitation, agents that neutralize receptors for angiogenic factors or agents that interfere with vascular basement membrane and extracellular matrix, including, without limitation, metalloprotease inhibitors and angiostatic steroids. Another group of anti- angiogenic compounds that may be used according to the methods of the disclosure includes, without limitation, anti-adhesion molecules, such as antibodies to integrin alpha v beta 3. Stillother anti-angiogenic compounds or compositions that may be used according to the methods of the disclosure include, without limitation, kinase inhibitors, thalidomide, itraconazole, carboxyamidotriazole, CM101, IFN-a, IL-12, SU5416, thrombospondin, cartilage-derived angiogenesis inhibitory factor, 2-methoxyestradiol, tetrathiomolybdate, thrombospondin, prolactin, and linomide. In one particular embodiment, the anti-angiogenic compound that may be used according to the methods of the disclosure is an antibody to VEGF, such as AvastinO / bevacizumab (Genentech).

[0196] In some embodiments, the anti-cancer therapy comprises an anti-DNA repair therapy, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the anti-DNA repair therapy is a PARP inhibitor (e.g., talazoparib, rucaparib, olaparib), a RAD51 inhibitor (e.g., RL1), 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).

[0197] In some embodiments, the anti-cancer therapy comprises a radiosensitizer, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Exemplary radiosensitizers include hypoxia radiosensitizers such as misonidazole, metronidazole, and trans-sodium crocetinate, a compound that helps to increase the diffusion of oxygen into hypoxic tumor tissue. The radiosensitizer can also be a DNA damage response inhibitor interfering with base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), recombinational repair comprising homologous recombination (HR) and non-homologous end-joining (NHEJ), and direct repair mechanisms. Single strand break (SSB) repair mechanisms include BER, NER, or MMR pathways, while double stranded break (DSB) repair mechanisms consist of HR and NHEJ pathways. Radiation causes DNA breaks that, if not repaired, are lethal. SSBs are repaired through a combination of BER, NER and MMR mechanisms using the intact DNA strand as a template. The predominant pathway of SSB repair is BER, utilizing a family of related enzymes termed poly-(ADP-ribose) polymerases (PARP). Thus, the radiosensitizer can include DNA damage response inhibitors such as PARP inhibitors.

[0198] In some embodiments, the anti-cancer therapy comprises an anti-inflammatory agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. 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 one or more of any 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; interferons (IFNs), e.g., IFNa, IFNP, IFNy, IFN-y inducing factor (IGIF); transforming growth factor-P (TGF-P); transforming growth factor-a (TGF-a); tumor necrosis factors, e.g., TNF-a, TNF-p, TNF-RI, TNF-RII; CD23; CD30; CD40L; EGF; G- CSF; GDNF; PDGF-BB; RANTES / CCL5; IKK; NF-KB; TLR2; TLR3; TLR4; TL5; TLR6; TLR7; TLR8; TLR8; TLR9; and / or any cognate receptors thereof. In some embodiments, the anti-inflammatory agent is an IL-1 or IL-1 receptor antagonist, such as anakinra (e.g., 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 (e.g., ACTEMRA®), olokizumab, clazakizumab, sarilumab, sirukumab, siltuximab, or ALX-0061. In some embodiments, the anti-inflammatory agent is a TNF-a antagonist, e.g., an anti-TNFa antibody, such as infliximab (Remicade®), golimumab (Simponi®), adalimumab (e.g., Humira®), certolizumab pegol (e.g., Cimzia®) or etanercept. In some embodiments, the anti-inflammatory agent is a corticosteroid. Exemplary corticosteroids include, but are not limited to, cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, e.g., Ala-Cort®, Hydrocort Acetate®, hydrocortone phosphate Lanacort®, Solu-Cortef®), decadron (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, e.g., Dexasone®, Diodex®, Hexadrol®, Maxidex®), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, e.g., Duralone®, Medralone®, Medrol®, M- Prednisol®, Solu-Medrol®), prednisolone (e.g., Delta-Cortef®, ORAPRED®, Pediapred®, Prezone®), and prednisone (e.g., Deltasone®, Liquid Pred®, Meticorten®, Orasone®), and bisphosphonates (e.g., pamidronate (Aredia®), and zoledronic acid (e.g., Zometac®).

[0199] In some embodiments, the anti-cancer therapy comprises an antimetabolite chemotherapeutic agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Antimetabolite chemotherapeutic agents are agents that are structurally similar to a metabolite, but cannot be used by the body in a productive manner. Many antimetabolite chemotherapeutic agents interfere with the production of RNA or DNA. Examples of antimetabolite chemotherapeutic agents include gemcitabine (e.g., GEMZAR®), 5 -fluorouracil (5-FU), capecitabine (e.g., XELODA™), 6-mercaptopurine, methotrexate, 6- thioguanine, pemetrexed, raltitrexed, arabinosylcytosine ARA-C cytarabine (e.g., CYTOSAR-U®), dacarbazine (DTIC-DOMED), azacytosine, deoxycitidine, pyrimidine, fludarabine (e.g., FLUDARA®), cladrabine, and 2-deoxy-D-glucose. In some embodiments, an antimetabolite chemotherapeutic agent is gemcitabine. Gemcitabine HC1 is sold by Eli Lilly under the trademark GEMZAR®.

[0200] In some embodiments, the anti-cancer therapy comprises a platinum-based chemotherapeutic agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Platinum-based chemotherapeutic agents are chemotherapeutic agents that comprise an organic compound containing platinum as an integral part of the molecule. In some embodiments, a 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.

[0201] In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options further comprise an additional anti-cancer therapy. In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options further comprise administering an additional anti-cancer therapy to the individual. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anticancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the additional anti-cancer therapy is an immunotherapy. In some embodiments, the additional anti-cancer therapy is a heat shock protein 90 inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018; Pall, Current opinion in oncology vol. 27,2 (2015): 118-24), an EGFR inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018), a SHP2 inhibitor (Dardaei et al., Nature medicine vol.24,4 (2018): 512-517), a MEK inhibitor (Shrestha et al., Scientific reports vol. 9,1 18842, 2019; Shrestha et al., The Journal of pharmacology and experimental therapeutics vol. 374,1 (2020): 134-140), an IGF-1R inhibitor (George, Journal of hematology & oncology vol. 12,1 80, 2019), a vascular endothelial growth factor (VEGF)-targeted therapy (Makimoto et al., Acta medica Okayama vol. 74,5 (2020): 371-379; Gristina et al., Pharmaceuticals (Basel, Switzerland) vol. 13,12474, 2020), an mTOR inhibitor (Kim et al., Anticancer research vol. 40,3 (2020): 1395-1403), or any combination thereof.

[0202] In some aspects, provided herein are therapeutic formulations comprising an anticancer therapy provided herein (e.g., an immunotherapy), and a pharmaceutically acceptable carrier, excipient, or stabilizer. A formulation provided herein may contain more than oneactive compound, e.g., an anti-cancer therapy provided herein and one or more additional agents (e.g., anti-cancer agents).

[0203] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, for example, one or more of: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium 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); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); surfactants such as non-ionic surfactants; or polymers such as polyethylene glycol (PEG).

[0204] The active ingredients may be entrapped in microcapsules. Such microcapsules may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin-microcapsules and poly-( methyl methacrylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nano-capsules); or in macroemulsions. Such techniques are known in the art.

[0205] Sustained-release compositions may be prepared. Suitable examples of sustained- release compositions include semi-permeable matrices of solid hydrophobic polymers containing an anti-cancer therapy of the disclosure. Such matrices may be in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0206] A formulation provided herein may also contain more than one active compound, for example, those with complementary activities that do not adversely affect each other. The type and effective amounts of such medicaments depend, for example, on the amount andtype of active compound(s) present in the formulation, and clinical parameters of the subjects.

[0207] For general information concerning formulations, see, e.g., 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: Tablets 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 Dekker, 2002.

[0208] Formulations to be used for in vivo administration are sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods known in the art.

[0209] In some embodiments, an anti-cancer therapy (e.g., immunotherapy) of the disclosure 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, e.g., as described herein. 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 comprise administration of any combination of any of the anti-cancer therapies provided herein. In some embodiments, the additional anti-cancer therapy comprises one or more of surgery, radiotherapy, chemotherapy, anti-angiogenic therapy, anti-DNA repair therapy, and anti-inflammatory therapy. In some embodiments, the additional anti-cancer therapy comprises an anti- neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, a radiation therapy, a cytotoxic agent, or combinations thereof. In some embodiments, an anti-cancer therapy may be administered in conjunction with a chemotherapy or chemotherapeutic agent. In some embodiments, the chemotherapy or chemotherapeutic agent is a platinum-based agent (including, without limitation cisplatin, carboplatin, oxaliplatin, and staraplatin). In some embodiments, an anti-cancer therapy may be administered in conjunction with a radiation therapy. In some embodiments, the anti-cancer therapy for use in any of the methods described herein (e.g., as 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 by Wang et al., Cancers (2020) 12(2):426.Reporting

[0210] In some embodiments, the methods provided herein comprise generating a report, and / or providing a report to party.

[0211] In some embodiments, a report according to the present disclosure comprises information about one or more of: one or more ESRI mutations of the disclosure or a variant allele frequency or clonal fraction thereof; a cancer of the disclosure, e.g., comprising one or more ESRI mutations of the disclosure; or a treatment, a therapy, or one or more treatment options for an individual having a cancer, such as a cancer of the disclosure (e.g., comprising one or more ESRI mutations as described herein).

[0212] In some embodiments, a report according to the present disclosure comprises information about the presence or absence of one or more ESRI mutations of the disclosure in a sample obtained from an individual, such as an individual having a cancer, e.g., a cancer provided herein. In one embodiment, a report according to the present disclosure indicates that one or more ESRI mutations of the disclosure are present in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates that one or more ESRI mutations of the disclosure are not present in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates that one or more ESRI mutations of the disclosure have been detected in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates that one or more ESRI mutations of the disclosure have not been detected in a sample obtained from the individual. In some embodiments, the report indicates a variant allele frequency or clonal fraction of the one or more ESRI mutations. In some embodiments, the report comprises an identifier for the individual from which the sample was obtained.

[0213] In some embodiments, the report includes information on the role of one or more ESRI mutations of the disclosure, or their wild type counterparts, in disease, such as in cancer. Such information can include one or more of: information on prognosis of a cancer, such as a cancer provided herein, e.g., comprising one or more ESRI mutations described herein; information on resistance of a cancer, such as a cancer provided herein, e.g., comprising one or more ESRI mutations described herein, to one or more treatments e.g., an endocrine therapy); information on potential or suggested therapeutic options (e.g., such as an anti-cancer agent other than a SERM or an aromatase inhibitor provided herein, or an endocrine therapy provided herein, or a treatment selected or identified according to themethods provided herein); or information on therapeutic options that should be avoided. In some embodiments, the report includes information on the likely effectiveness, acceptability, and / or advisability of applying a therapeutic option (e.g., such as an anti-cancer agent other than a SERM or an aromatase inhibitor provided herein, or an endocrine therapy provided herein, or a treatment selected or identified according to the methods provided herein) to an individual having a cancer, such as a cancer provided herein, e.g., comprising one or more ESRI mutations described herein and identified in the report. In some embodiments, the report includes information or a recommendation on the administration of a treatment (e.g., an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein). In some embodiments, the information or recommendation includes the dosage of the treatment and / or a treatment regimen (e.g., in combination with other treatments, such as a second therapeutic agent). In some embodiments, the report comprises information or a recommendation for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more treatments.

[0214] Also provided herein are methods of generating a report according to the present disclosure. In some embodiments, a report according to the present disclosure is generated by a method comprising one or more of the following steps: measuring a variant allele frequency of one or more ESRI mutations in a liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report. In some embodiments, a report generated according to the methods provided herein comprises one or more of: information about the presence or absence of one or more ESRI mutations of the disclosure or a clonal fraction thereof in the sample; an identifier for the individual from which the sample was obtained; information on the role of the one or more ESRI mutations of the disclosure, or their wild type counterparts, in disease (e.g., such as in cancer); information on prognosis, resistance, or potential or suggested therapeutic options (such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein); information on the likely effectiveness, acceptability, or the advisability of applying a therapeutic option (e.g., such as an anti-cancer agent other than a SERM or an aromatase inhibitor provided herein, or an endocrine therapy provided herein, or a treatment selected or identified according to the methods provided herein) to the individual; arecommendation or information on the administration of a treatment (e.g., such as an anticancer agent other than a SERM or an aromatase inhibitor provided herein, or an endocrine therapy provided herein, or a treatment selected or identified according to the methods provided herein); or a recommendation or information on the dosage or treatment regimen of a treatment (e.g., such as an anti-cancer agent other than a SERM or an aromatase inhibitor provided herein, or an endocrine therapy provided herein, or a treatment selected or identified according to the methods provided herein), e.g., in combination with other treatments (e.g., a second therapeutic agent). In some embodiments, the report generated is a personalized cancer report.

[0215] A report according to the present disclosure may be in an electronic, web-based, or paper form. The report may be provided to an individual or a patient (e.g., an individual or a patient with a cancer, such as a cancer provided herein), or to an individual or entity other than the individual or patient (e.g., other than the individual or patient with the cancer), such as one or more of a caregiver, a physician, an oncologist, a hospital, a clinic, a third party payor, an insurance company, or a government entity. In some embodiments, the report is provided or delivered to the individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from obtaining a sample from an individual (e.g., an individual having a cancer). In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from detecting one or more ESRI mutations of the disclosure in a sample obtained from an individual (e.g., an individual having a cancer). In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from acquiring knowledge of the presence of one or more ESRI mutations of the disclosure in a sample obtained from an individual (e.g., an individual having a cancer). In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal.Software, Systems, and Devices

[0216] In some other aspects, provided herein are non-transitory computer-readable storage media. In some embodiments, the non-transitory computer-readable storage media compriseone or more programs for execution by one or more processors of a device, the one or more programs including instructions which, when executed by the one or more processors, cause the device to perform a method according to any of the embodiments described herein.

[0217] FIG. 6 illustrates an example of a computing device or system in accordance with one embodiment. Device 600 can be a host computer connected to a network. Device 600 can be a client computer or a server. As shown in FIG. 6, device 600 can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s) 610, input devices 620, output devices 630, memory or storage devices 640, communication devices 660, and nucleic acid sequencers 670. Software 650 residing in memory or storage device 640 may comprise, e.g., an operating system as well as software for executing the methods described herein, e.g., for measuring clonal fraction of one or more ESRI mutations. Input device 620 and output device 630 can generally correspond to those described herein, and can either be connectable or integrated with the computer.

[0218] Input device 620 can be any suitable device that provides input, such as a touch screen, keyboard or keypad, mouse, or voice-recognition device. Output device 630 can be any suitable device that provides output, such as a touch screen, haptics device, or speaker.

[0219] Storage 640 can be any suitable device that provides storage (e.g., an electrical, magnetic or optical memory including a RAM (volatile and non-volatile), cache, hard drive, or removable storage disk). Communication device 660 can include any suitable device capable of transmitting and receiving signals over a network, such as a network interface chip or device. The components of the computer can be connected in any suitable manner, such as via a wired media (e.g., a physical system bus 680, Ethernet connection, or any other wire transfer technology) or wirelessly (e.g., Bluetooth®, Wi-Fi®, or any other wireless technology).

[0220] Software module 650, which can be stored as executable instructions in storage 640 and executed by processor(s) 610, can include, for example, an operating system and / or the processes that embody the functionality of the methods of the present disclosure, e.g., for measuring clonal fraction of one or more ESRI mutations (e.g., as embodied in the devices as described herein).

[0221] Software module 650 can also be stored and / or transported within any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described herein, that can fetch instructionsassociated with the software from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a computer-readable storage medium can be any medium, such as storage 640, that can 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 like hard drives, flash drives and distribute modules that operate as a single functional unit. Also, various processes described herein may be embodied as modules configured to operate in accordance with the embodiments and techniques described above. Further, while processes may be shown and / or described separately, those skilled in the art will appreciate that the above processes may be routines or modules within other processes.

[0222] Software module 650 can also be propagated within any transport medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described above, that can fetch instructions associated with the software from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a transport medium can be any medium that can communicate, propagate or transport programming for use by or in connection with an instruction execution system, apparatus, or device. The transport readable medium can include, but is not limited to, an electronic, magnetic, optical, electromagnetic or infrared wired or wireless propagation medium.

[0223] Device 600 may be connected to a network (e.g., network 704, as shown in FIG. 7 and described below), which can be any suitable type of interconnected communication system. The network can implement any suitable communications protocol and can be secured by any suitable security protocol. The network can comprise network links of any suitable arrangement that can implement the transmission and reception of network signals, such as wireless network connections, T1 or T3 lines, cable networks, DSL, or telephone lines.

[0224] Device 600 can be implemented using any operating system, e.g., an operating system suitable for operating on the network. Software module 650 can be written in any suitable programming language, such as C, C++, Java or Python. In various embodiments, application software embodying the functionality of the present disclosure can be deployed in different configurations, such as in a client / server arrangement or through a Web browser as a Webbased application or Web service, for example. In some embodiments, the operating system is executed by one or more processors, e.g., processor(s) 610.

[0225] Device 600 can further include a sequencer 670, which can be any suitable nucleic acid sequencing instrument. Exemplary sequencers can include, without limitation, 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.

[0226] FIG. 7 illustrates an example of a computing system in accordance with one embodiment. In computing system 700, device 600 (e.g., as described above and illustrated in FIG. 6) is connected to network 704, which is also connected to device 706. In some embodiments, device 706 is a sequencer. Exemplary sequencers can include, without limitation, 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.

[0227] Devices 600 and 706 may communicate, e.g., using suitable communication interfaces via network 704, such as a Local Area Network (LAN), Virtual Private Network (VPN), or the Internet. In some embodiments, network 704 can be, for example, the Internet, an intranet, a virtual private network, a cloud network, a wired network, or a wireless network. Devices 600 and 706 may communicate, in part or in whole, via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. Additionally, devices 600 and 706 may communicate, e.g., using suitable communication interfaces, via a second network, such as a mobile / cellular network. Communication between devices 600 and 706 may further include or communicate with various servers such as a mail server, mobile server, media server, telephone server, and the like. In some embodiments, devices 600 and 706 can communicate directly (instead of, or in addition to, communicating via network 704), e.g., via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. In some embodiments, devices 600 and 706 communicate via communications 708, which can be a direct connection or can occur via a network (e.g., network 704).

[0228] One or all of devices 600 and 706 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, for providing and / or receiving information via network 704 according to various examples described herein.

[0229] FIG. 8 illustrates an exemplary process 800 for measuring clonal fraction of one or more ESRI mutations (e.g.. in a liquid biopsy sample), in accordance with some embodiments of the present disclosure. Process 800 is performed, for example, using one or more electronic devices implementing a software program. In some examples, process 800 is performed using a client-server system, and the blocks of process 800 are divided up in any manner between the server and a client device. In other examples, the blocks of process 800 are divided up between the server and multiple client devices. Thus, while portions of process 800 are described herein as being performed by particular devices of a client-server system, it will be appreciated that process 800 is not so limited. In some embodiments, the executed steps can be executed across many systems, e.g., in a cloud environment. In other examples, process 800 is performed using only a client device or only multiple client devices. In process 800, 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 the process 800. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature and, as such, should not be viewed as limiting.

[0230] At block 802, a plurality of sequence reads of one or more nucleic acid molecules is obtained, wherein the one or more nucleic acid molecules are derived from a liquid biopsy sample obtained from an individual, e.g., as described herein. In some embodiments, the one or more nucleic acid molecues is obtained or isolated directly from the liquid biopsy sample. In other embodiments, the one or more nucleic acid molecules are obtained indirectly from the liquid biopsy sample, e.g., the one or more nucleic acid molecules are synthesized based on nucleic acid molecules obtained from the one or more samples (such as cDNA from mRNA, or PCR amplicons from nucleic acids). In some embodiments, the liquid biopsy sample is obtained from an individual having, suspected of having, or being tested for a cancer, such as a cancer described herein. In some embodiments, the sequence reads are obtained using a sequencer, e.g., as described herein or otherwise known in the art. In some embodiments, the nucleic acid molecules comprise one or more nucleic acid molecules corresponding to ESRI mutations and optionally one or more additional genes such as one or more cancer-related genes, ALK, EGFR, or a panel of known / suspected oncogenes and / or tumor suppressors, or any combination thereof, or fragments thereof. Optionally, prior to obtaining the sequence reads, the sample(s) are purified, enriched e.g., for nucleic acid(s) corresponding to: ESRI and optionally one or more genes such as one or more cancer-related genes, ALK, EGFR, or a panel of known / suspected oncogenes and / or tumor suppressors, orany combination thereof, or fragments thereof), and / or subjected to PCR amplification. In some embodiments, the nucleic acid molecules represent tumor nucleic acid molecules from the liquid biopsy sample, which can comprise both tumor nucleic acid molecules and nontumor nucleic acid molecules.

[0231] At block 804, an exemplary system (e.g., one or more electronic devices) analyzes the plurality of sequence reads for one or more ESRI mutations. Optionally, the plurality of sequence reads can further be analyzed, e.g., for calculation of a tumor fraction of the liquid biopsy sample. At block 806, the system measures (e.g., based on the analysis) a variant allele frequency of the one or more ESRI mutations (e.g., in the liquid biopsy sample). For example, the variant allele frequency of the one or more ESRI mutations can be measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom (e.g., based on the plurality of sequence reads) in the liquid biopsy sample. In some embodiments, an ESRI mutation is detected by comparing one or more sequence reads to a reference genome (e.g., a portion of a human genome encoding ESRI or a portion thereof). At block 808, the system measures a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. Optionally, the tumor fraction can be measured based on a plurality of sequence reads, e.g., as described in reference to block 804. At block 810, the system calculates a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. Optionally, the ratio can be further compared with a threshold ratio, e.g., as described herein.

[0232] Optionally, the system can further analyze the plurality of sequence reads for one or more oncogenic or tumor suppressor mutations present in tumor nucleic acid molecules from the liquid biopsy sample and measure frequency (e.g., variant allele frequency) of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample. Optionally, the system can further calculate a ratio of the frequency of the one or more oncogenic or tumor suppressor mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments, the frequency of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations.

[0233] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample (e.g., a clonal fraction of one or more ESRI mutations) greater than or equal to a threshold ratio identifies an individual for treatment with an anti-cancer agent other than a SERM or an aromatase inhibitor. In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample (e.g., a clonal fraction of one or more ESRI mutations) greater than or equal to a threshold ratio predicts the individual having the cancer to be resistant to an endocrine therapy. In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample (e.g., a clonal fraction of one or more ESRI mutations) less than a threshold ratio identifies an individual for treatment with an endocrine therapy. In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample (e.g., a clonal fraction of one or more ESRI mutations) less than a threshold ratio predicts the individual having the cancer to be sensitive and / or responsive to an endocrine therapy.

[0234] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the plurality of sequence reads is obtained by sequencing nucleic acids obtained from any of the samples described herein, e.g., a liquid biopsy. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the liquid biopsy sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the liquid biopsy sample comprises circulating tumor cells (CTCs). In some embodiments, the liquid biopsy sample comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0235] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the plurality of sequence reads is obtained by sequencing. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the massively parallel sequencing technique comprises next generation sequencing (NGS).

[0236] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the cancer is breast cancer (e.g., HR+ breast cancer) or endometrial cancer.

[0237] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and non-tumor nucleic acid molecules in the liquid biopsy sample. In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations, e.g., as described herein.

[0238] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the disclosed methods for measuring clonal fraction of one or more ESRI mutations may be implemented as part of a genomic profiling process that comprises identification of the presence of variant sequences at one or more gene loci in a sample derived from an individual as part of detecting, monitoring, predicting a risk factor, or selecting a treatment for a particular disease, e.g., cancer. In some instances, the variant panel selected for genomic profiling may comprise the detection of variant sequences at a selected set of gene loci. In some instances, the variant panel selected for genomic profiling may comprise detection of variant sequences at a number of gene loci through comprehensive genomic profiling (CGP), a next- generation sequencing (NGS) approach used to assess hundreds of genes (including relevant cancer biomarkers) in a single assay. Inclusion of the disclosed methods for determining the presence or absence of one or more ESRI mutations as part of a genomic profiling process can improve the validity of, e.g., disease detection calls, made on the basis of the genomic profile by, for example, independently confirming the presence of the one or more ESRI mutations in a given patient sample(s). In some embodiments, the comprehensive genomic profiling includes detecting, determining, or acquiring information on the presence of genes (or variant sequences thereof), copy number variations, epigenetic traits, proteins (or modifications thereof), and / or other biomarkers in an individual’s genome and / or proteome, as well as the individual’s corresponding phenotypic traits and the interaction between genetic or genomic traits, phenotypic traits, and environmental factors. In some instances, the comprehensive genomic profiling includes results from a nucleic acid sequencing-based test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof.

[0239] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a molecular profile is generated for the individual or the sample, based, at least in part, on measuring clonal fraction of one or more ESRI mutations. In some instances, the molecular profile may comprise information on the presence of genes (or variant sequences thereof, such as oncogenes and / or tumor suppressor genes), copy number variations, epigenetic traits, proteins (or modifications thereof), and / or other biomarkers in an individual’ s genome and / or proteome, as well as information on the individual’s corresponding phenotypic traits and the interaction between genetic or genomic traits, phenotypic traits, and environmental factors. In some instances, the molecular profile may comprise results from a comprehensive genomic profiling (CGP) test (e.g., as describe above), a nucleic acid sequencing-based test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile further comprises / indicates / comprises information on presence, absence, or clonal fraction of mutations in one or more additional genes, e.g., a panel of known / suspected oncogenes and / or tumor suppressors. In some embodiments, the one or more additional genes comprise one or more cancer-related genes, ALK, EGFR, CD274, or any combination thereof. In some embodiments, the molecular profile is obtained from a genomic profiling assay (such as a cancer- or tumor-related genomic profiling assay), e.g., as obtained using any of the sequencing methodologies described herein. In some embodiments, the molecular profile includes information from whole-genome or whole-exome sequencing. In some embodiments, the molecular profile includes information from targeted sequencing. In some embodiments, the molecular profile includes information from NGS. In some embodiments, the molecular profile comprises / indicates / comprises information on presence or absence of mutations such as short variant alterations (e.g., a base substitution, insertion, or deletion), copy-number alterations (e.g., an amplification or a homozygous deletion), and / or rearrangements (e.g., a gene fusion or other genomic or chromosomal rearrangement) of one or more genes, e.g., a panel of known / suspected oncogenes and / or tumor suppressors, one or more cancer-related genes, ALK, EGFR, CD274, or any combination thereof. In some embodiments, the individual is administered a treatment based at least in part on the molecular profile.

[0240] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a report is generated, e.g., as described in further detail above. In some embodiments, the reportcomprises / indicates / comprises information on ESRI mutation(s) and / or a clonal fraction thereof in the cancer in an individual (e.g., in a liquid biopsy sample from the individual). In some embodiments, the report comprises / indicates / comprises information on results of a genomic profiling process of a cancer in an individual (e.g., in one or more samples from the individual), e.g., as described above. In some embodiments, the report comprises / indicates / comprises information on results of comprehensive genomic profiling of a cancer in an individual (e.g., in one or more samples from the individual), e.g., as described above. In some embodiments, the report comprises / indicates / comprises information on a molecular profile generated for the individual or the sample, e.g., as described above. In some embodiments, the report comprises / indicates / comprises information on a treatment or one or more treatment options selected or identified for the individual, based, at least in part, on the clonal fraction of one or more ESRI mutations (e.g., as compared with a threshold ratio) in the cancer in an individual (e.g., in a liquid biopsy sample from the individual), and optionally based on results of a genomic profiling process, comprehensive genomic profiling, and / or a molecular profile generated for the individual or a sample, e.g., as described above. In some embodiments, the treatment or one or more treatment options comprise an anticancer agent other than a SERM or an aromatase inhibitor, or an endocrine therapy, e.g., as described herein. In some embodiments, the report is provided or transmitted to the individual, a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office, e.g., as described in further detail above. In some embodiments, the report is transmitted via a computer network or a peer-to-peer connection. In some embodiments, an individual is administered a treatment based, at least in part, on the report. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal.

[0241] The method steps of the methods described herein are intended to include any suitable method of causing one or more other parties or entities to perform the steps, unless a different meaning is expressly provided or otherwise clear from the context. Such parties or entities need not be under the direction or control of any other party or entity, and need not be located within a particular jurisdiction. Thus, for example, a description or recitation of "adding a first number to a second number" includes causing one or more parties or entities to add the two numbers together. For example, if person X engages in an arm's length transaction with person Y to add the two numbers, and person Y indeed adds the two numbers, then both persons X and Y perform the step as recited: person Y by virtue of the fact that he actuallyadded the numbers, and person X by virtue of the fact that he caused person Y to add the numbers. Furthermore, if person X is located within the United States and person Y is located outside the United States, then the method is performed in the United States by virtue of person X's participation in causing the step to be performed.IV. Exemplary Embodiments

[0242] The following exemplary embodiments are representative of some aspects of the invention:Embodiment 1. A method of measuring clonal fraction of one or more estrogen receptor 1 (ESRE) mutations in a liquid biopsy sample from an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.Embodiment 2. A method of selecting a therapy for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is greater than or equal to a threshold ratio identifies the individual as one who may benefit from a treatment comprising an anti-cancer agent other than a selective estrogen receptor modulator (SERM) or an aromatase inhibitor.Embodiment 3. A method of identifying one or more treatment options for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report comprising one or more treatment options identified for the individual based, at least in part, on the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction, wherein the one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor, and wherein the calculated ratio is greater than or equal to a threshold ratio.Embodiment 4. A method of treating or delaying progression of cancer, comprising:(a) acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual; and(b) responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor.Embodiment 5. A method of treating or delaying progression of cancer, comprising administering to an individual having cancer an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor, wherein the anti-cancer agent is administered responsive to acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual.Embodiment 6. A method of treating or delaying progression of cancer, comprising:(a) measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;(b) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample;(c) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(d) administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor.Embodiment 7. The method of any one of embodiments 4-6, wherein the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is greater than or equal to a threshold ratio.Embodiment 8. A method of monitoring resistance to endocrine therapy in an individual having cancer, the method comprising:measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is greater than or equal to a threshold ratio identifies the individual as one who may be resistant to endocrine therapy.Embodiment 9. A method of selecting a therapy for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is less than a threshold ratio identifies the individual as one who may benefit from an endocrine therapy.Embodiment 10. A method of identifying one or more treatment options for an individual having cancer, the method comprising:measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report comprising one or more treatment options identified for the individual based, at least in part, on the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction, wherein the one or more treatment options comprise an endocrine therapy, and wherein the calculated ratio is less than a threshold ratio.Embodiment 11. A method of predicting responsiveness to endocrine therapy in an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is less than a threshold ratio identifies the individual as one who may be responsive to endocrine therapy.Embodiment 12. A method of treating or delaying progression of cancer, comprising:(a) acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual; and(b) responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an endocrine therapy.Embodiment 13. A method of treating or delaying progression of cancer, comprising administering to an individual having cancer an effective amount of a treatment that comprises an endocrine therapy, wherein the endocrine therapy is administered responsive to acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual.Embodiment 14. A method of treating or delaying progression of cancer, comprising:(a) measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;(b) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample;(c) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(d) administering to the individual an effective amount of a treatment that comprises an endocrine therapy.Embodiment 15. The method of any one of embodiments 12-14, wherein the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is less than a threshold ratio.Embodiment 16. A method of selecting a therapy for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and identifying the individual as one who may benefit from a treatment comprising an anti-cancer agent other than a SERM or an aromatase inhibitor when the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is greater than or equal to a threshold ratio; or identifying the individual as one who may benefit from a treatment comprising an endocrine therapy when the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is less than the threshold ratio.Embodiment 17. A method for monitoring resistance to endocrine therapy in an individual with cancer, the method comprising:(a) measuring a first variant allele frequency of the one or more ESRI mutations in a first liquid biopsy sample obtained from the individual at a first timepoint, wherein the first liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the first variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the first liquid biopsy sample;(b) measuring a first tumor fraction of tumor nucleic acid molecules in the first liquid biopsy sample;(c) calculating a ratio of the first variant allele frequency of the one or more ESRI mutations to the first tumor fraction of tumor nucleic acid molecules in the first liquid biopsy sample, thereby measuring a first clonal fraction of the one or more ESRI mutations in the first liquid biopsy sample;(d) measuring a second variant allele frequency of the one or more ESRI mutations in a second liquid biopsy sample obtained from the individual at a second timepoint after the first timepoint, wherein the second liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the second variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the second liquid biopsy sample;(e) measuring a second tumor fraction of tumor nucleic acid molecules in the second liquid biopsy sample;(f) calculating a ratio of the second variant allele frequency of the one or more ESRI mutations to the second tumor fraction of tumor nucleic acid molecules in the second liquid biopsy sample, thereby measuring a second clonal fraction of the one or more ESRI mutations in the second liquid biopsy sample; and(h) providing an assessment of resistance to endocrine therapy in the individual based, at least in part, on the first and second clonal fractions of the one or more ESRI mutations; wherein the second clonal fraction is greater than the first clonal fraction.Embodiment 18. The method of embodiment 17, wherein the second timepoint is after administration of an endocrine therapy to the individual.Embodiment 19. The method of embodiment 17 or embodiment 18, further comprising, after providing the assessment of resistance to endocrine therapy, administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor; wherein optionally the method further comprises halting administration of an endocrine therapy to the individual based, at least in part, on the first and second ratios of the clonal fraction of the one or more ESRI mutations.Embodiment 20. The method of any one of embodiments 8-19, wherein the endocrine therapy comprises a SERM or an aromatase inhibitor.Embodiment 21. A method of measuring a clonal fraction of one or more ESRI mutations in a liquid biopsy sample, the method comprising:(a) providing a plurality of nucleic acid molecules obtained from a liquid biopsy sample from an individual having cancer, wherein the plurality of nucleic acid molecules comprises tumor nucleic acid molecules;(b) optionally, ligating one or more adapters onto one or more tumor nucleic acid molecules from the plurality of nucleic acid molecules;(c) optionally, amplifying the one or more ligated tumor nucleic acid molecules from the plurality of nucleic acid molecules;(d) optionally, capturing amplified tumor nucleic acid molecules from the amplified tumor nucleic acid molecules;(e) sequencing, by a sequencer, the captured tumor nucleic acid molecules to obtain a plurality of sequence reads that represent the captured tumor nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to one or more ESRI mutations;(f) analyzing the plurality of sequence reads for presence of the one or more ESRI mutations;(g) based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations;(h) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(i) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.Embodiment 22. The method of embodiment 21, wherein the sequencer comprises a next-generation sequencer.Embodiment 23. A method of measuring a clonal fraction of one or more ESRI mutations in a liquid biopsy sample, the method comprising:(a) providing a liquid biopsy sample from an individual having cancer, wherein the sample comprises a plurality of nucleic acid molecules comprising tumor nucleic acid molecules;(b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample;(c) amplifying said library;(d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to one or more ESRI mutations in said library to produce an enriched sample;(e) sequencing the enriched sample, thereby producing a plurality of sequence reads;(f) analyzing the plurality of sequence reads for presence of the one or more ESRI mutations;(g) based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations;(h) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(i) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.Embodiment 24. The method of embodiment 21 or embodiment 23, wherein the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences.Embodiment 25. The method of embodiment 23, wherein the selectively enriching comprises: (a) combining one or more bait molecules with the library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more ESRI mutations, and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.Embodiment 26. The method of any one of embodiments 21-25, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.Embodiment 27. The method of any one of embodiments 21-26, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non- PCR amplification technique, or an isothermal amplification technique.Embodiment 28. The method of any one of embodiments 1-22, 26, and 27, further comprising selectively enriching for one or more tumor nucleic acid molecules in the liquid biopsy sample comprising nucleotide sequences corresponding to the one or more ESRI mutations; wherein the selectively enriching produces an enriched sample.Embodiment 29. The method of embodiment 28, wherein the selectively enriching comprises: (a) combining one or more bait molecules with the liquid biopsy sample, thereby hybridizing the one or more bait molecules to one or more tumor nucleic acid molecules in the sample comprising nucleotide sequences corresponding to the one or more ESRI mutations and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.Embodiment 30. The method of any one of embodiments 25, 26, and 29, wherein the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the one or more ESRI mutations.Embodiment 31. The method of embodiment 30, wherein the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides.Embodiment 32. The method of any one of embodiments 25, 26, and 29-31, wherein the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent.Embodiment 33. The method of embodiment 32, wherein the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.Embodiment 34. The method of any one of embodiments 30-33, wherein the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.Embodiment 35. The method of embodiment 28, wherein the selectively enriching comprises amplifying the one or more tumor nucleic acid molecules comprising nucleotidesequences corresponding to the one or more ESRI mutations using a polymerase chain reaction (PCR) to produce an enriched sample.Embodiment 36. The method of any one of embodiments 27-35, further comprising sequencing the enriched sample.Embodiment 37. The method of any one of embodiments 21-36, wherein the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; optionally wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing techniqu...

Claims

CLAIMSWhat is claimed is:

1. A method of measuring clonal fraction of one or more estrogen receptor 1 (ESRI) mutations in a liquid biopsy sample from an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

2. A method of selecting a therapy for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is greater than or equal to a threshold ratio identifies the individualas one who may benefit from a treatment comprising an anti-cancer agent other than a selective estrogen receptor modulator (SERM) or an aromatase inhibitor.

3. A method of identifying one or more treatment options for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report comprising one or more treatment options identified for the individual based, at least in part, on the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction, wherein the one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor, and wherein the calculated ratio is greater than or equal to a threshold ratio.

4. A method of treating or delaying progression of cancer, comprising:(a) acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual; and(b) responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor.

5. A method of treating or delaying progression of cancer, comprising administering to an individual having cancer an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor, wherein the anti-cancer agent is administered responsive to acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual.

6. A method of treating or delaying progression of cancer, comprising:(a) measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;(b) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample;(c) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(d) administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor.

7. The method of any one of claims 4-6, wherein the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is greater than or equal to a threshold ratio.

8. A method of monitoring resistance to endocrine therapy in an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is greater than or equal to a threshold ratio identifies the individual as one who may be resistant to endocrine therapy.

9. A method of selecting a therapy for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is less than a threshold ratio identifies the individual as one who may benefit from an endocrine therapy.

10. A method of identifying one or more treatment options for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and generating a report comprising one or more treatment options identified for the individual based, at least in part, on the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction, wherein the one or more treatment options comprise an endocrine therapy, and wherein the calculated ratio is less than a threshold ratio.

11. A method of predicting responsiveness to endocrine therapy in an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, wherein a calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction that is less than a threshold ratio identifies the individual as one who may be responsive to endocrine therapy.

12. A method of treating or delaying progression of cancer, comprising:(a) acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual; and(b) responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an endocrine therapy.

13. A method of treating or delaying progression of cancer, comprising administering to an individual having cancer an effective amount of a treatment that comprises an endocrine therapy, wherein the endocrine therapy is administered responsive to acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual.

14. A method of treating or delaying progression of cancer, comprising:(a) measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;(b) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample;(c) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(d) administering to the individual an effective amount of a treatment that comprises an endocrine therapy.

15. The method of any one of claims 12-14, wherein the ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is less than a threshold ratio.

16. A method of selecting a therapy for an individual having cancer, the method comprising: measuring a variant allele frequency of the one or more ESRI mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the oneor more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample; measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and identifying the individual as one who may benefit from a treatment comprising an anti-cancer agent other than a SERM or an aromatase inhibitor when the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is greater than or equal to a threshold ratio; or identifying the individual as one who may benefit from a treatment comprising an endocrine therapy when the calculated ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction is less than the threshold ratio.

17. A method for monitoring resistance to endocrine therapy in an individual with cancer, the method comprising:(a) measuring a first variant allele frequency of the one or more ESRI mutations in a first liquid biopsy sample obtained from the individual at a first timepoint, wherein the first liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the first variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the first liquid biopsy sample;(b) measuring a first tumor fraction of tumor nucleic acid molecules in the first liquid biopsy sample;(c) calculating a ratio of the first variant allele frequency of the one or more ESRI mutations to the first tumor fraction of tumor nucleic acid molecules in the first liquid biopsy sample, thereby measuring a first clonal fraction of the one or more ESRI mutations in the first liquid biopsy sample;(d) measuring a second variant allele frequency of the one or more ESRI mutations in a second liquid biopsy sample obtained from the individual at a secondtimepoint after the first timepoint, wherein the second liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the second variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the second liquid biopsy sample;(e) measuring a second tumor fraction of tumor nucleic acid molecules in the second liquid biopsy sample;(f) calculating a ratio of the second variant allele frequency of the one or more ESRI mutations to the second tumor fraction of tumor nucleic acid molecules in the second liquid biopsy sample, thereby measuring a second clonal fraction of the one or more ESRI mutations in the second liquid biopsy sample; and(h) providing an assessment of resistance to endocrine therapy in the individual based, at least in part, on the first and second clonal fractions of the one or more ESRI mutations; wherein the second clonal fraction is greater than the first clonal fraction.

18. The method of claim 17, wherein the second timepoint is after administration of an endocrine therapy to the individual.

19. The method of claim 17 or claim 18, further comprising, after providing the assessment of resistance to endocrine therapy, administering to the individual an effective amount of a treatment that comprises an anti-cancer agent other than a SERM or an aromatase inhibitor; wherein optionally the method further comprises halting administration of an endocrine therapy to the individual based, at least in part, on the first and second ratios of the clonal fraction of the one or more ESRI mutations.

20. The method of any one of claims 8-19, wherein the endocrine therapy comprises a SERM or an aromatase inhibitor.

21. A method of measuring a clonal fraction of one or more ESRI mutations in a liquid biopsy sample, the method comprising:(a) providing a plurality of nucleic acid molecules obtained from a liquid biopsy sample from an individual having cancer, wherein the plurality of nucleic acid molecules comprises tumor nucleic acid molecules;(b) optionally, ligating one or more adapters onto one or more tumor nucleic acidmolecules from the plurality of nucleic acid molecules;(c) optionally, amplifying the one or more ligated tumor nucleic acid molecules from the plurality of nucleic acid molecules;(d) optionally, capturing amplified tumor nucleic acid molecules from the amplified tumor nucleic acid molecules;(e) sequencing, by a sequencer, the captured tumor nucleic acid molecules to obtain a plurality of sequence reads that represent the captured tumor nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to one or more ESRI mutations;(f) analyzing the plurality of sequence reads for presence of the one or more ESRI mutations;(g) based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations;(h) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(i) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

22. The method of claim 21, wherein the sequencer comprises a next- generation sequencer.

23. A method of measuring a clonal fraction of one or more ESRI mutations in a liquid biopsy sample, the method comprising:(a) providing a liquid biopsy sample from an individual having cancer, wherein the sample comprises a plurality of nucleic acid molecules comprising tumor nucleic acid molecules;(b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample;(c) amplifying said library;(d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to one or more ESRI mutations in said library to produce an enriched sample;(e) sequencing the enriched sample, thereby producing a plurality of sequence reads;(f) analyzing the plurality of sequence reads for presence of the one or more ESRI mutations;(g) based on the analyzing step, measuring a variant allele frequency of the one or more ESRI mutations;(h) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(i) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

24. The method of claim 21 or claim 23, wherein the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences.

25. The method of claim 23, wherein the selectively enriching comprises: (a) combining one or more bait molecules with the library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more ESRI mutations, and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.

26. The method of any one of claims 21-25, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.

27. The method of any one of claims 21-26, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique.

28. The method of any one of claims 1-22, 26, and 27, further comprising selectively enriching for one or more tumor nucleic acid molecules in the liquid biopsy sample comprising nucleotide sequences corresponding to the one or more ESRI mutations; wherein the selectively enriching produces an enriched sample.

29. The method of claim 28, wherein the selectively enriching comprises: (a) combining one or more bait molecules with the liquid biopsy sample, thereby hybridizing the one or more bait molecules to one or more tumor nucleic acid molecules in the sample comprising nucleotide sequences corresponding to the one or more ESRI mutations and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.

30. The method of any one of claims 25, 26, and 29, wherein the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the one or more ESRI mutations.

31. The method of claim 30, wherein the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides.

32. The method of any one of claims 25, 26, and 29-31, wherein the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent.

33. The method of claim 32, wherein the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.

34. The method of any one of claims 30-33, wherein the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.

35. The method of claim 28, wherein the selectively enriching comprises amplifying the one or more tumor nucleic acid molecules comprising nucleotide sequences corresponding to the one or more ESRI mutations using a polymerase chain reaction (PCR) to produce an enriched sample.

36. The method of any one of claims 27-35, further comprising sequencing the enriched sample.

37. The method of any one of claims 21-36, wherein the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; optionally wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS).

38. The method of any one of claims 1-37, wherein the one or more ESRI mutations comprise one or more base substitutions or short insertion / deletions.

39. The method of any one of claims 1-38, wherein the one or more ESRI mutations comprise one or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1.

40. The method of any one of claims 1-39, wherein the one or more ESRI mutations comprise one or more gene fusions, rearrangements, or ESRI gene amplifications.

41. The method of any one of claims 1-40, wherein the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations.

42. The method of claim 41, wherein the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations that comprises two or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H,Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1.

43. The method of any one of claims 1-42, wherein the one or more ESRI mutations comprise a first ESRI mutation present in a first cell or set of cells of the cancer and a second ESRI mutation present in a different cell or set of cells of the cancer.

44. The method of any one of claims 1-43, wherein the tumor nucleic acid molecules in the liquid biopsy sample are circulating tumor DNA (ctDNA) molecules.

45. The method of any one of claims 1-44, wherein the tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and nontumor nucleic acid molecules in the liquid biopsy sample.

46. The method of any one of claims 1-45, wherein the tumor fraction is greater than or equal to about 1%.

47. The method of any one of claims 4, 5, 12, 13, and 28-46, wherein acquiring knowledge of a ratio of variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in a liquid biopsy sample obtained from the individual comprises:(a) measuring a variant allele frequency of the one or more ESRI mutations in the liquid biopsy sample, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the variant allele frequency of the one or more ESRI mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample;(b) measuring a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(c) calculating a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

48. The method of any one of claims 1-47, further comprising obtaining the liquid biopsy sample from the individual.

49. The method of any one of claims 1-48, wherein the liquid biopsy sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

50. The method of any one of claims 1-49, wherein the tumor nucleic acid molecules comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer.

51. The method of any one of claims 1-49, wherein the liquid biopsy sample comprises circulating tumor cells (CTCs).

52. The method of any one of claims 1-51, further comprising detecting one or more oncogenic or tumor suppressor mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the one or more oncogenic or tumor suppressor mutations are detected in tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample.

53. The method of claim 52, wherein the one or more oncogenic or tumor suppressor mutations are detected in the same liquid biopsy sample from which the variant allele frequency of the one or more ESRI mutations is measured.

54. The method of any one of claims 1-53, further comprising measuring frequency of one or more oncogenic or tumor suppressor mutations in a liquid biopsy sample obtained from the individual, wherein the liquid biopsy sample comprises tumor nucleic acid molecules, and wherein the frequency of the one or more oncogenic or tumor suppressor mutations is measured from tumor nucleic acid molecules or nucleic acid molecules derived therefrom in the liquid biopsy sample.

55. The method of claim 54, wherein the frequency of the one or more oncogenic or tumor suppressor mutations is measured in the same liquid biopsy sample from which the variant allele frequency of the one or more ESRI mutations is measured.

56. The method of claim 54 or claim 55, further comprising calculating a ratio of the frequency of the one or more oncogenic or tumor suppressor mutations to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample.

57. The method of claim 56, wherein the tumor fraction used to measure the clonal fraction of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample is the same tumor fraction used to measure the clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

58. The method of any one of claims 52-57, wherein the one or more oncogenic or tumor suppressor mutations comprise a mutation in one or more of P1K3CA, RBI, PTEN, NF1, ARID1A, AKT1, FGFR2, FGFR3, KRAS, ERBB2, EGFR, and BRAF.

59. The method of any one of claims 54-58, wherein the frequency of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations.

60. The method of any one of claims 2, 3, 7-11, 15, 16, and 28-59, wherein the threshold ratio is a ratio of a threshold variant allele frequency of one or more ESRI mutations to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

61. The method of claim 60, wherein the threshold ratio is the ratio of a threshold variant allele frequency of the one or more ESRI mutations that is greater than zero to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample that is greater than or equal to 1%.

62. The method of any one of claims 2, 3, 7-11, 15, 16, and 28-59, wherein the threshold ratio is a ratio of a threshold frequency of one or more oncogenic or tumor suppressor mutations in tumor nucleic acid molecules in the liquid biopsy sample to a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

63. The method of any one of claims 52-62, wherein the one or more oncogenic or tumor suppressor mutations and the one or more ESRI mutations are present in different cells or sets of cells of the cancer.

64. The method of any one of claims 52-63, wherein the anti-cancer agent other than a SERM or aromatase inhibitor is a targeted therapy targeting one of the one or more oncogenic or tumor suppressor mutations.

65. The method of any one of claims 1-64, wherein the individual has received one or more prior treatments with an endocrine therapy.

66. The method of any one of claims 1-65, further comprising, prior to measuring or acquiring knowledge of the variant allele frequency of the one or more ESRI mutations, administering an effective amount of a treatment that comprises an endocrine therapy to the individual.

67. The method of claim 65 or claim 66, wherein the endocrine therapy comprises a SERM or an aromatase inhibitor.

68. The method of any one of claims 2-7, 16, 19, 20, and 28-67, wherein the SERM is tamoxifen, raloxifene, EM652, GW7604, keoxifene, toremifene, bazedoxifene, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, ospemifene, or a pharmaceutically acceptable salt thereof.

69. The method of any one of claims 2-7, 16, 19, 20, and 28-68, wherein the aromatase inhibitor is aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), 4- Androstene-3, 6, 17-trione (“6-OXO”), or a pharmaceutically acceptable salt thereof.

70. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises one or more of a small molecule inhibitor, an antibody, a cellular therapy, a nucleic acid, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), a treatment for cancer comprising an ESRI mutation, an anti-cancer agent being tested in a clinical trial, a treatment for cancer comprising an ESRI mutation being tested in a clinical trial, or any combination thereof.

71. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises a selective estrogen receptor covalent antagonist (SERCA).

72. The method of claim 71, wherein the anti-cancer agent is H3B-5942 or H3B-6545.

73. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises a selective estrogen receptor degrader (SERD).

74. The method of claim 73, wherein the anti-cancer agent is fulvestrant, elacestrant, amcenestrant, camizestrant, giredestrant, rintodestrant, imlunestrant, ZB-716, Zn-c5, LSZ102, LY3484356, or D-0502, or a pharmaceutically acceptable salt thereof.

75. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises a PROTAC.

76. The method of claim 75, wherein the PROTAC is ARV-471.

77. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises a CDK4 / 6 inhibitor.

78. The method of claim 77, wherein the CDK4 / 6 inhibitor is palbociclib, abemaciclib, ribociclib, or a pharmaceutically acceptable salt thereof.

79. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises a PI3K inhibitor.

80. The method of claim 79, wherein the PI3K inhibitor is GSK2636771, buparlisib, AZD8186, copanlisib, LY294002, PX-866, TGX115, TGX126, BEZ235, SF1126, idelalisib, pictilisib, GDC0032, IPI145, INK1117, SAR260301, KIN-193, duvelisib, GS-9820, GSK2636771, GDC-0980, AMG319, paxalisib, or alpelisib, or a pharmaceutically acceptable salt thereof.

81. The method of any one of claims 2-7, 16, 19, 20, and 28-69, wherein the anticancer agent comprises an mTOR inhibitor.

82. The method of claim 81, wherein the mTOR inhibitor is temsirolimus, everolimus, ridaforolimus, dactolisib, GSK2126458, XL765, AZD8055, AZD2014, MLN128, PP242, NVP-BEZ235, LY3023414, PQR309, PKI587, or OSI027, or a pharmaceutically acceptable salt thereof.

83. The method of claim 70, wherein the nucleic acid inhibits the expression of an ESRI nucleic acid molecule, or an ESRI polypeptide encoded by the ESRI nucleic acid molecule.

84. The method of claim 83, wherein the nucleic acid is a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA).

85. The method of claim 70, wherein the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy.

86. The method of any one of claims 1-85, wherein the cancer is breast cancer.

87. The method of claim 86, wherein the breast cancer is hormone receptor positive(HR+) breast cancer.

88. The method of any one of claims 1-85, wherein the cancer is endometrial cancer.

89. The method of any one of claims 1-88, wherein the cancer is advanced or metastatic.

90. The method of any one of claims 4-7, 16, 19, 20, and 28-89, wherein the treatment or the one or more treatment options further comprise an additional anti-cancer therapy.

91. The method of claim 90, wherein the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virusbased therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof.

92. The method of claim 91, wherein the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophagebased therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy.

93. The method of claim 91, wherein the nucleic acid comprises a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA).

94. The method of any one of claims 1, 2, 4-9, 16, 19, 20, and 28-93, further comprising generating a report, wherein the report: (a) indicates the presence of the one or more ESRI mutations in the sample from the individual; and / or (b) indicates a treatment orone or more treatment options identified or selected for the individual based, at least in part, on the presence of the one or more ESRI mutations in the sample from the individual, wherein the treatment or the one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor.

95. The method of any one of claims 1-94, further comprising generating a molecular profile for the individual, based, at least in part, on detecting or acquiring knowledge of the one or more ESRI mutations in the sample from the individual.

96. The method of claim 95, wherein the molecular profile for the individual further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof.

97. The method of claim 95 or claim 96, wherein the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test.

98. The method of any one of claims 95-97, wherein the molecular profile for the individual further indicates presence of the one or more ESRI mutations in the liquid biopsy sample.

99. The method of any one of claims 94-98, wherein the report or the molecular profile for the individual further indicates the variant allele frequency or the clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

100. The method of any one of claims 95-99, further comprising selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an anti-cancer agent other than a SERM or an aromatase inhibitor.

101. The method of any one of claims 95-99, further comprising selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an endocrine therapy.

102. The method of any one of claims 95-101, further comprising generating a report, wherein the report comprises the molecular profile for the individual.

103. The method of claim 102, wherein the report further comprises information on a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the treatment or one or more treatment options comprise an anti-cancer agent other than a SERM or an aromatase inhibitor.

104. The method of claim 102, wherein the report further comprises information on a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the treatment or one or more treatment options comprise an endocrine therapy.

105. The method of any one of claims 3, 10, and 94-104, further comprising providing the report to the individual, a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office.

106. The method of any one of claims 1-105, wherein the individual is a human.

107. A system for measuring clonal fraction of one or more ESRI mutations in a liquid biopsy sample from an individual having cancer, the system comprising: a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to:(a) obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from a liquid biopsy sample obtained from an individual having cancer, wherein the liquid biopsy sample comprises tumor nucleic acid molecules;(b) analyze the plurality of sequence reads for one or more ESRI mutations present in tumor nucleic acid molecules from the liquid biopsy sample;(c) measure, based on the analyzing, a variant allele frequency of the one or more ESRI mutations in the liquid biopsy sample;(d) measure a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(e) calculate a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

108. The system of claim 107, wherein the one or more ESRI mutations comprise one or more base substitutions or short insertion / deletions.

109. The system of claim 107 or claim 108, wherein the one or more ESRI mutations comprise one or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1.

110. The system of any one of claims 107-109, wherein the one or more ESRI mutations comprise one or more gene fusions, rearrangements, or ESRI gene amplifications.

111. The system of any one of claims 107-110, wherein the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations.

112. The system of claim 111, wherein the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations that comprises two or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R,L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1.

113. The system of any one of claims 107-112, wherein the tumor nucleic acid molecules in the liquid biopsy sample are circulating tumor DNA (ctDNA) molecules.

114. The system of any one of claims 107-113, wherein the tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and non-tumor nucleic acid molecules in the liquid biopsy sample.

115. The system of any one of claims 107-114, wherein the tumor fraction is greater than or equal to about 1%.

116. The system of any one of claims 107-115, wherein the liquid biopsy sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

117. The system of any one of claims 107-116, wherein the tumor nucleic acid molecules comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell- free RNA from the cancer.

118. The system of any one of claims 107-116, wherein the liquid biopsy sample comprises circulating tumor cells (CTCs).

119. The system of any one of claims 107-118, wherein the one or more program instructions when executed by the one or more processors are further configured to: analyze the plurality of sequence reads for one or more oncogenic or tumor suppressor mutations present in tumor nucleic acid molecules from the liquid biopsy sample; and measure, based on the analyzing, frequency of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample.

120. The system of claim 119, wherein the one or more program instructions when executed by the one or more processors are further configured to calculate a ratio of thefrequency of the one or more oncogenic or tumor suppressor mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

121. The system of claim 119 or claim 120, wherein the one or more oncogenic or tumor suppressor mutations comprise a mutation in one or more of P1K3CA, RBI, PTEN, NF1, ARID1A, AKT1, FGFR2, FGFR3, KRAS, ERBB2, EGFR, and BRAF.

122. The system of any one of claims 119-121, wherein the frequency of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations.

123. The system of any one of claims 107-122, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene- targeted sequencing, or next-generation sequencing.

124. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for measuring clonal fraction of one or more ESRI mutations in a liquid biopsy sample from an individual having cancer, the method comprising:(a) obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from a liquid biopsy sample obtained from an individual having cancer, wherein the liquid biopsy sample comprises tumor nucleic acid molecules;(b) analyzing, using the one or more processors, the plurality of sequence reads for one or more ESRI mutations present in tumor nucleic acid molecules from the liquid biopsy sample;(c) measuring, using the one or more processors, a variant allele frequency of the one or more ESRI mutations in the liquid biopsy sample;(d) measuring, using the one or more processors, a tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample; and(e) calculating, using the one or more processors, a ratio of the variant allele frequency of the one or more ESRI mutations to the tumor fraction of tumor nucleic acidmolecules in the liquid biopsy sample, thereby measuring a clonal fraction of the one or more ESRI mutations in the liquid biopsy sample.

125. The non-transitory computer readable storage medium of claim 124, wherein the one or more ESRI mutations comprise one or more base substitutions or short insertion / deletions .

126. The non-transitory computer readable storage medium of claim 124 or claim 125, wherein the one or more ESRI mutations comprise one or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I, Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1.

127. The non-transitory computer readable storage medium of any one of claims 124-126, wherein the one or more ESRI mutations comprise one or more gene fusions, rearrangements, or ESRI gene amplifications.

128. The non-transitory computer readable storage medium of any one of claims 124-127, wherein the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations.

129. The non-transitory computer readable storage medium of claim 128, wherein the variant allele frequency is based on a total frequency representing a plurality of ESRI mutations that comprises two or more of D538G, Y537S, Y537N, E380Q, Y537C, L536H, L536P, V422del, L536R, S463P, Y537D, H524L, M357I, H356D, L536Q, H356Y, E380K, L549P, V418E, D351N, G442R, L469V, V533M, L370F, T585M, G344_L345insC, L379I, Y328_S329del, P535H, Y537H, Y537P, E247K, L536_Y537>S, G344D, T311M, D538Y, Y537G, D538N, S341L, L536_Y537del, L536V, L536K, P535_L536insP, D538E, Y537_L539del, Y537_D538insY, Y537T, H196R, L536_D538>P, L536F, R256Q, L536_Y537>QN, D538>NL, P535_Y537>QERG, D538H, Y537del, N532_L536>I,Y537_D538>N, L536_Y537insH, L536I, L536_Y537>P, L536_Y537>H, F209del, D538V, P535_L539del, Y537>SN, L536_M543>T, Y191H, V534E, V533_L536del, and D538P mutations, numbering according to SEQ ID NO:1.

130. The non-transitory computer readable storage medium of any one of claims 124-129, wherein the tumor nucleic acid molecules in the liquid biopsy sample are circulating tumor DNA (ctDNA) molecules.

131. The non-transitory computer readable storage medium of any one of claims 124-130, wherein the tumor fraction is based on an abundance of tumor nucleic acid molecules relative to tumor nucleic acid molecules and non-tumor nucleic acid molecules in the liquid biopsy sample.

132. The non-transitory computer readable storage medium of any one of claims 124-131, wherein the tumor fraction is greater than or equal to about 1%.

133. The non-transitory computer readable storage medium of any one of claims 124-132, wherein the liquid biopsy sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

134. The non-transitory computer readable storage medium of any one of claims 124-133, wherein the tumor nucleic acid molecules comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer.

135. The non-transitory computer readable storage medium of any one of claims 124- 133, wherein the liquid biopsy sample comprises circulating tumor cells (CTCs).

136. The non-transitory computer readable storage medium of any one of claims 124- 135, wherein the method further comprises: analyzing, using the one or more processors, the plurality of sequence reads for one or more oncogenic or tumor suppressor mutations present in tumor nucleic acid molecules from the liquid biopsy sample; and measuring, using the one or more processors, frequency of the one or more oncogenic or tumor suppressor mutations in the liquid biopsy sample.

137. The non-transitory computer readable storage medium of claim 136, wherein the method further comprises calculating, using the one or more processors, a ratio of the frequency of the one or more oncogenic or tumor suppressor mutations to the tumor fraction of tumor nucleic acid molecules in the liquid biopsy sample.

138. The non-transitory computer readable storage medium of claim 136 or claim 137, wherein the one or more oncogenic or tumor suppressor mutations comprise a mutation in one or more of PIK3CA, RBI, PTEN, NF1, ARID1A, AKT1, FGFR2, FGFR3, KRAS, ERBB2, EGFR, and BRAF.

139. The non-transitory computer readable storage medium of any one of claims 136-138, wherein the frequency of the one or more oncogenic or tumor suppressor mutations is based on a total frequency of a plurality of oncogenic or tumor suppressor mutations.

140. The non-transitory computer readable storage medium of any one of claims 124-139, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or next-generation sequencing.

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