Methods for treating HR-positive her2-negative breast cancer

Abstract

Provided herein are methods related to selecting a first-line treatment for an individual having HR+HER2- breast cancer, as well as methods, uses, systems, and non-transitory computer readable storage media related thereto. In some embodiments, the methods comprise detecting presence or absence of an ESRI mutation in a sample from the individual; and selecting a treatment, generating a report, or administering a treatment based thereon (e.g., a first- line treatment that comprises a SERD and a CD Ki based on the presence of an ESRI mutation, or a first-line treatment that comprises an Al and a CDKi based on the absence of an ESRI mutation).

Description

METHODS FOR TREATING HR-POSITIVE HER2-NEGATIVE BREAST CANCERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit of U.S. Provisional Application No. 63 / 606,018, 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 (197102017340seqlist.xml; Size: 9,675 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 selecting a first-line treatment for an individual having HR+HER2- breast cancer, as well as methods, uses, systems, and non-transitory computer readable storage media related thereto. In some embodiments, the methods comprise detecting presence or absence of an ESRI mutation in a sample from the individual; and selecting a treatment, generating a report, or administering a treatment based thereon (e.g., a first- line treatment that comprises a SERD and a CD Ki based on the presence of an ESRI mutation, or a first-line treatment that comprises an Al and a CDKi based on the absence of an ESRI mutation).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, E’SRJmut 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] Endocrine therapy (ET) in combination with CDK4 / 6 inhibitors (CKDi) is the first- line standard of care (SOC) for patients with metastatic breast cancer (mBC) hormone receptor (HR) positive (+) and HER2 negative (-). An aromatase inhibitor (Al) is usually the first ET option for de novo or ET-naive mBC, followed by a selective estrogen receptor degrader (SERD) upon clinical radiological disease progression. Although most HR(+)HER2(-) mBC benefit from first-line ET, second-line ET monotherapy has limited benefit, possibly due to acquired resistance mechanisms [1].

[0006] Mutations in ESRI (ES7? / mut) have been identified as the main acquired resistance mechanism to ET [2], and have been reported in approximately 20-40% of patients pretreated with Al depending on the duration and setting of ET [3]. ESRI codes for the estrogen receptor (ER) alpha, and ES7? / mut usually result in a constitutively activated ER and is thus unaffected by Al depletion of estrogen [1]. ASCO guidelines were updated recently to recommend ES7? / mut tissue or liquid testing at recurrence or progression on ET [4] after subgroup analysis in EMERALD trial indicated that elacestrant have superior outcomes over SOC only for patients with ESRlmut detected by ctDNA [5].

[0007] Several completed and ongoing clinical trials attempt to investigate the role of acquired ESRlmut in patients with HR(+)HER2(-) mBC receiving ET treatment and its implications for optimizing first and subsequent lines of therapy. For instance, in the PADA- 1 trial, patients receiving Al + CD Ki were screened every two months for ESRlmut, and those with an ESRlmut detected and switched to another ET backbone before clinical radiological disease progression had better outcomes than those who only shifted treatment upon clinical progression [1].

[0008] The role of intrinsic ESRlmut in de novo or recurrent HR(+)HER2(-) mBC is much less explored. Previous studies have shown that ESRlmut prevalence is only 1.5-7% in recurrent BC after prior adjuvant or neoadjuvant Al, and less than 1% in ET-naive mBC [3] and the clinical utility of detected ESRlmut before first-line mBC treatment is not well- defined.

[0009] Thus, there is a need in the art for improved first-line treatments and methods for selecting a first-line treatment for HR(+)HER2(-) breast cancer.

[0010] 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

[0011] In some aspects, provided herein is a method of selecting a first- line treatment for an individual having hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer, the method comprising detecting an estrogen receptor 1 (ESRI) mutation in a sample from the individual, wherein detection of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a selective estrogen receptor degrader (SERD) and a cyclin-dependent kinase inhibitor (CDKi).

[0012] In some aspects, provided herein is a method of selecting a first- line treatment for an individual having HR+HER2- breast cancer, the method comprising detecting absence of an ESRI mutation in a sample from the individual, wherein detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an aromatase inhibitor (Al) and a CDKi.

[0013] In some aspects, provided herein is a method of selecting a first- line treatment for an individual having HR+HER2- breast cancer, the method comprising detecting presence or absence of an ESRI mutation in a sample from the individual, wherein detection of the presence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi, or wherein detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.

[0014] In some aspects, provided herein is a method of selecting a first- line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi.

[0015] In some aspects, provided herein is a method of selecting a first- line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

[0016] In some aspects, provided herein is a method of selecting a first- line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of knowledge of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the acquisition of knowledge of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

[0017] In some aspects, provided herein is a method of identifying one or more first- line treatment options for an individual having HR+HER2- breast cancer, the method comprising detecting an ESRI mutation in a sample from the individual; and generating a report comprising one or more first-line treatment options identified for the individual based at least in part on the detection of an ESRI mutation in the sample, wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi. In some aspects, provided herein is a method of identifying one or more first-line treatment options for an individual having HR+HER2- breast cancer, the method comprising detecting absence of an ESRI mutation in a sample from the individual; and generating a report comprising one or more first-line treatment options identified for the individual based at least in part on the detection of the absence of an ESRI mutation in the sample, wherein the one or more first- line treatment options comprise first-line treatment with an Al and a CDKi. In some aspects, provided herein is a method of identifying one or more first-line treatment options for an individual having HR+HER2- breast cancer, the method comprising detecting presence or absence of an ESRI mutation in a sample from the individual; and generating a report comprising one or more first-line treatment options identified for the individual, wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi based at least in part on the detection of the presence an ESRI mutation in the sample, or wherein the one or more first-line treatment options comprise first-line treatment with an Al and a CDKi based at least in part on the detection of the absence of an ESRI mutation in the sample. In some embodiments, the report further indicates the presence or absence of the ESRI mutation in the sample.

[0018] In some aspects, provided herein is a method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising acquiring knowledge of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and responsive to said knowledge, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi.

[0019] In some aspects, provided herein is a method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and responsive to said knowledge, administering to the individual an effective amount of a first- line treatment that comprises an Al and a CDKi.

[0020] In some aspects, provided herein is a method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and responsive to knowledge of the presence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi; or responsive to knowledge of the absence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

[0021] In some aspects, provided herein is a method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising detecting an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi.

[0022] In some aspects, provided herein is a method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising detecting absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

[0023] In some aspects, provided herein is a method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising detecting presence or absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and responsive to detecting the presence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi; or responsive to detecting the absence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

[0024] In some aspects, provided herein is a method of identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies an ESRI mutation in the sample; and selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises a SERD and a CDKi. In some aspects, provided herein is a method of identifying a candidate first- line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies absence of an ESRI mutation in the sample; and selecting a first- line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises an Al and a CDKi. In some aspects, provided herein is a method of identifying a candidate first- line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies presence or absence of an ESRI mutation in the sample; and selecting a first- line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises treatment with a SERD and a CDKi when the sequencing mutation profile identifies the presence of an ESRI mutation in the sample, or wherein the first- line treatment comprises treatment with an Al and a CDKi when the sequencing mutation profile identifies the absence of an ESRI mutation in the 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. In some embodiments, the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS).

[0025] In some aspects, provided herein is a method of predicting survival of an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to survival of the individual when treated with a first-line treatment comprising an Al and a CDKi. In some aspects, provided herein is a method of predicting survival of an individual having HR+HER2- breast cancer, comprisingacquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to survival when treated with a first-line treatment comprising an Al and a CDKi of an individual whose sample comprises an ESRI mutation. In some aspects, provided herein is a method of screening an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a first-line treatment comprising a SERD and a CDKi and / or longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to response and / or survival when treated with a first-line treatment comprising an Al and a CDKi. In some aspects, provided herein is a method of screening an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a first-line treatment comprising an Al and a CDKi and / or longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to response and / or survival of an individual whose sample comprises an ESRI mutation. In some embodiments, the survival comprises progression-free survival (PFS). In some embodiments, the survival comprises overall survival (OS). In some embodiments, the improved response comprises longer time-to- treatment discontinuation (TTD). In some embodiments, the improved response comprises longer time-to-next treatment (TTNT).

[0026] In some embodiments according to any of the embodiments described herein, the treatment or treatment option is a first-line metastatic treatment. In some embodiments according to any of the embodiments described herein, the HR+HER2- breast cancer is first- line metastatic HR+HER2- breast cancer. In some embodiments, the individual has received a prior adjuvant therapy. In some embodiments, the individual has not received a prior treatment (e.g., non-adjuvant treatment) for HR+HER2- breast cancer (e.g., first- line metastatic HR+HER2- breast cancer). In some embodiments, the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.

[0027] In some embodiments according to any of the embodiments described herein, the SERD is fulvestrant. In some embodiments, the Al is aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4- hydroxyandrostenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), or 4-Androstene-3,6,17-trione (“6-OXO”). In some embodiments, the CDKi is a CDK4 / CDK6 inhibitor. In some embodiments, the CDKi is palbociclib, ribociclib, abemaciclib, or dalpiciclib. In some embodiments, the CDKi is a CDK4-selective inhibitor. In some embodiments, the CDKi is PF-07220060 or 2-94. In some embodiments, the CDKi is a CDK2 inhibitor. In some embodiments, the CDKi is tagtociclib (PF-07104091).

[0028] In some embodiments according to any of the embodiments described herein, the ESRI mutation comprises a base substitution or short insertion / deletion. In some embodiments, the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1. In some embodiments, the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1. In some embodiments, the ESRI mutation comprises a rearrangement or gene fusion. In some embodiments, detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof.

[0029] In some embodiments according to any of the embodiments described herein, the methods further comprise obtaining the sample from the individual. In some embodiments, the sample is obtained or derived from the HR+HER2- breast cancer. In some embodiments, the sample comprises a tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample. In some embodiments, the sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises circulating tumor cells (CTCs). In some embodiments, the sample comprises cell- free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell. In some embodiments, the sample comprises cells and / or nucleic acids from the HR+HER2- breast cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the HR+HER2- breast cancer. In some embodiments, the ESRI mutation or absence thereof is detected by immunohistochemistry analysis (IHC), comprehensive genomic profiling (CGP), comparative genomic hybridization (CGH), sequencing, or any combination thereof. 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).

[0030] In some embodiments according to any of the embodiments described herein, detecting the ESRI mutation or absence thereof comprises: providing a plurality of nucleic acid molecules obtained from the sample from the individual, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an ESRI gene, or a portion thereof; optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; optionally, amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; optionally, capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; 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 sequence reads of the plurality of sequence reads correspond to an ESRI gene, or a portion thereof; analyzing the plurality of sequence reads for the presence or absence of an ESRI mutation; and based on the analyzing step, detecting the presence or absence of an ESRI mutation in the sample. In some embodiments, the sequencer comprises a next-generation sequencer. In some embodiments, detecting the ESRI mutation or absence thereof comprises: providing the sample from the individual, wherein the sample comprises a plurality of 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 an ESRI gene or a portion thereof 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 the presence or absence of an ESRI mutation; and detecting, based on the analyzing step, the presence or absence of an ESRI alteration in the sample from the individual. In some embodiments, 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 an ESRI gene or a portion thereof 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. In someembodiments, the methods further comprise selectively enriching for one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof; wherein 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 more bait molecules to one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof 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 an ESRI gene or a portion thereof. 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 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 nucleic acid molecules comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof 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 plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules. In some embodiments, the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample, and wherein the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample, and the non-cancer nucleic acid molecules are derived from a nontumor fraction of the liquid biopsy sample. In some embodiments, the methods further comprise generating a report, wherein the report: indicates the presence or absence of the ESRI mutation in the sample from the individual; and / or indicates a first- line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the presence of an ESRI mutation in the sample from the individual, wherein thefirst-line treatment or the one or more first-line treatment options comprise a SERD and a CDKi, or indicates a first-line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the absence of an ESRI mutation in the sample from the individual, wherein the first-line treatment or the one or more first-line treatment options comprise an Al and a CDKi. 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 presence or absence of the ESRI mutation. 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 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 first-line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the first-line treatment or one or more first-line treatment options comprise a SERD and a CDKi when the molecular profile indicates the presence of an ESRI mutation in the sample from the individual, or wherein the first-line treatment or one or more first-line treatment options comprise an Al and a CDKi when the molecular profile indicates the absence of an ESRI mutation in the sample from the individual. 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.

[0031] In some embodiments according to any of the embodiments described herein, the individual is a human.

[0032] In some aspects, provided herein is a system for detecting presence or absence of an ESRI mutation, or, identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising a SERD and a CDKi or an Al and a CDKi, 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 someembodiments, 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 sample obtained from an individual having HR+HER2- breast cancer; analyze the plurality of sequence reads for presence of an ESRI mutation; and detect, based on the analyzing, presence of the ESRI mutation in the sample; wherein detecting the presence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi. 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 sample obtained from an individual having HR+HER2- breast cancer; analyze the plurality of sequence reads for absence of an ESRI mutation; and detect, based on the analyzing, absence of the ESRI mutation in the sample; wherein detecting the absence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi. 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 sample obtained from an individual having HR+HER2- breast cancer; analyze the plurality of sequence reads for presence or absence of an ESRI mutation; and detect, based on the analyzing, presence of the ESRI mutation in the sample; wherein responsive to the detection of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the detection of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

[0033] 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 detecting presence or absence of an ESRI mutation, or, identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising a SERD and a CD Ki or an Al and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer; analyzing, using the one or more processors, the plurality of sequence reads for presence of an ESRI mutation; and detecting, using the one or more processors and based on the analyzing, the presence of the ESRI mutation in the sample; wherein detecting the presence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi. 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 sample obtained from an individual having HR+HER2- breast cancer; analyzing, using the one or more processors, the plurality of sequence reads for absence of an ESRI mutation; and detecting, using the one or more processors and based on the analyzing, the absence of the ESRI mutation in the sample; wherein detecting the absence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi. 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 sample obtained from an individual having HR+HER2- breast cancer; analyzing, using the one or more processors, the plurality of sequence reads for presence or absence of an ESRI mutation; and detecting, using the one or more processors and based on the analyzing, the presence or absence of the ESRI mutation in the sample; wherein responsive to the detection of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the detection of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first- line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

[0034] In some embodiments according to any of the embodiments described herein, the ESRI mutation comprises a base substitution or short insertion / deletion. In some embodiments, the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1. In some embodiments, the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1. In some embodiments, the ESRI mutation comprises a rearrangement or gene fusion. In some embodiments, detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof. In some embodiments, the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer. In some embodiments, the treatment is a first-line metastatic treatment. 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.

[0035] 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

[0036] FIG. 1 is a schematic showing the prevalence of ESRI mutations (ESRlmut) detected in tissue specimens obtained from HR+HER2- metastatic breast cancer patients in first 3 metastatic lines of therapy, as indicated. Al: aromatase inhibitors; chemo: chemotherapy; CDKi: CDK 4 / 6 inhibitors; ET: endocrine therapy; HR: hormone receptor; LBx: liquid biopsy; mBC: metastatic breast cancer; mut: mutations; SERD: selective estrogen receptor degrader (fulvestrant); TBx: tissue biopsy; TF: ctDNA tumor fraction; Tx: therapy.

[0037] FIG. 2 is a schematic showing the prevalence of ESRI mutations (ESRlmut) detected in liquid specimens obtained from HR+HER2- metastatic breast cancer patients in first 3 metastatic lines of therapy, as indicated. Al: aromatase inhibitors; chemo: chemotherapy; CDKi: CDK 4 / 6 inhibitors; ET: endocrine therapy; HR: hormone receptor; LBx: liquid biopsy; mBC: metastatic breast cancer; mut: mutations; SERD: selective estrogen receptor degrader (fulvestrant); TBx: tissue biopsy; TF: ctDNA tumor fraction; Tx: therapy.

[0038] FIGS. 3A-4 show outcomes for HR(+)HER2(-) breast cancer patients receiving first line aromatase inhibitor (Al) and CDK4 / 6 inhibitor (CDKi) in 1st line metastatic setting, comparing patients with ESRI wild-type (WT) tissue specimens (*) vs. those with ESR1- mutated (ESRlmut) tissue specimens (#). FIG. 3A shows real-world time-to-treatment discontinuation (rwTTD). FIG. 3B shows real world progression-free survival (rwPFS). FIG. 3C shows real world overall survival (rwOS). Kaplan-Meier plots show outcomes by ES7? / mut status. FIG. 4 shows rwTTD and rwPFS in patients with ESE / mut specimens tissue receiving first line Al + CDKi treatment. Swimmer plot shows rwTTD and rwPFS for patients with ES7? / mut ordered by specific ESRlmut, as indicated. Al: aromatase inhibitors; ESRlmut: ESRI mutations; ESRI WT: ESRI wild-type; HR: hazard ratio; OS: overall survival; PFS: progression-free survival; rw: real-world; TBx: tissue biopsy; TTD: time to treatment discontinuation.

[0039] FIGS. 5A-6 show outcomes for HR(+)HER2(-) breast cancer patients receiving first line fulvestrant and CDK4 / 6 inhibitor (CDKi) in 1st line metastatic setting, comparing patients with ESRI wild-type (WT) tissue specimens (*) vs. those with ESRI- mutated (ESRlmut) tissue specimens (#). FIG. 5A shows real-world time-to-treatment discontinuation (rwTTD). FIG. 5B shows real world progression-free survival (rwPFS). FIG. 5C shows real world overall survival (rwOS). Kaplan-Meier plots show outcomes by ESRlmut status. FIG. 6 shows rwTTD and rwPFS in patients with ESRlmut specimens tissue receiving first line fulvestrant + CDKi treatment. Swimmer plot shows rwTTD and rwPFS for patients with ESRlmut ordered by specific ESRlmut, as indicated. ESRlmut: ESRI mutations; ESRI WT: ESRI wild-type; HR: hazard ratio; OS: overall survival; PFS: progression-free survival; rw: real-world; TBx: tissue biopsy; TTD: time to treatment discontinuation.

[0040] FIGS. 7A-7D show multivariate models for HR+HER2- metastatic breast cancer patients receiving Al + CDKi or fulvestrant + CDKi. Multivariable models for ESRlmut vs. ESRlwt in patients receiving Al + CDKi: rwPFS (FIG. 7A) and rwOS (FIG. 7B). Multivariable models for ESRlmut vs. ESRIwt in patients receiving fulvestrant + CDKi: rwPFS (FIG. 7C) and rwOS (FIG. 7D). Al: aromatase inhibitors; CI: confidence interval; ECOG PS: Eastern Cooperative Oncology Group performance score; ET: endocrine therapy; ESRlmut: ESRI mutations; ESRlwt: ESRI wild type; HR: hazard ratio; IDC: Invasive ductal carcinoma; IEC: Invasive lobular carcinoma; Met; metastasis; OS: overall survival; PFS: progression-free survival; rw: real-world; TBx: tissue biopsy; Tx: therapy.

[0041] FIG. 8A shows baseline patient characteristics (patients included in the outcome analyses). Al: aromatase inhibitors; CDKi: CDK 4 / 6 inhibitors; CNS: central nervous system; ECOG PS: Eastern Cooperative Oncology Group performance score; ES7? / mut: ESRI mutations; ESRIwt ESRI wild type; ET: endocrine therapy; Tx: therapy.

[0042] FIG. 8B depicts an exemplary device, in accordance with some embodiments.

[0043] FIG. 9 depicts an exemplary system, in accordance with some embodiments.

[0044] FIG. 10 depicts a block diagram of an exemplary process for detecting an ESRI mutation, in accordance with some embodiments.DETAILED DESCRIPTION

[0045] The present disclosure relates generally to selecting a first- line treatment (e.g., first- line metastatic treatment) for an individual having HR+HER2- breast cancer (e.g., HR+HER2- MBC), as well as related methods and uses.

[0046] The present disclosure demonstrates that ESRI mutations are prevalent in 8-11% of baseline tissue or liquid samples from patients with HR+HER2- mBC. ES7? / mut detected at the start of metastatic treatment was associated with less favorable outcomes in patients receiving Al + CDKi 1stline therapy, but not in patients receiving fulvestrant + CDKi. ESRlmut prevalence increased during treatment course. As such, it is thought that presence or absence of an ESRI mutation could stratify first-line treatment in patients HR+HER2- mBC to provide more effective first- line treatment options.I. General Techniques

[0047] 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 Eaboratory 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 Celland 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

[0048] 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.

[0049] 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 or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0050] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0051] 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.

[0052] 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.

[0053] As used herein, the term "ESRl" refers to a gene encoding an estrogen receptor 1 polypeptide. The human ESRI gene is located on chromosome 6q25.1-q25.2. ESRI is alsoknown as ER, ESR, Era, ESRA, ESTRR, and NR3AL 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 AAYEFNAAAAANA QVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQ VPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGY HYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRML KHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLA DRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAP NLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRV LDKITDTLIHLM AKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV (SEQ ID NO:1)

[0054] “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 or RNA 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.

[0055] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted beforeor 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 as arabinose, 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.

[0056] “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.

[0057] 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.

[0058] “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.

[0059] 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, short insertion / 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.

[0060] “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 sequencethat is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.

[0061] 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.

[0062] 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, or by 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)).

[0063] 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.

[0064] 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 thatwould 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).

[0065] 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.

[0066] 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 Markov methods, Bayesian methods, long-range correlation methods, change point methods, or any combination thereof.

[0067] 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.

[0068] 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 beperformed. 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.

[0069] “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.

[0070] 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.

[0071] 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 infiltration into 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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, intraarterial, 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.

[0077] 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).

[0078] 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.

[0079] 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.

[0080] 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

[0081] In some aspects, provided herein are methods for selecting a first- line treatment for an individual having HR+HER2- breast cancer. In other aspects, provided herein are methods for identifying one or more first-line treatment options for an individual having HR+HER2- breast cancer. In other aspects, provided herein are methods for treating or delaying progression of HR+HER2- breast cancer in an individual. In other aspects, provided herein are methods for identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof. In other aspects, provided herein are methods for predicting survival of an individual having HR+HER2- breast cancer. In other aspects, provided herein are methods for screening an individual having HR+HER2- breast cancer.

[0082] In some embodiments, the individual is a human. In some embodiments according to any of the embodiments described herein, the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer. In some embodiments, the treatment is a first-line metastatic treatment.

[0083] In some embodiments of any of the methods provided herein, the methods comprise detecting or acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual. In some embodiments, detection of an ESRI mutation (or presence thereof) in the sample identifies the individual as one who may benefit from, is identified as likely to respond to, or is predicted to have longer survival and / or improved response when treated with, a first-line treatment comprising a selective estrogen receptor degrader (SERD)and a cyclin-dependent kinase inhibitor (CDKi). In some embodiments, detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from, is identified as likely to respond to, or is predicted to have longer survival and / or improved response when treated with, a first-line treatment comprising an aromatase inhibitor (Al) and a CDKi.

[0084] 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) 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 sample obtained from an individual having HR+HER2- breast cancer; analyzing, using the one or more processors, the plurality of sequence reads for presence or absence of an ESRI mutation; and detecting, using the one or more processors and based on the analyzing, the presence or absence of the ESRI mutation in the sample. 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.ESRI mutations and detection methods

[0085] Certain aspects of the present disclosure relate to ESRI mutations. In some embodiments, an ESRI mutation includes, without limitation, a base substitution, short insertion / deletion, missense mutation, truncation mutation, rearrangement, or gene fusion.

[0086] As demonstrated herein, ESRI mutations (E.S7? / mut) are detected in a certain portion of patients with HR+HER2- mBC, and E.S7? / mut detected at the start of metastatic treatment was associated with less favorable outcomes in patients receiving Al + CDKi 1stline therapy, but not in patients receiving fulvestrant + CDKi. E.S7? / mut prevalence increased during treatment course. As such, it is thought that presence or absence of an ESRI mutation could stratify first-line treatment in patients HR+HER2- mBC to provide more effective first-line treatment options.

[0087] As used herein '^ESRl" refers to a gene encoding an ESRI mRNA or ESRI polypeptide. 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 gene is representedby 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 CTTCACCGGACCCGCAGGCTCCCGGGGCAGGGCCGGGGCCAGAGCTCGCGTGTCGGCGGGACATGCGCT GCGTCGCCTCTAACCTCGGGCTGTGCTCTTTTTCCAGGTGGCCCGCCGGTTTCTGAGCCTTCTGCCCTGCGG GGACACGGTCTGCA CCCTGCCCGCGGCCACGGACCATGACCATGACCCTCCACACCAAAGCATCTGGG ATGGCCCTACTGCATCAGATCCAAGGGAACGAGCTGGAGCCCCTGAACCGTCCGCAGCTCAAGATCCCCC TGGAGCGGCCCCTGGGCGAGGTGTACCTGGACAGCAGCAAGCCCGCCGTGTACAACTACCCCGAGGGCGC CGCCTACGAGTTCAAC GCCGCGGCCGCCGCCAACGCGCAGGTCTACGGTCAGACCGGCCTCCCCTACGGC CCCGGGTCTGAGGCTGCGGCGTTCGGCTCCAACGGCCTGGGGGGTTTCCCCCCACTCAACAGCGTGTCTCC GAGCCCGCTGATGCTACTGCACCCGCCGCCGCAGCTGTCGCCTTTCCTGCAGCCCCACGGCCAGCAGGTG CCCTACTACCTGGAG AACGAGCCCAGCGGCTACACGGTGCGCGAGGCCGGCCCGCCGGCATTCTACAGG CCAAATTCAGATAATCGACGCCAGGGTGGCAGAGAAAGATTGGCCAGTACCAATGACAAGGGAAGTATG GCTATGGAATCTGCCAAGGAGACTCGCTACTGTGCAGTGTGCAATGACTATGCTTCAGGCTACCATTATGGA GTCTGGTCCTGTGAGGGCTGCAAGGCCTTCTTCAAGAGAAGTATTCAAGGACATAACGACTATATGTGTC CAGCCACCAACCAGTGCACCATTGATAAAAACAGGAGGAAGAGCTGCCAGGCCTGCCGGCTCCGTAAAT GCTACGAAGTGGGAATGATGAAAGGTGGGATACGAAAAGACCGAAGAGGAGGGAGAATGTTGAAACACA AGCGCCAGAGAGATGATGGGGAGGGCAGGGGTGAAGTGGGGTCTGCTGGAGACATGAGAGCTGCCAACCTT TGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGA TGGTCAGTGCCTTGTT GGATGCTGAGCCCCCGATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGT GAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCG AAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGA GATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGGGAAGCTACTGTTTGCTCCTAACTTG CTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACA TCATCTCGGTTCCGCA 1TGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACGGTCTGAGAGCTCCCTGGCTCCCACACGGTTCAGATAATCCCTGCTGCATTTTACCCTCATCATGCACCACTTTAGCCAAATTCTGTCTCCTGCATACACTCCGGCATGCATCCAACACCAATGGCTTTCTAGATGAGTGGCCATTCATTTGCTTGCTCAGTTCTTAGTGGCACATCTTCTGTCTTCTGTTGGGAACAGCCAAAGGGATTCCAAGGCTAAATCTTTGTAACAGCTCTCTTTCCCCCTTGCTATGTTACTAAGCGTGAGGATTCCCGTAGCTCTTCACAGCTGAACTCAGTCTATGGGTTGGGGCTCAGATAACTCTGTGCATTTAAGCTACTTGTAGAGACCCAGGCCTGGAGAGTAGACATTTTGCCTCTGATAAGCACTTTTTAAATGGCTCTAAGAATAAGCCACAGCAAAGAATTTAAAGTGGCTCCTTTAATTGGTGACTTGGAGAAAGCTAGGTCAAGGGTTTATTATAGCACCCTCTTGTATTCCTATGGCAATGCATCCTTTTATGAAAGTGGTACACCTTAAAGCTTTTATATGACTGTAGCAGAGTATCTGGTGATTGTCAATTCATTCCCCCTATAGGAATACAAGGGGCACACAGGGAAGGCAGATCCCCTAGTTGGCAAGACTATTTTAACTTGATACACTGCAGATTCAGATGTGCTGAAAGCTCTGCCTCTGGCTTTCCGGTCATGGGTTCCAGTTAATTCATGCCTCCCATGGACCTATGGAGAGCAGCAAGTTGATCTTAGTTAAGTCTCCCTATATGAGGGATAAGTTCCTGATTTTTGTTTTTATTTTTGTGTTACAAAAGAAAGCCCTCCCTCCCTGAACTTGCAGTAAGGTCAGCTTCAGGACCTGTTCCAGTGGGCACTGTACTTGGATCTTCCCGGCGTGTGTGTGCCTTACACAGGGGTGAACTGTTCACTGTGGTGATGCATGATGAGGGTAAATGGTAGTTGAAAGGAGCAGGGGCCCTGGTGTTGCATTTAGCCCTGGGGCATGGAGCTGAACAGTACTTGTGCAGGATTGTTGTGGCTACTAGAGAACAAGAGGGAAAGTAGGGCAGAAACTGGATACAGTTCTGAGGCACAGCCAGACTTGCTCAGGGTGGCCCTGCCACAGGCTGCAGCTACCTAGGAACATTCCTTGCAGACCCCGCATTGCCCTTTGGGGGTGCCCTGGGATCCCTGGGGTAGTCCAGCTCTTCTTCATTTCCCAGCGTGGCCCTGGTTGGAAGAAGCAGCTGTCACAGCTGCTGTAGACAGCTGTGTTCCTACAATTGGCCCAGCACCCTGGGGCACGGGAGAAGGGTGGGGACCGTTGCTGTCACTACTCAGGCTGACTGGGGCCTGGTCAGATTACGTATGCCCTTGGTGGTTTAGAGATAATCCAAAATCAGGGTTTGGTTTGGGGAAGAAAATCCTCCCCCTTCCTCCCCCGCCCCGTTCCCTACCGCCTCCACTCCTGCCAGCTCATTTCCTTCAATTTCCTTTGACCTATAGGCTAAAAAAGAAAGGCTCATTCCAGCCACAGGGCAGCCTTCCCTGGGCCTTTGCTTCTCTAGCACAATTATGGGTTACTTCCTTTTTCTTAACAAAAAAGAATGTTTGATTTCCTCTGGGTGACCTTATTGTCTGTAATTGAAACCCTATTGAGAGGTGATGTCTGTGTTAGCCAATGACCCAGGTGAGCTGCTCGGGCTTCTCTTGGTATGTCTTGTTTGGAAAAGTGGATTTCATTCATTTCTGATTGTCCAGTTAAGTGATCACCAAAGGACTGAGAATCTGGGAGGGCAAAAAAAAAAAAAAAGTTTTTATGTGCACTTAAATTTGGGGACAATTTTATGTATCTGTGTTAAGGATATGTTTAAGAACATAATTCTTTTGTTGCTGTTTGTTTAAGAAGCACCTTAGTTTGTTTAAGAAGCACCTTATATAGTATAATATATATTTTTTTGAAATTACATTGCTTGTTTATCAGACAATTGAATGTAGTAATTCTGTTCTGGATTTAATTTGACTGGGTTAACATGCAAAAACCAAGGAAAAATATTTAGTTTTTTTTTTTTTTTTTGTATACTTTTCAAGCTACCTTGTCATGTATACAGTCATTTATGCCTAAAGCCTGGTGATTATTCATTTAAATGAAGATCACATTTCATATCAACTTTTGTATCCACAGTAGACAAAATAGCACTAATCCAGATGCCTATTGTTGGATACTGAATGACAGACAATCTTATGTAGCAAAGATTATGCCTGAAAAGGAAAATTATTCAGGGCAGCTAATTTTGCTTTTACCAAAATATCAGTAGTAATATTTTTGGACAGTAGCTAATGGGTCAGTGGGTTCTTTTTAATGTTTATACTTAGATTTTCTTTTAAAAAAATTAAAATAAAACAAAAAAAAATTTCTAGGACTAGACGATGTAATACCAGCTAAAGCCAAACAATTATACAGTGGAAGGTTTTACATTATTCATCCAATGTGTTTCTATTCATGTTAAGATACTACTACATTTGAAGTGGGCAGAGAACATCAGATGATTGAAATGTTCGCCCAGGGGTCTCCAGCAACTTTGGAAATCTCTTTGTATTTTTACTTGAAGTGCCACTAATGGACAGCAGATATTTTCTGGCTGATGTTGGTATTGGGTGTAGGAACATGATTTAAAAAAAAACTCTTGCCTCTGCTTTCCCCCACTCTGAGGCAAGTTAAAATGTAAAAGATGTGATTTATCTGGGGGGCTCAGGTATGGTGGGGAAGTGGATTCAGGAATCTGGGGAATGGCAAATATATTAAGAAGAGTATTGAAAGTATTTGGAGGAAAATGGTTAATTCTGGGTGTGCACCAGGGTTCAGTAGAGTCCACTTCTGCCCTGGAGACCACAAATCAACTAGCTCCATTTACAGCCATTTCTAAAATGGCAGCTTCAGTTCTAGAGAAGAAAGAACAACATCAGCAGTAAAGTCCATGGAATAGCTAGTGGTCTGTGTTTCTTTTCGCCATTGCCTAGCTTGCCGTAATGATTCTATAATGCCATCATGCAGCAATTATGAGAGG CTAGGTCATCCAAAG AGAAGACCCTATCAATGTAGGTTGCAAAATCTAACCCCTAAGGAAGTGCAGTCT TTGATTTGATTTCCCTAGTAACCTTGCAGATATGTTTAACCAAGCCATAGCCCATGCCTTTTGAGGGCTGA ACAAATAAGGGACTTACTGATAATTTACTTTTGATCACATTAAGGTGTTCTCACCTTGAAATCTTATACAC TGAAATGGCCATTGATTTAGGCCACTGGCTTAGAGTACTCCTTCCCCTGCATGACACTGATTACAAATA CTTTCCTATTCATACTTTCCAATTATGAGATGGACTGTGGGTACTGGGAGTGATCACTAACACCATAGTA ATGTCTAATATTCACAGGCAGATCTGCTTGGGGAAGCTAGTTATGTGAAAGGCAAATAGAGTCATACAG TAGCTCAAAAGGCAACCATAATTCTCTTTGGTGCAGGTCTTGGGAGCGTGATCTAGATTACACTGCACCAT TCCCAAGTTAATCCC CTGAAAACTTACTCTCAACTGGAGCAAATGAACTTTGGTCCCAAATATCCATCTT TTCAGTAGCGTTAATTATGCTCTGTTTCCAACTGCATTTCCTTTCCAATTGAATTAAAGTGTGGCCTCGTT TTTAGTCATTTAAAATTGTTTTCTAAGTAATTGCTGCCTCTATTATGGCACTTCAATTTTGCACTGTCTTT TGAGATTCAAGAAAAATTTCTATTCTTTTTTTTGCATCCAATTGTGCCTGAACTTTTAAAATATGTAAA TGCTGCCATGTTCCAAACCCATCGTCAGTGTGTGTGTTTAGAGCTGTGCACCCTAGAAACAACATATTG TCCCATGAGCAGGTGCCTGAGACACAGACCCCTTTGCATTCACAGAGAGGTCATTGGTTATAGAGACTTG AATTAATAAGTGACATTATGCCAGTTTCTGTTCTCTCACAGGTGATAAACAATGCTTTTTGTGCACTACAT ACTCTTCAGTGTAGAGCTCTTGTTTTATGGGAAAAGGCTCAAATGCCAAATTGTGTTTGATGGATTAAT ATGCCCTTTTGCCGA TGCATACTATTACTGATGTGACTCGGTTTTGTCGCAGCTTTGCTTTGTTTAATGAA ACACACTTGTAAACCTCTTTTGCACTTTGAAAAAGAATCCAGCGGGATGCTCGAGCACCTGTAAACAAT TTTCTCAACCTATTTGATGTTCAAATAAAGAATTAAACTAAA(SEQ ID NO: 2)

[0088] 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 VPYYEENEPSGYTVREAGPPAFYRPNSDNRRQGGREREASTNDKGSMAMESAKETRYCAVCNDYASGY HYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRML KHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAP NLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRV LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLD AHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV (SEQ ID NO:1)

[0089] In some embodiments, the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1. In some embodiments, the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1.

[0090] In some embodiments, detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof, or a wild-type ESRI polypeptide or portion thereof.

[0091] ESRI mutations may be assessed using any suitable method known in the art, e.g., for detecting specific polynucleotides and / or polypeptides. 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, SDS-PAGE, mass spectrometry, antibody-based detection such as immunohistochemistry (IHC), 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, ESRI mutations in a cancer (e.g., in a sample from a cancer) are assessed using an array-based method, such as array-based comparative genomic hybridization (CGH) methods.

[0093] In array-based CGH methods, a first sample of nucleic acids (e.g., from a sample, such as from a cancer, a tumor, or a tissue or liquid biopsy) is labeled with a first label, while a second sample of nucleic acids (e.g., a control, such as from a healthy cell / tissue) is labeled with a second label. In some embodiments, equal quantities of the two samples are mixed and co-hybridized to a DNA microarray of several thousand evenly spaced cloned DNA fragments or oligonucleotides, e.g., which have been spotted on the array. In someembodiments, the microarray is a whole genome microarray, e.g., it comprises DNA fragments or oligonucleotides with nucleotide sequences that cover the entire genome. In some embodiments, the microarray is a targeted microarray, e.g., it comprises DNA fragments or oligonucleotides with nucleotide sequences that cover a particular segment of the genome, such as a particular chromosome or chromosomal segment, such as chromosome 6 or a segment of chromosome 6 including the ESRI locus, or hybridize to particular ESRI mutations. After hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores.

[0094] In some embodiments, ESRI mutations in a cancer (e.g., in a sample from a cancer) 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 mutation 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 mutations. In some embodiments, ESRI mutations in a cancer (e.g., in a sample from a cancer) are assessed using a qPCR or ddPCR method.

[0095] In some embodiments, ESRI mutations in a cancer (e.g., in a sample from a cancer) 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. 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). 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.

[0096] 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, withoutlimitation, 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 sample from a cancer) 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 alterations in a cancer (e.g., in a sample from a cancer) include, without limitation, the Genome Sequencer (GS) FEX 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).

[0097] In some embodiments, methods for detecting ESRI mutations in a cancer (e.g., in a sample from a cancer) comprise providing a sample from an individual (e.g., an individual having cancer), wherein the sample comprises one or more nucleic acids. In some embodiments, an ESRI mutation is detected directly from one or more nucleic acids from the sample. In some embodiments, an ESRI mutations is detected from an amplicon, sequence read, or other nucleic acid otherwise derived from one or more nucleic acids from the sample.

[0098] 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 AMPureXP 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.

[0099] 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.

[0100] In some embodiments, the methods further comprise analyzing sequence data obtained from the sequencing, e.g., a plurality of sequence reads, for the presence or absence of 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 the presence or absence of ESRI mutations comprises one or more, or all, of the steps as described in Frampton et al., (2013) Nat Biotechnol, 31:1023- 1031.

[0101] 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., an individual 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 non-tumor 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 aregion 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.

[0102] 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, suspected of having or determined to have 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 the plurality of sequence reads correspond to an ESRI gene / coding sequence or portion thereof; (f) analyzing the plurality of sequence reads to determine presence or absence of an ESRI mutation, e.g., as described above; and (g) based on the analysis, detecting the presence or absence of an ESRI mutation in the 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.

[0103] 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 to determine presence or absence of an ESRI alteration, e.g., as described above; (g) detecting, based on the analyzing step, the presence or absence of the ESRI alteration in the sample from the individual.

[0104] In some embodiments of any of the methods provided herein, the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules. In some embodiments, the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample, and the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample; and the non-cancer 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.

[0105] 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 theoriginal sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0106] 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.

[0107] 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 more bait 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.

[0108] In some embodiments of any of the methods for detection of ESRI mutations 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., one or more cancer-related genes such as EGFR, ALK and / or FGFR3, or a panel of known / suspected oncogenes and / or tumor suppressors, orany 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).

[0109] In some embodiments of any of the methods for detection of ESRI mutations provided herein, the methods further comprise generating a molecular profile for the individual or the sample, based, at least in part, on detecting the presence or absence of the ESRI mutation. 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 DNA methylation 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 sequencingbased 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.

[0110] In some embodiments of any of the methods for detection of ESRI mutations 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 anti-cancer therapy, e.g., as described herein, e.g., an immunotherapy. In some embodiments of any of the methods for detection of ESRImutations provided herein, the methods further comprise generating a report indicating the presence or absence of an ESRI mutation in the sample. In some embodiments of any of the methods for detection of ESRI mutation 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. 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.

[0111] In some embodiments of any of the methods for detection of ESRI mutations provided herein), the methods for determining the presence or absence of an ESRI mutation 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 improve the validity of, e.g., disease detection calls by, for example, independently confirming the presence of the ESRI mutation in a given patient sample.

[0112] 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 tissue biopsy sample, a liquid biopsy sample, or a normal control. 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 nontumor or non-cancer nucleic acid molecules. In some instances, the tumor nucleic acidmolecules may be derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-tumor nucleic acid molecules may be derived from a normal portion of the heterogeneous tissue biopsy sample. In some instances, the sample may comprise a liquid biopsy sample, and 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.

[0113] Also provided herein are probes, baits and oligonucleotides suitable for the detection of an ESRI mutation, e.g., according to any methods of detection known in the art and / or described herein.

[0114] Provided herein are probes suitable for the detection of an ESRI mutation, e.g., according to any methods of detection known in the art and / or described herein.

[0115] In some embodiments, a probe provided herein comprises a nucleic acid sequence configured to hybridize to a target nucleic acid molecule (e.g., corresponding to one or more genes, such as ESRI), or a portion thereof. In some embodiments, the probe comprises a nucleotide sequence configured to hybridize to a nucleotide sequence in an intron or an exon of a target gene, such as an ESRI gene. In some embodiments, the probe comprises a nucleic acid molecule which is a DNA, RNA, or a DNA / RNA molecule. In some embodiments, the probe comprises a nucleic acid molecule comprising any of between about 10 and about 20 nucleotides, between about 12 and about 20 nucleotides, between about 10 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 probe comprises a nucleic acid molecule comprising any of 10 nucleotides, 11 nucleotides,12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides,18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides,24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides. In some embodiments, the probe comprises a nucleic acid molecule comprising any of between about 40 nucleotides and about 50 nucleotides, 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 andabout 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 probe comprises a nucleic acid molecule comprising between about 12 and about 20 nucleotides. In some embodiments, a probe provided herein includes a label or a tag. In some embodiments, the label or tag is a radiolabel (e.g., a radioisotope), a fluorescent label (e.g., a fluorescent compound), an enzymatic label, an enzyme co-factor, a sequence tag, biotin, or another ligand. In some embodiments, a probe provided herein includes a detection reagent such as a fluorescent marker. In some embodiments, a probe provided herein includes (e.g., is conjugated to) an affinity tag, e.g., that allows capture and isolation of a hybrid formed by a probe and a nucleic acid molecule hybridized to the probe. In some embodiments, the affinity tag is an antibody, an antibody fragment, biotin, or any other suitable affinity tag or reagent known in the art. In some embodiments, a probe is suitable for solution phase hybridization. In some embodiments, probes provided herein may be used according to the methods of detection of an ESRI mutation provided herein. For example, a probe provided herein may be used for detecting an ESRI mutation in a sample, e.g., a sample obtained from an individual. In some embodiments, the probe may be used for identifying cells or tissues that comprise an ESRI mutation. In some embodiments, one or more probes provided herein are suitable for use in in situ hybridization methods, e.g., as described above, such as FISH.

[0116] Provided herein are baits suitable for the detection of an ESRI mutation, e.g., according to any methods of detection known in the art and / or described herein.

[0117] In some embodiments of the methods provided herein, nucleic acid molecules (e.g., corresponding to one or more genes, such as ESRI) 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 ESRI). 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 andabout 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 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 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 nucleicacid 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.

[0118] 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 acid molecule 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.

[0119] 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.

[0120] 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 150nucleotides 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.

[0121] 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.

[0122] 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 / or described herein.

[0123] 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.

[0124] 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 oligonucleotidespecifically hybridizes to a nucleic acid molecule comprising the target nucleotide sequence under conditions that allow a polymerization reaction (e.g., PCR) to occur.

[0125] 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 to hybridize 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.

[0126] 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.

[0127] 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 providedherein 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 some embodiments, 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.

[0128] In one aspect, provided herein is a primer or primer set for amplifying a nucleic acid molecule comprising a cytogenetic abnormality that is or results in an ESRI mutation. The cytogenetic abnormality may be any cytogenetic abnormality that is or results in an ESRI mutation. Examples of such cytogenetic abnormalities include, without limitation, deletions (e.g., deletions of entire chromosomes or deletions of fragments of one or more chromosomes), duplications (e.g., of entire chromosomes, or of regions smaller than an entire chromosome), translocations (e.g., non-reciprocal translocations, balanced translocations, reciprocal translocations), intra-chromosomal inversions, rearrangements e.g., gene fusions), point mutations, insertions, deletions, gene copy number changes, germ-line mutations, and gene expression level changes.Samples

[0129] A variety of materials can be the source of, or serve as, samples for use in any of the methods of the disclosure, such as any of the methods for detection of ESRI mutations. In some embodiments, any of the methods of the present disclosure further comprise obtaining one or more samples of the present disclosure from an individual. In some embodiments, asample of the present disclosure is obtained or derived from a cancer or tumor. In some embodiments, the individual is a human, e.g., with breast cancer.

[0130] For example, the sample can be, or be derived from: solid tissue such as from a fresh, frozen and / or preserved organ, tissue sample, biopsy (e.g., tumor, tissue or liquid biopsy), resection, smear, or aspirate; scrapings; bone marrow or bone marrow specimens; a bone marrow aspirate; blood or any blood constituents; blood cells; bodily fluids such as cerebrospinal fluid, amniotic fluid, urine, saliva, sputum, peritoneal fluid or interstitial fluid; pleural fluid; ascites; tissue or fine needle biopsy samples; surgical specimens; cellcontaining body fluids; free-floating nucleic acids; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as ductal lavages or bronchoalveolar lavages; cells from any time in gestation or development of an individual; cells from a cancer or tumor; other body fluids, secretions, and / or excretions, and / or cells therefrom. In some embodiments, a sample is or comprises cells obtained from an individual. In some embodiments, the sample is or is derived from blood or blood constituents, e.g., obtained from a liquid biopsy. In some embodiments, the sample is or is derived from a tumor sample. In some embodiments, the sample is or comprises biological tissue or fluid. In some embodiments, the sample can contain compounds that are not naturally intermixed with the source of the sample in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. In some embodiments, the sample is preserved as a frozen sample or as a formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation. In some embodiments, the sample comprises circulating tumor cells (CTCs).

[0131] In one embodiment, the sample comprises one or more cells associated with a tumor, e.g., tumor cells or tumor-infiltrating lymphocytes (TIL). In one embodiment, the sample includes one or more premalignant or malignant cells. In one embodiment, the sample is acquired from a hematologic malignancy (or pre-malignancy), e.g., a hematologic malignancy (or pre-malignancy) described herein. In one embodiment, the sample is acquired from a cancer, such as a cancer described herein. In some embodiments, the sample is acquired from a solid tumor, a soft tissue tumor or a metastatic lesion. In other embodiments, the sample includes tissue or cells from a surgical margin. In one embodiment, the sample is or is acquired from a liquid biopsy of blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample includes cell-free DNA (cfDNA) and / or circulating tumor DNA (ctDNA), e.g., from a biopsy of blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In another embodiment, the sample includes one or more circulating tumor cells (CTCs) e.g., a CTC acquired from a bloodsample). In one embodiment, the sample is a cell not associated with a tumor or cancer, e.g., a non-tumor or non-cancer cell or a peripheral blood lymphocyte.

[0132] In some embodiments, a sample is a primary sample obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by a method chosen from biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, or collection of body fluid (e.g., blood, lymph, or feces). In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. Such a processed sample may comprise, for example, nucleic acids (e.g., for use in any of the methods for detection of ESRI mutations) or proteins (e.g., for use in any of the methods for detection of ESRI mutations) extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification methods, reverse transcription of mRNA, or isolation and / or purification of certain components such as nucleic acids and / or proteins.

[0133] In some embodiments, the sample comprises nucleic acids, e.g., genomic DNA, cDNA, or mRNA. In some embodiments, the sample comprises cell-free DNA (cfDNA). In some embodiments, the sample comprises cell-free RNA (cfRNA). In some embodiments, the sample comprises circulating tumor DNA (ctDNA). In certain embodiments, the nucleic acids are purified or isolated (e.g., removed from their natural state). In some embodiments, the sample comprises tumor or cancer nucleic acids, such as nucleic acids from a tumor or cancer sample, e.g., genomic DNA, RNA, or cDNA derived from RNA, or from a liquid biopsy, e.g., ctDNA from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In certain embodiments, a tumor or cancer nucleic acid sample, or a ctDNA sample, is purified or isolated (e.g., it is removed from its natural state).

[0134] In some embodiments, the sample comprises tumor or cancer proteins or polypeptides, such as proteins or polypeptides from a tumor or a cancer sample, or from a liquid biopsy, e.g., from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In certain embodiments, the proteins or polypeptides are purified or isolated (e.g., removed from their natural state).

[0135] 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 acancer (e.g., a cancer in an individual). In some embodiments, the one or more samples comprise at least 20% tumor cell nuclear area.

[0136] In some embodiments, the sample is a control sample or a reference sample, e.g., not containing an ESRI mutation. In certain embodiments, the reference sample is purified or isolated e.g., it is removed from its natural state). In certain embodiments, the control or reference sample is from a non-tumor or cancer sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different individual.

[0137] In some embodiments, an ESRI mutation IS detected in a sample comprising genomic or subgenomic DNA fragments, or RNA (e.g., mRNA), isolated from a sample, e.g., a tumor or cancer sample, a normal adjacent tissue (NAT) sample, a tissue sample, or a blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva sample obtained from an individual. In some embodiments, the sample comprises cDNA derived from an mRNA sample or from a sample comprising mRNA. In some embodiments, an ESRI mutation is detected in a sample comprising cell-free DNA (cfDNA), cell-free RNA, and / or circulating tumor DNA (ctDNA). In some embodiments, an ESRI mutation is detected in a sample comprising cell-free DNA (cfDNA) and / or circulating tumor DNA (ctDNA). In some embodiments, an ESRI mutation is detected in a sample comprising circulating tumor DNA (ctDNA).

[0138] In some embodiments, any of the methods of the present disclosure comprise acquiring knowledge of or detecting any of the biomarkers described herein (e.g., 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 treatments

[0139] 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; or identifying a candidate treatment for a cancer in an individual in need thereof.

[0140] In some embodiments of any of the methods provided herein, the methods comprise acquiring knowledge of or detecting in one or more samples from an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, an ESRI mutation. In other embodiments, the methods comprise acquiring knowledge of or detecting in one or more samples from an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, an ESRI mutation.

[0141] In some embodiments of any of the methods provided herein, detection of an ESRI mutation or presence thereof in one or more samples from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) identifies the individual as one who may benefit from a first-line treatment comprising a selective estrogen receptor degrader (SERD) and a cyclin-dependent kinase inhibitor (CDKi). In some embodiments, responsive to acquiring knowledge of an ESRI mutation or presence thereof in a sample from the individual, the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi. In some embodiments, one or more first-line treatment options are identified for an individual based at least in part on the detection of an ESRI mutation or presence thereof in a sample (e.g., from the individual), wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi. In some embodiments, responsive to knowledge of an ESRI mutation or presence thereof in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi. In some embodiments, responsive to detection of an ESRI mutation or presence thereof in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi. In some embodiments, the methods comprise performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies an ESRI mutation or presence thereof in the sample; and selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises a SERD and a CDKi.

[0142] In some embodiments, responsive to acquiring knowledge of or detecting an ESRI mutation or presence thereof in a sample from the individual, the individual is predicted to have longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to survival of the individual when treated with a first-line treatment comprisingan Al and a CDKi. In some embodiments, responsive to acquiring knowledge of or detecting an ESRI mutation or presence thereof in a sample from the individual, the individual is predicted to have an improved response to a first- line treatment comprising a SERD and a CDKi and / or longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to response and / or survival when treated with a first-line treatment comprising an Al and a CDKi. In some embodiments, the survival comprises progression- free survival (PFS) and / or overall survival (OS). In some embodiments, an improved response comprises longer time-to-treatment discontinuation (TTD) and / or longer time-to- next treatment (TTNT).

[0143] In some embodiments of any of the methods provided herein, detection of the absence of an ESRI mutation in one or more samples from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) identifies the individual as one who may benefit from a first-line treatment comprising an aromatase inhibitor (Al) and a CDKi. In some embodiments, responsive to acquiring knowledge of the absence of an ESRI mutation in a sample from the individual, the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi. In some embodiments, one or more first-line treatment options are identified for an individual based at least in part on the detection of the absence of an ESRI mutation in a sample (e.g., from the individual), wherein the one or more first-line treatment options comprise first-line treatment with an Al and a CDKi. In some embodiments, responsive to knowledge of the absence of an ESRI mutation in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi. In some embodiments, responsive to detection of the absence of an ESRI mutation in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi. In some embodiments, the methods comprise performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies the absence of an ESRI mutation in the sample; and selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises an Al and a CDKi.

[0144] In some embodiments, responsive to acquiring knowledge of or detecting the absence of an ESRI mutation in a sample from the individual, the individual is predicted to havelonger survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to survival when treated with a first-line treatment comprising an Al and a CDKi of an individual whose sample comprises an ESRI mutation. In some embodiments, responsive to acquiring knowledge of or detecting the absence of an ESRI mutation in a sample from the individual, the individual is predicted to have an improved response to a first-line treatment comprising an Al and a CDKi and / or longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to response and / or survival of an individual whose sample comprises an ESRI mutation. In some embodiments, the survival comprises progression-free survival (PFS) and / or overall survival (OS). In some embodiments, an improved response comprises longer time-to-treatment discontinuation (TTD) and / or longer time-to-next treatment (TTNT).

[0145] In some embodiments according to any of the embodiments described herein, the cancer is HR+HER2- breast cancer. In some embodiments, the cancer is advanced or metastatic HR+HER2- breast cancer. In some embodiments, the treatment or treatment option is a first-line metastatic treatment or treatment option.

[0146] In some embodiments according to any of the embodiments described herein, the SERD is fulvestrant (FASLODEX®). Other SERDs known in the art include, without limitation, 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).

[0147] In some embodiments according to any of the embodiments described herein, the Al is aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), or 4- Androstene-3, 6, 17-trione (“6-OXO”). In some embodiments, the Al is anastrozole, letrozole, or exemestane.

[0148] In some embodiments according to any of the embodiments described herein, the CDKi is a CDK4 / CDK6 inhibitor. Exemplary CDK4 / CDK6 inhibitors include, without limitation, palbociclib, ribociclib, abemaciclib, or dalpiciclib. In some embodiments, the CDKi is a CDK4-selective inhibitor. Exemplary CDK4-selective inhibitors include, without limitation, PF-07220060 or 2-94. In some embodiments, the CDKi is a CD K2- selective inhibitor. Exemplary CDK2-selective inhibitors include, without limitation, tagtociclib (PF- 07104091).Reporting

[0149] In some embodiments, the methods provided herein comprise generating a report, and / or providing a report to party.

[0150] In some embodiments, a report according to the present disclosure comprises information about one or more of: an ESRI mutation; a cancer of the disclosure, e.g., comprising an ESRI mutation; 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., a SERD and a CDKi, or an Al and a CDKi).

[0151] In some embodiments, a report according to the present disclosure comprises information about the presence or absence of an ESRI mutation in one or more samples 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 an ESRI mutation is present in one or more samples obtained from the individual. In one embodiment, a report according to the present disclosure indicates that an ESRI mutation is not present in one or more samples obtained from the individual. In one embodiment, a report according to the present disclosure indicates that an ESRI mutation has been detected in one or more samples obtained from the individual. In one embodiment, a report according to the present disclosure indicates that an ESRI mutation has not been detected in one or more samples obtained from the individual. In some embodiments, the report comprises an identifier for the individual from which the sample(s) was obtained.

[0152] In some embodiments, the report includes information on the role of an ESRI mutation 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 an ESRI mutation; information on resistance of a cancer, such as a cancer provided herein (e.g., HR+HER2- breast cancer, such as HR+HER2- MBC) to one or more treatments; information on potential or suggested therapeutic options (e.g., such as a SERD and a CDKi, or an Al and a CDKi, as described 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 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 an ESRI mutation) and identified in the report. In some embodiments, the report includes information or a recommendation on the administration of a treatment (e.g., a SERDand a CD Ki, or an Al and a CD Ki). 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.

[0153] 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: obtaining one or more samples, such as sample(s) described herein, from an individual, e.g., an individual having a cancer, such as a cancer provided herein; detecting an ESRI mutation in the sample(s), or acquiring knowledge of the presence of an ESRI mutation in the sample(s); 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 an ESRI mutation in the sample(s); an identifier for the individual from which the sample(s) was obtained; information on the role of the ESRI mutation, 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 (such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein) to the individual; a recommendation or information on the administration of a treatment (such as an anti-cancer 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 (such as a SERD and a CDKi, or an Al and a CDKi), e.g., in combination with other treatments (e.g., a second therapeutic agent). In some embodiments, the report generated is a personalized cancer report.

[0154] 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 having, suspected of having, or being tested for a cancer, such as a cancer provided herein, e.g., comprising an ESRI mutation), or to an individual or entity other than the individual or patient, 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 ofabout 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 the individual. 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 an ESRI mutation in one or more samples obtained from the individual. 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 an ESRI mutation in one or more samples obtained from the individual. 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

[0155] In some other aspects, provided herein are non-transitory computer-readable storage media. In some embodiments, the non-transitory computer-readable storage media comprise one 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.

[0156] FIG. 8B illustrates an example of a computing device or system in accordance with one embodiment. Device 800 can be a host computer connected to a network. Device 800 can be a client computer or a server. As shown in FIG. 8B, device 800 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) 810, input devices 820, output devices 830, memory or storage devices 840, communication devices 860, and nucleic acid sequencers 870. Software 850 residing in memory or storage device 840 may comprise, e.g., an operating system as well as software for executing the methods described herein, e.g., for detecting an ESRI mutation. Input device 820 and output device 830 can generally correspond to those described herein, and can either be connectable or integrated with the computer.

[0157] Input device 820 can be any suitable device that provides input, such as a touch screen, keyboard or keypad, mouse, or voice-recognition device. Output device 830 can be any suitable device that provides output, such as a touch screen, haptics device, or speaker.

[0158] Storage 840 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 860 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 880, Ethernet connection, or any other wire transfer technology) or wirelessly (e.g., Bluetooth®, Wi-Fi®, or any other wireless technology).

[0159] Software module 850, which can be stored as executable instructions in storage 840 and executed by processor(s) 810, 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 detecting an ESRI mutation (e.g., as embodied in the devices as described herein).

[0160] Software module 850 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 instructions associated 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 840, 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.

[0161] Software module 850 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.

[0162] Device 800 may be connected to a network (e.g., network 904, as shown in FIG. 9 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.

[0163] Device 800 can be implemented using any operating system, e.g., an operating system suitable for operating on the network. Software module 850 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) 810.

[0164] Device 800 can further include a sequencer 870, 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.

[0165] FIG. 9 illustrates an example of a computing system in accordance with one embodiment. In computing system 900, device 800 (e.g., as described above and illustrated in FIG. 8B) is connected to network 904, which is also connected to device 906. In some embodiments, device 906 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.

[0166] Devices 800 and 906 may communicate, e.g., using suitable communication interfaces via network 904, such as a Local Area Network (LAN), Virtual Private Network (VPN), or the Internet. In some embodiments, network 904 can be, for example, the Internet, an intranet, a virtual private network, a cloud network, a wired network, or a wireless network. Devices 800 and 906 may communicate, in part or in whole, via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. Additionally, devices 800 and 906 may communicate, e.g., using suitable communication interfaces, via a second network, such as a mobile / cellular network. Communication between devices 800 and 906 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 800 and 906 can communicate directly (instead of, or in addition to, communicating via network 904), e.g., via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. In some embodiments, devices 800 and 906 communicate via communications 1008, which can be a direct connection or can occur via a network (e.g., network 1004).

[0167] One or all of devices 800 and 906 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 904 according to various examples described herein.

[0168] FIG. 10 illustrates an exemplary process 1000 for detecting an ESRI mutation in one or more samples, in accordance with some embodiments of the present disclosure. Process 1000 is performed, for example, using one or more electronic devices implementing a software program. In some examples, process 1000 is performed using a client-server system, and the blocks of process 1000 are divided up in any manner between the server and a client device. In other examples, the blocks of process 1000 are divided up between the server and multiple client devices. Thus, while portions of process 1000 are described herein as being performed by particular devices of a client-server system, it will be appreciated that process 1000 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 1000 is performed using only a client device or only multiple client devices. In process 1000, 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 1000. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature and, as such, should not be viewed as limiting.

[0169] At block 1002, 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 one or more samples 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 one or more samples. In other embodiments, the one or more nucleic acid molecules are obtained indirectly from the one or more samples, 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 one or more samples are 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 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, or any combination thereof, or fragments thereof), and / or subjected to PCR amplification.

[0170] At block 1004, an exemplary system (e.g., one or more electronic devices) analyzes the plurality of sequence reads for the presence of an ESRI mutation. At block 1006, the system detects (e.g., based on the analysis) an ESRI mutation, in the one or more samples. 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). In some embodiments, the presence of an ESRI mutation is detected based on analysis of sequence reads obtained from the sample from the individual. In some embodiments, the absence of an ESRI mutation is detected based on analysis of sequence reads obtained from the sample from the individual.

[0171] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, detection of an ESRI mutation or presence thereof in one or more samples from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) identifies the individual as one who may benefit from a first-line treatment comprising a selective estrogen receptor degrader (SERD) and a cyclin-dependent kinaseinhibitor (CDKi). In some embodiments, responsive to acquiring knowledge of an ESRI mutation or presence thereof in a sample from the individual, the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi. In some embodiments, one or more first-line treatment options are identified for an individual based at least in part on the detection of an ESRI mutation or presence thereof in a sample (e.g., from the individual), wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi. In some embodiments, responsive to knowledge of an ESRI mutation or presence thereof in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi. In some embodiments, responsive to detection of an ESRI mutation or presence thereof in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi. In some embodiments, the methods comprise performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies an ESRI mutation or presence thereof in the sample; and selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises a SERD and a CDKi.

[0172] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, responsive to acquiring knowledge of or detecting an ESRI mutation or presence thereof in a sample from the individual, the individual is predicted to have longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to survival of the individual when treated with a first-line treatment comprising an Al and a CDKi. In some embodiments, responsive to acquiring knowledge of or detecting an ESRI mutation or presence thereof in a sample from the individual, the individual is predicted to have an improved response to a first-line treatment comprising a SERD and a CDKi and / or longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to response and / or survival when treated with a first-line treatment comprising an Al and a CDKi. In some embodiments, the survival comprises progression-free survival (PFS) and / or overall survival (OS). In some embodiments, an improved response comprises longer time-to-treatment discontinuation (TTD) and / or longer time-to-next treatment (TTNT).

[0173] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, detection of the absence of an ESRI mutation in one or more samples from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) identifies the individual as one who may benefit from a first-line treatment comprising an aromatase inhibitor (Al) and a CDKi. In some embodiments, responsive to acquiring knowledge of the absence of an ESRI mutation in a sample from the individual, the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi. In some embodiments, one or more first-line treatment options are identified for an individual based at least in part on the detection of the absence of an ESRI mutation in a sample (e.g., from the individual), wherein the one or more first-line treatment options comprise first-line treatment with an Al and a CDKi. In some embodiments, responsive to knowledge of the absence of an ESRI mutation in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi. In some embodiments, responsive to detection of the absence of an ESRI mutation in a sample from an individual, the methods comprise administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi. In some embodiments, the methods comprise performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies the absence of an ESRI mutation in the sample; and selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises an Al and a CDKi.

[0174] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, responsive to acquiring knowledge of or detecting the absence of an ESRI mutation in a sample from the individual, the individual is predicted to have longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to survival when treated with a first-line treatment comprising an Al and a CDKi of an individual whose sample comprises an ESRI mutation. In some embodiments, responsive to acquiring knowledge of or detecting the absence of an ESRI mutation in a sample from the individual, the individual is predicted to have an improved response to a first-line treatment comprising an Al and a CDKi and / or longer survival when treated with a first-line treatment comprising an Al and a CDKi, ascompared to response and / or survival of an individual whose sample comprises an ESRI mutation. In some embodiments, the survival comprises progression-free survival (PFS) and / or overall survival (OS). In some embodiments, an improved response comprises longer time-to-treatment discontinuation (TTD) and / or longer time-to-next treatment (TTNT).

[0175] 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., tissue and / or liquid biopsies, etc. In some embodiments, the sample is obtained from the cancer. In some embodiments, the sample comprises a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some embodiments, the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell. 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 sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0176] 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).

[0177] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the cancer is HR+HER2- breast cancer. In some embodiments, the cancer is advanced or metastatic HR+HER2- breast cancer. In some embodiments, the treatment or treatment option is a first-line metastatic treatment or treatment option.

[0178] 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 determining the presence or absence of an ESRI mutation may be implemented as part of a genomic profiling process that comprises identification of the presence of variant sequencesat 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 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 ESRI mutation 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.

[0179] 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 detecting the presence or absence of an ESRI mutation. In some instances, the 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. 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 or absence of mutations in one ormore 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, e.g., as discussed herein.

[0180] 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 report comprises / indicates / comprises information on the presence or absence of an ESRI mutation in the cancer in an individual (e.g., in one or more samples 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 presence of an ESRI mutation in the cancer in an individual (e.g., in one or more samples 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 asample, e.g., as described above. 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.

[0181] 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 actually added 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

[0182] The following exemplary embodiments are representative of some aspects of the invention:Embodiment 1. A method of selecting a first-line treatment for an individual having hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer, the method comprising detecting an estrogen receptor 1 (ESRI) mutation in a sample from the individual, wherein detection of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a selective estrogen receptor degrader (SERD) and a cyclin-dependent kinase inhibitor (CDKi).Embodiment 2. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, the method comprising detecting absence of an ESRI mutation in a sample from the individual, wherein detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an aromatase inhibitor (Al) and a CDKi.Embodiment 3. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, the method comprising detecting presence or absence of an ESRI mutation in a sample from the individual, wherein detection of the presence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi, or wherein detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.Embodiment 4. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi.Embodiment 5. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.Embodiment 6. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of knowledge of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the acquisition of knowledge of the absence of anESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first- line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.Embodiment 7. A method of identifying one or more first- line treatment options for an individual having HR+HER2- breast cancer, the method comprising:(a) detecting an ESRI mutation in a sample from the individual; and(b) generating a report comprising one or more first-line treatment options identified for the individual based at least in part on the detection of an ESRI mutation in the sample, wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi.Embodiment 8. A method of identifying one or more first- line treatment options for an individual having HR+HER2- breast cancer, the method comprising:(a) detecting absence of an ESRI mutation in a sample from the individual; and(b) generating a report comprising one or more first-line treatment options identified for the individual based at least in part on the detection of the absence of an ESRI mutation in the sample, wherein the one or more first-line treatment options comprise first- line treatment with an Al and a CDKi.Embodiment 9. A method of identifying one or more first- line treatment options for an individual having HR+HER2- breast cancer, the method comprising:(a) detecting presence or absence of an ESRI mutation in a sample from the individual; and(b) generating a report comprising one or more first-line treatment options identified for the individual, wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi based at least in part on the detection of the presence an ESRI mutation in the sample, or wherein the one or more first-line treatment options comprise first-line treatment with an Al and a CDKi based at least in part on the detection of the absence of an ESRI mutation in the sample.Embodiment 10. The method of any one of embodiments 7-9, wherein the report further indicates the presence or absence of the ESRI mutation in the sample.Embodiment 11. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) acquiring knowledge of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) responsive to said knowledge, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi.Embodiment 12. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) acquiring knowledge of absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) responsive to said knowledge, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.Embodiment 13. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(bl) responsive to knowledge of the presence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi; or(b2) responsive to knowledge of the absence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.Embodiment 14. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) detecting an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi.Embodiment 15. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) detecting absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.Embodiment 16. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) detecting presence or absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(bl) responsive to detecting the presence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi; or(b2) responsive to detecting the absence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.Embodiment 17. A method of identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies an ESRI mutation in the sample; and(b) selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises a SERD and a CDKi.Embodiment 18. A method of identifying a candidate first-line treatment forHR+HER2- breast cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies absence of an ESRI mutation in the sample; and(b) selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises an Al and a CDKi.Embodiment 19. A method of identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies presence or absence of an ESRI mutation in the sample; and(b) selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises treatment with a SERD and a CDKi when the sequencing mutation profile identifies the presence of an ESRI mutation in the sample, or wherein the first-line treatment comprises treatment with an Al and a CDKi when the sequencing mutation profile identifies the absence of an ESRI mutation in the sample.Embodiment 20. The method of any one of embodiments 17-19, 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.Embodiment 21. The method of embodiment 20, wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS).Embodiment 22. A method of predicting survival of an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a first-line treatment comprising a SERDand a CDKi, as compared to survival of the individual when treated with a first-line treatment comprising an Al and a CDKi.Embodiment 23. A method of predicting survival of an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to survival when treated with a first-line treatment comprising an Al and a CDKi of an individual whose sample comprises an ESRI mutation.Embodiment 24. A method of screening an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a first- line treatment comprising a SERD and a CDKi and / or longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to response and / or survival when treated with a first-line treatment comprising an Al and a CDKi.Embodiment 25. A method of screening an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a first- line treatment comprising an Al and a CDKi and / or longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to response and / or survival of an individual whose sample comprises an ESRI mutation.Embodiment 26. The method of any one of embodiments 22-25, wherein the survival comprises progression-free survival (PFS).Embodiment 27. The method of any one of embodiments 22-26, wherein the survival comprises overall survival (OS).Embodiment 28. The method of any one of embodiments 22-27, wherein the improved response comprises longer time-to-treatment discontinuation (TTD).Embodiment 29. The method of any one of embodiments 22-28, wherein the improved response comprises longer time-to-next treatment (TTNT).Embodiment 30. The method of any one of embodiments 1, 3, 4, 6, 7, 9-11, 13, 14, 16, 17, 19-22, 24, and 26-29, wherein the SERD is fulvestrant.Embodiment 31. The method of any one of embodiments 2, 3, 5, 6, 8-10, 12, 13, 15, 16, and 18-29, wherein the Al is aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1,4,6-androstatrien- 3, 17-dione (ATD), or 4- Androstene-3, 6, 17-trione (“6-OXO”).Embodiment 32. The method of any one of embodiments 1-31, wherein the CD Ki is a CDK4 / CDK6 inhibitor.Embodiment 33. The method of embodiment 32, wherein the CDKi is palbociclib, ribociclib, abemaciclib, or dalpiciclib.Embodiment 34. The method of any one of embodiments 1-31, wherein the CDKi is a CD K4- selective inhibitor.Embodiment 35. The method of embodiment 34, wherein the CDKi is PF-07220060 or2-94.Embodiment 36. The method of any one of embodiments 1-31, wherein the CDKi is aCDK2 inhibitor.Embodiment 37. The method of embodiment 34, wherein the CDKi is tagtociclib (PF-07104091).Embodiment 38. The method of any one of embodiments 1-37, wherein the ESRI mutation comprises a base substitution or short insertion / deletion.Embodiment 39. The method of embodiment 38, wherein the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1.Embodiment 40. The method of embodiment 38, wherein the ESRI mutation results in aV422 deletion, relative to SEQ ID NO:1.Embodiment 41. The method of any one of embodiments 1-37, wherein the ESRI mutation comprises a rearrangement or gene fusion.Embodiment 42. The method of any one of embodiments 2, 3, 5, 6, 8-10, 12, 13, 15, 16, and 18-41, wherein detecting the absence of an ESRI mutation comprises detecting a wildtype ESRI gene or portion thereof.Embodiment 43. The method of any one of embodiments 1-42, wherein the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.Embodiment 44. The method of embodiment 43, wherein the treatment or treatment option is a first-line metastatic treatment or treatment option.Embodiment 45. The method of any one of embodiments 1-44, further comprising obtaining the sample from the individual.Embodiment 46. The method of any one of embodiments 1-45, wherein the sample is obtained or derived from the HR+HER2- breast cancer.Embodiment 47. The method of any one of embodiments 1-46, wherein the sample comprises a tissue biopsy sample.Embodiment 48. The method of any one of embodiments 1-46, wherein the sample comprises a liquid biopsy sample.Embodiment 49. The method of embodiment 48, wherein the sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.Embodiment 50. The method of embodiment 48 or embodiment 49, wherein the sample comprises circulating tumor cells (CTCs).Embodiment 51. The method of any one of embodiments 48-50, wherein the sample comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.Embodiment 52. The method of any one of embodiments 1-46, wherein the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell.Embodiment 53. The method of any one of embodiments 1-46, wherein the sample comprises cells and / or nucleic acids from the HR+HER2- breast cancer.Embodiment 54. The method of embodiment 53, wherein the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the HR+HER2- breast cancer.Embodiment 55. The method of any one of embodiments 1-54, wherein the ESRI mutation or absence thereof is detected by immunohistochemistry analysis (IHC), comprehensive genomic profiling (CGP), comparative genomic hybridization (CGH), sequencing, or any combination thereof.Embodiment 56. The method of embodiment 55, 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.Embodiment 57. The method of embodiment 56, wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS).Embodiment 58. The method of any one of embodiments 1-57, wherein detecting the ESRI mutation or absence thereof comprises:(a) providing a plurality of nucleic acid molecules obtained from the sample from the individual, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an ESRI gene, or a portion thereof;(b) optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules;(c) optionally, amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules;(d) optionally, 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 sequence reads of the plurality of sequence reads correspond to an ESRI gene, or a portion thereof;(f) analyzing the plurality of sequence reads for the presence or absence of an ESRI mutation; and(g) based on the analyzing step, detecting the presence or absence of an ESRI mutation in the sample.Embodiment 59. The method of embodiment 58, wherein the sequencer comprises a next-generation sequencer.Embodiment 60. The method of any one of embodiments 1-57, wherein detecting the ESRI mutation or absence thereof comprises:(a) providing the sample from the individual, 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 or a 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 the presence or absence of an ESRI mutation; and(g) detecting, based on the analyzing step, the presence or absence of an ESRI alteration in the sample from the individual.Embodiment 61. The method of any one of embodiments 58-60, 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 62. The method of embodiment 60, 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 an ESRI gene or a portion thereof and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.Embodiment 63. The method of any one of embodiments 58-62, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.Embodiment 64. The method of any one of embodiments 58-63, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non- PCR amplification technique, or an isothermal amplification technique.Embodiment 65. The method of any one of embodiments 1-64, further comprising selectively enriching for one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof; wherein the selectively enriching produces an enriched sample.Embodiment 66. The method of embodiment 65, wherein the selectively enriching comprises: (a) combining one or more bait molecules with the sample, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.Embodiment 67. The method of any one of embodiments 62-66, wherein the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to an ESRI gene or a portion thereof.Embodiment 68. The method of embodiment 67, wherein the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides.Embodiment 69. The method of any one of embodiments 62-68, wherein the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent.Embodiment 70. The method of embodiment 69, wherein the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.Embodiment 71. The method of any one of embodiments 67-70, wherein the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.Embodiment 72. The method of embodiment 71, wherein the selectively enriching comprises amplifying the one or more nucleic acid molecules comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof using a polymerase chain reaction (PCR) to produce an enriched sample.Embodiment 73. The method of any one of embodiments 65-72, further comprising sequencing the enriched sample.Embodiment 74. The method of any one of embodiments 58-73, wherein the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules.Embodiment 75. The method of embodiment 74, wherein the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample.Embodiment 76. The method of embodiment 74, wherein the sample comprises a liquid biopsy sample, and wherein the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample, and the non-cancer nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample.Embodiment 77. The method of any one of embodiments 1-6 and 11-76, further comprising generating a report, wherein the report:(a) indicates the presence or absence of the ESRI mutation in the sample from the individual; and / or(bl) indicates a first-line treatment or one or more first- line treatment options identified or selected for the individual based, at least in part, on the presence of an ESRI mutation in the sample from the individual, wherein the first-line treatment or the one or more first-line treatment options comprise a SERD and a CDKi, or(b2) indicates a first-line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the absence of an ESRI mutation in the sample from the individual, wherein the first-line treatment or the one or more first-line treatment options comprise an Al and a CDKi.Embodiment 78. The method of any one of embodiments 1-77, further comprising generating a molecular profile for the individual, based, at least in part, on detecting or acquiring knowledge of the presence or absence of the ESRI mutation.Embodiment 79. The method of embodiment 78, 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.Embodiment 80. The method of embodiment 78 or embodiment 79, wherein the molecular profile for the individual further comprises results from a nucleic acid sequencingbased test.Embodiment 81. The method of any one of embodiments 78-80, further comprising generating a report, wherein the report comprises the molecular profile for the individual.Embodiment 82. The method of embodiment 81, wherein the report further comprises information on a first-line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the first-line treatment or one or more first-line treatment options comprise a SERD and a CDKi when the molecular profile indicates the presence of an ESRI mutation in the sample from the individual, or wherein the first-line treatment or one or more first-linetreatment options comprise an Al and a CDKi when the molecular profile indicates the absence of an ESRI mutation in the sample from the individual.Embodiment 83. The method of any one of embodiments 7-10, 81, and 82, 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.Embodiment 84. The method of any one of embodiments 1-83, wherein the individual is a human.Embodiment 85. A system for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising a SERD and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyze the plurality of sequence reads for presence of an ESRI mutation; and(c) detect, based on the analyzing, presence of the ESRI mutation in the sample; wherein detecting the presence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi.Embodiment 86. A system for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising an Al and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyze the plurality of sequence reads for absence of an ESRI mutation; and(c) detect, based on the analyzing, absence of the ESRI mutation in the sample; wherein detecting the absence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.Embodiment 87. A system for selecting a first-line treatment for an individual having HR+HER2- breast cancer, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyze the plurality of sequence reads for presence or absence of an ESRI mutation; and(c) detect, based on the analyzing, presence of the ESRI mutation in the sample;wherein responsive to the detection of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the detection of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.Embodiment 88. The system of any one of embodiments 85-87, wherein the ESRI mutation comprises a base substitution or short insertion / deletion.Embodiment 89. The system of embodiment 88, wherein the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1.Embodiment 90. The system of embodiment 88, wherein the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1.Embodiment 91. The system of any one of embodiments 85-87, wherein the ESRI mutation comprises a rearrangement or gene fusion.Embodiment 92. The system of any one of embodiments 86-91, wherein detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof.Embodiment 93. The system of any one of embodiments 85-92, wherein the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.Embodiment 94. The system of embodiment 93, wherein the treatment is a first-line metastatic treatment.Embodiment 95. The system of any one of embodiments 86-94, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or next-generation sequencing.Embodiment 96. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method foridentifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising a SERD and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for presence of an ESRI mutation; and(c) detecting, using the one or more processors and based on the analyzing, the presence of the ESRI mutation in the sample; wherein detecting the presence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi.Embodiment 97. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising an Al and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for absence of an ESRI mutation; and(c) detecting, using the one or more processors and based on the analyzing, the absence of the ESRI mutation in the sample; wherein detecting the absence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.Embodiment 98. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method forselecting a first-line treatment for an individual having HR+HER2- breast 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for presence or absence of an ESRI mutation; and(c) detecting, using the one or more processors and based on the analyzing, the presence or absence of the ESRI mutation in the sample; wherein responsive to the detection of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the detection of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.Embodiment 99. The non-transitory computer readable storage medium of any one of embodiments 96-98, wherein the ESRI mutation comprises a base substitution or short insertion / deletion.Embodiment 100. The non-transitory computer readable storage medium of embodiment99, wherein the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQID NO:1.Embodiment 101. The non-transitory computer readable storage medium of embodiment100, wherein the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1.Embodiment 102. The non-transitory computer readable storage medium of any one of embodiments 96-98, wherein the ESRI mutation comprises a rearrangement or gene fusion.Embodiment 103. The non-transitory computer readable storage medium of any one of embodiments 97-102, wherein detecting the absence of an ESRI mutation comprises detecting a wild- type ESRI gene or portion thereof.Embodiment 104. The non-transitory computer readable storage medium of any one of embodiments 96-103, wherein the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.Embodiment 105. The non-transitory computer readable storage medium of embodiment 104, wherein the treatment is a first- line metastatic treatment.Embodiment 106. The non-transitory computer readable storage medium of any one of embodiments 96-105, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or nextgeneration sequencing.

[0183] The method steps of the invention(s) 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 actually added 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.

[0184] The terminology used in the description of the various described embodiments herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the description of the various described embodiments and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term“and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “includes,” “including,” “comprises,” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0185] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. To the extent that any reference incorporated by reference conflicts with the instant disclosure, the instant disclosure shall control.EXAMPLES

[0186] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.Example 1: Comprehensive genomic profiling (CGP) of ESRI mutations (ESRlmut) in H R(+)H ER2(-)metastatic breast cancer (MBC): prevalence along treatment course and predictive value for endocrine therapy (ET) resistance in real-world practiceBackground

[0187] Endocrine therapy (ET) in combination with CDK4 / 6 inhibitors (CKDi) is the first- line standard of care (SOC) for patients with metastatic breast cancer (mBC) hormone receptor (HR) positive (+) and HER2 negative (-). An aromatase inhibitor (Al) is usually the first ET option for de novo or ET-naive mBC, followed by a selective estrogen receptor degrader (SERD) upon clinical radiological disease progression. Although most HR(+)HER2(-) mBC benefit from first-line ET, second-line ET monotherapy has limited benefit, possibly due to acquired resistance mechanisms [1].

[0188] Mutations in ESRI ESRlmut) have been identified as the main acquired resistance mechanism to ET [2], and have been reported in approximately 20-40% of patients pretreated with Al depending on the duration and setting of ET [3]. ESRI codes for the estrogen receptor (ER) alpha, and ES7? / mut usually result in a constitutively activated ER and is thus unaffected by Al depletion of estrogen [1]. ASCO guidelines were updated recently to recommend ES7? / mut tissue or liquid testing at recurrence or progression on ET [4] after subgroup analysis in EMERALD trial indicated that elacestrant have superior outcomes over SOC only for patients with ESRlmut detected by ctDNA [5].

[0189] Several completed and ongoing clinical trials attempt to investigate the role of acquired ESRlmut in patients with HR(+)HER2(-) mBC receiving ET treatment and its implications for optimizing first and subsequent lines of therapy. For instance, in the PADA- 1 trial, patients receiving Al + CD Ki were screened every two months for ESRlmut, and those with an ESRlmut detected and switched to another ET backbone before clinical radiological disease progression had better outcomes than those who only shifted treatment upon clinical progression [1].

[0190] The role of intrinsic ESRlmut in de novo or recurrent HR(+)HER2(-) mBC is much less explored. Previous studies have shown that ESRlmut prevalence is only 1.5-7% in recurrent BC after prior adjuvant or neoadjuvant Al, and less than 1% in ET-naive mBC [3] and the clinical utility of detected ESRlmut before first-line mBC treatment is not well- defined.

[0191] ESRlmut prevalence is known to depend on prior duration of Al therapy, but the prevalence of ESRlmut along well-defined points in the patient journey remains unknown. This Example characterizes the prevalence of ESRlmut at the start of successive lines of therapy and evaluates clinical outcomes of ET by ESRI status in 1stline therapy in real- world practice. In addition, detailed ESRI genomic alterations (GAs) detected by tissue and liquid biopsies (TBx, LBx) of mBC patients with HR(+)HER2(-) are reported.Methods

[0192] This study included patients with mBC who underwent genomic testing using Foundation Medicine tissue or liquid comprehensive genomic profiling (CGP) assays during routine care. Clinical data was obtained by a nationwide (US-based) de-identified real-world clinicogenomic breast database between January 2015 and March 2023. Retrospective deidentified longitudinal clinical data were derived from electronic health records (EHR) fromapproximately 280 US cancer clinics (-800 sites of care) and comprises patient-level structured and unstructured data, curated via technology-enabled abstraction of clinical notes and radiology / pathology reports. Clinical data included demographics, clinical and laboratory features, time of therapy exposure, and survival. These were linked to genomic data by deidentified, deterministic matching [6]

[0193] Hybrid capture-based NGS assays were performed on patient tumor specimens in Clinical Laboratory Improvement Amendments (CLIA)-certified, College of American Pathologists (CAP)-accredited laboratory. The level of ctDNA shed in the liquid assay for each specimen was quantified by calculating an investigational measure that merges two methods for estimation of ctDNA TF [7]. When aneuploidy is detectable, ctDNA TF is estimated based on a computational model of tumor purity, ploidy, and copy number alterations, excluding potential clonal hematopoiesis (CH)-derived aneuploid genomic segments [8]. This aneuploidy-based approach avoids erroneously inferring elevated ctDNA TF due to the presence of germline variants detected at high variant allele frequency. For samples lacking detectable tumor aneuploidy, a variant-based calculation is made by identifying the highest allele fraction non-germline variant, excluding alterations that are determined to be derived from CH based on a multiomic analysis of the alterations.

[0194] Real-world (rw)PFS and rwOS were the primary endpoints, and time to treatment discontinuation (rwTTD) was the secondary endpoint. rwPFS was calculated from the treatment start date until the time of disease progression or death, and patients not yet reaching progression or death were right-censored at the date of the last clinical note. rwOS was calculated from the start of treatment to death from any cause, and patients with no record of mortality were right-censored at the date of the last clinic visit or structured EHR activity. rwOS risk intervals were left truncated to the date of the CGP report to account for immortal time, as patients cannot enter the database until a CGP report is provided [9, 10]. rwTTD was calculated from the treatment start date until the treatment discontinuation for any reason or death, and patients not yet reaching treatment discontinuation or death were right-censored at the date of last clinical visit, laboratory result, or medication use. The mortality information in the Flatiron Health database is a composite derived from deidentified patient-level data within the EHR, the public Social Security Death Index, and a commercial death dataset mining data from obituaries and funeral homes. This mortality information has been externally validated in comparison to the National Death Index[l 1]. Inaddition, the real-world clinicogenomic database has replicated associations with survival observed in biomarker subgroup analyses of randomized controlled trials [12, 13].

[0195] The full spectrum of predicted pathogenic ESRI GAs detected in TBx and LBx of patients with HR(+)HER2(-) mBCA in CGDB was assessed. E’SRJmut prevalence was calculated in TBx and LBx collected in the 1st, 2nd, and 3rd metastatic lines of therapy (up to 60 days before or after line start). rwPFS, rwOS, and rwTTD were compared between patients with TBx who received Al + CDKi and between patients receiving fulvestrant + CDKi 1st line therapy | S7? / mut vs. ESRI wild-type (wt)] by Cox models. Multivariable analyses adjusted for age, ECOG, histology, menopausal status, adjuvant therapy, bone only vs. visceral metastasis, and number of metastatic sites were performed. %2 tests and Wilcoxon rank sum tests were used to assess differences between groups of categorical and continuous variables, respectively. R version 4.1.3 software was used for outcome analyses.Results

[0196] A total of 5,848 and 1,228 HR(+)HER2(-) mBC patients with TBx and LBx, respectively, were included in this study. Out of those, 1,084 TBx and 361 LBx had a predicted pathogenic ESRI alteration detected.ESRlmut is prevalent in about 8-12% of TBx and. LBx collected proximal to 1stline therapy and the prevalence increases during the patient journey

[0197] Evaluating specimens collected around the time of therapy initiation, it was observed that the prevalence of S7? / mut in TBx collected in 1st, 2nd, and 3rd lines of therapy was 7.9% (n=159 / 2004), 26.4% (n=66 / 250), and 34.0% (n=68 / 200), respectively. Patients with S7? / mut detected around the 1stline initiation tend to receive therapies other than Al. Previous Al use is more common in patients with S7? / mut detected in 2ndand 3rd lines than patients with ESRlwt (FIG. 1).

[0198] The prevalence of S7? / mut in LBx collected proximal to initiation of 1st, 2nd, and 3rd lines of therapy was 11.9% (n=26 / 218), 37.0% (n=54 / 146), and 38.1% (n=32 / 84), respectively. The prevalence of ES7? / mut detected is higher for samples with ctDNA TF >1% (FIG. 2).Clinical characteristic of patients with HR(+)HER2(-) mBC receiving Al + CDKi orFulvestrant + CDKi in 1sttherapy

[0199] Out of the 2004 HR(+)HER2(-) mBC patients with TBx collected at 1stline of therapy, a total of 807 patients were included in the outcome analyses. 62.6% received first- line Al + CDKi, and 37.4% received Fulvestrant + CD Ki. Baseline ES7? / mut was detected in TBx of 69 patients (8.6%), and 27.5% of those received first-line Al + CDKi and 72.5% received Fulvestrant + CDKi. The median age observed for patients with ESRIwt and ES7? / mut was 63.0 (IQR 54-70) and 66 (59-73), respectively. Most of patients had ECOG of 0 (53.3%) and bone metastasis (53.0%). Most of the patients (62.3%) with an ES7?Jmut detected had received adjuvant ET with Al or tamoxifen, while only 34.4% had received adjuvant therapy with ET among those with ESRIwt. Finally, menopause status and histology data were unknown in many cases, but for those cases with data available, the majority were postmenopausal and had an invasive ductal carcinoma (FIG. 8A).Baseline ESRlmut is associated with less favorable outcomes in patients receiving 1stline Al + CDKi, but not in patients receiving fulvestrant + CDKi

[0200] Among patients receiving Al + CDKi 1st line therapy (n=505), those with ES7? / mut vs. ESRIwt had less favorable rwPFS [median 6.1 vs. 20.8 months, hazard ratio (HR) 2.36, 95% CI 1.35-4.14, p=0.003], rwOS (median 29.9 vs. 53.5 months, HR 1.93, 95%CI 0.94- 3.96, p=0.072), and rwTTD (5.0 vs. 18.9 months, HR 4.13, 95% CI 2.48-6.9, p<0.0001) (FIGS. 3A-3C). Among patients receiving fulvestrant + CDKi 1st line therapy (n=302), those with ES7? / mut vs. ESRlv had less favorable rwPFS (median 10.5 vs. 14.5 months, HR 1.5, 95% CI 1.05-2.15, p=0.025), but no difference was observed for rwOS and rwTTD (p>0.05) (FIGS. 5A-5C). Specific ES7? / mut did not seem to be associated with different outcomes to Al + CKDi or fulvestrant + CDKi (FIGS. 4 & 6).

[0201] Notably, the multivariable analyses found the independent association of ES7? / mut and less favorable rwPFS (HR 2.70, 95% CI 1.44-5.06, p=0.002, FIG. 7A) and rwOS (HR 3.04, 95% CI 1.28-7.21, p=0.012, FIG. 7B) in patients receiving Al + CDKi, but not in patients receiving Fulvestrant + CDKi (p>0.05 for rwPFS and rwOS) (FIGS. 7C & 7D). The rwTTD multivariable analyses were consistent with rwPFS and rwOS.Conclusion

[0202] In this dataset, ESE / mut was prevalent in about 8-11% of baseline tissue and liquid samples of patients with HR(+)HER2(-) mBC. ESRlmut detected at the start of metastatic treatment was associated with less favorable outcomes in patients receiving Al + CDKi 1stline therapy, but not in patients receiving fulvestrant + CDKi. ESRlmut prevalence increasedduring treatment course. CGP detected a wide spectrum of activating GAs in ESRI, including missense mutations, insertion / deletions, and amplifications. Without wishing to be bound to theory, these results may suggest that the evaluation of ESRlmut at this timepoint, with tissue or liquid, has clinical utility.References1. Bidard, F.C., et al., Switch to fulve strant and. palbociclib versus no switch in advanced breast cancer with rising ESRI mutation during aromatase inhibitor and palbociclib therapy (PADA-1): a randomised, open-label, multicentre, phase 3 trial. Lancet Oncol, 2022. 23(11): p. 1367-1377.2. Razavi, R, et al., The Genomic Landscape of Endocrine-Resistant Advanced Breast Cancers. Cancer Cell, 2018. 34(3): p. 427-438 e6.3. Brett, J.O., et al., ESRI mutation as an emerging clinical biomarker in metastatic hormone receptor-positive breast cancer. Breast Cancer Research, 2021. 23(1): p. 85.4. Burstein, H.J., et al., Testing for ESRI Mutations to Guide Therapy for Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Metastatic Breast Cancer: ASCO Guideline Rapid Recommendation Update. J Clin Oncol, 2023. 41(18): p. 3423- 3425.5. Bidard, F.C., et al., Elacestrant (oral selective estrogen receptor degrader) Versus Standard Endocrine Therapy for Estrogen Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer: Results From the Randomized Phase HI EMERALD Trial. J Clin Oncol, 2022. 40(28): p. 3246-3256.6. Singal, G., et al., Association of Patient Characteristics and Tumor Genomics With Clinical Outcomes Among Patients With Non-Small Cell Lung Cancer Using a Clinicogenomic Database. JAMA, 2019. 321(14): p. 1391-1399.7. Tukachinsky, H., et al., Genomic Analysis of Circulating Tumor DNA in 3,334 Patients with Advanced Prostate Cancer Identifies Targetable BRCA Alterations and AR Resistance Mechanisms. Clin Cancer Res, 2021. 27(11): p. 3094-3105.8. Li, M., et al., Abstract 2231: Utility of plasma tumor fraction (TF) to inform sensitivity of FoundationOne Liquid CDx (FILCDx). Cancer Research, 2021. 81(13_Supplement): p. 2231-2231.9. McGough, S.F., et al., Penalized regression for left-truncated and right-censored survival data. Stat Med, 2021. 40(25): p. 5487-5500.10. Brown, S., et al., Implications of Selection Bias Due to Delayed Study Entry in Clinical Genomic Studies. JAMA Oncol, 2022. 8(2): p. 287-291.11. Zhang, Q., et al., Validation analysis of a composite real-world mortality endpoint for patients with cancer in the United States. Health Serv Res, 2021. 56(6): p. 1281-1287.12. Quintanilha, J.C.F., et al., Comparative Effectiveness of Immune Checkpoint Inhibitors vs Chemotherapy in Patients With Metastatic Colorectal Cancer With Measures of Microsatellite Instability, Mismatch Repair, or Tumor Mutational Burden. JAMA Netw Open, 2023. 6(1): p. e2252244.13. Graf, R.R, et al., Comparative Effectiveness of Immune Checkpoint Inhibitors vs Chemotherapy by Tumor Mutational Burden in Metastatic Castration-Resistant Prostate Cancer. JAMA Netw Open, 2022. 5(3): p. e225394.

Claims

CLAIMSWhat is claimed is:

1. A method of selecting a first-line treatment for an individual having hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer, the method comprising detecting an estrogen receptor 1 (ESRI) mutation in a sample from the individual, wherein detection of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a selective estrogen receptor degrader (SERD) and a cyclin-dependent kinase inhibitor (CD Ki).

2. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, the method comprising detecting absence of an ESRI mutation in a sample from the individual, wherein detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an aromatase inhibitor (Al) and a CDKi.

3. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, the method comprising detecting presence or absence of an ESRI mutation in a sample from the individual, wherein detection of the presence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi, or wherein detection of the absence of an ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.

4. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi.

5. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and aCDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

6. A method of selecting a first-line treatment for an individual having HR+HER2- breast cancer, comprising acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of knowledge of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the acquisition of knowledge of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

7. A method of identifying one or more first-line treatment options for an individual having HR+HER2- breast cancer, the method comprising:(a) detecting an ESRI mutation in a sample from the individual; and(b) generating a report comprising one or more first-line treatment options identified for the individual based at least in part on the detection of an ESRI mutation in the sample, wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi.

8. A method of identifying one or more first- line treatment options for an individual having HR+HER2- breast cancer, the method comprising:(a) detecting absence of an ESRI mutation in a sample from the individual; and(b) generating a report comprising one or more first-line treatment options identified for the individual based at least in part on the detection of the absence of an ESRI mutation in the sample, wherein the one or more first-line treatment options comprise first- line treatment with an Al and a CDKi.

9. A method of identifying one or more first-line treatment options for an individual having HR+HER2- breast cancer, the method comprising:(a) detecting presence or absence of an ESRI mutation in a sample from the individual; and(b) generating a report comprising one or more first-line treatment options identified for the individual, wherein the one or more first-line treatment options comprise first-line treatment with a SERD and a CDKi based at least in part on the detection of the presence an ESRI mutation in the sample, or wherein the one or more first-line treatment options comprise first-line treatment with an Al and a CDKi based at least in part on the detection of the absence of an ESRI mutation in the sample.

10. The method of any one of claims 7-9, wherein the report further indicates the presence or absence of the ESRI mutation in the sample.

11. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) acquiring knowledge of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) responsive to said knowledge, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi.

12. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) acquiring knowledge of absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) responsive to said knowledge, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

13. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) acquiring knowledge of presence or absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(bl) responsive to knowledge of the presence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi; or(b2) responsive to knowledge of the absence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

14. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) detecting an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi.

15. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) detecting absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(b) administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

16. A method of treating or delaying progression of HR+HER2- breast cancer in an individual, comprising:(a) detecting presence or absence of an ESRI mutation in a sample from the individual having HR+HER2- breast cancer; and(bl) responsive to detecting the presence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises a SERD and a CDKi; or(b2) responsive to detecting the absence of an ESRI mutation in the sample, administering to the individual an effective amount of a first-line treatment that comprises an Al and a CDKi.

17. A method of identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies an ESRI mutation in the sample; and(b) selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises a SERD and a CDKi.

18. A method of identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies absence of an ESRI mutation in the sample; and(b) selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises an Al and a CDKi.

19. A method of identifying a candidate first-line treatment for HR+HER2- breast cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on a sample obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies presence or absence of an ESRI mutation in the sample; and(b) selecting a first-line treatment for the individual based at least in part on the sequencing mutation profile, wherein the first-line treatment comprises treatment with a SERD and a CDKi when the sequencing mutation profile identifies the presence of an ESRI mutation in the sample, or wherein the first-line treatment comprises treatment with an Al and a CDKi when the sequencing mutation profile identifies the absence of an ESRI mutation in the sample.

20. The method of any one of claims 17-19, 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.

21. The method of claim 20, wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises nextgeneration sequencing (NGS).

22. A method of predicting survival of an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to survival of the individual when treated with a first-line treatment comprising an Al and a CDKi.

23. A method of predicting survival of an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a first-line treatment comprising an Al and a CDKi, as compared to survival when treated with a first-line treatment comprising an Al and a CDKi of an individual whose sample comprises an ESRI mutation.

24. A method of screening an individual having HR+HER2- breast cancer, comprising acquiring knowledge of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a first- line treatment comprising a SERD and a CDKi and / or longer survival when treated with a first-line treatment comprising a SERD and a CDKi, as compared to response and / or survival when treated with a first-line treatment comprising an Al and a CDKi.

25. A method of screening an individual having HR+HER2- breast cancer, comprising acquiring knowledge of absence of an ESRI mutation in a sample from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a first- line treatment comprising an Al and a CDKi and / or longer survival when treated with a first-line treatment comprising an Al and aCDKi, as compared to response and / or survival of an individual whose sample comprises an ESRI mutation.

26. The method of any one of claims 22-25, wherein the survival comprises progression-free survival (PFS).

27. The method of any one of claims 22-26, wherein the survival comprises overall survival (OS).

28. The method of any one of claims 22-27, wherein the improved response comprises longer time-to -treatment discontinuation (TTD).

29. The method of any one of claims 22-28, wherein the improved response comprises longer time-to-next treatment (TTNT).

30. The method of any one of claims 1, 3, 4, 6, 7, 9-11, 13, 14, 16, 17, 19-22, 24, and 26-29, wherein the SERD is fulvestrant.

31. The method of any one of claims 2, 3, 5, 6, 8-10, 12, 13, 15, 16, and 18-29, wherein the Al is aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), or 4-Androstene-3, 6, 17-trione (“6-OXO”).

32. The method of any one of claims 1-31, wherein the CDKi is a CDK4 / CDK6 inhibitor.

33. The method of claim 32, wherein the CDKi is palbociclib, ribociclib, abemaciclib, or dalpiciclib.

34. The method of any one of claims 1-31, wherein the CDKi is a CD K4- selective inhibitor.

35. The method of claim 34, wherein the CDKi is PF-07220060 or 2-94.

36. The method of any one of claims 1-31, wherein the CDKi is a CDK2 inhibitor.

37. The method of claim 34, wherein the CDKi is tagtociclib (PF-07104091).

38. The method of any one of claims 1-37, wherein the ESRI mutation comprises a base substitution or short insertion / deletion.

39. The method of claim 38, wherein the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1.

40. The method of claim 38, wherein the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1.

41. The method of any one of claims 1-37, wherein the ESRI mutation comprises a rearrangement or gene fusion.

42. The method of any one of claims 2, 3, 5, 6, 8-10, 12, 13, 15, 16, and 18-41, wherein detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof.

43. The method of any one of claims 1-42, wherein the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.

44. The method of claim 43, wherein the treatment or treatment option is a first- line metastatic treatment or treatment option.

45. The method of any one of claims 1-44, further comprising obtaining the sample from the individual.

46. The method of any one of claims 1-45, wherein the sample is obtained or derived from the HR+HER2- breast cancer.

47. The method of any one of claims 1-46, wherein the sample comprises a tissue biopsy sample.

48. The method of any one of claims 1-46, wherein the sample comprises a liquid biopsy sample.

49. The method of claim 48, wherein the sample comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

50. The method of claim 48 or claim 49, wherein the sample comprises circulating tumor cells (CTCs).

51. The method of any one of claims 48-50, wherein the sample comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

52. The method of any one of claims 1-46, wherein the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell.

53. The method of any one of claims 1-46, wherein the sample comprises cells and / or nucleic acids from the HR+HER2- breast cancer.

54. The method of claim 53, wherein the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the HR+HER2- breast cancer.

55. The method of any one of claims 1-54, wherein the ESRI mutation or absence thereof is detected by immunohistochemistry analysis (IHC), comprehensive genomic profiling (CGP), comparative genomic hybridization (CGH), sequencing, or any combination thereof.

56. The method of claim 55, 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.

57. The method of claim 56, wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises nextgeneration sequencing (NGS).

58. The method of any one of claims 1-57, wherein detecting the ESRI mutation or absence thereof comprises:(a) providing a plurality of nucleic acid molecules obtained from the sample from the individual, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an ESRI gene, or a portion thereof;(b) optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules;(c) optionally, amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules;(d) optionally, 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 sequence reads of the plurality of sequence reads correspond to an ESRI gene, or a portion thereof;(f) analyzing the plurality of sequence reads for the presence or absence of an ESRI mutation; and(g) based on the analyzing step, detecting the presence or absence of an ESRI mutation in the sample.

59. The method of claim 58, wherein the sequencer comprises a next-generation sequencer.

60. The method of any one of claims 1-57, wherein detecting the ESRI mutation or absence thereof comprises:(a) providing the sample from the individual, 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 or a 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 the presence or absence of an ESRI mutation; and(g) detecting, based on the analyzing step, the presence or absence of an ESRI alteration in the sample from the individual.

61. The method of any one of claims 58-60, 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.

62. The method of claim 60, 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 an ESRI gene or a portion thereof and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.

63. The method of any one of claims 58-62, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.

64. The method of any one of claims 58-63, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique.

65. The method of any one of claims 1-64, further comprising selectively enriching for one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof; wherein the selectively enriching produces an enriched sample.

66. The method of claim 65, wherein the selectively enriching comprises: (a) combining one or more bait molecules with the sample, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.

67. The method of any one of claims 62-66, wherein the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to an ESRI gene or a portion thereof.

68. The method of claim 67, 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.

69. The method of any one of claims 62-68, wherein the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent.

70. The method of claim 69, wherein the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.

71. The method of any one of claims 67-70, wherein the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.

72. The method of claim 71, wherein the selectively enriching comprises amplifying the one or more nucleic acid molecules comprising nucleotide sequences corresponding to an ESRI gene or a portion thereof using a polymerase chain reaction (PCR) to produce an enriched sample.

73. The method of any one of claims 65-72, further comprising sequencing the enriched sample.

74. The method of any one of claims 58-73, wherein the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules.

75. The method of claim 74, wherein the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample.

76. The method of claim 74, wherein the sample comprises a liquid biopsy sample, and wherein the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample, and the non-cancer nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample.

77. The method of any one of claims 1-6 and 11-76, further comprising generating a report, wherein the report:(a) indicates the presence or absence of the ESRI mutation in the sample from the individual; and / or(bl) indicates a first-line treatment or one or more first- line treatment options identified or selected for the individual based, at least in part, on the presence of an ESRI mutation in the sample from the individual, wherein the first-line treatment or the one or more first-line treatment options comprise a SERD and a CDKi, or(b2) indicates a first-line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the absence of an ESRI mutation in the sample from the individual, wherein the first-line treatment or the one or more first-line treatment options comprise an Al and a CDKi.

78. The method of any one of claims 1-77, further comprising generating a molecular profile for the individual, based, at least in part, on detecting or acquiring knowledge of the presence or absence of the ESRI mutation.

79. The method of claim 78, 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.

80. The method of claim 78 or claim 79, wherein the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test.

81. The method of any one of claims 78-80, further comprising generating a report, wherein the report comprises the molecular profile for the individual.

82. The method of claim 81, wherein the report further comprises information on a first-line treatment or one or more first-line treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the first- line treatment or one or more first-line treatment options comprise a SERD and a CDKi when the molecular profile indicates the presence of an ESRI mutation in the sample from the individual, or wherein the first-line treatment or one or more first-line treatment options comprise an Al and a CDKi when the molecular profile indicates the absence of an ESRI mutation in the sample from the individual.

83. The method of any one of claims 7-10, 81, and 82, 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.

84. The method of any one of claims 1-83, wherein the individual is a human.

85. A system for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising a SERD and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyze the plurality of sequence reads for presence of an ESRI mutation; and(c) detect, based on the analyzing, presence of the ESRI mutation in the sample; wherein detecting the presence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi.

86. A system for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising an Al and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyze the plurality of sequence reads for absence of an ESRI mutation; and(c) detect, based on the analyzing, absence of the ESRI mutation in the sample; wherein detecting the absence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.

87. A system for selecting a first-line treatment for an individual having HR+HER2- breast cancer, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyze the plurality of sequence reads for presence or absence of an ESRI mutation; and(c) detect, based on the analyzing, presence of the ESRI mutation in the sample; wherein responsive to the detection of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; orwherein responsive to the detection of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

88. The system of any one of claims 85-87, wherein the ESRI mutation comprises a base substitution or short insertion / deletion.

89. The system of claim 88, wherein the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1.

90. The system of claim 88, wherein the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1.

91. The system of any one of claims 85-87, wherein the ESRI mutation comprises a rearrangement or gene fusion.

92. The system of any one of claims 86-91, wherein detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof.

93. The system of any one of claims 85-92, wherein the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.

94. The system of claim 93, wherein the treatment is a first-line metastatic treatment.

95. The system of any one of claims 86-94, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or next-generation sequencing.

96. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising a SERD and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for presence of an ESRI mutation; and(c) detecting, using the one or more processors and based on the analyzing, the presence of the ESRI mutation in the sample; wherein detecting the presence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising a SERD and a CDKi.

97. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for identifying an individual having HR+HER2- breast cancer who may benefit from a first-line treatment comprising an Al and a CDKi, 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for absence of an ESRI mutation; and(c) detecting, using the one or more processors and based on the analyzing, the absence of the ESRI mutation in the sample; wherein detecting the absence of the ESRI mutation in the sample identifies the individual as one who may benefit from a first-line treatment comprising an Al and a CDKi.

98. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for selecting a first-line treatment for an individual having HR+HER2- breast 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 sample obtained from an individual having HR+HER2- breast cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for presence or absence of an ESRI mutation; and(c) detecting, using the one or more processors and based on the analyzing, the presence or absence of the ESRI mutation in the sample; wherein responsive to the detection of the presence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising a SERD and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises a SERD and a CDKi; or wherein responsive to the detection of the absence of an ESRI mutation in the sample: (i) the individual is classified as a candidate to receive a first-line treatment comprising an Al and a CDKi; and / or (ii) the individual is identified as likely to respond to a first-line treatment that comprises an Al and a CDKi.

99. The non-transitory computer readable storage medium of any one of claims 96-98, wherein the ESRI mutation comprises a base substitution or short insertion / deletion.

100. The non-transitory computer readable storage medium of claim 99, wherein the ESRI mutation results in a Y537S, D538G, E380Q, L536R, L536H, Y537C, Y537D, Y537N, L549P, S463P, G442R, or M357I amino acid substitution, relative to SEQ ID NO:1.

101. The non-transitory computer readable storage medium of claim 100, wherein the ESRI mutation results in a V422 deletion, relative to SEQ ID NO:1.

102. The non-transitory computer readable storage medium of any one of claims 96-98, wherein the ESRI mutation comprises a rearrangement or gene fusion.

103. The non-transitory computer readable storage medium of any one of claims 97-102, wherein detecting the absence of an ESRI mutation comprises detecting a wild-type ESRI gene or portion thereof.

104. The non-transitory computer readable storage medium of any one of claims 96-103, wherein the HR+HER2- breast cancer is advanced or metastatic HR+HER2- breast cancer.

105. The non-transitory computer readable storage medium of claim 104, wherein the treatment is a first-line metastatic treatment.

106. The non-transitory computer readable storage medium of any one of claims 96- 105, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or next-generation sequencing.

Images