Use of fibroblast growth factor receptor 3 mutations and tumor mutational burden to predict response to immunotherapies

Abstract

Provided herein are methods related to selecting an individual having cancer for treatment with an immunotherapy, as well as uses, systems, and computer readable storage media related thereto. In some embodiments, the methods comprise detecting an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer; and / or administering to the individual an effective amount of a treatment that comprises an immunotherapy, e.g., responsive to said detection.

Description

USE OF FIBROBLAST GROWTH FACTOR RECEPTOR 3 MUTATIONS AND TUMOR MUTATIONAL BURDEN TO PREDICT RESPONSE TO IMMUNOTHERAPIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit of U.S. Provisional Application No. 63 / 607,996, filed on December 8, 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 (197102016340seqlist.xml; Size: 7,919 bytes; and Date of Creation: December 5, 2024) are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0003] Provided herein are methods related to selecting an individual having cancer for treatment with an immunotherapy, as well as uses, systems, and computer readable storage media related thereto. In some embodiments, the methods comprise detecting a fibroblast growth factor receptor 3 (FGFR3) alteration and a high TMB in one or more samples from an individual having a cancer; and / or administering to the individual an effective amount of a treatment that comprises an immunotherapy, e.g., responsive to said detection.BACKGROUND

[0004] Immunotherapies, such as immune checkpoint inhibitors (ICPIs) have been approved for use in multiple tumor types, and are incorporated into the National Comprehensive Cancer Network (NCCN) guidelines, influencing real world clinical management of patients with cancer (Vaddepally et al., Cancers (2020) 12, 738). Despite this, only an estimated 12.5% of eligible patients (based on PD-L1 positivity) are reported to respond to ICPI treatment (Haslam et al., JAMA Netw Open 2 (2019) el92535).

[0005] Other biomarkers, such as high tumor mutational burden (TMB -High) and micro satellite instability-high (MSLH) have also been used to select patients for treatment with ICPIs. In particular, TMB-High (e.g., with a TMB greater than or equal to 10 mutations / Megabase [mut / Mb]) and MSLH solid tumor patients are eligible to receive ICPItreatment based on two pan-solid tumor approvals (Subbiah et al., Ann. Oncol. (2020) 31, 1115-1118; Marcus et al., Clin. Cancer Res (2019) 25, 3753-3758). However, the clinical outcomes of ICPI treatment in these biomarker positive patients is varied (Huang et al., Mod. Pathol. (2020); Strickler et al., Clin. Cancer Res. (2021) 27, 1236 LP - 1241).

[0006] In some types of cancer, characteristic gene fusions are thought to predict a negative response to ICPI treatment. For example, in lung cancer, ICPI treatment has been associated with poor outcomes in patients with ALK, ROS1 , or RET rearrangements (Mazieres, J et al., Ann. Oncol. (2019) 30(8): 1321- 1328). Some evidence has suggested that FGFR3 alterations could predict lack of response to ICPI treatment in some tumor types, such as bladder cancer (Huang, R.S.P. et al. Oncologist (2019) 26(5):375-382; Loriot, Y et al. N. Engl. J. Med.(2019) 381(4):338-348). Therefore, FGFR3 fusions / alterations might also be associated with poor responses to ICPI in, for example, bladder cancers, but studies on the association between FGFR3 alterations / fusions and response to ICPI treatment are lacking.

[0007] Thus, there is a need in the art for improved predictive biomarkers of response to immunotherapies, such as ICPIs, in cancer to guide the treatment of cancer patients.

[0008] 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

[0009] In some aspects, provided herein is a method of identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, the method comprising detecting in one or more samples from the individual an alteration in a fibroblast growth factor receptor 3 (FGFR3) gene and a high tumor mutational burden (TMB), wherein detection of the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy.

[0010] In some aspects, provided herein is a method of selecting a treatment for an individual having a cancer, the method comprising detecting in one or more samples from the individual an FGFR3 alteration and a high TMB, wherein detection of the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy.

[0011] In some aspects, provided herein is a method of identifying one or more treatment options for an individual having a cancer, the method comprising: detecting in one or more samples from the individual an FGFR3 alteration and a high TMB; and generating a report comprising one or more treatment options identified for the individual based at least in part on detection of the FGFR3 alteration and the high TMB in the one or more samples, wherein the one or more treatment options comprise an immunotherapy. In some embodiments, the report further indicates presence or absence of the FGFR3 alteration and the high TMB in the one or more samples.

[0012] In some aspects, provided herein is a method of identifying one or more treatment options for an individual having a cancer, the method comprising: acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual; and generating a report comprising one or more treatment options identified for the individual based at least in part on said knowledge, wherein the one or more treatment options comprise an immunotherapy. In some embodiments, the report further indicates presence or absence of the FGFR3 alteration and the high TMB in the one or more samples.

[0013] In some aspects, provided herein is a method of selecting a treatment for an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a treatment comprising an immunotherapy; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an immunotherapy.

[0014] In some aspects, provided herein is a method of predicting survival of an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB. In some embodiments, the individual is predicted to have longer overall survival (OS) when treated with a treatment comprising an immunotherapy, as compared to OS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB. In some embodiments, the individual is predicted to have longer progression-free survival (PFS) when treated with a treatment comprising an immunotherapy, as compared to PFS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

[0015] In some aspects, provided herein is a method of predicting survival of an individual having a cancer treated with a treatment comprising an immunotherapy, the method comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to an individual whose cancer does not exhibit an FGFR3 alteration and a high TMB. In some embodiments, the individual is predicted to have longer overall survival (OS) when treated with a treatment comprising an immunotherapy, as compared to OS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB. In some embodiments, the individual is predicted to have longer progression-free survival (PFS) when treated with a treatment comprising an immunotherapy, as compared to PFS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

[0016] In some aspects, provided herein is a method of monitoring, evaluating, or screening an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a treatment comprising an immunotherapy and / or longer survival when treated with a treatment comprising an immunotherapy, as compared to an individual whose cancer does not comprise an FGFR3 alteration and a high TMB. In some embodiments, the individual is predicted to have longer overall survival (OS) when treated with a treatment comprising an immunotherapy, as compared to OS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB. In some embodiments, the individual is predicted to have longer progression-free survival (PFS) when treated with a treatment comprising an immunotherapy, as compared to PFS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

[0017] In some aspects, provided herein is a method of selecting a treatment for an individual having a cancer, the method comprising detecting in one or more samples from the individual an FGFR3 alteration and a TMB, wherein detection of the FGFR3 alteration in the one or more samples identifies the individual as one who is more likely to benefit from a treatment comprising an immunotherapy at the detected TMB than an individual whose cancer shows the same TMB but lacks an FGFR3 alteration.

[0018] In some aspects, provided herein is a method of predicting survival of an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a TMB in one or more samples from the individual, wherein responsive to the acquisition of saidknowledge, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer shows the same TMB but lacks an FGFR3 alteration.

[0019] In some aspects, provided herein is a method of treating or delaying progression of a cancer in an individual, comprising: acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer; and responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an immunotherapy.

[0020] In some aspects, provided herein is a method of treating or delaying progression of a cancer in an individual, comprising administering to an individual having a cancer an effective amount of a treatment that comprises an immunotherapy, wherein the immunotherapy is administered responsive to acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual.

[0021] In some aspects, provided herein is a method of treating or delaying progression of a cancer in an individual, comprising: detecting an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer; and administering to the individual an effective amount of a treatment that comprises an immunotherapy.

[0022] In some aspects, provided herein is a method of identifying a candidate treatment for a cancer in an individual in need thereof, comprising: performing DNA sequencing on one or more samples obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies the presence of an FGFR3 alteration and a high TMB in the one or more samples; and selecting a treatment (e.g., a candidate treatment) for the individual based at least in part on the sequencing mutation profile, wherein the treatment comprises an immunotherapy. In some embodiments, the presence of the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy. In some embodiments, the presence of the FGFR3 alteration and the high TMB in the one or more samples predicts the individual to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB. 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).

[0023] In some embodiments according to any of the embodiments described herein, the cancer is bladder cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is advanced or metastatic. In some embodiments, the immunotherapy is an immune checkpoint inhibitor (ICPI), a cancer vaccine, a cell-based therapy, a T cell receptor (TCR)-based therapy, an adjuvant immunotherapy, a cytokine immunotherapy, or an oncolytic virus therapy. In some embodiments, the immunotherapy is an ICPI. In some embodiments, the ICPI is a PD-1 -targeted agent or a PD-L1 -targeted agent. In some embodiments, the ICPI is a PD-1 inhibitor. In some embodiments, the ICPI comprises one or more of nivolumab, pembrolizumab, cemiplimab, or dostarlimab. In some embodiments, the ICPI is a PD-L1 inhibitor. In some embodiments, the ICPI comprises one or more of atezolizumab, avelumab, or durvalumab. In some embodiments, the ICPI is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor comprises ipilimumab. In some embodiments, the immunotherapy is a first-line immunotherapy or treatment. In some embodiments, the immunotherapy is a monotherapy (e.g., is administered as a monotherapy). In some embodiments, the treatment or one or more treatment options do not comprise a chemotherapy, e.g., comprising gemcitabine and / or carboplatin.

[0024] In some embodiments according to any of the embodiments described herein, the FGFR3 alteration is a base substitution, short insertion / deletion, or rearrangement. In some embodiments, the FGFR3 alteration results in an S249C amino acid substitution, relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an FGFR3-TACC3 gene fusion. In some embodiments, the FGFR3 alteration results in a Y373C, R248C, G370C, G380R, A391E, K650E, S371C, K650N, or K650M amino acid substitution, relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an E768* mutation, relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an FGFR3- TNIP2, FGFR3-COL17A1, or FGFR3-CCDC149 gene fusion.

[0025] In some embodiments according to any of the embodiments described herein, the high TMB comprises a TMB of greater than or equal to about 5 mutations / Megabase (mut / Mb), a TMB of greater than or equal to about 10 mut / Mb, or a TMB of greater than or equal to about 20 mut / Mb. In some embodiments, TMB is assessed based on number of synonymous and non-driver non-synonymous mutations. In some embodiments, TMB is assessed across at least about 0.8 Mb. In some embodiments, TMB is assessed across between about 0.8 and 1.2 Mb. In some embodiments, TMB is assessed on sequenced DNA.

[0026] In some embodiments according to any of the embodiments described herein, the methods further comprise assessing expression of PD-L1 protein in a sample from the individual. In some embodiments, the report further indicates the PD-L1 protein expression status of the cancer. In some embodiments, the molecular profile further indicates the PD-L1 protein expression status of the cancer. In some embodiments, the cancer is PD-L1 negative. In some embodiments, the cancer is PD-L1 positive. In some embodiments, PD-L1 protein expression is determined using an immunohistochemistry assay. In some embodiments, the immunohistochemistry assay is a DAKO PD-L1 22C3 assay. In some embodiments, PD-L1 positivity is defined as a combined positive score (CPS) of greater than or equal to 10. In some embodiments, the immunohistochemistry assay is a VENTANA SP 142 assay. In some embodiments, PD-L1 expression is assessed based on the proportion of tumor area occupied by PD-Ll-expressing tumor-infiltrating immune cells of any intensity (IC), or the percentage of PD-Ll-expressing tumor cells of any intensity (TC). In some embodiments, PD-L1 positivity is defined as an immune cell score of greater than or equal to 5.

[0027] In some embodiments according to any of the embodiments described herein, acquiring knowledge of an FGFR3 alteration and a high TMB comprises detecting the FGFR3 alteration and the high TMB in the one or more samples. In some embodiments, the FGFR3 alteration and the high TMB are detected in nucleic acids, or sequence reads derived therefrom, from the same sample. In some embodiments, the FGFR3 alteration and the high TMB are detected in nucleic acids, or sequence reads derived therefrom, from different samples. In some embodiments, the methods further comprise obtaining the one or more samples from the individual. In some embodiments, the one or more samples are obtained or derived from the cancer. In some embodiments, the one or more samples from the individual comprise a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the one or more samples from the individual are from a tumor biopsy, tumor specimen, or circulating tumor cell. In some embodiments, the one or more samples from the individual are a liquid biopsy sample comprising blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the one or more samples from the individual are a liquid biopsy sample comprising circulating tumor cells (CTCs). In some embodiments, the one or more samples from the individual are a liquid biopsy sample comprising cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the one or more samples from the individual comprise cells and / or nucleic acids from the cancer. In some embodiments, the one or more samples from the individual comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In someembodiments, the FGFR3 alteration is detected by fluorescence in situ hybridization (FISH), comprehensive genomic profiling (CGP), comparative genomic hybridization (CGH), sequencing, or any combination thereof. In some embodiments, the high TMB is detected by sequencing, e.g., NGS sequencing and / or whole-genome or whole-exome 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 sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS). In some embodiments, detecting the FGFR3 alteration comprises: (a) providing a plurality of nucleic acid molecules obtained from a sample from an individual having a cancer, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an FGFR3 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 FGFR3 gene, or a portion thereof; (f) analyzing the plurality of sequence reads for the presence or absence of an FGFR3 alteration; and (g) based on the analyzing step, detecting the presence or absence of an FGFR3 alteration in the sample. In some embodiments, the sequencer comprises a nextgeneration sequencer. In some embodiments, detecting the FGFR3 alteration comprises: (a) providing a sample from an individual having a 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 FGFR3 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 FGFR3 alteration; and (g) detecting, based on the analyzing step, the presence or absence of an FGFR3 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. Insome 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 FGFR3 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 some embodiments, the methods further comprise selectively enriching for one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an FGFR3 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 FGFR3 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 FGFR3 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 FGFR3 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 biopsysample. 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 non-tumor fraction of the liquid biopsy sample. In some embodiments, the methods further comprise generating a report, wherein the report: (a) indicates the presence of the FGFR3 alteration and high TMB in the one or more samples from the individual; and / or (b) indicates a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the presence of the FGFR3 alteration and high TMB in the one or more samples from the individual, wherein the treatment or the one or more treatment options comprise an immunotherapy. 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 FGFR3 alteration and / or high TMB. 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 treatment or one or more treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the treatment or one or more treatment options comprise an immunotherapy. 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.

[0028] In some embodiments according to any of the embodiments described herein, the individual is a human.

[0029] In some aspects, provided herein is a system for identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, 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 one or more samples obtained from an individualhaving a cancer; (b) analyze the plurality of sequence reads for presence of an FGFR3 alteration and presence of a high TMB; and (c) detect, based on the analyzing, presence of the FGFR3 alteration and presence of the high TMB in the one or more samples; wherein detecting the presence of the FGFR3 alteration and the presence of the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy. In some aspects, provided herein is a system for identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, 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 perform the method according to any one of the embodiments described above.

[0030] In some aspects, provided herein is a non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, 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 one or more samples obtained from an individual having a cancer; (b) analyzing, using the one or more processors, the plurality of sequence reads for presence of an FGFR3 alteration and presence of a high TMB; and (c) detecting, using the one or more processors and based on the analyzing, the FGFR3 alteration and the high TMB in the one or more samples; wherein detecting the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy. In some aspects, provided herein is a non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method according to any one of the embodiments described above.

[0031] In some embodiments according to any of the embodiments described herein, the cancer is bladder cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is advanced or metastatic. In some embodiments according to any of the embodiments described herein, the FGFR3 alteration is a base substitution, short insertion / deletion, or rearrangement. In some embodiments, the FGFR3 alteration results in an S249C amino acid substitution, relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an FGFR3-TACC3 gene fusion. In some embodiments, the FGFR3 alteration results in a Y373C, R248C, G370C, G380R, A391E, K650E, S371C,K650N, or K650M amino acid substitution, relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an E768* mutation, relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an FGFR3-TNIP2, FGFR3-COL17A1, or FGFR3-CCDC149 gene fusion. In some embodiments according to any of the embodiments described herein, the high TMB comprises a TMB of greater than or equal to about 5 mutations / Megabase (mut / Mb), a TMB of greater than or equal to about 10 mut / Mb, or a TMB of greater than or equal to about 20 mut / Mb. In some embodiments, TMB is assessed based on number of synonymous and non-driver non- synonymous mutations. In some embodiments, TMB is assessed across at least about 0.8 Mb. In some embodiments, TMB is assessed across between about 0.8 and 1.2 Mb. In some embodiments, the presence of FGFR3 and the presence of high TMB are detected based on analysis of sequence reads obtained from the same sample from the individual. In some embodiments, the presence of FGFR3 and the presence of high TMB are detected based on analysis of sequence reads obtained from different samples from the individual. In some embodiments, the one or more samples are obtained or derived from the cancer. In some embodiments, the one or more samples from the individual comprise a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the one or more samples from the individual are from a tumor biopsy, tumor specimen, or circulating tumor cell. In some embodiments, the one or more samples from the individual are a liquid biopsy sample comprising blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the one or more samples from the individual are a liquid biopsy sample comprising circulating tumor cells (CTCs). In some embodiments, the one or more samples from the individual are a liquid biopsy sample comprising cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the one or more samples from the individual comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or nextgeneration sequencing.

[0032] 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

[0033] FIG. 1 shows a diagram of inclusion criteria for the cohort used in this study.

[0034] FIGS. 2A-2C show outcomes on first-line ICPI based on FGFR3 and TMB status. Presented are unadjusted Kaplan-Meier curves for real-world overall survival (rwOS) (FIG. 2A), real-world progression-free survival (rwPFS) (FIG. 2B), and real-world time to next treatment (rwTTNT) (FIG. 2C) stratified by FGFR3 and TMB status. Patients with TMB < 10 had worse outcomes overall compared to TMB > 10, but among TMB > 10 patients, FGFR3-alterations conferred better rwOS, rwPFS, and rwTTNT compared to FGFR3-wt.

[0035] FIGS. 3A-3C show outcomes for FGFR3- altered, TMB>10 patients on ICPI vs. chemotherapy. Adjusted Kaplan-Meier curves for rwOS (FIG. 3A), rwPFS (FIG. 3B), and rwTTNT (FIG. 3C) for FGFR3-altered and TMB > 10 patients who received either first-line carboplatin + gemcitabine (chemo) or first-line ICPI show more favorable rwOS, rwPFS, and rwTTNT for patients receiving ICPI . Kaplan-Meier curves were adjusted with propensity weights for imbalances with therapy selection.

[0036] FIG. 4 shows the frequency of specific FGFR3 alterations observed in the patient cohort, as indicated.

[0037] FIGS. 5A-5C show outcomes for FGFR3-altered vs FGFR3- wildtype patients on ICPI. FG / 7 / ?.? -altered and wild-type (wt) patients had similar outcomes on first-line mono or doublet ICPI. FIGS. 5A-5C each present unadjusted Kaplan-Meier curves on the left and univariable and multivariable Cox models on the right for rwOS (FIG. 5A), rwPFS (FIG. 5B), and rwTTNT (FIG. 5C).

[0038] FIG. 6 shows the pre and post-adjustment balance for clinical factors included in the propensity weighting for patients receiving first-line ICPI or chemotherapy.

[0039] FIGS. 7A-7C show outcomes for FGFR3-altered patients on ICPI vs. chemotherapy. Presented are adjusted Kaplan-Meier curves for rwOS (FIG. 7A), rwPFS (FIG. 7B), and rwTTNT (FIG. 7C) for FGFR3-altered patients who received either first-line carboplatin + gemcitabine (chemo) or first-line ICPI. Kaplan-Meier curves were adjusted with propensity weights for imbalances with therapy selection.

[0040] FIG. 8 depicts an exemplary device, in accordance with some embodiments.

[0041] FIG. 9 depicts an exemplary system, in accordance with some embodiments.

[0042] FIG. 10 depicts a block diagram of an exemplary process for detecting an FGFR3 alteration and a high TMB, in accordance with some embodiments.DETAILED DESCRIPTION

[0043] The present disclosure relates generally to identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, as well as methods of treatment, systems, non-transitory computer readable storage media, and uses related thereto. In some aspects, provided herein are methods of identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, selecting a treatment (e.g., comprising an immunotherapy) for an individual having a cancer, identifying one or more treatment options (e.g., comprising an immunotherapy) for an individual having a cancer, predicting survival of an individual having a cancer (e.g., treated with an immunotherapy), monitoring, evaluating, or screening an individual having a cancer, treating or delaying progression of a cancer, or identifying a candidate treatment (e.g., comprising an immunotherapy) for a cancer in an individual in need thereof. In some embodiments, the methods comprise: (a) acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, or detecting an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer; and (b) responsive to said knowledge or detection, administering to the individual an effective amount of a treatment that comprises an immunotherapy.

[0044] The present disclosure describes the results of analysis of FGFR3 alteration(s) and their effect on immunotherapy (e.g., comprising an ICPI) in cancer, e.g., urothelial carcinoma. Contrary to a hypothesis that FGFR3 alteration(s) might portend limited response to ICPI, it was shown that cancers that were both TMB high (e.g., TMB >=10) and exhibited an FGFR3 alteration portended a better response to ICPI than cancers with either factor alone. As such, this combination showed value as a potential predictive biomarker to decide between ICPI and chemotherapy in the 1st line, even though the presence of FGFR3 alteration(s) alone did not show that it could distinguish between ICPI response versus no response, or whether a patient would fare better on ICPI versus a chemotherapy regimen.I. General Techniques

[0045] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, etal. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).II. Definitions

[0046] 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.

[0047] 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.

[0048] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0049] 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.

[0050] 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.

[0051] As used herein, the term “FGFR3” refers to a gene encoding a fibroblast growth factor receptor 3. The human FGFR3 gene is located on chromosome 4pl6.3. FGFR3 is also known as ACH, CEK2, JTK4, CD333, and HSFGFR3EX. In some embodiments, an FGFR3 gene is a human FGFR3 gene.

[0052] “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.

[0053] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins,antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl-, 2'-fluoro-, or 2'- azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such 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.

[0054] “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.

[0055] 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.

[0056] “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.

[0057] 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 FGFR3 alteration (e.g., an FGFR3 base substitution, missense mutation, truncation mutation, short insertion / deletion, rearrangement, or gene fusion) and / or tumor mutational burden status (e.g., high tumor mutational burden). 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.

[0058] “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.

[0059] 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 toamplify 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.

[0060] 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)).

[0061] 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.

[0062] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebro- spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. In some instances, the sample is a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is from a tumor e.g., a “tumorsample”), such as from a biopsy. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] By ‘ ‘correlate” or “correlating” is meant comparing, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocol and / or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0067] ‘ ‘Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, ofdisease 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 courseof 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.

[0073] 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.

[0074] 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.

[0075] 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).

[0076] 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.

[0077] 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 certainembodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

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

[0079] In some aspects, provided herein are methods for identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy. In other aspects, provided herein are methods for selecting a therapy or treatment for an individual having a cancer. In other aspects, provided herein are methods for identifying one or more treatment options for an individual having a cancer. In other aspects, provided herein are methods for predicting survival of an individual having a cancer. In other aspects, provided herein are methods for predicting survival of an individual having a cancer treated with a treatment comprising an immunotherapy. In other aspects, provided herein are methods for treating or delaying progression of cancer. In other aspects, provided herein are methods for monitoring, evaluating or screening an individual having a cancer. In other aspects, provided herein are methods for assessing an FGFR3 alteration and / or a high tumor mutational burden in a cancer in an individual. In other aspects, provided herein are methods for detecting an FGFR3 alteration and / or a high tumor mutational burden in one or more samples from an individual having a cancer. In other aspects, provided herein are methods for detecting the presence or absence of a cancer comprising an FGFR3 alteration and / or a high tumor mutational burden in an individual. In other aspects, provided herein are methods for monitoring progression or recurrence of a cancer in an individual. In other aspects, provided herein are methods for identifying a candidate treatment for a cancer in an individual in need thereof.

[0080] In some embodiments of any of the methods provided herein, the methods comprise detecting the presence or absence of an FGFR3 alteration and a high tumor mutational burden in one or more samples from an individual. In other embodiments of any of the methods provided herein, the methods comprise acquiring knowledge of the presence or absence of an FGFR3 alteration and a high tumor mutational burden in one or more samples from an individual. In some embodiments, detection of an FGFR3 alteration and a high tumor mutational burden in the sample(s) identifies the individual as one who may benefit from the treatment comprising an immunotherapy. In some embodiments, the methods further comprise generating a report comprising one or more treatment options identified for theindividual based at least in part on detection of the FGFR3 alteration and high tumor mutational burden in the sample(s), wherein the one or more treatment options comprise an immunotherapy, e.g., as a monotherapy. In some embodiments, the one or more treatment options do not include a chemotherapy (in some embodiments, administered as a monotherapy or otherwise in the absence of an immunotherapy of the present disclosure), e.g., gemcitabine and / or carboplatin. In some embodiments, the methods further comprise generating a report comprising one or more treatment options identified for the individual based at least in part on knowledge of the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual, wherein the one or more treatment options comprise an immunotherapy. In some embodiments, responsive to the acquisition of knowledge of the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual: (i) the individual is classified as a candidate to receive a treatment comprising an immunotherapy; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an immunotherapy. In some embodiments, responsive to the acquisition of knowledge of the presence of the an FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer does not comprise an FGFR3 alteration and a high tumor mutational burden. In some embodiments, responsive to the acquisition of knowledge of the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual, the method comprises administering to the individual an effective amount of a treatment that comprises an immunotherapy. In some embodiments, responsive to the acquisition of knowledge of the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual, the individual is predicted to have decreased risk of cancer progression or cancer recurrence when treated with a treatment comprising an immunotherapy, as compared to an individual whose cancer does not comprise an FGFR3 alteration and a high tumor mutational burden. In some embodiments, responsive to the acquisition of knowledge of the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual, the individual is predicted to have an improved response to a treatment comprising an immunotherapy and / or longer survival when treated with a treatment comprising an immunotherapy, as compared to an individual whose cancer does not comprise an FGFR3 alteration and a high tumor mutational burden. In some embodiments, the methods provided herein comprise providing an assessment of an FGFR3 alteration and a high tumor mutational burden, e.g., in an individual or in one or moresamples from an individual. In some embodiments, the methods provided herein comprise detecting the FGFR3 alteration and high tumor mutational burden, in one or more samples from an individual, and administering to the individual an effective amount of a treatment that comprises an immunotherapy.

[0081] In some embodiments according to any of the embodiments described herein, the methods comprise acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual. In other embodiments, the methods comprise acquiring knowledge of an FGFR3 alteration in one or more samples from a cancer or tumor already known to possess a high TMB. In other embodiments, the methods comprise acquiring knowledge of a high TMB in one or more samples from a cancer or tumor already known to possess an FGFR3 alteration. In some embodiments according to any of the embodiments described herein, the methods comprise detecting an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer. In other embodiments, the methods comprise detecting an FGFR3 alteration in one or more samples from a cancer or tumor already known to possess a high TMB. In other embodiments, the methods comprise detecting a high TMB in one or more samples from a cancer or tumor already known to possess an FGFR3 alteration.

[0082] 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) identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy. In some embodiments, the systems and non-transitory computer readable storage media provided herein are for (e.g., are configured for) detecting, in one or more samples from an individual having cancer, an FGFR3 alteration and a high tumor mutational burden. In some embodiments, detecting the FGFR3 alteration and high tumor mutational burden in the sample(s) from the individual identifies the individual as one who may benefit from a treatment comprising an immunotherapy.A. FGFR3 Alterations and Detection Methods

[0083] Certain aspects of the present disclosure relate to FGFR3 alterations, including without limitation FGFR3 base substitutions, missense mutations, truncation mutations, short insertion / deletions, rearrangements, or gene fusions.

[0084] As demonstrated herein, FGFR3 alterations in cancer with high tumor mutational burden may be predictive of increased survival and / or increased likelihood of response when treated with an immunotherapy, such as an immune checkpoint inhibitor. Accordingly, in some embodiments, provided herein are methods that comprise acquiring knowledge of or detecting an FGFR3 alteration in a cancer. In some embodiments, the methods of the disclosure also comprise acquiring knowledge of or detecting a high tumor mutational burden in the cancer (see, e.g., Section B, below).

[0085] As used herein “FGFR3” refers to a gene encoding an FGFR3 mRNA or an FGFR3 polypeptide. FGFR3 is also known as ACH, CEK2, JTK4, CD333, and HSFGFR3EX. In some embodiments, an FGFR3 gene is a human FGFR3 gene. An exemplary FGFR3 gene is represented by NCBI Gene ID No. 2261. In some embodiments, an FGFR3 gene is located at chromosomal coordinates chr4: 1,793,293-1,808,872 forward strand. An exemplary FGFR3 polynucleotide sequence is represented by NCBI Ref. Seq. NM_000142.5, provided below as SEQ ID NO: 1:AGTGCGCGGTGGCGGCGGCGTCGCGGGCAGCTGGCGCCGCGCGGTCCTGCTCTG CCGGTCGCACGGACGCACCGGCGGGCCGCCGGCCGGAGGGACGGGGCGGGAGCTGGGCCCGCGGACAGC GAGCCGGAGCGGGAGCC GCGCGTAGCGAGCCGGGCTCCGGCGCTCGCCAGTCTCCCGAGCGGCGCCCGCCT CCCGCCGGTGCCCGCG CCGGGCCGTGGGGGGCAGCATGCCCGCGCGCGCTGCCTGAGGACGCCGCGGCCC CCGCCCCCGCCATGGG CGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCC TCCTCGGAGTCCTTG GGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCC CGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTG GTCCCATGGGGCCCAC TGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAG GTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTC ACGCAGCGCGTACTGT GCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACG GGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGA CAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTC CCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGC GGCATCAGCAGTGGAG CCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGCTCCAGCTCCTCAGGGGACGACTCCGTGTTTGCCCACGACCTGCTGCCCCCGGCCCCACCCAGCAGTGGGGGCTCGCGGACGTGAAGGGCCACTGGTCCCCAACAATGTGAGGGGTCCC 1TAGCAGCCCACCCTGCTGCTGGTGCACAGCCACTCCCCGGCATGAGACTCAGTGCAGATGGAGAGACAGCTACACAGAGCTTTGGTCTGTGTGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGCACATCCGCGTGTGCCTGTGTGCGTGCGCATCTTGCCTCCAGGTGCAGAGGTACCCTGGGTGTCCCCGCTGCTGTGCAACGGTCTCCTGACTGGTGCTGCAGCACCGAGGGGCCTTTGTTCTGGGGGGACCCAGTGCAGAATGTAAGTGGGCCCACCCGGTGGGACCCCCGTGGGGCAGGGAGCTGGGCCCGACATGGCTCCGGCCTCTGCCTTTGCACCACGGGACATCACAGGGTGGGCCTCGGCCCCTCCCACACCCAAAGCTGAGCCTGCAGGGAAGCCCCACATGTCCAGCACCTTGTGCCTGGGGTGTTAGTGGCACCGCCTCCCCACCTCCAGGCTTTCCCACTTCCCACCCTGCCCCTCAGAGACTGAAATTACGGGTACCTGAAGATGGGAGCCTTTACCTTTTATGCAAAAGGTTTATTCCGGAAACTAGTGTACATTTCTATAAATAGATGCTGTGTATATGGTATATATACATATATATATATAACATATATGGAAGAGGAAAAGGCTGGTACAACGGAGGCCTGCGACCCTGGGGGCACAGGAGGCAGGCATGGCCCTGGGCGGGGCGTGGGGGGGCGTGGAGGGAGGCCCCAGGGGGTCTCACCCATGCAAGCAGAGGACCAGGGCCTTTTCTGGCACCGCAGTTTTGTTTTAAAACTGGACCTGTATATTTGTAAAGCTATTTATGGGCCCCTGGCACTCTTAGT TT TC TTC TC AA AC CA TC TC AC TC TA GACACTTCCAGCATTTAGCTGGCCACATGGCGGAGAGTTTTAACAACCGAGAAGGTTTATCCCGCCGATAGAGGGACGGCCAAGAATGTACGTCCAGCCTGCCCCGGAGCTGGAGGATCCCCTCCAAGCCTAAAAGGTTGTTAATAGTTGGAGGTGATTCCAGTGAAGATATTTTATTTCCTTTGTCCTTTTTCAGGAGAATTAGATTTCTATAGGATTTTTCTTTAGGAGATTTATTTTTTGGACTTCAAAGCAAGCTGGTATTTTCATACAAATTCTTCTAATTGCTGTGTGTCCCAGGCAGGGAGACGGTTTCCAGGGAGGGGCCGGCCCTGTGTGCAGGTTCCGATGTTATTAGATGTTACAAGTTTATATATATCTATATATATAATTTATTGAGTTTTTACAAGATGTATTTGTTGTAGACTTAACACTTCTTACGCAATGCTTCTAGAGTTTTATAGCCTGGACTGCTACCTTTCAAAGCTTGGAGGGAAGCCGTGAATTCAGTTGGTTCGTTCTGTACTGTTACTGGGCCCTGAGTCTGGGCAGCTGTCCCTTGCTTGCCTGCAGGGCCATGGCTCAGGGTGGTCTCTTCTTGGGGCCCAGTGCATGGTGGCCAGAGGTGTCACCCAAACCGGCAGGTGCGATTTTGTTAACCCAGCGACGAACTTTCCGAAAAATAAAGACACCTGGTTGCTAA(SEQ ID NO: 1)

[0086] An exemplary amino acid sequence of a PD-L1 polypeptide is represented by NCBIRef. Seq. NP_000133.1, provided below as SEQ ID NO: 2:MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDA VELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVT DAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFR GEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVL GSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGE YTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLG SPTVHKISRFPLK RQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPL GEGCFGQVVMAE AIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGAC TQGGPLYVLVEYAAK GNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRD LAARNVLVTEDNV MKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLL WEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTS TDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPSSGGSRT(SEQ ID NO: 2)

[0087] In some embodiments, the FGFR3 alteration is a base substitution, short insertion / deletion, or rearrangement. In some embodiments, the FGFR3 alteration results in an S249C amino acid substitution, e.g., relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in a Y373C, R248C, G370C, G380R, A391E, K650E, S371C, K650N, or K650M amino acid substitution, e.g., relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an E768* mutation, e.g., relative to SEQ ID NO:2. In some embodiments, the FGFR3 alteration results in an FGFR3-TACC3 gene fusion. In some embodiments, the FGFR3 alteration results in an FGFR3-TNIP2, FGFR3- COL17A1, or FGFR3-CCDC149 gene fusion.

[0088] FGFR3 alterations 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 FGFR3 alterations include fluorescence in situ hybridization (FISH), 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 FGFR3 alteration is detected using amassively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. Certain methods for detecting FGFR3 alterations are described in further detail below as nonlimiting examples.(i) FISH

[0089] In some embodiments, FISH analysis is used to assess FGFR3 alterations in a cancer (e.g., in a sample from a cancer). Methods for performing FISH are known in the art and can be used in nearly any type of tissue. In FISH analysis, nucleic acid probes which are detectably labeled, e.g. fluorescently labeled, are allowed to bind to specific regions of DNA, e.g., a chromosome, or an RNA, e.g., an mRNA, and then examined, e.g., through a microscope. See, for example, U.S. Patent No. 5,776,688. DNA or RNA molecules are first fixed onto a slide, the labeled probe is then hybridized to the DNA or RNA molecules, and then visualization is achieved, e.g., using enzyme-linked label-based detection methods known in the art. Nucleic acid probes used in FISH analysis comprise single stranded nucleic acids. Such probes are typically at least about 50 nucleotides in length. In some embodiments, probes comprise about 100 to about 500 nucleotides. Probes that hybridize with centromeric DNA and locus- specific DNA or RNA are available commercially, for example, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively, probes can be made non-commercially from chromosomal or genomic DNA or other sources of nucleic acids through standard techniques. Examples of probes, labeling and hybridization methods are known in the art.

[0090] Several variations of FISH methods are known in the art and are suitable for use according to the methods of the disclosure, including single-molecule RNA FISH, Fiber FISH, Q-FISH, Flow-FISH, MA-FISH, break-away FISH, hybrid fusion-FISH, and multifluor FISH or mFISH. In some embodiments, FGFR3 alterations in a cancer (e.g., in a sample from a cancer) are assessed using FISH according to any suitable method known in the art, such as the methods described in Inoue et al., JAMA Network Open (2020) 3(9):e2011818.

[0091] In some embodiments, FGFR3 alterations, in a cancer (e.g., in a sample from a cancer) are assessed using a FISH probe that binds to an FGFR3 gene. In some embodiments, the FISH probe that binds to an FGFR3 gene is detectably labeled, e.g. fluorescently labeled.(ii) CGH

[0092] In some embodiments, FGFR3 alterations 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 some embodiments, 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 4 or a segment of chromosome 4 including the FGFR3 locus, or hybridize to particular FGFR3 alteration(s). After hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores.( Hi (Amplification-Based Methods

[0094] In some embodiments, FGFR3 alterations 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 FGFR3 alteration are used to specifically amplified altered FGFR3 polynucleotide(s). In some embodiments, general primers are used to amplify the FGFR3 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 FGFR3 alteration(s). In some embodiments, FGFR3 alterations in a cancer (e.g., in a sample from a cancer) are assessed using a qPCR or ddPCR method.(iv) Sequencing

[0095] In some embodiments, FGFR3 alterations 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 FGFR3 alteration(s). 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 capture-based 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, without limitation, massively parallel short-read sequencing, template-based sequencing, pyrosequencing, real-time sequencing comprising imaging the continuous incorporation of dye-labeling nucleotides during DNA synthesis, nanopore sequencing, sequencing by hybridization, nano-transistor array based sequencing, polony sequencing, scanning tunneling microscopy (STM)-based sequencing, or nanowire-molecule sensor based sequencing. See, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is hereby incorporated by reference. Exemplary NGS methods and platforms that may be used to detect FGFR3 alterations 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 FGFR3 alterations in a cancer (e.g., in a sample from a cancer) include, without limitation, the Genome Sequencer (GS) FLX System from Roche (Basel, CHE), the G.007 polonator system, the Solexa Genome Analyzer, HiSeq 2500, HiSeq3000, HiSeq 4000, and NovaSeq 6000 platforms from Illumina Inc. (San Diego, CA, USA).

[0097] In some embodiments, methods for detecting FGFR3 alterations in a cancer (e.g., in a sample from a cancer) comprise providing a sample from an individual (e.g., an individualhaving cancer), wherein the sample comprises one or more nucleic acids. In some embodiments, an FGFR3 alteration is detected directly from one or more nucleic acids from the sample. In some embodiments, an FGFR3 alteration 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 AMPure XP Beads (Agencourt) and / or solid phase reversible immobilization (SPRI) methods. In some embodiments, sequencing library construction using the purified nucleic acids is carried out using any suitable method, e.g., using commercially available library preparation kits, such as an NEBNext kit (e.g., available from New England Biolabs). In some embodiments, library preparation is performed using a “with-bead” protocol. See, e.g., Fisher et al., Genome Biol (2011) 12:R1. In some embodiments, the library preparation method is selected based on the sequencing method used, e.g., an NEBNext kit is suitable for use with NGS sequencing platforms from Illumina Inc. In some embodiments, a sequencing library indexed, e.g., with barcodes such as six base pair barcodes, is amplified, e.g., using any suitable method, such as PCR. In some embodiments, amplified nucleic acids are purified using any suitable method, such as SPRI purification. In some embodiments, the methods further comprise quantifying the amplified and / or purified nucleic acids, e.g., by qPCR. In some embodiments, the methods further comprise sizing the amplified and / or purified nucleic acids using any suitable method, such as using a LabChip GX system, e.g., available from Caliper Life Sciences. In some embodiments, size selection is not performed.

[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 FGFR3 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 isperformed 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 FGFR3 alteration(s). 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 FGFR3 alteration(s)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 a region of a captured nucleic acid molecule), (vi) sequencing the nucleic acid molecules extracted from the sample (or library proxies derived therefrom) using, e.g., a next-generation (massively parallel) sequencing technique, a whole genome sequencing (WGS) technique, a whole exome sequencing technique, a targeted sequencing technique, a direct sequencing technique, or a Sanger sequencing technique) using, e.g., a next-generation (massively parallel) sequencer, and (vii) generating, displaying, transmitting, and / or delivering a report (e.g., an electronic, web-based, or paper report) to the individual (or patient), a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office. In some instances, the report comprises output from the methods described herein. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal. In some instances, the report is transmitted via a computer network or peer-to-peer connection.

[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 FGFR3 gene / coding sequence or portion thereof; (b) ligating one or more adapters onto one or more nucleic acid molecules from the pluralityof 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 FGFR3 gene / coding sequence or portion thereof; (f) analyzing the plurality of sequence reads to determine presence or absence of an FGFR3 alteration, e.g., as described above; and (g) based on the analysis, detecting the presence or absence of an FGFR3 alteration 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 FGFR3 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 FGFR3 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 FGFR3 alteration, e.g., as described above; (g) detecting, based on the analyzing step, the presence or absence of the FGFR3r 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 biopsysample; 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 the original 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 FGFR3. In some embodiments, the selectivelyenriching 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 FGFR3 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 FGFR3 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 FGFR3 alterations provided herein (see, e.g., sections i-iv 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, or any combination thereof). In some embodiments, the presence or absence of one or more gene alterations of the disclosure is detected using any suitable method known in the art, e.g., as described in Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, base substitution alterations are detected using Bayesian methodology, which allows detection of novel somatic mutations at low mutant allele frequency (MAF) and increased sensitivity for mutations at hotspot sites through the incorporation of tissue- specific prior expectations. See, e.g., Kim et al., Cancer Discov (2011) 1:44-53 and Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, insertion / deletion (indel) alterations are detected using any suitable method, such as de novo local assembly, e.g., using the de Bruijn approach, see, e.g., Compeau et al., Nat Biotechnol (2011) 29:987-991 and Frampton et al., (2013) Nat Biotechnol, 31:1023- 1031. In some embodiments, gene fusion and genomic rearrangement alterations are detected using any suitable method, such as by analyzing chimeric read pairs (read pairs for which reads map to separate chromosomes, or at a distance of over 10 Mbp), see, e.g., Frampton et al., (2013) Nat Biotechnol, 31:1023-1031. In some embodiments, rearrangements are annotated for predicted function (e.g., creation of fusion gene or tumor suppressor inactivation).

[0109] In some embodiments of any of the methods for detection of FGFR3 alterations provided herein (see, e.g., sections i-iv herein), the methods further comprise generating amolecular profile for the individual or the sample, based, at least in part, on detecting the presence or absence of the FGFR3 alteration. 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 sequencing-based test. In some instances, a molecular profile may comprise information on the presence of genes (or variant sequences thereof), copy number variations, epigenetic traits, proteins (or modifications thereof), and / or other biomarkers in an individual’s genome and / or proteome, as well as information on the individual’s corresponding phenotypic traits and the interaction between genetic or genomic traits, phenotypic traits, and environmental factors.

[0110] In some embodiments of any of the methods for detection of FGFR3 alterations provided herein (see, e.g., sections i-iv 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 FGFR3 alterations provided herein (see, e.g., sections i-iv herein), the methods further comprise generating a report indicating the presence or absence of an FGFR3 alteration in the sample. In some embodiments of any of the methods for detection of FGFR3 alterations provided herein (see, e.g., sections i-iv herein), the methods further comprise generating, by one or more processors, a report indicating the presence or absence of an FGFR3 alteration 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 FGFR3 alterations provided herein (see, e.g., sections i-iv herein), the methods for determining the presence or absence of an FGFR3 alteration 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 variantpanel 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 FGFR3 alteration 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 FGFR3 alteration in a given patient sample. In some embodiments, the genomic profiling process further comprises measurement of TMB or detection of high TMB, e.g. , as disclosed herein.

[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 acid molecules 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.(v) Probes, Baits and Oligonucleotides

[0113] Also provided herein are probes, baits and oligonucleotides suitable for the detection of an FGFR3 alteration, e.g., according to any methods of detection known in the art and / or described herein.Probes

[0114] Provided herein are probes suitable for the detection of an FGFR3 alteration, 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 FGFR3), 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 FGFR3 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 about10 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 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 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 someembodiments, 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 FGFR3 alteration provided herein. For example, a probe provided herein may be used for detecting an FGFR3 alteration 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 FGFR3 alteration. 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] Chromosomal probes, e.g., for use in the FISH methods described herein, are typically about 50 to about 105nucleotides in length. Longer probes typically comprise smaller fragments of about 100 to about 500 nucleotides. Probes that hybridize with centromeric DNA and locus-specific DNA are available commercially, for example, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively, probes can be made non-commercially from chromosomal or genomic DNA through standard techniques. For example, sources of DNA that can be used include genomic DNA, cloned DNA sequences, somatic cell hybrids that contain one, or a part of one, chromosome (e.g., human chromosome) along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection. The region of interest can be isolated through cloning, or by site-specific amplification via the polymerase chain reaction (PCR). Probes of the disclosure may also hybridize to RNA molecules, e.g., mRNA, such as a CD274 gene product (e.g., a PD-L1 mRNA).

[0117] In some embodiments, probes, such as probes for use in the FISH methods described herein, are labeled such that a chromosomal region or a region on an RNA to which the probes hybridize can be detected. Probes typically are directly labeled with a fluorophore, allowing the probe to be visualized without a secondary detection molecule. Probes can also be labeled by nick translation, random primer labeling or PCR labeling. Labeling may be accomplished using fluorescent (direct)-or haptene (indirect)-labeled nucleotides. Representative, non-limiting examples of labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP, Fluorescein- 12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP, Cy5- dUTP, Biotin(BIO)-l l-dUTP, Digoxygenin(DIG)-l l-dUTP and Dinitrophenyl (DNP)-l l- dUTP. Probes can also be indirectly labeled with biotin or digoxygenin, or labeled with radioactive isotopes such as32P and3H, and secondary detection molecules may be used, or further processing may be performed, to visualize the probes. For example, a probe labeled with biotin can be detected by avidin conjugated to a detectable marker, e.g., avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. Enzymatic markers can be detected in standard colorimetric reactions using a substrate and / or a catalyst for the enzyme. Catalysts for alkaline phosphatase include 5-bromo-4-chloro-3- indolylphosphate and nitro blue tetrazolium. Diaminobenzoate can be used as a catalyst for horseradish peroxidase. Probes can also be prepared such that a fluorescent or other label is added after hybridization of the probe to its target to detect that the probe hybridized to the target. For example, probes can be used that have antigenic molecules incorporated into the nucleotide sequence. After hybridization, these antigenic molecules are detected, for example, using specific antibodies reactive with the antigenic molecules. Such antibodies can, for example, themselves incorporate a fluorochrome, or can be detected using a second antibody with a bound fluorochrome. For fluorescent probes, e.g., used in FISH techniques, fluorescence can be viewed with a fluorescence microscope equipped with an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the chromosomal probes.Baits

[0118] Provided herein are baits suitable for the detection of an FGFR3 alteration, e.g., according to any methods of detection known in the art and / or described herein.

[0119] In some embodiments of the methods provided herein, nucleic acid molecules (e.g., corresponding to one or more genes, such as FGFR3) 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 FGFR3). In some embodiments, the fragment comprises (or is) between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the fragment comprises (or is) about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, or about 300 nucleotides in length. In some embodiments, the capture nucleic acid molecule comprises (or is) between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises (or is) about 100 nucleotides, about 125 nucleotides, about 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 FGFR3 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. Insome embodiments, the capture nucleic acid molecule is about 150 nucleotides. In some embodiments, the capture nucleic acid molecule comprises about 170 nucleotides. In some embodiments, the capture nucleic acid molecule is about 170 nucleotides.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.Oligonucleotides

[0124] Provided herein are oligonucleotides, e.g., useful as primers, suitable for the detection of an FGFR3 alteration, e.g., according to any methods of detection known in the art and / or described herein.

[0125] 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 FGFR3 gene), or a fragment or portion thereof. In some embodiments, the oligonucleotide comprises a nucleotide sequence configured to hybridize to an FGFR3 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 FGFR3 gene), or a fragment thereof. In some embodiments, the oligonucleotide comprises a nucleotide sequence corresponding to a gene, such as an FGFR3 gene. In some embodiments, the oligonucleotide comprises a nucleotide sequence corresponding to a fragment or a portion of a gene, such as an FGFR3 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 FGFR3 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 FGFR3 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.

[0126] 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 thetarget nucleotide sequence, e.g., under high stringency conditions. In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence that is sufficiently complementary to its target nucleotide sequence such that the oligonucleotide specifically hybridizes to a nucleic acid molecule comprising the target nucleotide sequence under conditions that allow a polymerization reaction (e.g., PCR) to occur.

[0127] 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 FGFR3 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 FGFR3 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 FGFR3 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 FGFR3 gene, or a portion thereof), or fragment thereof, e.g., using a PCR reaction.

[0128] 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.

[0129] 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 providedherein comprises at least about 12 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 15 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 30 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 30 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 25 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 15 deoxyribonucleotides or ribonucleotides. In 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.

[0130] 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 FGFR3 alteration. The cytogenetic abnormality may be any cytogenetic abnormality that is or results in an FGFR3 alteration. 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.B. Tumor Mutational Burden

[0131] In some embodiments, the methods provided herein comprise acquiring knowledge of or detecting the level of tumor mutational burden in a cancer of the disclosure.

[0132] As demonstrated herein, FGFR3 alterations in cancer with high tumor mutational burden may be predictive of increased survival and / or increased likelihood of response when treated with an immunotherapy, such as an immune checkpoint inhibitor. Accordingly, in some embodiments, provided herein are methods that comprise acquiring knowledge of or detecting high tumor mutational burden in a cancer. In some embodiments, the methods of the disclosure also comprise acquiring knowledge of or detecting FGFR3 gene copy number alteration(s) in the cancer (see, e.g., Section A, above).

[0133] In some embodiments, acquiring knowledge of or detecting the level of tumor mutational burden in a cancer of the disclosure comprises measuring the level of tumor mutational burden in a sample, e.g., in a sample from a cancer or a tumor, obtained from an individual.

[0134] In some embodiments, tumor mutational burden is assessed in sample from an individual, such as sample described herein. In some embodiments, the sample from the individual comprises fluid, cells, or tissue. In some embodiments, the sample from the individual comprises a tumor biopsy or a circulating tumor cell. In some embodiments, the sample from the individual comprises nucleic acids. In some embodiments, the sample from the individual comprises mRNA, DNA, circulating tumor DNA, cell-free DNA, or cell-free RNA.

[0135] In some embodiments, tumor mutational burden is measured using any suitable method known in the art. For example, tumor mutational burden may be measured using whole-exome sequencing (WES), next-generation sequencing (NGS), whole genome sequencing, gene-targeted sequencing, or sequencing of a panel of genes, e.g., panels including cancer-related genes. See, e.g., Melendez et al., Transl Lung Cancer Res (2018) 7(6): 661-667. In some embodiments, tumor mutational burden is measured using gene- targeted sequencing, e.g., using a nucleic acid hybridization-capture method, e.g., coupled with sequencing. See, e.g., Fancello et al., J Immunother Cancer (2019) 7:183. In some embodiments, TMB 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. In some embodiments, TMB is detected usinga massively parallel sequencing (MPS) technique, and the massively parallel sequencing technique comprises next-generation sequencing (NGS).

[0136] In some embodiments, tumor mutational burden is measured according to the methods provided in WO2017151524A1. In some embodiments, tumor mutational burden is measured according to the methods described in Montesion, M., et al., Cancer Discovery (2021) ll(2):282-92.

[0137] In some embodiments, tumor mutational burden is measured according to the methods described in Chalmers et al., Genome Med (2017) 19;9( 1 ):34. In some embodiments, tumor mutational burden is assessed as the number of somatic, coding, base substitution, and indel mutations per megabase of genome examined. In some embodiments, all base substitutions and indels in the coding regions of targeted genes, including synonymous alterations, are counted. In some embodiments, assessment of tumor mutational burden further comprises filtering / counting of the base substitutions and indels, comprising one or more, or all, of the following steps: (a) synonymous mutations are counted in order to reduce sampling noise;(b) non-coding alterations are not counted; (c) alterations listed as known somatic alterations in COSMIC (see, e.g., cancer.sanger.ac.uk / cosmic) and truncations in tumor suppressor genes are not counted; (d) alterations predicted to be germline by a somatic-germline zygosity algorithm (Sun et al., Cancer Res. 2014;74(19S): 1893) are not counted; (e) alterations that are recurrently predicted to be germline are not counted; (f) known germline alterations in dbSNP (see, e.g., ncbi.nlm.nih.gov / snp / ) are not counted; and (g) germline alterations occurring with two or more counts in the ExAC database (Lek et al., Nature. 2016;536:285- 91.) are not counted. In some embodiments, to calculate the tumor mutational burden per megabase, the total number of mutations counted (e.g., as described above) is divided by the size of the coding region of the targeted territory. In some embodiments, the nonparametric Mann-Whitney U test is used to test for significance in difference of means between two populations.

[0138] In some embodiments, tumor mutational burden is assessed based on the number of non-driver somatic coding mutations / megabase (mut / Mb) of genome sequenced. In some embodiments, TMB is assessed based on number of synonymous and non-driver non- synonymous mutations.

[0139] In some embodiments, tumor mutational burden is measured in the sample by whole exome sequencing, e.g., assessed on sequenced DNA. In some embodiments, tumor mutational burden is measured in the sample using next-generation sequencing, e.g., assessed on sequenced DNA. In some embodiments, tumor mutational burden is measured in thesample using whole genome sequencing, e.g., assessed on sequenced DNA. In some embodiments, tumor mutational burden is measured in the sample by gene-targeted sequencing, e.g., assessed on sequenced DNA. In some embodiments, tumor mutational burden is measured on, or assessed across, at least about 0.8 Mb of sequenced DNA. In some embodiments, tumor mutational burden is measured on, or assessed across, between about 0.8 Mb and about 1.2 Mb of sequenced DNA. In some embodiments, tumor mutational burden is measured on, or assessed across, any of about 0.8 Mb, about 0.81 Mb, about 0.82 Mb, about 0.83 Mb, about 0.84 Mb, about 0.85 Mb, about 0.86 Mb, about 0.87 Mb, about 0.88 Mb, about 0.89 Mb, about 0.9 Mb, about 0.91 Mb, about 0.92 Mb, about 0.93 Mb, about 0.94 Mb, about 0.95 Mb, about 0.96 Mb, about 0.97 Mb, about 0.98 Mb, about 0.99 Mb, about 1 Mb, about 1.01 Mb, about 1.02 Mb, about 1.03 Mb, about 1.04 Mb, about 1.05 Mb, about 1.06 Mb, about 1.07 Mb, about 1.08 Mb, about 1.09 Mb, about 1.1 Mb, or about 1.2 Mb. In some embodiments, the amount of sequenced DNA on which TMB is measured or assessed across can be discontinuous. In some embodiments, the sequenced DNA on which TMB is measured or assessed across can represent exonic DNA. In some embodiments, TMB is only measured on or assessed across exonic DNA.

[0140] In some embodiments, a cancer of the disclosure has a high tumor mutational burden, wherein the cancer has a tumor mutational burden of at least about 5 mut / Mb. In some embodiments, a cancer of the disclosure has a high tumor mutational burden, wherein the cancer has a tumor mutational burden of at least about 10 mut / Mb. In some embodiments, the cancer has a tumor mutational burden of at least about 20 mut / Mb. In some embodiments, the cancer has a tumor mutational burden of any of between about 10 mut / Mb and about 15 mut / Mb, between about 10 mut / Mb and about 20 mut / Mb, between about 10 mut / Mb and about 30 mut / Mb, between about 10 mut / Mb and about 40 mut / Mb, between about 10 mut / Mb and about 50 mut / Mb, between about 10 mut / Mb and about 100 mut / Mb, between about 15 mut / Mb and about 20 mut / Mb, between about 20 mut / Mb and about 25 mut / Mb, between about 25 mut / Mb and about 30 mut / Mb, between about 30 mut / Mb and about 35 mut / Mb, between about 35 mut / Mb and about 40 mut / Mb, between about 40 mut / Mb and about 45 mut / Mb, between about 45 mut / Mb and about 50 mut / Mb, between about 50 mut / Mb and about 55 mut / Mb, between about 55 mut / Mb and about 60 mut / Mb, between about 60 mut / Mb and about 65 mut / Mb, between about 65 mut / Mb and about 70 mut / Mb, between about 70 mut / Mb and about 75 mut / Mb, between about 75 mut / Mb and about 80 mut / Mb, between about 80 mut / Mb and about 85 mut / Mb, between about 85 mut / Mb and about 90 mut / Mb, between about 90 mut / Mb and about 95 mut / Mb, or between about 95mut / Mb and about 100 mut / Mb. In some embodiments, the cancer has a tumor mutational burden of any of between about 100 mut / Mb and about 110 mut / Mb, between about 110 mut / Mb and about 120 mut / Mb, between about 120 mut / Mb and about 130 mut / Mb, between about 130 mut / Mb and about 140 mut / Mb, between about 140 mut / Mb and about 150 mut / Mb, between about 150 mut / Mb and about 160 mut / Mb, between about 160 mut / Mb and about 170 mut / Mb, between about 170 mut / Mb and about 180 mut / Mb, between about 180 mut / Mb and about 190 mut / Mb, between about 190 mut / Mb and about 200 mut / Mb, between about 210 mut / Mb and about 220 mut / Mb, between about 220 mut / Mb and about 230 mut / Mb, between about 230 mut / Mb and about 240 mut / Mb, between about 240 mut / Mb and about 250 mut / Mb, between about 250 mut / Mb and about 260 mut / Mb, between about 260 mut / Mb and about 270 mut / Mb, between about 270 mut / Mb and about 280 mut / Mb, between about 280 mut / Mb and about 290 mut / Mb, between about 290 mut / Mb and about 300 mut / Mb, between about 300 mut / Mb and about 310 mut / Mb, between about 310 mut / Mb and about 320 mut / Mb, between about 320 mut / Mb and about 330 mut / Mb, between about 330 mut / Mb and about 340 mut / Mb, between about 340 mut / Mb and about 350 mut / Mb, between about 350 mut / Mb and about 360 mut / Mb, between about 360 mut / Mb and about 370 mut / Mb, between about 370 mut / Mb and about 380 mut / Mb, between about 380 mut / Mb and about 390 mut / Mb, between about 390 mut / Mb and about 400 mut / Mb, or more than 400 mut / Mb. In some embodiments, the cancer has a TMB of at least about 100 mut / Mb, at least about 110 mut / Mb, at least about 120 mut / Mb, at least about 130 mut / Mb, at least about 140 mut / Mb, at least about 150 mut / Mb, or more.

[0141] In some embodiments, measuring tumor mutational burden comprises assessing mutations in a sample derived from a cancer in an individual. In some embodiments, measuring tumor mutational burden comprises assessing mutations in a sample derived from a cancer in an individual and in a matched normal sample, e.g., a sample from the individual derived from a tissue or other source that is free of the cancer. In some embodiments, TMB is detected directly from one or more nucleic acids from the sample. In some embodiments, TMB is detected from an amplicon, sequence read, or other nucleic acid otherwise derived from one or more nucleic acids from the sample.

[0142] In some embodiments, tumor mutational burden is obtained from a plurality of sequence reads, e.g., a plurality of sequence reads obtained by sequencing nucleic acids corresponding to at least a portion of a genome (such as from an enriched or unenriched sample), e.g., according to any sequencing method known in the art or described herein (e.g., as described above, in Section A). In some embodiments, tumor mutational burden isdetermined based on the number of non-driver somatic coding mutations per megabase of genome sequenced.

[0143] In some embodiments, any of the methods of the present disclosure comprise acquiring knowledge of one or more FGFR3 alterations (e.g., in a sample obtained from an individual), and acquiring knowledge of tumor mutational burden (e.g., in a sample obtained from an individual). In some embodiments, any of the methods of the present disclosure comprise detecting one or more FGFR3 alterations (e.g., in a sample obtained from an individual), and acquiring knowledge of tumor mutational burden (e.g., in a sample obtained from an individual). In some embodiments, any of the methods of the present disclosure comprise acquiring knowledge of one or more FGFR3 alterations (e.g., in a sample obtained from an individual), and detecting or determining tumor mutational burden (e.g., in a sample obtained from an individual). In some embodiments, any of the methods of the present disclosure comprise detecting one or more FGFR3 alterations (e.g., in a sample obtained from an individual), and detecting or determining tumor mutational burden (e.g., in a sample obtained from an individual).

[0144] In some embodiments of any of the methods of the disclosure, the samples used to detect / determine one or more FGFR3 alterations and tumor mutational burden are the same (i.e., FGFR3 alterations and tumor mutational burden are detected / determined in one sample), e.g., the FGFR3 alteration and the high TMB are detected in nucleic acids or sequence reads derived therefrom from the same sample. In some embodiments of any of the methods of the disclosure, the samples used to detect / determine one or more FGFR3 alterations and tumor mutational burden are different (i.e., FGFR3 alterations are detected / determined in one sample; and tumor mutational burden is detected / determined in another sample), e.g., the FGFR3 alteration and the high TMB are detected in nucleic acids or sequence reads derived therefrom from different samples.C. PD-L1 Expression

[0145] In some embodiments, the methods provided herein comprise acquiring knowledge of or detecting the level of PD-L1 expression in a cancer of the disclosure. In some embodiments, acquiring knowledge of or detecting the level of PD-L1 expression in a cancer of the disclosure comprises measuring PD-L1 expression in a sample, e.g., in a sample from a cancer obtained from an individual. In some embodiments, a report or molecular profile of the present disclosure further indicates PD-L1 expression status (e.g., protein expression) ofthe cancer, e.g., of an individual of the present discosure. In some embodiments, the cancer is PD-L1 negative. In other embodiments, the cancer is PD-L1 positive.

[0146] Any suitable method for measuring PD-L1 expression in a sample from an individual may be used. For example, the level of PD-L1 expression may be measured using immunohistochemistry (IHC), Western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), fluorescence activated cell sorting (FACS), MassARRAY, proteomics (e.g., mass spectrometry), quantitative blood based assays (as for example serum ELISA), biochemical enzymatic activity assays, in situ hybridization, Northern analysis, polymerase chain reaction (“PCR”) including quantitative real time PCR (qRT-PCR) and other amplification-based methods, RNA-sequencing (RNA-seq), FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression (“SAGE”). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.

[0147] In some embodiments, PD-L1 expression in a sample from an individual is measured based on the level of PD-L1 mRNA in the sample. Any suitable method for measuring mRNA expression in a sample from an individual may be used. For example, the level of PD- L1 mRNA expression may be measured using in situ hybridization, Northern analysis, polymerase chain reaction (“PCR”) including quantitative real time PCR (qRT-PCR) and other amplification-based methods, RNA-sequencing (RNA-seq), FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression (“SAGE”).

[0148] In some embodiments, PD-L1 expression in a sample from an individual is measured based on the level of PD-L1 protein in the sample. Any suitable method for measuring protein expression in a sample from an individual may be used. For example, the level of PD- L1 protein expression may be measured using immunohistochemistry (IHC), Western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), fluorescence activated cell sorting (FACS), proteomics (e.g., mass spectrometry), quantitative blood based assays (as for example serum ELISA), biochemical enzymatic activity assays, or multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”).

[0149] In some embodiments, PD-L1 expression is measured by immunohistochemistry using commercially available antibody clones 22C3 (Dako / Agilent) or SP142 (Ventana), e.g., according to methods known in the art and / or described herein.

[0150] In some embodiments, a cancer provided herein is determined to be positive for PD- L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of tumor infiltrating immune cells (ICs) and / or tumor cells (TCs), e.g., in a sample from an individual, express PD-L1 protein and / or PD-L1 mRNA (e.g., are positive for PD-L1 protein and / or PD-L1 mRNA). In some embodiments, a sample from an individual is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of tumor infiltrating immune cells (ICs) and / or tumor cells (TCs) in the sample express PD-L1 protein and / or PD-L1 mRNA (e.g., are positive for PD-L1 protein and / or PD-L1 mRNA). In some embodiments, a cancer provided herein is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the tumor area is occupied by PD-L1 -expressing tumor-infiltrating immune cells. In some embodiments, a sample from an individual is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the tumor area is occupied by PD-L1 -expressing tumor-infiltrating immune cells.

[0151] In some embodiments, the level of PD-L1 protein and / or PD-L1 mRNA is assessed in a sample from an individual, such as a sample described herein. In some embodiments, the sample from the individual comprises fluid, cells, or tissue. In some embodiments, the sample from the individual comprises a tumor biopsy or a circulating tumor cell. In some embodiments, the sample is obtained or derived from a cancer or the disclosure.

[0152] In some embodiments of any of the methods provided herein, a sample from an individual, e.g., an individual having a cancer, is determined to be PD-L1 -negative if less than 1% of tumor cells in the sample express PD-L1. In some embodiments of any of the methods provided herein, a sample from an individual having a cancer is determined to be PD-L1 positive if at least about 1% of tumor cells in the sample express PD-L1.

[0153] In some embodiments, the level of PD-L1 protein expression is measured using a VENTANA PD-L1 assay (SP142). In some embodiments, the level of PD-L1 protein expression is determined based on PD-L1 expression in tumor infiltrating immune cells (ICs) and / or tumor cells (TCs) using a VENTANA PD-L1 assay (SP142). Additional information about the VENTANA SP142 assay may be found in the website: www[dot]accessdata[dot]fda[dot]gov / cdrh_docs / pdfl6 / P160002c.pdf. In some embodiments, a cancer provided herein is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of tumor infiltrating immune cells (ICs) and / or tumor cells (TCs), e.g., in a sample from an individual, express PD-L1 protein (e.g., are positive for PD-L1 protein). In some embodiments, a sample from an individual is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of tumor infiltrating immune cells (ICs) and / or tumor cells (TCs) in the sample express PD-L1 protein (e.g., are positive for PD-L1 protein). In some embodiments, a cancer provided herein is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at leastabout 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the tumor area is occupied by PD-L1- expressing tumor- infiltrating immune cells of any intensity (IC). In some embodiments, a sample from an individual is determined to be positive for PD-L1 if at least about 1% (e.g., any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the tumor area is occupied by PD-L1 -expressing tumor-infiltrating immune cells of any intensity (IC).

[0154] In some embodiments, the level of PD-L1 protein expression is assessed based on a tumor proportion score (TPS). The TPS is the percentage of tumor cells showing partial or complete PD-L1 membrane staining (e.g., at a >1+ intensity on a 0, 1+, 2+, and 3 scale) relative to all tumor cells present in the sample. In some embodiments, the TPS is calculated as: the number of PD-L1 -positive tumor cells / Total number of PD-L1 -positive tumor cells + Total number of PD-L1 -negative tumor cells. A PD-L1 low positive status refers to a TPS of between 1% and 49%, PD-L1 high positive status refers to a TPS of 50% or greater, and a PD-L1 negative status refers to a TPS of less than 1%. In some embodiments, a cancer of the disclosure is determined to be PD-L1 positive if it has PD-L1 low positive status or a PD-L1 high positive status. In some embodiments, a cancer of the disclosure is PD-L1 positive (e.g., the cancer is determined have a TPS of any of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, in a sample obtained from an individual having the cancer). In some embodiments, a cancer of the disclosure is PD-L1 low positive (e.g., the cancer is determined have a TPS of any of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, or about 49%, in a sample obtained from an individual having the cancer). In some embodiments, a cancer of the disclosure is PD-L1 high positive (e.g., the cancer is determined have a TPS of any of about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, in a sample obtained from an individual having the cancer). In some embodiments, a cancer of the disclosure is PD-L1 negative (e.g., the cancer is determined have a TPS of less than 1%, in a sample obtained from an individual having the cancer). In some embodiments, the TPS is determined using a DAKO 22C3 assay. Additional information about the DAKO 22C3 assay and the TPS score may be found, e.g., in the website: www [dot] agilent[dot]com / cs / library / usermanuals / public / 29158_pd-l 1 -ihc-22C3-pharmdx- nsclc-interpretation-manual.pdf.

[0155] In some embodiments, PD-L1 expression is assessed based on a combined positive score (CPS). The CPS refers to the number of PD-L1 staining cells (e.g., tumor cells, lymphocytes, or macrophages) divided by the total number of viable tumor cells, and multiplied by 100. See, e.g., www[dot]agilent[dot]com / en / product / pharmdx / pd-ll-ihc-22c3- pharmdx-overview#pink3. In some embodiments, a cancer of the disclosure has a CPS of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10. In some embodiments, a cancer of the disclosure has high PD-L1 expression, e.g., with a CPS of at least about 1, such as between about 1 and about 5, between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, between about 25 and about 30, between about 30 and about 35, between about 35 and about 40, between about 40 and about 45, between about 45 and about 50, between about 50 and about 55, between about 55 and about 60, between about 60 and about 65, between about 65 and about 70, between about 70 and about 75, between about 75 and about 80, between about80 and about 85, between about 85 and about 90, between about 90 and about 95, or about 100. In some embodiments, PD-L1 expression based on CPS is assessed using a DAKO 22C3 assay. Additional information about the DAKO 22C3 assay and the CPS may be found, e.g., in the websites: www[dot]agilent[dot]com / en / product / pharmdx / pd-ll-ihc-22c3- pharmdx-overview#pink3 ; www[dot]agilent[dot]com / cs / library / usermanuals / public / 29171_22C3-ihc-pharmdx- interpretation-manual-eu.pdf; www[dot]agilent[dot]com / cs / library / usermanuals / public / 13350a_eu_urothelial_carcinoma_m terpretation_manual_r3v9_fin_150_single.pdf.pdf; and www[dot]agilent[dot]com / cs / library / usermanuals / public / 29314_22c3_pharmDx_hnscc_mter pretation_manual_us .pdf.

[0156] In some embodiments of any of the methods provided herein, PD-L1 expression is assessed using a companion diagnostic device, e.g., as provided in www[dot]fda[dot]gov / medical-devices / in-vitro-diagnostics / list-cleared-or-approved- companion-diagnostic-devices-in-vitro-and-imaging-tools.D. Samples

[0157] 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 FGFR3 alteration(s), tumor mutational burden, or PD-L1 expression. 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, a sample of the present disclosure is obtained or derived from a cancer or tumor. In some embodiments, the individual is a human.

[0158] 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).

[0159] 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 blood sample). 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.

[0160] 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 FGFR3 alterations, tumor mutational burden, or PD-L1 expression) or proteins (e.g., for use in any of the methods fordetection of FGFR3 alterations or PD-L1 expression provided herein) 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.

[0161] 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).

[0162] 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).

[0163] In some embodiments, the sample is obtained from an individual having a cancer, such as a cancer described herein. In some embodiments, the methods provided herein comprise obtaining one or more samples from the individual (e.g., the individual having a cancer). In some embodiments, the one or more samples are obtained or derived from a cancer (e.g., a cancer in an individual). In some embodiments, the one or more samples comprise at least 20% tumor cell nuclear area.

[0164] In some embodiments, the sample is a control sample or a reference sample, e.g., not containing an FGFR3 alteration and / or high tumor mutational burden. 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.

[0165] In some embodiments, an FGFR3 alteration and / or high tumor mutational burden are 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 FGFR3 alteration and / or high tumor mutational burden are detected in a sample comprising cell-free DNA (cfDNA), cell-free RNA, and / or circulating tumor DNA (ctDNA). In some embodiments, an FGFR3 alteration and / or high tumor mutational burden are detected in a sample comprising cell-free DNA (cfDNA) and / or circulating tumor DNA (ctDNA). In some embodiments, an FGFR3 alteration and / or high tumor mutational burden are detected in a sample comprising circulating tumor DNA (ctDNA).

[0166] 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., FGFR3 alterations, tumor mutational burden, and / or PD-L1 expression) in one or more samples (e.g., as described above) obtained from an individual (e.g., an individual having a cancer). In some embodiments, the samples used to acquire knowledge of or detect any of the biomarkers described herein (e.g., FGFR3 alterations, tumor mutational burden, and / or PD-L1 expression) are the same sample (i.e., one or more, or all, of FGFR3 alterations, tumor mutational burden, and / or PD-L1 expression are detected or determined in one sample). In some embodiments, the samples used to acquire knowledge of or detect any of the biomarkers described herein (e.g., FGFR3 alterations, tumor mutational burden, and / or PD- L1 expression) comprise more than one sample (e.g., some of the biomarkers may be detected or determined in one sample, and some of the biomarkers may be detected or determined in another sample). For example, in some embodiments, FGFR3 alterations may be detected in one sample, and tumor mutational burden may be detected or determined in another sample; or FGFR3 alterations and TMB may be detected or determined in one sample, and PD-L1 expression may be detected or determined in another sample. In some embodiments, an FGFR3 alteration and / or high TMB are detected in nucleic acids obtained from a sample of the present disclosure. In some embodiments, an FGFR3 alteration and / or high TMB are detected in sequence reads obtained or derived from nucleic acids obtained from a sample of the present disclosure. For example, the sequence reads can be obtained or derived directly from nucleic acids obtained from the sample, or the sequence reads can be obtained or derived from amplicons or a library generated from nucleic acids obtained from the sample.E. Cancers and Methods Related Thereto

[0167] 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.

[0168] 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 FGFR3 alteration and a high tumor mutational burden. 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 FGFR3 alteration and a high tumor mutational burden.

[0169] In some embodiments of any of the methods provided herein, detection of an FGFR3 alteration and a high tumor mutational burden 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 treatment comprising an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy .

[0170] In some embodiments, the methods comprise detecting, in one or more samples obtained from the individual at a first time point, the presence or absence of an FGFR3 alteration and a high tumor mutational burden. In some embodiments, the methods further comprise detecting, in one or more samples obtained from the individual at a second time point after the first time point, the presence or absence of an FGFR3 alteration and a high tumor mutational burden. In some embodiments, the methods further comprise providing an assessment of cancer progression or cancer recurrence in the individual based, at least in part, on the presence or absence of the FGFR3 alteration and high tumor mutational burden in the one or more samples obtained at the first and / or second time points. In some embodiments,the presence of the FGFR3 alteration and high tumor mutational burden in the one or more samples obtained from the individual at the first and / or second time points identifies the individual as having decreased risk of cancer progression or cancer recurrence when treated with a treatment comprising an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy. In some embodiments, the methods further comprise selecting a treatment, administering a treatment, adjusting a treatment, adjusting the dose of a treatment, or applying a treatment to the individual based, at least in part, on detecting the presence of the FGFR3 alteration and high tumor mutational burden in the one or more samples obtained from the individual at the first and / or second time points, wherein the treatment comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy.

[0171] In some embodiments, the methods comprise performing DNA sequencing on one or more samples obtained from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) to determine a sequencing mutation profile. In some embodiments, the group of genes comprises one or more cancer- related genes such as ALK, EGFR and / or FGFR3, or a panel of known / suspected oncogenes and / or tumor suppressors, or any combination thereof. In some embodiments, the sequencing mutation profile identifies the presence or absence of an FGFR3 alteration, and a high tumor mutational burden. In some embodiments, the methods further comprise identifying a candidate treatment for a cancer in an individual, based at least in part on the sequencing mutation profile. In some embodiments, the candidate treatment comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy. In some embodiments, the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) identifies the individual as one who may benefit from a treatment comprising an anti-cancer therapy, e.g., an anti-cancer therapy provided herein, such as an immunotherapy. In some embodiments, the presence of the FGFR3 alteration and high tumor mutational burden in the sample(s) predicts the individual to have longer survival when treated with a treatment comprising an anti-cancer therapy, e.g., an immunotherapy, as compared to survival of an individual whose cancer does not comprise an FGFR3 alteration and a high tumor mutational burden. In some embodiments, the sequencing comprises sequencing by any method known in the art or described herein, such as massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, a Sanger sequencing technique, or next-generation sequencing.

[0172] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based at least in part on detection of the FGFR3 alteration and high tumor mutational burden in the sample(s), wherein the one or more treatment options comprise an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy. In some embodiments, the report indicates the presence or absence of the FGFR3 alteration and high tumor mutational burden in the sample(s).

[0173] In some embodiments of any of the methods provided herein, responsive to acquisition of knowledge of an FGFR3 alteration and high tumor mutational burden 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): (i) the individual is classified as a candidate to receive a treatment comprising an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy. In some embodiments, responsive to acquisition of knowledge of the FGFR3 alteration and high tumor mutational burden in one or more samples from the individual, the individual is predicted to have longer survival when treated with a treatment comprising an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy, as compared to survival of an individual whose cancer does not comprise or exhibit an FGFR3 alteration and a high tumor mutational burden.

[0174] In some embodiments, responsive to acquisition of knowledge of an FGFR3 alteration and high tumor mutational burden 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), the methods comprise administering to the individual an effective amount of a treatment that comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy.

[0175] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based, at least in part, on knowledge of an FGFR3 alteration and high tumor mutational burden in one or more samples from the individual, wherein the one or more treatment options comprise an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an immunotherapy.

[0176] In some embodiments, acquiring knowledge of an FGFR3 alteration and high tumor mutational burden in one or more samples comprises detecting the FGFR3 alteration and hightumor mutational burden in the sample(s). In some embodiments, the methods of the disclosure further comprise providing an assessment of the FGFR3 alteration and high tumor mutational burden.

[0177] In some embodiments of any of the methods provided herein, the anti-cancer therapy is an immunotherapy, such as any immunotherapy described herein or known in the art. In some embodiments, the immunotherapy is an immune checkpoint inhibitor, a cancer vaccine, a cell-based therapy, a T cell receptor (TCR)-based therapy, an adjuvant immunotherapy, a cytokine immunotherapy, or an oncolytic virus therapy. In some embodiments, the anticancer therapy is an immune checkpoint inhibitor (ICPI), such as any immune checkpoint inhibitor described herein or known in the art. In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor; an antibody; a cellular therapy; a nucleic acid; a virusbased therapy; an antibody-drug conjugate; a recombinant protein; a fusion protein; a natural compound; a peptide; a PROteolysis-TArgeting Chimera (PROTAC); a targeted therapy; or any combination thereof, e.g., a described in further detail below. In some embodiments, the immunotherapy (e.g., an immune checkpoint inhibitor) is a monotherapy. In some embodiments, the immune checkpoint inhibitor is a first-line immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a second-line immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1- or a PD-L1- targeted agent. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor comprises one or more of nivolumab, pembrolizumab, cemiplimab, or dostarlimab. In some embodiments, the immune checkpoint inhibitor is a PD-L1 -inhibitor. In some embodiments, the immune checkpoint inhibitor comprises one or more of atezolizumab, avelumab, or durvalumab. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor comprises ipilimumab. In some embodiments, the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy. In some embodiments, the nucleic acid inhibits the expression of an immune checkpoint gene or protein. In some embodiments, the nucleic acid comprises a double- stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA), e.g., as described herein. In some embodiments, the anticancer therapy does not comprise a chemotherapy, e.g., gemcitabine and / or carboplatin.

[0178] In some embodiments of any of the methods provided herein, the methods further comprise acquiring knowledge of or detecting 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) the presence or absence of one or more alterations in one or more genes, such as a base substitution, a short insertion / deletion (indel), a copy number alteration, or a genomic rearrangement in one or more genes. In some embodiments, the one or more genes comprise one or more cancer-related genes such as ALK, EGFR and / or FGFR3, or a panel of known / suspected oncogenes and / or tumor suppressors, or any combination thereof. In some embodiments, the one or more alterations in the one or more genes are oncogenic.

[0179] In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options, e.g., the immunotherapy, further comprise an additional anticancer therapy, e.g., an immunotherapy in combination with an additional anti-cancer therapy. In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options, e.g., the immunotherapy, further comprise administering an additional anti-cancer therapy to the individual, e.g., administering an immunotherapy in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the additional anti-cancer therapy is an immunotherapy. In some embodiments, the additional anti-cancer therapy is a heat shock protein 90 inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018; Pall, Current opinion in oncology vol. 27,2 (2015): 118- 24), an EGFR inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018), a SHP2 inhibitor (Dardaei et al., Nature medicine vol. 24,4 (2018): 512-517), a MEK inhibitor (Shrestha et al., Scientific reports vol. 9,1 18842, 2019; Shrestha et al., The Journal of pharmacology and experimental therapeutics vol. 374,1 (2020): 134- 140), an IGF-1R inhibitor (George, Journal of hematology & oncology vol. 12,1 80, 2019), a vascular endothelial growth factor (VEGF) -targeted therapy (Makimoto et al., Acta medica Okayama vol. 74,5 (2020): 371-379; Gristina et al., Pharmaceuticals (Basel, Switzerland) vol. 13,12 474, 2020),an mTOR inhibitor (Kim et al., Anticancer research vol. 40,3 (2020): 1395-1403), or any combination thereof.

[0180] In some embodiments, the individual has been previously treated, or is being treated, for cancer with a treatment for cancer, e.g., an anti-cancer therapy described herein or any other anti-cancer therapy or treatment known in the art. In some embodiments, the individual has been previously treated, or is being treated, for cancer with an immune checkpoint inhibitor. In other embodiments, the individual has not been previously treated, or is not being treated, for cancer with a treatment for cancer, e.g., an anti-cancer therapy described herein or any other anti-cancer therapy or treatment known in the art. In certain embodiments, the individual has not been previously treated, or is not being treated, for cancer with an immune checkpoint inhibitor. In some embodiments, the cancer progressed on a prior treatment for cancer. In some embodiments, the individual, or the cancer, is immune checkpoint inhibitor naive. In some embodiments, the individual was previously treated, or is being treated, with a VEGF-targeted anti-cancer therapy, an EGFR-targeted anti-cancer therapy, a platinum-based chemotherapy, or a single agent chemotherapy. In some embodiments, the VEGF-targeted anti-cancer therapy is an anti-VEGF chemotherapy combination treatment. In some embodiments, the EGFR-targeted anti-cancer therapy is an EGFR tyrosine kinase inhibitor.

[0181] In some embodiments of any of the methods provided herein, the cancer is a carcinoma, a sarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a bladder cancer. In some embodiments, the cancer is a urothelial carcinoma. In some embodiments, the cancer is advanced or metastatic.

[0182] In some embodiments, the methods further comprise detecting the presence or absence of a cancer in a sample from the individual. In some embodiments, the methods further comprise administering an effective amount of anti-cancer therapy to the individual, e.g., an anti-cancer therapy described herein, such as an immunotherapy

[0183] In some embodiments, any of the cancers described herein may comprise an FGFR3 alteration and high tumor mutational burden. In some embodiments, any of the cancers described herein may be assessed for an FGFR3 alteration and / or a high tumor mutational burden using any of the methods described herein or known in the art.

[0184] In some embodiments, any of the cancers described herein may have a high tumor mutational burden. In some embodiments, any of the cancers described herein may have a high tumor mutational burden with a tumor mutational burden of at least about 5 mut / Mb, atleast about 10 mut / Mb, or at least about 20 mut / Mb. In some embodiments, any of the cancers described herein may have a high tumor mutational burden with a tumor mutational burden of at least about 10 mut / Mb. In some embodiments, any of the cancers described herein may have a tumor mutational burden of at least about 20 mut / Mb. In some embodiments, any of the cancers described herein may have a tumor mutational burden of any of between about 10 mut / Mb and about 15 mut / Mb, between about 10 mut / Mb and about 20 mut / Mb, between about 10 mut / Mb and about 30 mut / Mb, between about 10 mut / Mb and about 40 mut / Mb, between about 10 mut / Mb and about 50 mut / Mb, between about 10 mut / Mb and about 60 mut / Mb, between about 10 mut / Mb and about 70 mut / Mb, between about 10 mut / Mb and about 80 mut / Mb, between about 10 mut / Mb and about 90 mut / Mb, between about 10 mut / Mb and about 100 mut / Mb, between about 15 mut / Mb and about 20 mut / Mb, between about 20 mut / Mb and about 25 mut / Mb, between about 25 mut / Mb and about 30 mut / Mb, between about 30 mut / Mb and about 35 mut / Mb, between about 35 mut / Mb and about 40 mut / Mb, between about 40 mut / Mb and about 45 mut / Mb, between about 45 mut / Mb and about 50 mut / Mb, between about 50 mut / Mb and about 55 mut / Mb, between about 55 mut / Mb and about 60 mut / Mb, between about 60 mut / Mb and about 65 mut / Mb, between about 65 mut / Mb and about 70 mut / Mb, between about 70 mut / Mb and about 75 mut / Mb, between about 75 mut / Mb and about 80 mut / Mb, between about 80 mut / Mb and about 85 mut / Mb, between about 85 mut / Mb and about 90 mut / Mb, between about 90 mut / Mb and about 95 mut / Mb, or between about 95 mut / Mb and about 100 mut / Mb. In some embodiments, any of the cancers described herein may have a tumor mutational burden of any of between about 100 mut / Mb and about 110 mut / Mb, between about 110 mut / Mb and about 120 mut / Mb, between about 120 mut / Mb and about 130 mut / Mb, between about 130 mut / Mb and about 140 mut / Mb, between about 140 mut / Mb and about 150 mut / Mb, between about 150 mut / Mb and about 160 mut / Mb, between about 160 mut / Mb and about 170 mut / Mb, between about 170 mut / Mb and about 180 mut / Mb, between about 180 mut / Mb and about 190 mut / Mb, between about 190 mut / Mb and about 200 mut / Mb, between about 210 mut / Mb and about 220 mut / Mb, between about 220 mut / Mb and about 230 mut / Mb, between about 230 mut / Mb and about 240 mut / Mb, between about 240 mut / Mb and about 250 mut / Mb, between about 250 mut / Mb and about 260 mut / Mb, between about 260 mut / Mb and about 270 mut / Mb, between about 270 mut / Mb and about 280 mut / Mb, between about 280 mut / Mb and about 290 mut / Mb, between about 290 mut / Mb and about 300 mut / Mb, between about 300 mut / Mb and about 310 mut / Mb, between about 310 mut / Mb and about 320 mut / Mb, between about 320 mut / Mb and about 330 mut / Mb, between about 330mut / Mb and about 340 mut / Mb, between about 340 mut / Mb and about 350 mut / Mb, between about 350 mut / Mb and about 360 mut / Mb, between about 360 mut / Mb and about 370 mut / Mb, between about 370 mut / Mb and about 380 mut / Mb, between about 380 mut / Mb and about 390 mut / Mb, between about 390 mut / Mb and about 400 mut / Mb, or more than 400 mut / Mb. In some embodiments, any of the cancers described herein may have a tumor mutational burden of at least about 100 mut / Mb, at least about 110 mut / Mb, at least about 120 mut / Mb, at least about 130 mut / Mb, at least about 140 mut / Mb, at least about 150 mut / Mb, or more.

[0185] In some embodiments, overall survival (OS) refers to the length of time from the date of the start of a treatment for cancer in an individual (e.g., according to the methods provided herein), to the time of death from any cause or the time of loss of follow-up of the individual. Accordingly, in some embodiments, OS refers to the length of time from the date of the start of treatment for cancer in an individual with an anti-cancer therapy provided herein, such as an immunotherapy, to the time of death from any cause or the time of loss of follow-up of the individual. Median overall survival (e.g., of a plurality of individuals treated according to the methods of the disclosure) may be assessed using any suitable method known in the art, such as the Kaplan-Meier method, optionally in combination with a log-rank test.

[0186] In some embodiments of any of the methods provided herein, the sample is a sample described herein. In some embodiments, the sample is obtained from the individual or from the cancer. In some embodiments, the methods further comprise obtaining the sample, e.g., from the individual or 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. In some embodiments, the fusion nucleic acid molecule or polypeptide is detected in a tissue biopsy sample, in a liquid biopsy sample, or in both a tissue biopsy sample and a liquid biopsy sample, from the individual. In some embodiments, the samples used to detect / determine FGFR3 alterations and tumor mutational burden are the same (i.e., FGFR3 alterations andtumor mutational burden are detected / determined in one sample). In some embodiments of any of the methods of the disclosure, the samples used to detect / determine FGFR3 alterations and tumor mutational burden are different (i.e., FGFR3 alterations are detected / determined in one sample; and tumor mutational burden is detected / determined in another sample).F. Anti-Cancer Therapies

[0187] Certain aspects of the present disclosure relate to anti-cancer therapies (e.g., immunotherapies), as well as 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; detecting the presence or absence of a cancer in an individual; monitoring progression or recurrence of a cancer in an individual; or identifying a candidate treatment for a cancer in an individual in need thereof. The present disclosure also provides uses for anti-cancer therapies (e.g., in methods of treating or delaying progression of cancer in an individual, or in methods for manufacturing a medicament for treating or delaying progression of cancer). In some instances, the methods of the disclosure can include administering a treatment comprising an anti-cancer therapy or applying a treatment comprising an anti-cancer therapy to an individual based on a generated genomic, molecular, and / or sequencing mutation profile. An anti-cancer therapy can refer to an agent or compound that is effective in the treatment of cancer cells. Examples of anti-cancer agents, compounds, or anti-cancer therapies include, but are not limited to, alkylating agents, antimetabolites, natural products, hormones, chemotherapy, radiation therapy, immunotherapy, surgery, or a therapy configured to target a defect in a specific cell signaling pathway, e.g., a defect in a DNA mismatch repair (MMR) pathway.

[0188] In some embodiments, an anti-cancer therapy of the disclosure is a small molecule inhibitor; an antibody; a cellular therapy; a nucleic acid; a virus-based therapy; an antibodydrug conjugate; a recombinant protein; a fusion protein; a natural compound; a peptide; a PROteolysis-TArgeting Chimera (PROTAC); a targeted therapy; or any combination thereof, e.g., a described in further detail below. In some embodiments, the anti-cancer therapy is an immunotherapy, such as any immunotherapy known in the art or described herein (e.g., a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy,adjuvant immunotherapy, cytokine immunotherapy, or oncolytic virus therapy). In some embodiments, the anti-cancer therapy is an immune checkpoint inhibitor, such as any immune checkpoint inhibitor described herein or known in the art.

[0189] In some embodiments, the anti-cancer therapy comprises an immunotherapy (i.e., a cancer immunotherapy), such as a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy, as well as any combination thereof. In some embodiments, the cancer immunotherapy comprises a small molecule, nucleic acid, polypeptide, carbohydrate, toxin, cell-based agent, or cell-binding agent. Examples of cancer immunotherapies are described in greater detail herein but are not intended to be limiting. In some embodiments, the cancer immunotherapy activates one or more aspects of the immune system to attack a cell (e.g., a tumor cell) that expresses a neoantigen. The cancer immunotherapies of the present disclosure are contemplated for use as monotherapies, or in combination approaches comprising two or more in any combination or number, subject to medical judgement. Any of the cancer immunotherapies (optionally as monotherapies or in combination with another cancer immunotherapy or other therapeutic agent described herein) may find use in any of the methods described herein.

[0190] In some embodiments, the cancer immunotherapy comprises a cancer vaccine. A range of cancer vaccines have been tested that employ different approaches to promoting an immune response against a cancer (see, e.g., Emens L A, Expert Opin Emerg Drugs 13(2): 295-308 (2008) and US20190367613). Approaches have been designed to enhance the response of B cells, T cells, or professional antigen-presenting cells against tumors. Exemplary types of cancer vaccines include, but are not limited to, DNA-based vaccines, RNA-based vaccines, virus transduced vaccines, peptide-based vaccines, dendritic cell vaccines, oncolytic viruses, whole tumor cell vaccines, tumor antigen vaccines, etc. In some embodiments, the cancer vaccine can be prophylactic or therapeutic. In some embodiments, the cancer vaccine is formulated as a peptide-based vaccine, a nucleic acid-based vaccine, an antibody based vaccine, or a cell based vaccine. For example, a vaccine composition can include naked cDNA in cationic lipid formulations; lipopeptides (e.g., Vitiello, A. et al, J. Clin. Invest. 95:341, 1995); naked cDNA or peptides encapsulated, e.g., in poly(DL-lactide- co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et ah, Molec. Immunol. 28:287-294, 1991: Alonso et al, Vaccine 12:299- 306, 1994; Jones et al, Vaccine 13:675-681, 1995); peptide composition contained in immune stimulating complexes (ISCOMS) (e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin. Exp. Immunol. 113:235-243, 1998); ormultiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196: 17-32, 1996). In some embodiments, a cancer vaccine is formulated as a peptide-based vaccine, or nucleic acid based vaccine in which the nucleic acid encodes the polypeptides. In some embodiments, a cancer vaccine is formulated as an antibody-based vaccine. In some embodiments, a cancer vaccine is formulated as a cell based vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104: 14-21, 2013). In some embodiments, such cancer vaccines augment an anti-cancer response.

[0191] In some embodiments, the cancer vaccine comprises a polynucleotide that encodes a neoantigen, e.g., neoantigen(s) expressed by a cancer of the disclosure, such as a cancer in an individual. In some embodiments, the cancer vaccine comprises DNA that encodes the neoantigen(s). In some embodiments, the cancer vaccine comprises RNA that encodes the neoantigen(s). In some embodiments, the cancer vaccine comprises a polynucleotide that encodes the neoantigen(s). In some embodiments, the cancer vaccine further comprises one or more additional antigens, neoantigens, or other sequences that promote antigen presentation and / or an immune response. In some embodiments, the polynucleotide is complexed with one or more additional agents, such as a liposome or lipoplex. In some embodiments, the polynucleotide(s) are taken up and translated by antigen presenting cells (APCs), which then present the neoantigen(s) via MHC class I on the APC cell surface.

[0192] In some embodiments, the cancer vaccine is selected from sipuleucel-T (e.g., Provenge®, Dendreon / V aleant Pharmaceuticals), which has been approved for treatment of asymptomatic, or minimally symptomatic metastatic castrate -resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (e.g., Imlygic®, BioVex / Amgen, previously known as T-VEC), a genetically modified oncolytic viral therapy approved for treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma. In some embodiments, the cancer vaccine is selected from an oncolytic viral therapy such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF, for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep (e.g., Reolysin®, Oncolytics Biotech), a variant of respiratory enteric orphan virus (reovirus) which does not replicate in cells that are not RAS-activated, in numerous cancers, including colorectal cancer(NCT01622543), prostate cancer (NCT01619813), head and neck squamous cell cancer (NCT01166542), pancreatic adenocarcinoma (NCT00998322), and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAdl), an adenovirus engineered to express a full length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117), metastatic or advanced epithelial tumors such as in colorectal cancer, bladder cancer, head and neck squamous cell carcinoma and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF, in melanoma (NCT03003676), and peritoneal disease, colorectal cancer or ovarian cancer (NCT02963831); GL-ONC1 (GLV-lh68 / GLV-lhl53, Genelux GmbH), vaccinia viruses engineered to express beta-galactosidase (beta-gal) / beta-glucoronidase or beta-gal / human sodium iodide symporter (hNIS), respectively, were studied in peritoneal carcinomatosis (NCTO 1443260), fallopian tube cancer, ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818); anti-gplOO; STINGVAX; GV AX; DCVaxL; and DNX-2401. In some embodiments, the cancer vaccine is selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is able to convert the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TGO1 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapy agents targeted for difficult-to-treat RAS mutations; and TILT- 123 (TILT Biotherapeutics), an engineered adenovirus designated: Ad5 / 3-E2F-delta24-hTNFa-IRES-hIL20; and VSV-GP (ViraTherapeutics) a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express antigens designed to raise an antigen-specific CD8+ T cell response. In some embodiments, the cancer vaccine comprises a vector-based tumor antigen vaccine. Vectorbased tumor antigen vaccines can be used as a way to provide a steady supply of antigens to stimulate an anti-tumor immune response. In some embodiments, vectors encoding for tumor antigens are injected into an individual (possibly with pro-inflammatory or other attractants such as GM-CSF), taken up by cells in vivo to make the specific antigens, which then provoke the desired immune response. In some embodiments, vectors may be used to deliver more than one tumor antigen at a time, to increase the immune response. In addition, recombinant virus, bacteria or yeast vectors can trigger their own immune responses, which may also enhance the overall immune response.

[0193] In some embodiments, the cancer vaccine comprises a DNA-based vaccine. In some embodiments, DNA-based vaccines can be employed to stimulate an anti-tumor response. The ability of directly injected DNA that encodes an antigenic protein, to elicit a protective immune response has been demonstrated in numerous experimental systems. Vaccination through directly injecting DNA that encodes an antigenic protein, to elicit a protective immune response often produces both cell-mediated and humoral responses. Moreover, reproducible immune responses to DNA encoding various antigens have been reported in mice that last essentially for the lifetime of the animal (see, e.g., Yankauckas et al.(1993) DNA Cell Biol., 12: 771-776). In some embodiments, plasmid (or other vector) DNA that includes a sequence encoding a protein operably linked to regulatory elements required for gene expression is administered to individuals (e.g. human patients, non-human mammals, etc.). In some embodiments, the cells of the individual take up the administered DNA and the coding sequence is expressed. In some embodiments, the antigen so produced becomes a target against which an immune response is directed.

[0194] In some embodiments, the cancer vaccine comprises an RNA-based vaccine. In some embodiments, RNA-based vaccines can be employed to stimulate an anti-tumor response. In some embodiments, RNA-based vaccines comprise a self-replicating RNA molecule. In some embodiments, the self-replicating RNA molecule may be an alphavirus-derived RNA replicon. Self-replicating RNA (or "SAM") molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A selfreplicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded polypeptide, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.

[0195] In some embodiments, the cancer immunotherapy comprises a cell-based therapy. In some embodiments, the cancer immunotherapy comprises a T cell-based therapy. In some embodiments, the cancer immunotherapy comprises an adoptive therapy, e.g., an adoptive T cell-based therapy. In some embodiments, the T cells are autologous or allogeneic to the recipient. In some embodiments, the T cells are CD 8+ T cells. In some embodiments, the Tcells are CD4+ T cells. Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) cancer cells. In some embodiments, the immune response results in inhibition of tumor and / or metastatic cell growth and / or proliferation, and in related embodiments, results in neoplastic cell death and / or resorption. The immune cells can be derived from a different organism / host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells). In some embodiments, the immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, or NKT cells) can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, the host cells (e.g., autologous or allogeneic T-cells) are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen. In some embodiments, NK cells are engineered to express a TCR. The NK cells may be further engineered to express a CAR. Multiple CARs and / or TCRs, such as to different antigens, may be added to a single cell type, such as T cells or NK cells. In some embodiments, the cells comprise one or more nucleic acids / expression constructs / vectors introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g. chimeric). In some embodiments, a population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. In some embodiments, a population of immune cells can be obtained from a donor, such as a histocompatibility-matched donor. In some embodiments, the immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells reside in said subject or donor. In some embodiments, the immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood. In some embodiments, when the population of immune cells is obtained from a donor distinct from the subject, the donor may be allogeneic, provided the cells obtained are subject-compatible, in that they can be introduced into the subject. In some embodiments,allogeneic donor cells may or may not be human-leukocyte-antigen (HLA)-compatible. In some embodiments, to be rendered subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

[0196] In some embodiments, the cell-based therapy comprises a T cell-based therapy, such as autologous cells, e.g., tumor-infiltrating lymphocytes (TILs); T cells activated ex-vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non-tumor- specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibodylike tumor recognition capacity known as "T- bodies". Several approaches for the isolation, derivation, engineering or modification, activation, and expansion of functional anti-tumor effector cells have been described in the last two decades and may be used according to any of the methods provided herein. In some embodiments, the T cells are derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the cells may be allogeneic and / or autologous. In some embodiments, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0197] In some embodiments, the T cell-based therapy comprises a chimeric antigen receptor (CAR)-T cell-based therapy. This approach involves engineering a CAR that specifically binds to an antigen of interest and comprises one or more intracellular signaling domains for T cell activation. The CAR is then expressed on the surface of engineered T cells (CAR-T) and administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the CAR specifically binds a neoantigen, such as a neoantigen expressed in a cancer of a disclosure, e.g., in an individual.

[0198] In some embodiments, the T cell-based therapy comprises T cells expressing a recombinant T cell receptor (TCR). This approach involves identifying a TCR that specifically binds to an antigen of interest, which is then used to replace the endogenous or native TCR on the surface of engineered T cells that are administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the recombinant TCR specifically binds a neoantigen expressed in a cancer of a disclosure, e.g., in an individual.

[0199] In some embodiments, the T cell-based therapy comprises tumor-infiltrating lymphocytes (TILs). For example, TILs can be isolated from a tumor or cancer of the present disclosure, then isolated and expanded in vitro. Some or all of these TILs may specifically recognize an antigen expressed by the tumor or cancer of the present disclosure. In some embodiments, the TILs are exposed to one or more neoantigens, e.g., expressed in a cancer of a disclosure, in vitro after isolation. TILs are then administered to the patient (optionally in combination with one or more cytokines or other immune-stimulating substances).

[0200] In some embodiments, the cell-based therapy comprises a natural killer (NK) cellbased therapy. Natural killer (NK) cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are critical effectors of the early innate immune response toward transformed and virus-infected cells. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. NK cells do not express T-cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors. In some embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art.

[0201] In some embodiments, the cell-based therapy comprises a dendritic cell (DC)-based therapy, e.g., a dendritic cell vaccine. In some embodiments, the DC vaccine comprises antigen-presenting cells that are able to induce specific T cell immunity, which are harvested from the patient or from a donor. In some embodiments, the DC vaccine can then be exposed in vitro to a peptide antigen, for which T cells are to be generated in the patient. In some embodiments, dendritic cells loaded with the antigen are then injected back into the patient. In some embodiments, immunization may be repeated multiple times if desired. Methods for harvesting, expanding, and administering dendritic cells are known in the art; see, e.g.,W02019178081. Dendritic cell vaccines (such as Sipuleucel-T, also known as APC8015 andPROVENGE®) are vaccines that involve administration of dendritic cells that act as APCs to present one or more cancer- specific antigens to the patient’s immune system. In some embodiments, the dendritic cells are autologous or allogeneic to the recipient.

[0202] In some embodiments, the cancer immunotherapy comprises a TCR-based therapy. In some embodiments, the cancer immunotherapy comprises administration of one or more TCRs or TCR-based therapeutics that specifically bind an antigen expressed by a cancer of the present disclosure, e.g., a neoantigen expressed in a cancer of a disclosure, e.g., in an individual. The TCR-based therapeutic may further include a moiety that binds an immune cell (e.g., a T cell), such as an antibody or antibody fragment that specifically binds a T cell surface protein or receptor (e.g., an anti-CD3 antibody or antibody fragment).

[0203] In some embodiments, the immunotherapy comprises adjuvant immunotherapy. Adjuvant immunotherapy comprises the use of one or more agents that activate components of the innate immune system, e.g., HILTONOL® (imiquimod), which targets the TLR7 pathway.

[0204] In some embodiments, the immunotherapy comprises cytokine immunotherapy. Cytokine immunotherapy comprises the use of one or more cytokines that activate components of the immune system. Examples include, but are not limited to, aldesleukin (e.g., PROLEUKIN®; interleukin-2), interferon alfa-2a (e.g., ROFERON®-A), interferon alfa-2b (e.g., INTRON®-A), and peginterferon alfa-2b (e.g., PEGINTRON®).

[0205] In some embodiments, the immunotherapy comprises oncolytic virus therapy. Oncolytic virus therapy uses genetically modified viruses to replicate in and kill cancer cells, leading to the release of antigens that stimulate an immune response. In some embodiments, replication-competent oncolytic viruses expressing a tumor antigen comprise any naturally occurring (e.g., from a “field source”) or modified replication-competent oncolytic virus. In some embodiments, the oncolytic virus, in addition to expressing a tumor antigen, may be modified to increase selectivity of the virus for cancer cells. In some embodiments, replication-competent oncolytic viruses include, but are not limited to, oncolytic viruses that are a member in the family of myoviridae, siphoviridae, podpviridae, teciviridae, corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae, poxyiridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae, adnoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hcpadnaviridae, retroviridae, cyctoviridae, reoviridae, birnaviridae, paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, Leviviridae, picornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae,tombusviridae, coronaviridae, glaviviridae, togaviridae, and bamaviridae. In some embodiments, replication-competent oncolytic viruses include adenovirus, retrovirus, reovirus, rhabdovirus, Newcastle Disease virus (NDV), polyoma virus, vaccinia virus (VacV), herpes simplex virus, picomavirus, coxsackie virus and parvovirus. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be engineered to lack one or more functional genes in order to increase the cancer selectivity of the virus. In some embodiments, an oncolytic vaccinia virus is engineered to lack thymidine kinase (TK) activity. In some embodiments, the oncolytic vaccinia virus may be engineered to lack vaccinia virus growth factor (VGF). In some embodiments, an oncolytic vaccinia virus may be engineered to lack both VGF and TK activity. In some embodiments, an oncolytic vaccinia virus may be engineered to lack one or more genes involved in evading host interferon (IFN) response such as E3L, K3L, B18R, or B8R. In some embodiments, a replicative oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister or Wyeth strain and lacks a functional TK gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister or Wyeth strain lacking a functional B18R and / or B8R gene. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be locally or systemically administered to a subject, e.g. via intratumoral, intraperitoneal, intravenous, intra-arterial, intramuscular, intradermal, intracranial, subcutaneous, or intranasal administration.

[0206] In some embodiments, the anti-cancer therapy comprises an immune checkpoint inhibitor. In some embodiments, the methods provided herein comprise administering to an individual an effective amount of an immune checkpoint inhibitor. As is known in the art, a checkpoint inhibitor targets at least one immune checkpoint protein to alter the regulation of an immune response. Immune checkpoint proteins include, e.g., CTLA4, PD-L1, PD-1, PD- L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CEACAM, LAIR1, CD80, CD86, CD276, VTCN1, MHC class I, MHC class II, GALS, adenosine, TGFR, CSF1R, MICA / B, arginase, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, 0X40, and A2aR. In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to: PD-1 (CD279), PD-L1 (B7- Hl, CD274), PD-L2 (B7-CD, CD273), CTLA-4 (CD152), HVEM, BTLA (CD272), a killercell immunoglobulin-like receptor (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM-1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRbeta, A2AR, GITR (CD357), CD80 (B7-1), CD86 (B7-2), CD276 (B7-H3),VTCNI (B7-H4), MHC class I, MHC class II, GALS, adenosine, TGFR, B7-H1, 0X40 (CD134), CD94 (KLRD1), CD137 (4-1BB), CD137L (4-1BBL), CD40, IDO, CSF1R, CD40L, CD47, CD70 (CD27L), CD226, HHLA2, ICOS (CD278), ICOSL (CD275), LIGHT (TNFSF14, CD258), NKG2a, NKG2d, OX40L (CD134L), PVR (NECL5, CD155), SIRPa, MICA / B, and / or arginase. In some embodiments, an immune checkpoint inhibitor (i.e., a checkpoint inhibitor) decreases the activity of a checkpoint protein that negatively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In other embodiments, a checkpoint inhibitor increases the activity of a checkpoint protein that positively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In some embodiments, the checkpoint inhibitor is an antibody. Examples of checkpoint inhibitors include, without limitation, a PD- 1 axis binding antagonist, a PD-L1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., atezolizumab (MPDL3280A)), an antagonist directed against a co-inhibitory molecule (e.g., a CTLA4 antagonist (e.g., an anti-CTLA4 antibody), a TIM-3 antagonist (e.g., an anti- TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof. In some embodiments, the immune checkpoint inhibitors comprise drugs such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (see, e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). In some embodiments, known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.

[0207] In some embodiments, the checkpoint inhibitor is a PD-L1 axis binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1,” "PDCD1 LG1," "CD274," "B7- H," and "PDL1." An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1 LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

[0208] In some embodiments, the checkpoint inhibitor is a PD-1 binding antagonist / inhibitor. In some embodiments, the PD-1 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specificembodiment, the PD-1 ligand binding partners are PD-L1 and / or PD-L2. In some embodiments, the checkpoint inhibitor is a PD-L1 binding antagonist / inhibitor. In some embodiments, a PD-L1 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-L1 to its binding ligands. In a specific embodiment, PD-L1 binding partners are PD-1 and / or B7-1. In some embodiments, the checkpoint inhibitor is a PD-L2 binding antagonist / inhibitor. In some embodiments, the PD-L2 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-L2 to its ligand binding partners. In a specific embodiment, the PD-L2 binding ligand partner is PD- 1. The antagonist or inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1, PD-L1, or PD-L1 binding antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0209] In some instances, the PD-1 binding antagonist or inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), for example, as described below. In some instances, the anti-PD-1 antibody is one or more of MDX-1 106 (nivolumab), MK-3475 (pembrolizumab, e.g., Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, BGB-108, BGB-A317, JS-001, STI-Al l 10, INCSHR-1210, PF-06801591, TSR-042, AM0001, ENUM 244C8, ENUM 388D4, cemiplimab, or dostarlimab. In other instances, the PD-1 binding antagonist or inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some instances, the PD-1 binding antagonist or inhibitor is AMP- 224. Other examples of anti-PD-1 antibodies include, but are not limited to, MEDI- 0680 (AMP-514; AstraZeneca), PDR001 (CAS Registry No. 1859072-53-9; Novartis), REGN2810 (e.g., LIBTAYO® or cemiplimab-rwlc; Regeneron), BGB-108 (BeiGene), BGB- A317 (BeiGene), BI 754091, JS-001 (Shanghai Junshi), STI-Al l 10 (Sorrento), INCSHR- 1210 (Incyte), PF-06801591 (Pfizer), TSR-042 (also known as ANB011;Tesaro / AnaptysBio), AM0001 (ARMO Biosciences), ENUM 244C8 (Enumeral Biomedical Holdings), or ENUM 388D4 (Enumeral Biomedical Holdings). In some embodiments, the PD-1 axis binding antagonist or inhibitor comprises tislelizumab (BGB-A317), BGB-108, STI-Al l 10, AM0001, BI 754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP-514), MGA-012 (INCMGA 0012), nivolumab (BMS-936558, MDX1106, ONO-4538), spartalizumab (PDR001), pembrolizumab (MK-3475, SCH 900475, e.g., Keytruda®),PF-06801591, cemiplimab (REGN-2810, REGEN2810), dostarlimab (TSR-042, ANB011), FITC-YT-16 (PD-1 binding peptide), APL-501 or CBT-501 or genolimzumab (GB-226), AB- 122, AK105, AMG 404, BCD- 100, F520, HLX10, HX008, JTX-4014, LZM009, Sym021, PSB205, AMP-224 (fusion protein targeting PD-1), CX-188 (PD-1 probody), AGEN-2034, GLS-010, budigalimab (ABBV-181), AK-103, BAT-1306, CS-1003, AM- 0001, TILT-123, BH-2922, BH-2941, BH-2950, ENUM-244C8, ENUM-388D4, HAB-21, H EISCOI 11-003, IKT-202, MCLA-134, MT-17000, PEGMP-7, PRS-332, RXI-762, STI- 1110, VXM-10, XmAb-23104, AK-112, HLX-20, SSI-361, AT-16201, SNA-01, AB122, PD1-PIK, PF-06936308, RG-7769, CAB PD-1 Abs, AK-123, MEDI-3387, MEDI-5771, 4H1128Z-E27, REMD-288, SG-001, BY-24.3, CB-201, IBI-319, ONCR-177, Max-1, CS- 4100, JBI-426, CCC-0701, or CCX- 4503, or derivatives thereof, or an antibody that competes with any of the preceding.

[0210] In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-1. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-L1 and VISTA or PD-L1 and TIM3. In some embodiments, the PD-L1 binding antagonist or inhibitor is CA-170 (also known as AUPM- 170). In some embodiments, the PD-L1 binding antagonist or inhibitor is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody can bind to a human PD-L1, for example a human PD-L1 as described above herein and / or as shown in UniProtKB / Swiss- Prot Accession No.Q9NZQ7.1, or a variant thereof. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0211] In some instances, the PD-L1 binding antagonist or inhibitor is an anti-PD-Ll antibody, for example, as described below. In some instances, the anti-PD-Ll antibody is capable of inhibiting the binding between PD-L1 and PD-1, and / or between PD-L1 and B7-1. In some instances, the anti-PD-Ll antibody is a monoclonal antibody. In some instances, the anti-PD-Ll antibody is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some instances, the anti-PD-Ll antibody is a humanized antibody. In some instances, the anti-PD-Ll antibody is a human antibody. In some instances, the anti- PD-Ll antibody is selected from YW243.55.S70, MPDL3280A (atezolizumab), MDX-1 105, MEDI4736 (durvalumab), MSB0010718C (avelumab), LY3300054, STI-A1014, KN035, FAZ053, or CX-072. In some embodiments, the PD-L1 axis binding antagonist or inhibitor comprises atezolizumab, avelumab, durvalumab (imfinzi), BGB-A333, SHR-1316 (HTL1088), CK-301, BMS-936559, envafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS- 936559), LY3300054, STI-A1014, FAZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M-7824, HTI-1088 (HTI-131, SHR-1316), MSB-2311, AK- 106, AVA-004, BBI- 801, CA-327, CBA-0710, CBT-502, FPT-155, IKT-201, IKT-703, 10-103, JS-003, KD-033, KY-1003, MCLA-145, MT-5050, SNA-02, BCD-135, APL-502 (CBT-402 or TQB2450), IMC-001, KD-045, INBRX-105, KN-046, IMC-2102, IMC-2101, KD-005, IMM-2502, 89Zr-CX-072, 89Zr-DFO-6El l, KY-1055, MEDI-1109, MT-5594, SL-279252, DSP- 106, Gensci-047, REMD-290, N-809, PRS-344, FS-222, GEN-1046, BH-29xx, or FS-118, or a derivative thereof, or an antibody that competes with any of the preceding.

[0212] In some embodiments, the checkpoint inhibitor is an antagonist or inhibitor of CTLA4. In some embodiments, the checkpoint inhibitor is a small molecule antagonist or inhibitor of CTLA4. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody. CTLA4 is part of the CD28-B7 immunoglobulin superfamily of immune checkpoint molecules that acts to negatively regulate T cell activation, particularly CD28- dependent T cell responses. CTLA4 competes for binding to common ligands with CD28, such as CD80 (B7-1) and CD86 (B7-2), and binds to these ligands with higher affinity than CD28. Blocking CTLA4 activity (e.g., using an anti-CTLA4 antibody) is thought to enhance CD28-mediated costimulation (leading to increased T cell activation / priming), affect T cell development, and / or deplete Tregs (such as intratumoral Tregs). In some embodiments, the CTLA4 antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the CTLA-4 antagonist or inhibitor comprises ipilimumab (IBB 10, BMS-734016, MDX010, MDX- CTLA4, MEDI4736), tremelimumab (CP-675, CP-675,206), APL-509, AGEN1884, CS1002, AGEN1181, Abatacept (Orencia, BMS-188667, RG2077), BCD-145, ONC-392, ADU-1604, REGN4659, ADG116, KN044, KN046, or a derivative thereof, or an antibody that competes with any of the preceding.

[0213] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 antagonist or inhibitor (e.g., an antibody, an antibody conjugate, or an antigenbinding fragment thereof). In some embodiments, the LAG-3 antagonist or inhibitor comprises a small molecule, a nucleic acid, a polypeptide (e.g., an antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the LAG-3 antagonist or inhibitor comprises a small molecule. In some embodiments, the LAG-3 antagonist or inhibitor comprises a LAG-3 binding agent. In some embodiments, the LAG-3 antagonist or inhibitor comprises an antibody, an antibody conjugate, or an antigen-binding fragment thereof. Insome embodiments, the LAG-3 antagonist or inhibitor comprises eftilagimod alpha (IMP321, IMP-321, EDDP-202, EOC-202), relatlimab (BMS-986016), GSK2831781 (IMP-731), LAG525 (IMP701), TSR-033, EVIP321 (soluble LAG-3 protein), BI 754111, IMP761, REGN3767, MK-4280, MGD-013, XmAb22841, INCAGN-2385, ENUM-006, AVA-017, AM-0003, iOnctura anti-LAG-3 antibody, Arcus Biosciences LAG-3 antibody, Sym022, a derivative thereof, or an antibody that competes with any of the preceding.

[0214] In some embodiments, the immune checkpoint inhibitor is monovalent and / or monospecific. In some embodiments, the immune checkpoint inhibitor is multivalent and / or multispecific.

[0215] In some embodiments, an anti-cancer therapy of the disclosure (e.g., an immunotherapy) is administered in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti- neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof.

[0216] In some embodiments, an anti-cancer therapy of the disclosure comprises a cyclin- dependent kinase (CDK) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the CDK inhibitor inhibits CDK4. In some embodiments, the CDK inhibitor inhibits Cyclin D / CDK4. In some embodiments, the CDK inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of CDK4, (b) an antibody that inhibits one or more activities of CDK4 (e.g., by binding to and inhibiting one or more activities of CDK4, binding to and inhibiting expression of CDK4, and / or binding to and inhibiting one or more activities of a cell expressing CDK4, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of CDK4 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the CDK inhibitor inhibits CDK4 and CDK6. In some embodiments, the CDK inhibitor is a small molecule inhibitor of CDK4 (e.g., a competitiveor non-competitive inhibitor). Non-limiting examples of CDK inhibitors include palbociclib, ribociclib, and abemaciclib, as well as pharmaceutically acceptable salts thereof.

[0217] In some embodiments, an anti-cancer therapy of the disclosure comprises a murine double minute 2 homolog (MDM2) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the MDM2 inhibitor is (a) a small molecule that inhibits one or more activities of MDM2 (e.g., binding to p53), (b) an antibody that inhibits one or more activities of MDM2 (e.g., by binding to and inhibiting one or more activities of MDM2, binding to and inhibiting expression of MDM2, and / or binding to and inhibiting one or more activities of a cell expressing MDM2, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of MDM2 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the MDM2 inhibitor is a small molecule inhibitor of MDM2 (e.g., a competitive or noncompetitive inhibitor). Non-limiting examples of MDM2 inhibitors include nutlin-3a, RG7112, idasanutlin (RG7388), AMG-232, MI-63, MI-291, MI-391, MI-77301 (SAR405838), APG-115, DS-3032b, NVP-CGM097, and HDM-201 (siremadlin), as well as pharmaceutically acceptable salts thereof. In some embodiments, the MDM2 inhibitor inhibits or disrupts interaction between MDM2 and p53.

[0218] In some embodiments, an anti-cancer therapy of the disclosure comprises (alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor) one or more of an antimetabolite, DNA-damaging agent, or platinum-containing therapeutic (e.g., 5- azacitadine, 5-fluorouracil, acadesine, busulfan, carboplatin, cisplatin, chlorambucil, CPT-11, cytarabine, daunorubicin, decitabine, doxorubicin, etoposide, fludarabine, gemcitabine, idarubicin, radiation, oxaliplatin, temozolomide, topotecan, trabectedin, GSK2830371, or rucaparib); a pro-apoptotic agent (e.g., a BCL2 inhibitor or downregulator, SMAC mimetic, or TRAIL agonist such as ABT-263, ABT-737, oridonin, venetoclax, combination of venetoclax and an anti-CD20 antibody such as obinutuzumab or rituximab, 1396-11, ABT- 10, SM-164, D269H / E195R, or rhTRAIL); a tyrosine kinase inhibitor; an inhibitor of RAS, RAF, MEK, or the MAPK pathway (e.g., AZD6244, dabrafenib, LGX818, PD0325901, pimasertib, trametinib, or vemurafenib); an inhibitor of PI3K, mTOR, or Akt; a CDK inhibitor; a PKC inhibitor (e.g., LXS196 or sotrastaurin); an antibody-based therapeutic (e.g., an anti-PD-1 or anti-PDLl antibody such as atezolizumab, pembrolizumab, nivolumab, or spartalizumab; an anti-CD20 antibody such as obinutuzumab or rituximab; or an anti-DR5 antibody such as drozitumab); a proteasome inhibitor (e.g., bortezomib, carfilzomib,ixazomib, or MG- 132); an HD AC inhibitor (e.g., SAHA or VPA); an antibiotic (e.g., actinomycin D); a zinc-containing therapeutic (e.g., zinc or ZMC1); an HSP inhibitor (e.g., geldanamycin); an ATPase inhibitor (e.g., archazolid); a mitotic inhibitor (e.g., paclitaxel or vincristine); metformin; methotrexate; tanshinone IIA; and / or P5091.

[0219] In some embodiments, the anti-cancer therapy comprises an immunoregulatory molecule or a cytokine, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. An immunoregulatory profile is required to trigger an efficient immune response and balance the immunity in a subject. Examples of suitable immunoregulatory cytokines include, but are not limited to, interferons (e.g., IFNa, IFNP and IFNy), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL- 12 and IL-20), tumor necrosis factors (e.g., TNFa and TNFP), erythropoietin (EPO), FLT-3 ligand, glplO, TCA-3, MCP-1, MIF, MIP-la, MIP-ip, Rantes, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments thereof. In some embodiments, any immunomodulatory chemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, or CX3C chemokine receptor, can be used in the context of the present disclosure. Examples of chemokines include, but are not limited to, MIP-3a (Lax), MIP-3P, Hcc-1, MPIF-1, MPIF- 2, MCP-2, MCP-3, MCP-4, MCP-5, Eotaxin, Tare, Elc, 1309, IL-8, GCP-2 Groa, Gro-P, Nap-2, Ena-78, Ip-10, MIG, LTac, SDF-1, or BCA-1 (Bic), as well as functional fragments thereof. In some embodiments, the immunoregulatory molecule is included with any of the treatments provided herein.

[0220] In some embodiments, an anti-cancer therapy of the disclosure comprises a tyrosine kinase inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the tyrosine kinase inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of a tyrosine kinase, (b) an antibody that inhibits one or more activities of a tyrosine kinase (e.g., by binding to and inhibiting one or more activities of the tyrosine kinase, binding to and inhibiting expression, such as cell surface expression, of the tyrosine kinase, and / or binding to and inhibiting one or more activities of a cell expressing the tyrosine kinase, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of a tyrosine kinase (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the tyrosine kinase inhibitor is a small molecule inhibitor of a tyrosine kinase (e.g., a competitive or noncompetitive inhibitor). Non-limiting examples of tyrosine kinase inhibitors include imatinib,crenolanib, linifanib, ninetedanib, axitinib, dasatinib, imetelstat, midostaurin, pazopanib, sorafenib, sunitinb, motesanib, masitinib, vatalanib, cabozanitinib, tivozanib, OSI-930, K18751, telatinib, dovitinib, tyrphostin AG 1296, and amuvatinib, as well as pharmaceutically acceptable salts thereof.

[0221] In some embodiments, an anti-cancer therapy of the disclosure comprises a mitogen- activated protein kinase (MEK) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the MEK inhibitor inhibits one or more activities of MEK1 and / or MEK2. In some embodiments, the anti-cancer therapy / MEK inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of MEK, (b) an antibody that inhibits one or more activities of MEK (e.g., by binding to and inhibiting one or more activities of MEK, binding to and inhibiting expression of MEK, and / or binding to and inhibiting one or more activities of a cell expressing MEK, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of MEK (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the MEK inhibitor is a small molecule inhibitor of MEK (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of MEK inhibitors include trametinib, cobimetinib, binimetinib, CI-1040, PD0325901, selumetinib, AZD8330, TAK- 733, GDC-0623, refametinib, pimasertib, RO4987655, RO5126766, WX-544, and HL-085, as well as pharmaceutically acceptable salts thereof. In some embodiments, the anti-cancer therapy inhibits one or more activities of the Raf / MEK / ERK pathway, including inhibitors of Raf, MEK, and / or ERK.

[0222] In some embodiments, an anti-cancer therapy of the disclosure comprises a mammalian target of rapamycin (mTOR) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the mTOR inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of mTOR, (b) an antibody that inhibits one or more activities of mTOR (e.g., by binding to and inhibiting one or more activities of mTOR, binding to and inhibiting expression of mTOR, and / or binding to and inhibiting one or more activities of a cell expressing mTOR, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of mTOR (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the mTOR inhibitor is a small molecule inhibitor of mTOR (e.g., a competitive inhibitor, such as an ATP-competitive inhibitor, or a non-competitive inhibitor, such as a rapamycin analog).Non-limiting examples of mTOR inhibitors include temsirolimus, everolimus, ridaforolimus, dactolisib, GSK2126458, XL765, AZD8055, AZD2014, MLN128, PP242, NVP-BEZ235, LY3023414, PQR309, PKI587, and OSI027, as well as pharmaceutically acceptable salts thereof. In some embodiments, the anti-cancer therapy inhibits one or more activities of the Akt / mTOR pathway, including inhibitors of Akt and / or mTOR.

[0223] In some embodiments, an anti-cancer therapy of the disclosure comprises a PI3K inhibitor or Akt inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the PI3K inhibitor inhibits one or more activities of PI3K. In some embodiments, the anti-cancer therapy / PI3K inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of PI3K, (b) an antibody that inhibits one or more activities of PI3K (e.g., by binding to and inhibiting one or more activities of PI3K, binding to and inhibiting expression of PI3K, and / or binding to and inhibiting one or more activities of a cell expressing PI3K, such as by inducing antibodydependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of PI3K (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the PI3K inhibitor is a small molecule inhibitor of PI3K (e.g., a competitive or non-competitive inhibitor). Nonlimiting examples of PI3K inhibitors include GSK2636771, buparlisib (BKM120), AZD8186, copanlisib (BAY80-6946), LY294002, PX-866, TGX115, TGX126, BEZ235, SF1126, idelalisib (GS-1101, CAL-101), pictilisib (GDC-094), GDC0032, IPI145, INK1117 (MLN1117), SAR260301, KIN-193 (AZD6482), duvelisib, GS-9820, GSK2636771, GDC- 0980, AMG319, pazobanib, and alpelisib (BYL719, Piqray), as well as pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor inhibits one or more activities of AKT (e.g., AKT1). In some embodiments, the AKT inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of AKT1, (b) an antibody that inhibits one or more activities of AKT1 (e.g., by binding to and inhibiting one or more activities of AKT1, binding to and inhibiting expression of AKT1, and / or binding to and inhibiting one or more activities of a cell expressing AKT1, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of AKT1 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the AKT1 inhibitor is a small molecule inhibitor of AKT1 (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of AKT1 inhibitors include GSK690693, GSK2141795 (uprosertib), GSK2110183 (afuresertib), AZD5363, GDC-0068 (ipatasertib), AT7867, CCT128930, MK-2206, BAY1125976, AKT1 and AKT2-IN-1, perifosine, and VIII, as well as pharmaceutically acceptable salts thereof. In some embodiments, the AKT1 inhibitor is a pan-Akt inhibitor.

[0224] In some embodiments, an anti-cancer therapy of the disclosure comprises a hedgehog (Hh) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the Hh inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of Hh, (b) an antibody that inhibits one or more activities of Hh (e.g., by binding to and inhibiting one or more activities of Hh, binding to and inhibiting expression of Hh, and / or binding to and inhibiting one or more activities of a cell expressing Hh, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of Hh (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the Hh inhibitor is a small molecule inhibitor of Hh (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of Hh inhibitors include sonidegib, vismodegib, erismodegib, saridegib, BMS833923, PF-04449913, and LY2940680, as well as pharmaceutically acceptable salts thereof.

[0225] In some embodiments, an anti-cancer therapy of the disclosure comprises a heat shock protein (HSP) inhibitor, a MYC inhibitor, an HD AC inhibitor, an immunotherapy, a neoantigen, a vaccine, or a cellular therapy, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor.

[0226] In some embodiments, the anti-cancer therapy comprises one or more of a chemotherapy, a VEGF inhibitor, an Integrin P3 inhibitor, a statin, an EGFR inhibitor, an mTOR inhibitor, a PI3K inhibitor, a MAPK inhibitor, or a CDK4 / 6 inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor.

[0227] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the kinase inhibitor is crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, ensartinib (X-396), repotrectinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP- 37440, belizatinib (TSR-011), ASP3026, KRCA-0008, TQ-B3139, TPX-0131, or TAE684 (NVP-TAE684). In some embodiments, the kinase inhibitor is an ALK kinase inhibitor, e.g., as described herein and / or in examples 3-39 of W02005016894.

[0228] In some embodiments, the anti-cancer therapy comprises a heat shock protein (HSP) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the HSP inhibitor is a Pan-HSP inhibitor, such as KNK423. In some embodiments, the HSP inhibitor is an HSP70 inhibitor, such ascmHsp70.1, quercetin, VER155008, or 17-AAD. In some embodiments, the HSP inhibitor is a HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is 17-AAD, Debio0932, ganetespib (STA-9090), retaspimycin hydrochloride (retaspimycin, IPI-504), AUY922, alvespimycin (KOS- 1022, 17-DMAG), tanespimycin (KOS-953, 17-AAG), DS 2248, or AT13387 (onalespib). In some embodiments, the HSP inhibitor is an HSP27 inhibitor, such as Apatorsen (OGX-427).

[0229] In some embodiments, the anti-cancer therapy comprises a MYC inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the MYC inhibitor is MYCi361 (NUCC-0196361), MYCi975 (NUCC- 0200975), Omomyc (dominant negative peptide), ZINC16293153 (Min9), 10058-F4, JKY-2- 169, 7594-0035, or inhibitors of MYC / MAX dimerization and / or MYC / MAX / DNA complex formation.

[0230] In some embodiments, the anti-cancer therapy comprises a histone deacetylase(HD AC) inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the HDAC inhibitor is belinostat (PXD101, e.g., Beleodaq®), SAHA (vorinostat, suberoylanilide hydroxamine, e.g., Zolinza®), panobinostat (LBH589, LAQ-824), ACY1215 (Rocilinostat), quisinostat (JNJ-26481585), abexinostat (PCI-24781), pracinostat (SB939), givinostat (ITF2357), resminostat (4SC-201), trichostatin A (TSA), MS-275 (etinostat), Romidepsin (depsipeptide, FK228), MGCD0103 (mocetinostat), BML-210, CAY10603, valproic acid, MC1568, CUDC-907, CI-994 (Tacedinaline), Pivanex (AN-9), AR-42, Chidamide (CS055, HBI-8000), CUDC-101, CHR- 3996, MPT0E028, BRD8430, MRLB-223, apicidin, RGFP966, BG45, PCI-34051, C149 (NCC149), TMP269, Cpd2, T247, T326, LMK235, CIA, HPOB, Nexturastat A, Befexamac, CBHA, Phenylbutyrate, MC1568, SNDX275, Scriptaid, Merck60, PX089344, PX105684, PX117735, PX117792, PX117245, PX105844, compound 12 as described by Li et al., Cold Spring Harb Perspect Med (2016) 6(10):a026831, or PX117445.

[0231] In some embodiments, the anti-cancer therapy comprises a VEGF inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the VEGF inhibitor is Bevacizumab (e.g., Avastin®), BMS-690514, ramucirumab, pazopanib, sorafenib, sunitinib, golvatinib, vandetanib, cabozantinib, levantinib, axitinib, cediranib, tivozanib, lucitanib, semaxanib, nindentanib, regorafinib, or aflibercept.

[0232] In some embodiments, the anti-cancer therapy comprises an integrin P3 inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Insome embodiments, the integrin P3 inhibitor is anti-avb3 (clone LM609), cilengitide (EMD121974, NSC, 707544), an siRNA, GLPG0187, MK-0429, CNTO95, TN-161, etaracizumab (MEDI-522), intetumumab (CNTO95) (anti-alphaV subunit antibody), abituzumab (EMD 525797 / DI17E6) (anti-alphaV subunit antibody), JSM6427, SJ749, BCH- 15046, SCH221153, or SC56631. In some embodiments, the anti-cancer therapy comprises an allbp3 integrin inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the allbp3 integrin inhibitor is abciximab, eptifibatide (e.g., Integrilin®), or tirofiban (e.g., Aggrastat®).

[0233] In some embodiments, the anti-cancer therapy comprises an mTOR inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the mTOR inhibitor is temsirolimus (CCI-779), KU-006379, PP242, Torinl, Torin2, ICSN3250, Rapalink-1, CC-223, sirolimus (rapamycin), everolimus (RAD001), dactosilib (NVP-BEZ235), GSK2126458, WAY-001, WAY-600, WYE-687, WYE-354, SF1126, XL765, INK128 (MLN012), AZD8055, OSI027, AZD2014, or AP- 23573.

[0234] In some embodiments, the anti-cancer therapy comprises a statin or a statin-based agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the statin or statin-based agent is simvastatin, atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, or cerivastatin.

[0235] In some embodiments, the anti-cancer therapy comprises a MAPK inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the MAPK inhibitor is SB203580, SKF-86002, BIRB-796, SC-409, RJW-67657, BIRB-796, VX-745, RO3201195, SB-242235, or MW181.

[0236] In some embodiments, the anti-cancer therapy comprises an EGFR inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the EGFR inhibitor is cetuximab, panitumumab, lapatinib, gefitinib, vandetanib, dacomitinib, icotinib, osimertinib (AZD9291), afatanib, olmutinib, EGF816 (nazartinib), avitinib (AC0010), rociletinib (CO-1686), BMS-690514, YH5448, PF- 06747775, ASP8273, PF299804, AP26113, necitumumab (e.g., Portrazza®), or erlotinib. In some embodiments, the EGFR inhibitor is gefitinib or cetuximab.

[0237] In some embodiments, the anti-cancer therapy comprises a nucleic acid molecule, such as a dsRNA, an siRNA, or an shRNA, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. As is known in the art, dsRNAs having a duplex structure are effective at inducing RNA interference (RNAi). In someembodiments, the anti-cancer therapy comprises a small interfering RNA molecule (siRNA). dsRNAs and siRNAs can be used to silence gene expression in mammalian cells (e.g., human cells). In some embodiments, a dsRNA of the disclosure comprises any of between about 5 and about 10 base pairs, between about 10 and about 12 base pairs, between about 12 and about 15 base pairs, between about 15 and about 20 base pairs, between about 20 and 23 base pairs, between about 23 and about 25 base pairs, between about 25 and about 27 base pairs, or between about 27 and about 30 base pairs. As is known in the art, siRNAs are small dsRNAs that optionally include overhangs. In some embodiments, the duplex region of an siRNA is between about 18 and 25 nucleotides, e.g., any of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. siRNAs may also include short hairpin RNAs (shRNAs), e.g., with approximately 29-base-pair stems and 2-nucleotide 3’ overhangs. Methods for designing, optimizing, producing, and using dsRNAs, siRNAs, or shRNAs, are known in the art.

[0238] In some embodiments, the anti-cancer therapy comprises a chemotherapy, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor.Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclo sphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L- norleucine, doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6- mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0239] Some non-limiting examples of chemotherapeutic drugs which can be combined with anti-cancer therapies of the present disclosure are carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Folex,Mexate, Amethop terin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0240] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Examples of kinase inhibitors include those that target one or more receptor tyrosine kinases, e.g., BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-a, PDGFR- 0, cKit, Flt-4, Flt3, FGFR1, FGFR2, FGFR3, FGFR4, CSF1R, c-Met, ROS1, RON, c-Ret, or ALK; one or more cytoplasmic tyrosine kinases, e.g., c-SRC, c-YES, Abl, or JAK-2; one or more serine / threonine kinases, e.g., ATM, Aurora A & B, CDKs, mTOR, PKCi, PLKs, b-Raf, c- Raf, S6K, or STK11 / LKB 1 ; or one or more lipid kinases, e.g., PI3K or SKI. Small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sutent (SU1 1248), sorafenib (BAY 43-9006), or leflunomide (SU101). Additional non-limiting examples of tyrosine kinase inhibitors include imatinib (Gleevec / Glivec) and gefitinib (Iressa).

[0241] In some embodiments, the anti-cancer therapy comprises an anti-angiogenic agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors require to survive. Non-limiting examples of angiogenesismediating molecules or angiogenesis inhibitors which may be used in the methods of the present disclosure include soluble VEGF (for example: VEGF isoforms, e.g., VEGF121 and VEGF165; VEGF receptors, e.g., VEGFR1, VEGFR2; and co-receptors, e.g., Neuropilin-1 and Neuropilin-2), NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMP and CD Al, Meth-1 and Meth-2, IFNa, IFN-0 and IFN-y, CXCL10, IL-4, IL- 12 and IL- 18, prothrombin (kringle domain-2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin and drugs such as bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-a platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids and heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactina v 03 inhibitors, linomide, or tasquinimod. In some embodiments, known therapeutic candidates that may be used according to the methods of the disclosure include naturally occurring angiogenic inhibitors, including without limitation, angiostatin, endostatin, or platelet factor-4. In anotherembodiment, therapeutic candidates that may be used according to the methods of the disclosure include, without limitation, specific inhibitors of endothelial cell growth, such as TNP-470, thalidomide, and interleukin- 12. Still other anti- angiogenic agents that may be used according to the methods of the disclosure include those that neutralize angiogenic molecules, including without limitation, antibodies to fibroblast growth factor, antibodies to vascular endothelial growth factor, antibodies to platelet derived growth factor, or antibodies or other types of inhibitors of the receptors of EGF, VEGF or PDGF. In some embodiments, anti-angiogenic agents that may be used according to the methods of the disclosure include, without limitation, suramin and its analogs, and tecogalan. In other embodiments, anti- angiogenic agents that may be used according to the methods of the disclosure include, without limitation, agents that neutralize receptors for angiogenic factors or agents that interfere with vascular basement membrane and extracellular matrix, including, without limitation, metalloprotease inhibitors and angiostatic steroids. Another group of anti- angiogenic compounds that may be used according to the methods of the disclosure includes, without limitation, anti-adhesion molecules, such as antibodies to integrin alpha v beta 3. Still other anti-angiogenic compounds or compositions that may be used according to the methods of the disclosure include, without limitation, kinase inhibitors, thalidomide, itraconazole, carboxyamidotriazole, CM101, IFN-a, IL-12, SU5416, thrombospondin, cartilage-derived angiogenesis inhibitory factor, 2-methoxyestradiol, tetrathiomolybdate, thrombospondin, prolactin, and linomide. In one particular embodiment, the anti-angiogenic compound that may be used according to the methods of the disclosure is an antibody to VEGF, such as AvastinO / bevacizumab (Genentech).

[0242] In some embodiments, the anti-cancer therapy comprises an anti-DNA repair therapy, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the anti-DNA repair therapy is a PARP inhibitor (e.g., talazoparib, rucaparib, olaparib), a RAD51 inhibitor (e.g., RI-1), or an inhibitor of a DNA damage response kinase, e.g., CHCK1 (e.g., AZD7762), ATM (e.g., KU-55933, KU-60019, NU7026, or VE-821), and ATR (e.g., NU7026).

[0243] In some embodiments, the anti-cancer therapy comprises a radiosensitizer, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Exemplary radiosensitizers include hypoxia radiosensitizers such as misonidazole, metronidazole, and trans-sodium crocetinate, a compound that helps to increase the diffusion of oxygen into hypoxic tumor tissue. The radiosensitizer can also be a DNA damage response inhibitor interfering with base excision repair (BER), nucleotide excision repair (NER),mismatch repair (MMR), recombinational repair comprising homologous recombination (HR) and non-homologous end-joining (NHEJ), and direct repair mechanisms. Single strand break (SSB) repair mechanisms include BER, NER, or MMR pathways, while double stranded break (DSB) repair mechanisms consist of HR and NHEJ pathways. Radiation causes DNA breaks that, if not repaired, are lethal. SSBs are repaired through a combination of BER, NER and MMR mechanisms using the intact DNA strand as a template. The predominant pathway of SSB repair is BER, utilizing a family of related enzymes termed poly-(ADP-ribose) polymerases (PARP). Thus, the radiosensitizer can include DNA damage response inhibitors such as PARP inhibitors.

[0244] In some embodiments, the anti-cancer therapy comprises an anti-inflammatory agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the anti-inflammatory agent is an agent that blocks, inhibits, or reduces inflammation or signaling from an inflammatory signaling pathway In some embodiments, the anti-inflammatory agent inhibits or reduces the activity of one or more of any of the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23; interferons (ILNs), e.g., ILNa, IENP, ILNy, ILN-y inducing factor (IGIE); transforming growth factor-P (TGE-P); transforming growth factor-a (TGE-a); tumor necrosis factors, e.g., TNE-a, TNE-p, TNE-RI, TNE-RII; CD23; CD30; CD40L; EGE; G- CSE; GDNE; PDGE-BB; RANTES / CCL5; IKK; NL-KB; TLR2; TLR3; TLR4; TL5; TLR6; TLR7; TLR8; TLR8; TLR9; and / or any cognate receptors thereof. In some embodiments, the anti-inflammatory agent is an IL-1 or IL-1 receptor antagonist, such as anakinra (e.g., Kineret®), rilonacept, or canakinumab. In some embodiments, the anti-inflammatory agent is an IL-6 or IL-6 receptor antagonist, e.g., an anti-IL-6 antibody or an anti-IL-6 receptor antibody, such as tocilizumab (e.g., ACTEMRA®), olokizumab, clazakizumab, sarilumab, sirukumab, siltuximab, or ALX-0061. In some embodiments, the anti-inflammatory agent is a TNE-a antagonist, e.g., an anti-TNEa antibody, such as infliximab (Remicade®), golimumab (Simponi®), adalimumab (e.g., Humira®), certolizumab pegol (e.g., Cimzia®) or etanercept. In some embodiments, the anti-inflammatory agent is a corticosteroid. Exemplary corticosteroids include, but are not limited to, cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, e.g., Ala-Cort®, Hydrocort Acetate®, hydrocortone phosphate Lanacort®, Solu-Cortef®), decadron (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, e.g., Dexasone®, Diodex®,Hexadrol®, Maxidex®), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, e.g., Duralone®, Medralone®, Medrol®, M-Prednisol®, Solu-Medrol®), prednisolone (e.g., Delta-Cortef®, ORAPRED®, Pediapred®, Prezone®), and prednisone (e.g., Deltasone®, Liquid Pred®, Meticorten®, Orasone®), and bisphosphonates (e.g., pamidronate (Aredia®), and zoledronic acid (e.g., Zometac®).

[0245] In some embodiments, the anti-cancer therapy comprises an anti-hormonal agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Anti-hormonal agents are agents that act to regulate or inhibit hormone action on tumors. Examples of anti-hormonal agents include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and FARESTON® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGACE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® (anastrozole); anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H- Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0246] In some embodiments, the anti-cancer therapy comprises an antimetabolite chemotherapeutic agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Antimetabolite chemotherapeutic agents are agents that are structurally similar to a metabolite, but cannot be used by the body in a productive manner. Many antimetabolite chemotherapeutic agents interfere with the production of RNA or DNA. Examples of antimetabolite chemotherapeutic agents include gemcitabine (e.g., GEMZAR®), 5 -fluorouracil (5-FU), capecitabine (e.g., XELODA™), 6-mercaptopurine, methotrexate, 6- thioguanine, pemetrexed, raltitrexed, arabinosylcytosine ARA-C cytarabine (e.g., CYTOSAR-U®), dacarbazine (DTIC-DOMED), azocytosine, deoxycytosine, pyridmidene, fludarabine (e.g., FLUDARA®), cladrabine, and 2-deoxy-D-glucose. In some embodiments, an antimetabolite chemotherapeutic agent is gemcitabine. Gemcitabine HC1 is sold by Eli Lilly under the trademark GEMZAR®.

[0247] In some embodiments, the anti-cancer therapy comprises a platinum-based chemotherapeutic agent, e.g., alone or in combination with an immunotherapy, such as an immune checkpoint inhibitor. Platinum-based chemotherapeutic agents are chemotherapeutic agents that comprise an organic compound containing platinum as an integral part of the molecule. In some embodiments, a chemotherapeutic agent is a platinum agent. In some such embodiments, the platinum agent is selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin.

[0248] In some embodiments, acquiring knowledge of or detecting the presence of an FGFR3 alteration and a high TMB in cancer or in a sample from an individual (e.g., an individual having a cancer) may be a predictor of non-response to, or lesser response to (as compared with response to an immunotherapy), anti-cancer therapies such as chemotherapies or chemotherapeutic agents, targeted therapies, alkylating agents, antimetabolites (e.g., gemcitabine), platinum agents (e.g., carboplatin), natural products, hormones, radiation therapy, or a therapy configured to target a defect in a specific cell signaling pathway, e.g., a defect in a DNA mismatch repair (MMR) pathway. Accordingly, in some embodiments, acquiring knowledge of or detecting the presence of an FGFR3 alteration and a high TMB in cancer or in a sample from an individual (e.g., an individual having a cancer) may identify an individual having the cancer as one who may not benefit from an anti-cancer therapy (such as chemotherapies or chemotherapeutic agents, targeted therapies, alkylating agents, antimetabolites (e.g., gemcitabine), platinum agents (e.g., carboplatin), natural products, hormones, radiation therapy, or a therapy configured to target a defect in a specific cell signaling pathway), and optionally as one who may benefit from a treatment comprising an immunotherapy, such an immune checkpoint inhibitor.

[0249] In some aspects, provided herein are therapeutic formulations comprising an anticancer therapy provided herein (e.g., an immunotherapy), and a pharmaceutically acceptable carrier, excipient, or stabilizer. A formulation provided herein may contain more than one active compound, e.g., an anti-cancer therapy provided herein and one or more additional agents (e.g., anti-cancer agents).

[0250] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, for example, one or more of: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol, 3-pentanol, or m-cresol; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); surfactants such as non-ionic surfactants; or polymers such as polyethylene glycol (PEG).

[0251] The active ingredients may be entrapped in microcapsules. Such microcapsules may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nano-capsules); or in macroemulsions. Such techniques are known in the art.

[0252] Sustained-release compositions may be prepared. Suitable examples of sustained- release compositions include semi-permeable matrices of solid hydrophobic polymers containing an anti-cancer therapy of the disclosure. Such matrices may be in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0253] A formulation provided herein may also contain more than one active compound, for example, those with complementary activities that do not adversely affect each other. The type and effective amounts of such medicaments depend, for example, on the amount and type of active compound(s) present in the formulation, and clinical parameters of the subjects.

[0254] For general information concerning formulations, see, e.g., Gilman et al. (eds.) The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press, 1990; A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Pennsylvania, 1990; Avis et al. (eds.) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York, 1993; Lieberman et al. (eds.) Pharmaceutical Dosage Forms: Tablets Dekker, New York, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse SystemsDekker, New York, 1990; and Walters (ed.) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol 1 19, Marcel Dekker, 2002.

[0255] Formulations to be used for in vivo administration are sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods known in the art.

[0256] In some embodiments, an anti-cancer therapy (e.g., immunotherapy) of the disclosure is administered as a monotherapy. In some embodiments, the anti-cancer therapy is administered in combination with one or more additional anti-cancer therapies or treatments, e.g., as described herein. In some embodiments, the one or more additional anti-cancer therapies or treatments include one or more anti-cancer therapies described herein. In some embodiments, the methods of the present disclosure comprise administration of any combination of any of the anti-cancer therapies provided herein. In some embodiments, the additional anti-cancer therapy comprises one or more of surgery, radiotherapy, chemotherapy, anti-angiogenic therapy, anti-DNA repair therapy, and anti-inflammatory therapy. In some embodiments, the additional anti-cancer therapy comprises an anti- neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, a radiation therapy, a cytotoxic agent, or combinations thereof. In some embodiments, an anti-cancer therapy may be administered in conjunction with a chemotherapy or chemotherapeutic agent. In some embodiments, the chemotherapy or chemotherapeutic agent is a platinum-based agent (including, without limitation cisplatin, carboplatin, oxaliplatin, and staraplatin). In some embodiments, an anti-cancer therapy may be administered in conjunction with a radiation therapy. In some embodiments, the anti-cancer therapy for use in any of the methods described herein (e.g., as monotherapy or in combination with another therapy or treatment) is an anti-cancer therapy or treatment described by Pietrantonio et al., J Natl Cancer Inst (2017) 109(12) and / or by Wang et al., Cancers (2020) 12(2):426, which are hereby incorporated by reference.G. Reporting

[0257] In some embodiments, the methods provided herein comprise generating a report, and / or providing a report to party.

[0258] In some embodiments, a report according to the present disclosure comprises information about one or more of: an FGFR3 alteration and a high tumor mutational burden; a cancer of the disclosure, e.g., comprising an FGFR3 alteration and a high tumor mutationalburden; 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., an immunotherapy, such as immune checkpoint inhibitor). Optionally, the report further comprises information about PD-L1 status of the cancer.

[0259] In some embodiments, a report according to the present disclosure comprises information about the presence or absence of an FGFR3 alteration and / or a high tumor mutational burden 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 FGFR3 alteration and / or a high tumor mutational burden are present in one or more samples obtained from the individual. In one embodiment, a report according to the present disclosure indicates that an FGFR3 alteration and / or a high tumor mutational burden 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 FGFR3 alteration and / or a high tumor mutational burden 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 FGFR3 alteration and / or a high tumor mutational burden 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.

[0260] In some embodiments, the report includes information on the role of an FGFR3 alteration and / or a high tumor mutational burden 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 FGFR3 alteration and / or a high tumor mutational burden; information on resistance of a cancer, such as a cancer provided herein (e.g., comprising an FGFR3 gene copy number alteration and / or a high tumor mutational burden) to one or more treatments; information on potential or suggested therapeutic options (e.g., such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein); or information on therapeutic options that should be avoided. In some embodiments, the report includes information on the likely effectiveness, acceptability, and / or advisability of applying a therapeutic option (e.g., such as an anti-cancer 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 FGFR3 alteration and a high tumor mutational burden) and identified in the report. In some embodiments, the report includes information or a recommendation on the administration of a treatment (e.g., an anti-cancer therapy provided herein, or a treatmentselected or identified according to the methods provided herein). In some embodiments, the information or recommendation includes the dosage of the treatment and / or a treatment regimen (e.g., in combination with other treatments, such as a second therapeutic agent). In some embodiments, the report comprises information or a recommendation for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more treatments.

[0261] 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 FGFR3 alteration and / or a high tumor mutational burden in the sample(s), or acquiring knowledge of the presence of an FGFR3 alteration and / or a high tumor mutational burden 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 FGFR3 alteration and / or a high tumor mutational burden in the sample(s); an identifier for the individual from which the sample(s) was obtained; information on the role of the FGFR3 alteration and / or a high tumor mutational burden, 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 an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein), e.g., in combination with other treatments (e.g., a second therapeutic agent). In some embodiments, the report generated is a personalized cancer report.

[0262] 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 FGFR3 alteration and / or a high tumor mutational burden), or to anindividual 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 of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from obtaining a sample from 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 FGFR3 alteration and / or a high tumor mutational burden 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 FGFR3 alteration and / or a high tumor mutational burden 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.H. Software, Systems, and Devices

[0263] 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.

[0264] FIG. 8 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. 8, 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., fordetecting an FGFR3 alteration and / or a high tumor mutational burden. Input device 820 and output device 830 can generally correspond to those described herein, and can either be connectable or integrated with the computer.

[0265] 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.

[0266] 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).

[0267] 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 FGFR3 alteration and / or a high tumor mutational burden (e.g., as embodied in the devices as described herein).

[0268] 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.

[0269] 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 thosedescribed 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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. 8) 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 6000Sequencing 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.

[0274] 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).

[0275] 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.

[0276] FIG. 10 illustrates an exemplary process 1000 for detecting an FGFR3 alteration and / or a high tumor mutational burden 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 1200 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, additionalsteps 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.

[0277] 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 FGFR3 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: FGFR3 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.

[0278] At block 1004, an exemplary system (e.g., one or more electronic devices) analyzes the plurality of sequence reads for the presence of an FGFR3 alteration and a high tumor mutational burden. At block 1006, the system detects (e.g., based on the analysis) an FGFR3 alteration and a high tumor mutational burden, in the one or more samples. In some embodiments, an FGFR3 alteration is detected by comparing one or more sequence reads to a reference genome (e.g., a portion of a human genome encoding FGFR3 or a portion thereof). Exemplary methods for detecting TMB are d...

Claims

CLAIMSWhat is claimed is:

1. A method of identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, the method comprising detecting in one or more samples from the individual an alteration in a fibroblast growth factor receptor 3 (FGFR3) gene and a high tumor mutational burden (TMB), wherein detection of the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy.

2. A method of selecting a treatment for an individual having a cancer, the method comprising detecting in one or more samples from the individual an FGFR3 alteration and a high TMB, wherein detection of the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy .

3. A method of identifying one or more treatment options for an individual having a cancer, the method comprising:(a) detecting in one or more samples from the individual an FGFR3 alteration and a high TMB; and(b) generating a report comprising one or more treatment options identified for the individual based at least in part on detection of the FGFR3 alteration and the high TMB in the one or more samples, wherein the one or more treatment options comprise an immunotherapy .

4. A method of identifying one or more treatment options for an individual having a cancer, the method comprising:(a) acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual; and(b) generating a report comprising one or more treatment options identified for the individual based at least in part on said knowledge, wherein the one or more treatment options comprise an immunotherapy.

5. The method of claim 3 or claim 4, wherein the report further indicates presence or absence of the FGFR3 alteration and the high TMB in the one or more samples.

6. A method of selecting a treatment for an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge: (i) the individual is classified as a candidate to receive a treatment comprising an immunotherapy; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an immunotherapy.

7. A method of predicting survival of an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

8. A method of predicting survival of an individual having a cancer treated with a treatment comprising an immunotherapy, the method comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to an individual whose cancer does not exhibit an FGFR3 alteration and a high TMB.

9. A method of monitoring, evaluating, or screening an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have an improved response to a treatment comprising an immunotherapy and / or longer survival when treated with a treatment comprising an immunotherapy, as compared to an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

10. The method of any one of claims 7-9, wherein the individual is predicted to have longer overall survival (OS) when treated with a treatment comprising an immunotherapy, as compared to OS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

11. The method of any one of claims 7-10, wherein the individual is predicted to have longer progression-free survival (PFS) when treated with a treatment comprising an immunotherapy, as compared to PFS of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

12. A method of selecting a treatment for an individual having a cancer, the method comprising detecting in one or more samples from the individual an FGFR3 alteration and a TMB, wherein detection of the FGFR3 alteration in the one or more samples identifies the individual as one who is more likely to benefit from a treatment comprising an immunotherapy at the detected TMB than an individual whose cancer shows the same TMB but lacks an FGFR3 alteration.

13. A method of predicting survival of an individual having a cancer, comprising acquiring knowledge of an FGFR3 alteration and a TMB in one or more samples from the individual, wherein responsive to the acquisition of said knowledge, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer shows the same TMB but lacks an FGFR3 alteration.

14. A method of treating or delaying progression of a cancer in an individual, comprising:(a) acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer; and(b) responsive to said knowledge, administering to the individual an effective amount of a treatment that comprises an immunotherapy.

15. A method of treating or delaying progression of a cancer in an individual, comprising administering to an individual having a cancer an effective amount of a treatment that comprises an immunotherapy, wherein the immunotherapy is administered responsive to acquiring knowledge of an FGFR3 alteration and a high TMB in one or more samples from the individual.

16. A method of treating or delaying progression of a cancer in an individual, comprising:(a) detecting an FGFR3 alteration and a high TMB in one or more samples from an individual having a cancer; and(b) administering to the individual an effective amount of a treatment that comprises an immunotherapy.

17. A method of identifying a candidate treatment for a cancer in an individual in need thereof, comprising:(a) performing DNA sequencing on one or more samples obtained from the individual to determine a sequencing mutation profile, wherein the sequencing mutation profile identifies the presence of an FGFR3 alteration and a high TMB in the one or more samples; and(b) selecting a treatment for the individual based at least in part on the sequencing mutation profile, wherein the treatment comprises an immunotherapy.

18. The method of claim 17, wherein the presence of the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy.

19. The method of claim 17 or claim 18, wherein the presence of the FGFR3 alteration and the high TMB in the one or more samples predicts the individual to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer does not comprise an FGFR3 alteration and a high TMB.

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. The method of any one of claims 1-21, wherein the cancer is bladder cancer.

23. The method of claim 22, wherein the cancer is urothelial carcinoma.

24. The method of claim 22 or claim 23, wherein the cancer is advanced or metastatic.

25. The method of any one of claims 1-24, wherein the immunotherapy is an immune checkpoint inhibitor (ICPI), a cancer vaccine, a cell-based therapy, a T cell receptor (TCR)- based therapy, an adjuvant immunotherapy, a cytokine immunotherapy, or an oncolytic virus therapy.

26. The method of claim 25, wherein the immunotherapy is an ICPI.

27. The method of claim 26, wherein the ICPI is a PD-l-targeted agent or a PD-L1- targeted agent.

28. The method of claim 27, wherein the ICPI is a PD-1 inhibitor.

29. The method of claim 28, wherein the ICPI comprises one or more of nivolumab, pembrolizumab, cemiplimab, or dostarlimab.

30. The method of claim 27, wherein the ICPI is a PD-L1 inhibitor.

31. The method of claim 30, wherein the ICPI comprises one or more of atezolizumab, avelumab, or durvalumab.

32. The method of claim 26, wherein the ICPI is a CTLA-4 inhibitor.

33. The method of claim 32, wherein the CTLA-4 inhibitor comprises ipilimumab.

34. The method of any one of claims 1-33, wherein the immunotherapy is a first-line immunotherapy .

35. The method of any one of claims 1-34, wherein the immunotherapy is a monotherapy.

36. The method of any one of claims 1-35, wherein the treatment or one or more treatment options do not comprise a chemotherapy.

37. The method of claim 36, wherein the chemotherapy comprises gemcitabine and / or carboplatin.

38. The method of any one of claims 1-37, wherein the FGFR3 alteration is a base substitution, short insertion / deletion, or rearrangement.

39. The method of claim 38, wherein the FGFR3 alteration results in an S249C amino acid substitution, relative to SEQ ID NO:2.

40. The method of claim 38, wherein the FGFR3 alteration results in an FGFR3- TACC3 gene fusion.

41. The method of claim 38, wherein the FGFR3 alteration results in a Y373C, R248C, G370C, G380R, A391E, K650E, S371C, K650N, or K650M amino acid substitution, relative to SEQ ID NO:2.

42. The method of claim 38, wherein the FGFR3 alteration results in an E768* mutation, relative to SEQ ID NO:2.

43. The method of claim 38, wherein the FGFR3 alteration results in an FGFR3- TNIP2, FGFR3-COL17A1, or FGFR3-CCDC149 gene fusion.

44. The method of any one of claims 1-43, wherein the high TMB comprises a TMB of greater than or equal to about 5 mutations / Megabase (mut / Mb).

45. The method of any one of claims 1-43, wherein the high TMB comprises a TMB of greater than or equal to about 10 mut / Mb.

46. The method of any one of claims 1-43, wherein the high TMB comprises a TMB of greater than or equal to about 20 mut / Mb.

47. The method of any one of claims 1-46, wherein TMB is assessed based on number of synonymous and non-driver non- synonymous mutations.

48. The method of any one of claims 1-47, wherein TMB is assessed across at least about 0.8 Mb.

49. The method of any one of claims 1-48, wherein TMB is assessed across between about 0.8 and 1.2 Mb.

50. The method of any one of claims 1-49, wherein TMB is assessed on sequencedDNA.

51. The method of any one of claims 1-50, further comprising assessing expression of PD-L1 protein in a sample from the individual.

52. The method of any one of claims 3-5 and 22-51, wherein the report further indicates the PD-L1 protein expression status of the cancer.

53. The method of any one of claims 17-51, wherein the molecular profile further indicates the PD-L1 protein expression status of the cancer.

54. The method of any one of claims 1-53, wherein the cancer is PD-L1 negative.

55. The method of any one of claims 1-53, wherein the cancer is PD-L1 positive.

56. The method of any one of claims 51-55, wherein PD-L1 protein expression is determined using an immunohistochemistry assay.

57. The method of claim 56, wherein the immunohistochemistry assay is a DAKO PD-L1 22C3 assay.

58. The method of claim 57, wherein PD-L1 positivity is defined as a combined positive score (CPS) of greater than or equal to 10.

59. The method of claim 56, wherein the immunohistochemistry assay is a VENTANA SP 142 assay.

60. The method of claim 59, wherein PD-L1 expression is assessed based on the proportion of tumor area occupied by PD-L1 -expressing tumor- infiltrating immune cells of any intensity (IC), or the percentage of PD-L1 -expressing tumor cells of any intensity (TC).

61. The method of claim 59 or claim 60, wherein PD-L1 positivity is defined as an immune cell score of greater than or equal to 5.

62. The method of any one of claims 4-11, 13-15, and 22-61, wherein acquiring knowledge of an FGFR3 alteration and a high TMB comprises detecting the FGFR3 alteration and the high TMB in the one or more samples.

63. The method of any one of claims 1-3, 12, and 16-62, wherein the FGFR3 alteration and the high TMB are detected in nucleic acids from the same sample.

64. The method of any one of claims 1-3, 12, and 16-62, wherein the FGFR3 alteration and the high TMB are detected in nucleic acids from different samples.

65. The method of any one of claims 1-64, further comprising obtaining the one or more samples from the individual.

66. The method of any one of claims 1-65, wherein the one or more samples are obtained or derived from the cancer.

67. The method of any one of claims 1-66, wherein the one or more samples from the individual comprise a tissue biopsy sample or a liquid biopsy sample.

68. The method of any one of claims 1-66, wherein the one or more samples from the individual are from a tumor biopsy, tumor specimen, or circulating tumor cell.

69. The method of any one of claims 1-66, wherein the one or more samples from the individual are a liquid biopsy sample comprising blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

70. The method of any one of claims 1-66, wherein the one or more samples from the individual are a liquid biopsy sample comprising circulating tumor cells (CTCs).

71. The method of any one of claims 1-66, wherein the one or more samples from the individual are a liquid biopsy sample comprising cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

72. The method of any one of claims 1-71, wherein the one or more samples from the individual comprise cells and / or nucleic acids from the cancer.

73. The method of claim 72, wherein the one or more samples from the individual comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer.

74. The method of any one of claims 1-73, wherein the FGFR3 alteration is detected by fluorescence in situ hybridization (FISH), comprehensive genomic profiling (CGP), comparative genomic hybridization (CGH), sequencing, or any combination thereof.

75. The method of any one of claims 1-74, wherein the high TMB is detected by sequencing.

76. The method of claim 74 or claim 75, 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.

77. The method of claim 76, wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises nextgeneration sequencing (NGS).

78. The method of any one of claims 1-77, wherein detecting the FGFR3 alteration comprises:(a) providing a plurality of nucleic acid molecules obtained from a sample from an individual having a cancer, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an FGFR3 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 FGFR3 gene, or a portion thereof;(f) analyzing the plurality of sequence reads for the presence or absence of an FGFR3 alteration; and(g) based on the analyzing step, detecting the presence or absence of an FGFR3 alteration in the sample.

79. The method of claim 78, wherein the sequencer comprises a next- generation sequencer.

80. The method of any one of claims 1-77, wherein detecting the FGFR3 alteration comprises:(a) providing a sample from an individual having a 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 FGFR3 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 FGFR3 alteration; and(g) detecting, based on the analyzing step, the presence or absence of an FGFR3 alteration in the sample from the individual.

81. The method of any one of claims 78-80, 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.

82. The method of claim 80, 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 FGFR3 gene or a portion thereof and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.

83. The method of any one of claims 78-82, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.

84. The method of any one of claims 78-83, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique.

85. The method of any one of claims 1-84, further comprising selectively enriching for one or more nucleic acid molecules in the sample comprising nucleotide sequences corresponding to an FGFR3 gene or a portion thereof; wherein the selectively enriching produces an enriched sample.

86. The method of claim 85, 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 FGFR3 gene or a portion thereof and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample.

87. The method of any one of claims 82-86, wherein the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to an FGFR3 gene or a portion thereof.

88. The method of claim 87, 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.

89. The method of any one of claims 82-88, wherein the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent.

90. The method of claim 89, wherein the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.

91. The method of any one of claims 87-90, wherein the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.

92. The method of claim 85, wherein the selectively enriching comprises amplifying the one or more nucleic acid molecules comprising nucleotide sequences corresponding to an FGFR3 gene or a portion thereof using a polymerase chain reaction (PCR) to produce an enriched sample.

93. The method of any one of claims 85-92, further comprising sequencing the enriched sample.

94. The method of any one of claims 80-93, wherein the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules.

95. The method of claim 94, 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.

96. The method of claim 94, 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.

97. The method of any one of claims 1, 2, and 6-96, further comprising generating a report, wherein the report: (a) indicates the presence of the FGFR3 alteration and high TMB in the one or more samples from the individual; and / or (b) indicates a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the presence of the FGFR3 alteration and high TMB in the one or more samples from the individual, wherein the treatment or the one or more treatment options comprise an immunotherapy .

98. The method of any one of claims 1-97, further comprising generating a molecular profile for the individual, based, at least in part, on detecting or acquiring knowledge of the FGFR3 alteration and / or high TMB.

99. The method of claim 98, wherein the molecular profile for the individual further comprises results from a comprehensive genomic profiling (CGP) test, a gene expressionprofiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof.

100. The method of claim 98 or claim 99, wherein the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test.

101. The method of any one of claims 98-100, further comprising generating a report, wherein the report comprises the molecular profile for the individual.

102. The method of claim 101, wherein the report further comprises information on a treatment or one or more treatment options identified or selected for the individual based, at least in part, on the molecular profile for the individual, wherein the treatment or one or more treatment options comprise an immunotherapy.

103. The method of any one of claims 3-5, 101, and 102, 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.

104. The method of any one of claims 1-103, wherein the individual is a human.

105. A system for identifying an individual having a cancer who may benefit from a treatment comprising an immunotherapy, 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 one or more samples obtained from an individual having a cancer;(b) analyze the plurality of sequence reads for presence of an FGFR3 alteration and presence of a high TMB; and(c) detect, based on the analyzing, presence of the FGFR3 alteration and presence of the high TMB in the one or more samples; wherein detecting the presence of the FGFR3 alteration and the presence of the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy.

106. The system of claim 105, wherein the cancer is bladder cancer.

107. The system of claim 106, wherein the cancer is urothelial carcinoma.

108. The system of any one of claims 105-107, wherein the cancer is advanced or metastatic.

109. The system of any one of claims 105-108, wherein the FGFR3 alteration is a base substitution, short insertion / deletion, or rearrangement.

110. The system of claim 109, wherein the FGFR3 alteration results in an S249C amino acid substitution, relative to SEQ ID NO:2.

111. The system of claim 109, wherein the FGFR3 alteration results in an FGFR3- TACC3 gene fusion.

112. The system of claim 109, wherein the FGFR3 alteration results in a Y373C, R248C, G370C, G380R, A391E, K650E, S371C, K650N, or K650M amino acid substitution, relative to SEQ ID NO:2.

113. The system of claim 109, wherein the FGFR3 alteration results in an E768* mutation, relative to SEQ ID NO:2.

114. The system of claim 109, wherein the FGFR3 alteration results in an FGFR3- TNIP2, FGFR3-COL17A1, or FGFR3-CCDC149 gene fusion.

115. The system of any one of claims 105-114, wherein the high TMB comprises a TMB of greater than or equal to about 5 mutations / Megabase (mut / Mb).

116. The system of any one of claims 105-114, wherein the high TMB comprises a TMB of greater than or equal to about 10 mut / Mb.

117. The system of any one of claims 105-114, wherein the high TMB comprises a TMB of greater than or equal to about 20 mut / Mb.

118. The system of any one of claims 105-117, wherein TMB is assessed based on number of non-driver mutations.

119. The system of claim 118, wherein the non-driver mutations include synonymous and non- synonymous mutations.

120. The system of any one of claims 105-119, wherein TMB is assessed across at least about 0.8 Mb.

121. The system of any one of claims 105-120, wherein TMB is assessed across between about 0.8 and 1.2 Mb.

122. The system of any one of claims 105-121, wherein the presence of FGFR3 and the presence of high TMB are detected based on analysis of sequence reads obtained from the same sample from the individual.

123. The system of any one of claims 105-121, wherein the presence of FGFR3 and the presence of high TMB are detected based on analysis of sequence reads obtained from different samples from the individual.

124. The system of any one of claims 105-123, wherein the one or more samples are obtained or derived from the cancer.

125. The system of any one of claims 105-124, wherein the one or more samples from the individual comprise a tissue biopsy sample or a liquid biopsy sample.

126. The system of any one of claims 105-124, wherein the one or more samples from the individual are from a tumor biopsy, tumor specimen, or circulating tumor cell.

127. The system of any one of claims 105-124, wherein the one or more samples from the individual are a liquid biopsy sample comprising blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

128. The system of any one of claims 105-124, wherein the one or more samples from the individual are a liquid biopsy sample comprising circulating tumor cells (CTCs).

129. The system of any one of claims 105-124, wherein the one or more samples from the individual are a liquid biopsy sample comprising cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

130. The system of any one of claims 105-124, wherein the one or more samples from the individual comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer.

131. The system of any one of claims 105-130, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene- targeted sequencing, or next-generation sequencing.

132. 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 a cancer who may benefit from a treatment comprising an immunotherapy, 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 one or more samples obtained from an individual having a cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for presence of an FGFR3 alteration and presence of a high TMB; and(c) detecting, using the one or more processors and based on the analyzing, the FGFR3 alteration and the high TMB in the one or more samples; wherein detecting the FGFR3 alteration and the high TMB in the one or more samples identifies the individual as one who may benefit from a treatment comprising an immunotherapy .

133. The non-transitory computer readable storage medium of claim 132, wherein the cancer is bladder cancer.

134. The non-transitory computer readable storage medium of claim 133, wherein the cancer is urothelial carcinoma.

135. The non-transitory computer readable storage medium of any one of claims 132- 134, wherein the cancer is advanced or metastatic.

136. The non-transitory computer readable storage medium of any one of claims 132- 135, wherein the FGFR3 alteration is a base substitution, short insertion / deletion, or rearrangement.

137. The non-transitory computer readable storage medium of claim 136, wherein the FGFR3 alteration results in an S249C amino acid substitution, relative to SEQ ID NO:2.

138. The non-transitory computer readable storage medium of claim 136, wherein the FGFR3 alteration results in an FGFR3-TACC3 gene fusion.

139. The non-transitory computer readable storage medium of claim 136, wherein the FGFR3 alteration results in a Y373C, R248C, G370C, G380R, A391E, K650E, S371C, K650N, or K650M amino acid substitution, relative to SEQ ID NO:2.

140. The non-transitory computer readable storage medium of claim 136, wherein the FGFR3 alteration results in an E768* mutation, relative to SEQ ID NO:2.

141. The non-transitory computer readable storage medium of claim 136, wherein the FGFR3 alteration results in an FGFR3-TNIP2, FGFR3-COL17A1, or FGFR3-CCDC149 gene fusion.

142. The non-transitory computer readable storage medium of any one of claims 132- 141, wherein the high TMB comprises a TMB of greater than or equal to about 5 mutations / Megabase (mut / Mb).

143. The non-transitory computer readable storage medium of any one of claims 132- 141, wherein the high TMB comprises a TMB of greater than or equal to about 10 mut / Mb.

144. The non-transitory computer readable storage medium of any one of claims 132- 141, wherein the high TMB comprises a TMB of greater than or equal to about 20 mut / Mb.

145. The non-transitory computer readable storage medium of any one of claims 132- 144, wherein TMB is assessed based on number of non-driver mutations.

146. The non-transitory computer readable storage medium of claim 145, wherein the non-driver mutations include synonymous and non-synonymous mutations.

147. The non-transitory computer readable storage medium of any one of claims 132-146, wherein TMB is assessed across at least about 0.8 Mb.

148. The non-transitory computer readable storage medium of any one of claims 132-147, wherein TMB is assessed across between about 0.8 and 1.2 Mb.

149. The non-transitory computer readable storage medium of any one of claims 132-148, wherein the presence of FGFR3 and the presence of high TMB are detected based on analysis of sequence reads obtained from the same sample from the individual.

150. The non-transitory computer readable storage medium of any one of claims 132- 148, wherein the presence of FGFR3 and the presence of high TMB are detected based on analysis of sequence reads obtained from different samples from the individual.

151. The non-transitory computer readable storage medium of any one of claims 132-150, wherein the one or more samples are obtained or derived from the cancer.

152. The non-transitory computer readable storage medium of any one of claims 132-151, wherein the one or more samples from the individual comprise a tissue biopsy sample or a liquid biopsy sample.

153. The non-transitory computer readable storage medium of any one of claims 132- 151, wherein the one or more samples from the individual are from a tumor biopsy, tumor specimen, or circulating tumor cell.

154. The non-transitory computer readable storage medium of any one of claims 132- 151, wherein the one or more samples from the individual are a liquid biopsy sample comprising blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

155. The non-transitory computer readable storage medium of any one of claims 132- 151, wherein the one or more samples from the individual are a liquid biopsy sample comprising circulating tumor cells (CTCs).

156. The non-transitory computer readable storage medium of any one of claims 132- 151, wherein the one or more samples from the individual are a liquid biopsy samplecomprising cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

157. The non-transitory computer readable storage medium of any one of claims 132- 151, wherein the one or more samples from the individual comprise mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer.

158. The non-transitory computer readable storage medium of any one of claims 132- 157, wherein the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or next-generation sequencing.

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