Use of TP53 biomarker for treating ovarian cancer background

Abstract

The present disclosure provides using Azenosertib for treating ovarian cancer where the treatment includes using a TP53 biomarker as a method for determining clinical response and evaluating treatment.

Description

USE OF TP53 BIOMARKER FOR TREATING OVARIAN CANCER BACKGROUND INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby expressly incorporated by reference under 37 CFR 1.57, and Rules 4.18 and 20.6, including U.S. Provisional Application No. 63 / 776,522, filed March 24, 2025, and U.S. Provisional Application No.63 / 787.299, filed April 11, 2025, the contents of each are hereby incorporated by reference in their entireties including any drawings.Field

[0002] The present application relates generally to methods of using a biomarker in the evaluation of a WEE1 inhibitor for treating diseases or conditions, such as a cancer.Description

[0003] DNA damage is typically resolved by a variety of pathways and proteins that repair damaged DNA. However, incorrect replacement of nucleotides into DNA can cause mutations and other genetic alterations that may lead to cancer development and progression. Improper DNA repair can lead to cell death, tumor progression and cancer. Cell cycle checkpoints are important for proper DNA repair, ensuring that cells do not progress with cellular replication until their genomic integrity is restored. WEE1 is a nuclear kinase involved in the G2-M cell-cycle checkpoint arrest for DNA repair before mitotic entry and overexpressed in a variety of cancers.

[0004] The TP53 gene produces the p53 protein, which repairs damaged DNA in cells. TP53 mutations are an indicator of cancer.

[0005] Various therapies for cancer, including ovarian cancer, have improved response rates and survival for patients, however, there is a need for discrete, quantitative and highly sensitive methods for determination of clinical response and efficacy so that potential toxicities may be limited and healthcare costs can be reduced.SUMMARY

[0006] The present disclosure, among other things, provides methods of determining clinical response and thereby modulating treatment regimen for ovarian cancer {e.g., High Grade Serous Ovarian Cancer (HGSOC)) using a WEE1 inhibitor {e.g., azenosertib, including pharmaceutically acceptable salts thereof). Among other things, provided herein are improved methods for determining molecular response (MR), by measuring longitudinal changes of tumor DNA fraction in cell free DNA (cfDNA), which represent a cost-effective and early efficacy endpoint for HGSOC. The present disclosure provides a facile method with a clear and specific threshold for molecular response by measuring change in a single TP53 marker {e.g., 50% reduction of a TP53 variant allele fraction from baseline), providing early guidance on therapeutic regimen for patients with respect to continuing or discontinuing treatment in patients with HGSOC based on the measured molecular response. Among other things, the methods of the present disclosure, provide an early and effective indicator of clinical response and efficacy to treatment of HGSOC using azenosertib. Among other things, the present disclosure, provides a method of identifying patient populations with decreased responsiveness e.g., Stable Disease (SD) or Progressive Disease (PD) in RECIST 1.1 scan) to a therapy, facilitating early transition to alternative therapeutic regimes and / or interventions, limiting toxicides and / or avoiding overtreatment by discontinuing treatment. In some embodiments, the present disclosure provides for personalized therapy for a patient having HGSOC with azenosertib based on the percentage reduction of TP53 biomarker measured before and after commencement of treatment and optionally measured at additional timepoints during the course of treatment.

[0007] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point; and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0008] In some embodiments, the patient with less than 50% reduction in (b) shows Stable Disease (SD) in a RECIST 1.1 scan.

[0009] In some embodiments, the patient with less than 50% reduction in (b) shows Progressive Disease (PD) in a RECIST 1.1 scan.

[0010] In some embodiments, the treatment is continued at a daily dose of about 400 mg.

[0011] In some embodiments, the treatment is continued at a daily dose of about 350 mg.

[0012] In some embodiments, the treatment is continued at a daily dose of about 300 mg.

[0013] In some embodiments, the treatment is continued in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0014] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point; (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0015] In some embodiments, the therapeutically effective daily dose is about 350 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0016] In some embodiments, the therapeutically effective daily dose is about 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0017] In some embodiments, the therapeutically effective daily dose is about 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0018] In some embodiments, the treatment is continued at substantially the same therapeutically effective daily dose as on treatment initiation.

[0019] In some embodiments, the treatment is continued at a daily dose of about 400 mg.

[0020] In some embodiments, the treatment is continued at a daily dose of about 350 mg.

[0021] In some embodiments, the treatment is continued at a daily dose of about 300 mg.

[0022] In some embodiments, the baseline level is measured at least two weeks prior to treatment initiation.

[0023] In some embodiments, the baseline level is measured two weeks prior to treatment initiation.

[0024] In some embodiments, the baseline level is measured at treatment initiation.

[0025] In some embodiments, the pre-determined time point is within 6 weeks of treatment initiation.

[0026] In some embodiments, the pre-determined time point is within 3 weeks of treatment initiation.

[0027] In some embodiments, the pre-determined time point is day 1 of a treatment cycle.

[0028] In some embodiments, a treatment cycle is three weeks.

[0029] In some embodiments, the pre-determined time point is day 1 of cycle 2 (C2D1).

[0030] In some embodiments, the pre-determined time point is day 1 of cycle 3 (C3D1).

[0031] In some embodiments, the TP 53 variant comprises a mutation selected from R273H, R248W, Y220C, R175H, R248Q, R273C, G266R, H179R, K132R, R282W, R337H, S241F and T256fs.

[0032] In some embodiments, the level of TP 53 variant allele fraction is measured by profiling plasma cell free DNA (cfDNA).

[0033] In some embodiments, the cfDNA is profiled by next-generation sequencing (NGS) assay or digital polymerase chain reaction (dPCR).

[0034] In some embodiments, the cfDNA is profiled by the next-generation sequencing (NGS) assay.

[0035] In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has at least 60%, 70%, 80%, 90% or 99% reduction. In some embodiments, the patient with the at least 50% reduction in the level of TP53 variant allele fraction has at least 60% reduction. In some embodiments, the patient with the at least 50% reduction in the level of TP53 variant allele fraction has at least 70% reduction. In some embodiments, the patient with the at least 50% reduction in the level of TP 53 variant allele fraction has at least 80% reduction. In some embodiments, the patient with the at least 50% reduction in the level of TP53 variant allele fraction has at least 90% reduction. In some embodiments, the patient with the at least 50% reduction in the level of TP53 variant allele fraction has at least 99% reduction. In some embodiments, the patient with the at least 50% reduction in the level of TP53 variant has allele fraction greater than 99% reduction.

[0036] In some embodiments, the patient has 100% reduction in the level of TP 53 variant allele fraction.

[0037] In some embodiments, the patient with less than 50% reduction in the level of TP53 variant allele fraction has less than 40%, 30%, 20% or 10% reduction. In some embodiments, the patient with the less than 50% reduction in the level of TP53 variant allele fraction has less than 40% reduction. In some embodiments, the patient with the less than 50% reduction in the level of TP53 variant allele fraction has less than 30% reduction. In some embodiments, the patient with the less than 50% reduction in the level of TP53 variant allele fraction has less than 20% reduction. In some embodiments, the patient with the less than 50% reduction in the level of TP53 variant allele fraction has less than 10% reduction.

[0038] In some embodiments, the patient has substantially no reduction in the level of TP53 variant allele fraction. In some embodiments, the patient has 0% reduction in the level of TP53 variant allele fraction.

[0039] In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 20%. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 30%. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 40%. In some embodiments, the patient withat least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 50%. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 60%. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by greater than 60%.

[0040] In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has a >20% decrease in the sum of diameters of target lesions according to RECIST. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has a >30% decrease in the sum of diameters of target lesions according to RECIST. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has a >50% decrease in the sum of diameters of target lesions according to RECIST. In some embodiments, the patient with at least 50% reduction in the level of TP53 variant allele fraction has a >60% decrease in the sum of diameters of target lesions according to RECIST.

[0041] In some embodiments, the patients with at least 50% reduction in the level of TP53 variant allele fraction have time to progression (TTP) of 4 months. In some embodiments, the patients with at least 50% reduction in the level of TP53 variant allele fraction have time to progression (TTP) of greater than 4 months.

[0042] In some embodiments, the patients with at least 50% reduction in the level of TP53 variant allele fraction have progression free survival of at least 3 months.

[0043] In some embodiments, the patients with at least 50% reduction in the level of TP 53 variant allele fraction have objective response rate (ORR) of at least 20%.

[0044] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0045] In some embodiments, the azenosertib is administered as a monotherapy.

[0046] In some embodiments, the azenosertib is administered in combination with a second therapeutic agent.BRIEF DESCRIPTION OF.DRA WINGS

[0047] These drawings are for illustrative purposes and are not meant to be limiting.

[0048] FIG. 1 describes the exemplary workflow for determining molecular response in azenosertib-treated HGSOC in a clinical setting. High grade serous ovarian cancer (HGSOC)patients enrolled in single-agent azenosertib studies received 300 mg or higher dose of azenosertib once a day. Plasma samples were collected at the start of treatment and every 3 weeks after and analyzed by Tempus xF+ Liquid Biopsy Assay to calculate TP53 variant allele fraction in cell-free DNA (cfDNA). Radiological tumor assessment (RECIST1.1) was conducted every 6 weeks.

[0049] FIG. 2 shows that exemplary RECIST responders that had a higher molecular response rate (MRR) showed better overall response rate. A high MRR of 80% was observed with confirmed objective response (cOR), about 56% MRR was correlated with stable disease and the lowest MRR of 35% was correlated with progressive disease (PD). Dashed lines indicate the staged MR cutoffs. The lines indicate molecular response stages.

[0050] FIG. 3A and FIG. 3B show a comparison of RECIST response rate between Molecular Response positive (MR+) and Molecular Response negative (MR-) groups. FIG.3A is a graph that showed that clinical response was enriched in the MR+ cohort. FIG.3B is a table that shows the actual values. Statistical significance was calculated using Chi-squared test and Fisher’s exact test (two-sided), respectively. PPV: positive predictive value, NPV: negative predictive value, CI: confidence interval.

[0051] FIG. 4A and FIG. 4B depict that exemplary molecular responders to azenosertib treatment showed greater tumor size reduction at target lesions. FIG. 4A is a graph that shows size reduction of the tumor (target lesion) from baseline with indications of the molecular response stage. FIG. 4B is a table that shows the median tumor size change across molecular response stages. Statistical significance from Kruskal-Wallis test. Dashed lines indicated 20% and -30% RECIST criteria cutoffs.

[0052] FIG. 5A and FIG. 5B show that exemplary molecular responders exhibited a longer progression-free survival benefit. FIG.5A is a graph where time to progression is compared between MR+ and MR- groups in the entire evaluable cohort. FIG. 5B is a graph where time to progression is compared in patients whose first overall response was stable disease. Median time to progression (mTTP) and hazard ratio (HR) with 95% confidence interval are shown in inset tables. P-values are from log-ranked test.

[0053] FIG. 6A and FIG.6B show that exemplary complete and confirmed molecular responses, respectively, further stratify patient groups with a longer time to progression and a shorter time to progression compared to molecular response shown in FIG.5A. FIG.6A illustrates Time to progression across staged MR groups in the entire evaluable cohort (n = 123). FIG. 6Billustrates Time to progression across confirmed MR groups in patients where two on-treatment samples (C2D1 and C3D1) were available and analyzed (n = 65). Confirmed MR positivity (cMR+) is defined as MR+ at both C2D1 and C3D1 timepoints, unconfirmed MR positivity (uMR+) as MR+ at either timepoint, and confirmed MR negativity (cMR-) as MR- at both timepoints. Median time to progression (mTTP) and hazard ratio (HR) with 95% confidence interval are shown in inset tables. P- values are from log-ranked test.

[0054] FIG. 7 A is a graph that show that molecular response predicts exemplary clinical response ahead of time. Timepoints of MR and RECIST scores are shown for the SD1A cohort (n = 69) defined in FIG.5B. FIG.7B is a table where patients were classified into 5 groups based on MR and earlier overall response. For the groups in which MR predicted clinical response, median time benefit was calculated.DETAILED DESCRIPTIONDefinitions

[0055] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the disclosure and to provide additional detail regarding its practice are hereby incorporated by reference.

[0056] Any numerical values used in this application are meant to cover any variations within the standard deviation or normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0057] About: As used herein, the term “about” has its usual meaning as understood by those skilled in the art and thus indicates that a value includes the inherent variation of error (such as ±10%. ±5%. ±1%, ±0.1%) for the method being employed to determine a value, or the variation that exists among multiple determinations.

[0058] Administering: As used herein, the terms “administering,” and “introducing” are used interchangeably in the context of delivering azenosertib described herein to a patient or subject in need thereof. Various methods are known in the art for administering therapeutics (such as azenosertib) to patients and subjects, including, for example, administering the therapeutic to a patient or subject in need thereof by subcutaneous methods.

[0059] Allele: As used herein, "allele" or " variant allelic " refers to a specific genetic variant at a defined genomic location or locus. A variant allelic is usually presented at a frequency of 50% (0.5) or 100%, depending on whether the allele is heterozygous or homozygous. For example, germline variants are inherited and usually have a frequency of 0.5 or 1. Somatic variants; however, are acquired variants and usually have a frequency of < 0.5. Major and minor alleles of a genetic locus refer to nucleic acids harboring the locus in which the locus is occupied by a nucleotide of a reference sequence, and a variant nucleotide different than the reference sequence, respectively. Measurements at a locus can take the form of allelic fractions (AFs), which measure the frequency with which an allele is observed in a sample.

[0060] Amplify: As used herein, “amplify” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide {e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.

[0061] Baseline: As used herein, the term “baseline” is defined as a minimum or starting point used for comparisons. The baseline, in the context of this disclosure, may be a timepoint prior to treatment for testing of TP53 variant levels.

[0062] Cell-Free Nucleic Acid: As used herein, “cell-free nucleic acid” refers to nucleic acids not contained within or otherwise bound to a cell or, in some embodiments, nucleic acids remaining in a sample following the removal of intact cells. Cell-free nucleic acid can include, for example, all non-encapsulated nucleic acids sourced from a bodily fluid {e.g., blood, plasma, serum, urine, cerebrospinal fluid (CSF), etc.) from a subject. Cell-free nucleic acids include DNA (cfDNA), RNA (cfRNA), and hybrids thereof, including genomic DNA, mitochondrial DNA. circulating DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA) and / or fragments of any of these. Cell-free nucleic acids can be double-stranded, singlestranded or a hybrid thereof. A cell-free nucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis, apoptosis, or the like.

[0063] Detect: As used herein, “detect”, “detecting,” or “detection” refers to an act of determining the existence or presence of one or more target nucleic acids e.g.. nucleic acids having targeted mutations or other markers) in a sample.

[0064] Dosing schedule or Intermittent dosing schedule: As used herein, “dosing schedule” or intermittent dosing schedule” refers to an intermittent dosing schedule wherein each dosing week comprises consecutive dosing days (e.g., two to seven days) followed by days without dosing (e.g., one to seven days). In some embodiments, the intermittent dosing schedule comprises one or more dosing weeks with each dosing week comprising at least two consecutive dosing days and at least one rest day. In some embodiments, the intermittent dosing schedule comprises one or more dosing weeks with each dosing week comprising at least three consecutive dosing days and at least one rest day.

[0065] Dosing Week: As used herein, “Dosing week” refers to a week of an intermittent dosing schedule comprising dosing days and days without dosing. For example, a dosing week has 2 days on, 5 days off; 3 days on, 4 days off; 4 days on, 3 days off; 5 days on, 2 days off; 6 days on, 1 day off; or 7 days on, 7 days off. As described herein, “days on” refers to days when azenosertib (alone or in combination) is administered to a patient and “days off’ refers to days azenosertib (alone or in combination) is not administered.

[0066] Molecular Response: As used herein, the term “molecular response” refers to a decrease that reaches a pre-determined threshold, e.g., at least 50% reduction at a pre-determined timepoint as compared to a baseline level, in one or more cell free DNA (cfDNA) variant allele frequencies, levels or amounts observed in samples taken from a given patient at different timepoints. Complete molecular response refers to the patient showing substantially no detectable levels of a specific cancer biomarker, e.g., TP53 variant. Partial molecular response refers to the patient showing some detectable levels (e.g., at least 50% but not complete reduction at a predetermined timepoint as compared to a baseline level) of a specific cancer biomarker, e.g., TP 53 variant.

[0067] Molecular Responder: As used herein, the terms “molecular responder” and “responder” refer to a subject having a molecular response (MR) score that indicates a predetermined decrease threshold, e.g., at least 50% reduction, in one or more cell free DNA (cfDNA) variant allele frequencies, levels or amounts observed in between samples taken from the patient at different time points.

[0068] Molecular Non-Responder: As used herein, the terms “molecular nonresponder” and “non-responder” refer to patients having a Molecular Response score that indicates a decrease that does not meet a pre-determined threshold (e.g., less than 50% reduction), or nochange, in one or more cell free DNA (cfDNA) variant allele frequencies, levels or amounts observed in between samples taken from the subject at different time points.

[0069] A threshold specifying a level of decrease (or increase) may be utilized to determine whether the subject is a molecular responder or a molecular non-responder. For example, a molecular responder refers to a subject associated with a decrease of more than a certain percentage change in TP53 variant allele fraction, and a non-responder refers to a subject associated with an increase, no change or a decrease by less than a certain percentage change in TP53 variant allele fraction, e.g., less than 50% reduction.

[0070] Next Generation Sequencing: As used herein, the term “next generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based approaches, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation and sequencing by hybridization.

[0071] Objective Response Rate: As used herein, the term “objective response rate” or “ORR” refers to the percentage of people in a study or treatment group who have a partial response or complete response to the treatment within a certain period of time {e.g., baseline prior to treatment to pre-determined time of measuring TP53 levels after commencing treatment).

[0072] Patient: As used herein, the terms “patient”, “subject” or “individual” refer to a human to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic and / or therapeutic purposes. In some embodiments, a human includes prenatal and postnatal forms. As used herein, the terms “a patient” or “the patient” encompasses a single patent and a patient population.

[0073] Polynucleotide: As used herein, "polynucleotide", "nucleic acid", "nucleic acid molecule" and "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides or analogs thereof) joined by intemucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5' - + 3' order from left to right and that in the case of DNA, "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T"denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.

[0074] Progression Free Survival: As used herein, the term “progression free survival” or “PFS” is the length of time from treatment initiation until disease progression measured by increase in tumor size, new lesions and / or death from any cause. It is typically reported as median time to progression or death. In other words, PFS is the period during and after the treatment of a disease, such as cancer, that a patient lives with the disease but it does not get worse. In a clinical trial, measuring the progression-free survival is one way to measure how well a treatment works. A higher PFS indicates that the treatment is more effective at delaying disease progression. PFS is a surrogate endpoint for overall survival (OS), and a useful predictor of clinical efficacy and response by predicting how well a treatment might extend patients’ survival. Exemplary methods for determining progression free survival of a subject having cancer include assessing evaluating tumor size, tumor number and / or metastasis.

[0075] Sequencing: As used herein, the term sequencing refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, shortread sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing and a combination thereof. In some embodiments, sequencing can be performer by a gene analyzer such as, forexample, gene analyzers commercially available from Illumina, Tnc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo Fisher Scientific, among many others.

[0076] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder and / or condition (e.g., cancer).

[0077] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder and / or condition, to treat, diagnose, prevent measure infection and / or delay the onset of the symptom(s) of the disease, disorder and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0078] Time to Progression: As used herein, the terms “time to progression” and “TTP” refer to the length of time from the date of diagnosis or the start of treatment for a disease until the disease starts to get worse or spread to other parts of the body.

[0079] TP53: As used herein, “TP53”, “cellular tumor antigen p53” and “p53,” are used interchangeably herein, and refer to a potent tumor suppressor protein encoding a 393 amino acid phosphoprotein. TP53 is negatively regulated or mutated in many cancers. Absence or inactivation of TP53 may contribute to cancer. A wide variety of TP53 mutations exist. In some cases, a cancer may overexpress TP53, in particular, mutant versions of TP53. A “wild type” TP53 is TP 53 found in normal (z.e., non-cancerous cells) or TP 53 that does not have a mutation correlated to a cancer. The TP 53 status of a sample e.g., whether the sample includes wild-type or mutant TP53) may be assessed as using standard techniques such as described herein or known in the art. A TP53 wild-type protein may include, without limitation, polypeptides containing sequences substantially identical to that set forth in, for example, UniProt Accession No. P04637.

[0080] Treating: As used herein, the terms “treat”, “treatment” and “treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0081] Tumor: As used herein, the term “tumor” is used in its usual meaning as understood by those skilled in the art and refers to an abnormal growth of cells or tissue. In some embodiments, the tumor is benign. In some embodiments, the tumor is malignant. A tumor becomes a cancer when it metastasizes, or spreads to other areas of the body. The term “solid tumor” as used herein has its usual meaning as understood by those skilled in the art and refers to an abnormal mass of tissue that does not contain liquid areas or cysts. The terms “cancer” and “tumor” may generally be used interchangeably unless the context clearly indicates that a more specific meaning is intended.

[0082] Value As used herein, the term “value” generally refers to an entry in a dataset and can be anything that characterizes the feature to which the value refers. This includes, without limitation, numbers, words or phrases, symbols (e.g., + or -) or degrees.

[0083] Variant allele fraction: As used herein, “variant allele fraction”, “mutant allele frequency”, “variant allele frequency”, “mutant allele fraction”, “VAF” or “MAF," refers to the frequency or fraction at which variant or mutant alleles, such as TP53 variant alleles, occur in a given population of nucleic acids, such as a sample obtained from a subject. MAF or VAF is generally expressed as a fraction or a percentage.

[0084] The present disclosure provides, among other things, methods for determination of clinical response and efficacy for modulation of a treatment regimen comprising use of azenosertib, a WEE1 inhibitor, in the treatment of high grade serous ovarian cancer (HGSOC) by using cell-free DNA, specifically TP53 variant alleles, as a biomarker for clinical response and efficacy. Among other things, TP53 is used as a biomarker in early detection and relapse monitoring of ovarian cancer. The present disclosure provides, among other things, a clear and specific threshold for Molecular Response by measuring change in a single TP53 marker e.g., 50% reduction of a TP53 variant allele fraction from a baseline level), providing early guidance on a therapeutic regimen for patients with respect to continuing or discontinuing treatment in patients with HGSOC based on the measured Molecular Response. Among other things, the methods of the present disclosure, provide an early and effective indicator of clinical response and efficacy to treatment of high grade serous ovarian cancer using azenosertib. Among other things, the present disclosure, provides a method of identifying patient populations with decreased responsiveness (e.g.. Progressive Disease or Stable Disease on RECIST scan) to therapy (such as treatment with azenosertib) and / or limiting toxicides by discontinuing treatment.

[0085] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective amount, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant at a pre-determined time point; (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level. In some embodiments, the therapeutically effective amount is about 300 mg, about 350 mg or about 400 mg, in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0086] In some embodiments, the patient with less than 50% reduction in (b) shows Stable Disease (SD) in a RECIST 1.1 scan. In some embodiments, the patient with less than 50% reduction in (b) subsequently shows Stable Disease (SD) in a RECIST 1.1 scan or equivalent. In some embodiments, the patient with less than 50% reduction in (b) shows Stable Disease (SD) in a RECIST 1.1 scan or equivalent, if treatment is discontinued. In some embodiments, the patient with less than 50% reduction in (b) shows Stable Disease (SD) in a RECIST 1.1 scan or equivalent, if treatment is not discontinued. In some embodiments, the patient with less than 50% reduction in (b) is MR- and shows a disease status of Stable Disease (SD) in a RECIST 1.1 scan or equivalent.

[0087] In some embodiments, the patient with less than 50% reduction in (b) shows Progressive Disease (PD) in a RECIST 1.1 scan. In some embodiments, the patient with less than 50% reduction in (b) subsequently shows Progressive Disease (PD) in a RECIST 1.1 scan or equivalent. In some embodiments, the patient with less than 50% reduction in (b) shows Progressive Disease (PD) in a RECIST 1.1 scan or equivalent, if treatment is discontinued. In some embodiments, the patient with less than 50% reduction in (b) shows Progressive Disease (PD) in a RECIST 1.1 scan or equivalent, if treatment is not discontinued. In some embodiments, the patient with less than 50% reduction in (b) is MR- and shows a disease status of Progressive Disease (PD) in a RECIST 1.1 scan or equivalent.

[0088] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allelefraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0089] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 350 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0090] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level. Ovarian Cancer

[0091] In some embodiments of the present disclosure, the cancer is ovarian cancer.

[0092] Ovarian cancer is responsible for significant morbidity and mortality in populations around the world. It is also the leading cause of gynecologic cancer deaths in the United States. Ovarian cancer is classified, on the basis of clinical and pathological features, in three groups, namely epithelial ovarian cancer (EOC; >90% of ovarian cancer in western countries), germ cell tumors (roughly 2-3% of ovarian cancer) and stromal ovarian cancer (roughly5% of ovarian cancer; Ozols et al., Cancer: Principles and Oncology, 1997, 5thed., DeVita et al., Eds. pp. 1502).

[0093] Numerous types of ovarian tumors exist, both benign and others malignant. Such tumors are named according to the type of cells from which the tumor is derived and whether the tumor is benign or malignant. Recognized histological tumor types include serous, mucinous, endometrioid and clear cell tumors.

[0094] In addition to the above, ovarian cancer tumors are classified according to recognized grade and stage scales known in the art. In grade I, the tumor tissue is well differentiated from normal ovarian tissue. In grade II, tumor tissue is moderately well differentiated. In grade III, the tumor tissue is poorly differentiated from normal tissue, and this grade correlates with a less favorable prognosis than grades I and II. Stage I is generally confined within the capsule surrounding one (stage IA) or both (stage IB) ovaries, although in some stage I (i.e., stage IC) cancers, malignant cells may be detected in ascites, in peritoneal rinse fluid or on the surface of the ovaries. Stage II involves extension or metastasis of the tumor from one or both ovaries to other pelvic structures. In stage IIA, the tumor extends or has metastasized to the uterus, the fallopian tubes or both. Stage IIB involves extension of the tumor to the pelvis. Stage IIC is stage IIA or IIB in which malignant cells may be detected in ascites, in peritoneal rinse fluid or on the surface of the ovaries. In stage III, the tumor comprises at least one malignant extension to the small bowel or the omentum, has formed extrapelvic peritoneal implants of microscopic (stage IIIA) or macroscopic (<2 -centimeter diameter, stage IIIB; >2-centimeter diameter, stage IIIC) size, or has metastasized to a retroperitoneal or inguinal lymph node (an alternate indicator of stage IIIC). In stage IV, distant (i.e., non-peritoneal) metastases of the tumor can be detected.High Grade Serous Ovarian Cancer

[0095] In some embodiments, the ovarian cancer is high grade serous ovarian cancer (HGSOC). HGSOC is a type of epithelial ovarian cancer that originates in the lining of the ovaries or the fallopian tubes, accounting for approximately 75% of epithelial ovarian cancers. HGSOC is the most common form of epithelial ovarian cancer.Platinum-resistant or Refractory Ovarian Cancer

[0096] Approximately 80% of women with advanced stage disease who respond to first-line chemotherapy relapse. Single agent chemotherapy response for heavily pretreatedplatinum resistant / refractory ovarian cancer is 10-15%. (NCI Int J Gynaecol Obstet. 2021 Oct; 155(Suppl 1): 61-85).TP53 Variants in Ovarian Cancer

[0097] TP53 is a gene that encodes for the tumor suppressor protein p53, which has been identified as a key regulator of cell growth, division and death. The TP53 gene is one of the more commonly mutated genes in human cancers. Mutations in the TP53 gene can lead to the loss or inactivation of p53 function, increasing the risk of cancer development. This is because damaged cells are no longer properly suppressed, allowing them to proliferate and form tumors. More than 80% of somatic and germline TP53 mutations lead to the synthesis of a stable mutant protein that can accumulate in the nucleus of tumor cells. Some TP53 mutations may result in a loss of wild-type functions either by loss of DNA-binding activity or by a dominant-negative effect whereby the mutated allele inhibits function of the wild-type allele.

[0098] Mutant p53 proteins can be expressed in any of a variety of human cancers, including, but not limited to, ovarian cancer, cholangiocarcinoma, melanoma, colon cancer, rectal cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer and bladder cancer.

[0099] Typically, HGSOC harbors a mutation in the TP53 gene, and such mutations are universal in this type of cancer. Germline TP53 mutation can also cause adult females to become predisposed to breast cancer. In the context of ovarian cancer, the risk of germline TP53 mutation is not significant enough to recommend prophylactic surgery, despite the prevalence of high grade serous ovarian cancer. The majority of mutations in TP53 in HGSOC are missense mutations occurring in exons 4-8, wherein the most frequently mutated codon is codon 273, wherein the mutations comprise R273C, R273H and R273L mutations (Ahmed et al., “Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary” J. Pathol. (2010) 221(l):49-56). A clinical study performed by Tuna and colleagues in 2019 (Tuna et al.. “Clinical relevance of TP53 hotspot mutations in high-grade serous ovarian cancers” British Journal of Cancer (2020) 122:405-412) revealed that the most frequent types of mutations in TP53 in HGSOC were missense mutations (60.52%), followed by frameshift (15.24%), splice site (10.52%), nonsense (10.73%) and in-frame mutations (3.22%). Nine of the most common “hotspot” mutation sites observed (in order of occurrence) were R273. R248, R175, Y220, 1195, C176, G245. S241 and Y163.

[0100] In some embodiments, the HGSOC is associated with a mutation or variant in TP53.Use of Change in TP53 Variant Allele Fraction as a Molecular Response Biomarker Continuing or Discontinuing Treatment

[0101] In some embodiments, detection of TP53 and its variants serve as a biomarker for clinical response to cancer therapeutic treatments. In some embodiments, measuring a level of TP53 variant allele fraction allows for modulation of treatment regimen, for example, continuing or discontinuing treatment based on a cut-off of at least 50% reduction in the level of a TP53 variant allele fraction as compared to a baseline level. In some embodiments, the disclosure provides methods of determining the level of TP53 variant allele fraction in a sample obtained from the patient at baseline and another pre-determined time point; and comparing the level of TP53 variant allele fraction to the baseline level of TP53 variant allele fraction, wherein an increase or decrease in the level of TP53 variant allele fraction in the patient sample relative to the baseline level provides information regarding continuing, changing or discontinuing treatment for the patient having a cancer, by predicting clinical responsiveness and thereby increasing safety and efficacy.

[0102] In some embodiments, the change or reduction in TP53 variant allele fraction is a biomarker for early detection and screening. In some embodiments, the change or reduction in TP53 variant allele fraction is a biomarker prior to diagnosis.

[0103] In some embodiments, the change or reduction in TP53 variant allele fraction is a biomarker for molecular response to treatment. In some embodiments, the change or reduction in TP53 variant allele fraction is a biomarker during treatment. In some embodiments, the change or reduction in TP53 variant allele fraction is a biomarker for relapse monitoring. In some embodiments, the change or reduction in TP 53 variant allele fraction is a biomarker post-treatment. In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring the level of TP53 variant allele fraction at baseline and another pre-determined time point, and if the patient has at least 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level, continuing the treatment; or if the patient has less than 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level, discontinuing the treatment.

[0104] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective daily dose, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point; (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level. In some embodiments, the treatment is continued at a daily dose of about 400 mg.

[0105] In some embodiments, the treatment is continued at a daily dose of about 350 mg.

[0106] In some embodiments, the treatment is continued at a daily dose of about 300 mg.

[0107] In some embodiments, the treatment is continued in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0108] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective daily dose, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point; (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0109] In some embodiments, the therapeutically effective daily dose is about 350 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0110] In some embodiments, the therapeutically effective daily dose is about 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

[0111] In some embodiments, the treatment is continued at the same therapeutically effective daily dose as on treatment initiation.

[0112] In some embodiments, the treatment is continued at about 350 mg daily dose.

[0113] In some embodiments, the treatment is continued at about 300 mg daily dose.

[0114] In some embodiments, the baseline is at least two weeks prior to treatment initiation.

[0115] In some embodiments, the baseline is two weeks prior to treatment initiation. In some embodiments, the baseline is three weeks prior to treatment initiation. In some embodiments, the baseline is four weeks prior to treatment initiation.

[0116] In some embodiments, the baseline is at treatment initiation.

[0117] In some embodiments, the pre-determined time point is within 10 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 9 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 8 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 7 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 6 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 5 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 4 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 3 weeks of treatment initiation. In some embodiments, the pre-determined time point is within 2 weeks of treatment initiation. In some embodiments, the pre-determined time point is within one week of treatment initiation.

[0118] In some embodiments, the pre-determined time point is day 1 of a treatment cycle.

[0119] In some embodiments, each treatment cycle is two weeks. In some embodiments, each treatment cycle is three weeks. In some embodiments, each treatment cycle is four weeks.

[0120] In some embodiments, the pre-determined time point is day 1 of cycle 2 (C2D1). In some embodiments, the pre-determined time point is day 1 of cycle 3 (C3D1). In some embodiments, the pre-determined time point is day 1 of cycle 4 (C4D1). In some embodiments, the pre-determined time point is day 1 of cycle 5 (C4D1). In some embodiments, the predetermined time point is day 1 of cycle 6 (C4D1).

[0121] In some embodiments, the first on-treatment level is measured at least 2 weeks prior to a subsequent on-treatment level of TP53. In some embodiments, the first on-treatment level is measured 2 weeks prior to a subsequent on-treatment level of TP53. In some embodiments, the first on-treatment level is measured 3 weeks prior to a subsequent on-treatment level of TP53. In some embodiments, the first on-treatment level is measured 4 weeks prior to a subsequent on-treatment level of TP53. In some embodiments, the first on-treatment level is measured 5 weeks prior to a subsequent on-treatment level of TP53. In some embodiments, the first on-treatment level is measured 6 weeks prior to a subsequent on-treatment level of TP 53.

[0122] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to a baseline.

[0123] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 350 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0124] In one aspect, the present disclosure provides, among other things, a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variantallele fraction at a pre-determined time point, and (a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level. Adjusting Dose

[0125] In some embodiments, measuring a level of TP 53 variant allele fraction allows for modulation of treatment regimen, for example, increasing or decreasing treatment (e.g., by adjusting dose and / or dosing frequency) based on a cut-off of at least 50% reduction in the level of a TP53 variant allele fraction as compared to a measured baseline level.

[0126] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, then (a) reducing the dosing frequency by increasing the days without dosing to 4:3 (4 days on:3 days off) for one or more treatment cycles until end of treatment if the patient has at least 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0127] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a daily dose of about 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a predetermined time point, then (a) reducing the dose of treatment to about 350 mg with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week until end of treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0128] In some aspects, provided herein is a method of treating ovarian cancer, comprising administering azenosertib to a patient at a daily dose of about 350 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allele fraction, measuring a first on-treatment level of TP 53 variant allele fraction at a pre-determined time point, then (a) reducing the dose treatment to about 300 mg with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week until end of treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP 53 variant allele fraction as compared to the baseline level.

[0129] In some aspects, provided herein is a method of treating ovarian cancer, comprising administering azenosertib to a patient at a daily dose of about 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP53 variant allele fraction, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, then (a) increasing the dose to about 350 mg with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week until the end of treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0130] In some aspects, provided herein is a method of treating ovarian cancer, comprising administering azenosertib to a patient at a daily dose of about 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week, measuring a baseline level of TP 53 variant allele fraction, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point, then (a) increasing the dose to about 400 mg with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week until the end of treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or (b) discontinuing the treatment if the patient has less than 50%reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

[0131] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring the level of TP53 variant allele fraction at a pre-determined time point, and if the patient has at least 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level, continuing administering azenosertib at the same therapeutically effective dose while maintaining the initial dosing schedule until the end of treatment; or if the patient has less than 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level, discontinuing the treatment.

[0132] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring the level of TP53 variant allele fraction at a pre-determined time point, and adjusting the therapeutically effective dose by increasing the dose while maintaining the initial dosing schedule until the end of treatment if the patient has at least 50% reduction in the level of TP53 variant allele fraction as compared to a baseline level; or discontinuing the treatment if the patient has less than 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level.

[0133] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring the level of TP 53 variant allele fraction at a pre-determined time point, increasing the dose to above 300 mg while maintaining the initial dosing schedule until the end of treatment if the patient has at least 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level; or discontinuing the treatment if the patient has less than 50% reduction in the level of TP 53 variant allele fraction as compared to the baseline level.

[0134] In some embodiments, the dose is increased to about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg or about 800 mg. In some embodiments, the dose is increased to about 350 mg. In some embodiments, the dose is increased to about 400 mg. In some embodiments, the dose is increased to about 450 mg. In some embodiments, the dose is increased to about 500 mg. In some embodiments, the dose is increased to about 550 mg. In some embodiments, the dose is increased to about 600 mg. In some embodiments, the dose is increased to about 650 mg. In someembodiments, the dose is increased to about 700 mg. In some embodiments, the dose is increased to about 750 mg. In some embodiments, the dose is increased to about 800 mg. In some embodiments, the dose is increased to about 850 mg. In some embodiments, the dose is increased to about 900 mg. In some embodiments, the dose is increased to about 950 mg. In some embodiments, the dose is increased to about 1000 mg.

[0135] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring the level of TP53 variant allele fraction at a pre-determined time point, adjusting the therapeutically effective dose by decreasing the dose while maintaining the initial dosing schedule until the end of treatment if the patient has at least 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level; or discontinuing the treatment if the patient has less than 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level.

[0136] In some aspects, provided herein is a method of treating ovarian cancer, comprising: administering azenosertib to a patient at a therapeutically effective dose, measuring the level of TP53 variant allele fraction at a pre-determined time point, decreasing the dose to below 300 mg while maintaining the initial dosing schedule until the end of treatment if the patient has at least 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level; or discontinuing the treatment if the patient has less than 50% reduction in the level of TP53 variant allele fraction as compared to the baseline level.

[0137] In some embodiments, the dose is decreased to about 250 mg, about 200 mg, about 150 mg or about 100 mg. In some embodiments, the dose is decreased to about 250 mg. In some embodiments, the dose is decreased to about 200 mg. In some embodiments, the dose is decreased to about 150 mg. In some embodiments, the dose is decreased to about 100 mg.

[0138] In some embodiments, the therapeutically effective dose is between 300 mg and 400 mg. In some embodiments, the therapeutically effective dose is about 300 mg. In some embodiments, the therapeutically effective dose is about 350 mg. In some embodiments, the therapeutically effective dose is about 400 mg.

[0139] In some embodiments, the baseline measured is prior to treatment initiation.

[0140] In some embodiments, the pre-determined time point is day 1 of each treatment cycle. In some embodiments, each treatment cycle is two weeks. In some embodiments, each treatment cycle is three weeks. In some embodiments, each treatment cycle is four weeks. In someembodiments, each treatment cycle is five weeks. In some embodiments, each treatment cycle is six weeks. In some embodiments, each treatment cycle is seven weeks.

[0141] In some embodiments, the subject is selected to have a TP53 biomarker level below a predetermined threshold. In some embodiments, the subject is selected to have a TP53 biomarker level above a predetermined threshold, hi some embodiments, the predetermined threshold is 50% reduction in level of TP53 variant allele fraction.

[0142] In some embodiments, the TP53 variant comprises a mutation selected from R175H. H179R, K132R, R237H, Y220C, R248Q, R248W, G266R, R273C, R273H. R282W, R337H, S241F and T256fs (“fs” indicates frameshift mutation). In some embodiments, the TP53 variant comprises the R175H mutation. In some embodiments, the TP53 variant comprises the H179R mutation. In some embodiments, the TP53 variant comprises the K132R mutation. In some embodiments, the TP53 variant comprises the R237H mutation. In some embodiments, the TP53 variant comprises the Y220C mutation. In some embodiments, the TP 53 variant comprises the R248Q mutation. In some embodiments, the TP53 variant comprises the R248W mutation. In some embodiments, the TP53 variant comprises the G266R mutation. In some embodiments, the TP 53 variant comprises the R237C mutation. In some embodiments, the TP 53 variant comprises the R273H mutation. In some embodiments, the TP53 variant comprises the R337H mutation. In some embodiments, the TP53 variant comprises the S241F mutation. In some embodiments, the TP53 variant comprises the T256fs (frameshift) mutation.Profiling Cell-Free DNA (cfDNA)

[0143] In some embodiments, the level of TP 53 variant allele fraction is measured by profiling plasma cell free DNA (cfDNA).

[0144] Isolation and extraction of cell free polynucleotides may be performed through collection of samples using a variety of techniques as described below or by another method known in the art. A sample can be any biological sample isolated from a patient. Samples can include body tissues, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies e.g., biopsies from known or suspected solid tumors), cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid (e.g., fluid from intercellular spaces), gingival fluid, crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat and urine. Samples are preferably body fluids, particularly blood and fractions thereof, and / or urine. Such samples includenucleic acids shed from tumors. The nucleic acids include DNA and RNA in double and / or singlestranded forms. A sample can be in the form originally isolated from a patient or can have been subjected to further processing to remove or add components, such as cells, enrich for one component relative to another, or convert one form of nucleic acid to another, such as RNA to DNA or single-stranded nucleic acids to double- stranded. Thus, for example, a body fluid sample for analysis is plasma or serum containing cell-free nucleic acids, e.g., cell-free DNA (cfDNA).

[0145] The sample can comprise various amounts of nucleic acid. Typically, the amount of nucleic acid in a given sample is equated with multiple genome equivalents. For example, a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cfDNA, about 200 billion (2xlOu) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.

[0146] In some embodiments, a sample comprises nucleic acids from various sources, e.g., from cells and from cell-free sources (e.g., blood samples, etc.). Typically, a sample includes nucleic acids carrying mutations. For example, a sample optionally comprises DNA carrying germline mutations and / or somatic mutations. Typically, a sample comprises DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). In some embodiments of the present disclosure, cell free nucleic acids in a subject may derive from a tumor.

[0147] Exemplary amounts of cell-free nucleic acids in a sample before amplification typically range from about 1 femtogram (fg) to about 1 microgram (pg), e.g., about 1 picogram (pg) to about 200 nanogram (ng), about 1 ng to about 100 ng, about 10 ng to about 1000 ng. hi some embodiments, a sample includes up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng or up to about 20 ng of cell-free nucleic acid molecules. Optionally, the amount is at least about 1 fg, at least about 10 fg, at least about 100 fg, at least about 1 pg, at least about 10 pg, at least about 100 pg. at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 150 ng or at least about 200 ng of cell-free nucleic acid molecules. In certain embodiments, the amount is up to about 1 fg, about 10 fg, about 100 fg, about 1 pg, about 10 pg, about 100 pg, about 1 ng, about 10 ng, about 100 ng, about 150 ng or about 200 ng of cell-free nucleic acid molecules. In some embodiments, methods include obtaining between about 1 fg to about 200 ng cell-free nucleic acid molecules from samples.

[0148] Cell-free nucleic acids typically have a size distribution of between about 100 nucleotides in length and about 500 nucleotides in length, with molecules of about 110 nucleotides in length to about 230 nucleotides in length representing about 90% of molecules in the sample, with a mode of about 168 nucleotides length and a second minor peak in a range between about 240 to about 440 nucleotides in length. In certain embodiments, cell-free nucleic acids are from about 160 to about 180 nucleotides in length, or from about 320 to about 360 nucleotides in length, or from about 440 to about 480 nucleotides in length.

[0149] In some embodiments, cell-free nucleic acids are isolated from bodily fluids through a partitioning step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. In some of these embodiments, partitioning includes techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids are lysed, and cell-free and cellular nucleic acids processed together. Generally, after addition of buffers and wash steps, cell-free nucleic acids are precipitated with, for example, an alcohol. In certain embodiments, additional clean up steps are used, such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, for example, are optionally added throughout the reaction to optimize certain aspects of the exemplary procedure, such as yield. After such processing, samples typically include various forms of nucleic acids including double-stranded DNA, single-stranded DNA and / or single-stranded RNA. Optionally, single stranded DNA and / or single stranded RNA are converted to double stranded forms so that they are included in subsequent processing and analysis steps.

[0150] Cell-free DNA (cfDNA) consists of short DNA fragments that are secreted and / or released into the bloodstream via apoptosis (programmed cell death) or necrosis (death of most or all cells in an organ or tissue due to disease or injury). Such fragments have a half-life of approximately one to two hours in circulation. The majority of cfDNA is derived from normal cells, but in individuals with cancer, tumor cells also release cfDNA.

[0151] The proportion of cfDNA from a tumor is fairly low, and thus, sensitive and specific sequencing methods are needed to detect mutations and copy-number changes. Analytical methods for detection and analysis of such DNA ranges from PCR-based approaches to NGS platforms. Most commonly used analytical techniques used in studies of cfDNA from tumors are digital PCR (dPCR) and next generation sequencing (NGS). The level of TP53 variant allelefraction is measured as a fraction of cfDNA molecules sequenced at a particular locus that carry the specific variant.

[0152] In some embodiments, the level of TP 53 variant allele fraction is measured by Next Generation Sequencing (NGS) or digital polymerase chain reaction (dPCR). In some embodiments, the level of TP53 variant allele fraction is measured by NGS. In some embodiments, the level of TP53 variant allele fraction is measured by dPCR.Nucleic Acid Sequencing

[0153] Sequencing methods or commercially available formats that are optionally utilized include, for example, Sanger sequencing, high-throughput sequencing, bisulfite sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanoporebased sequencing, semiconductor sequencing, sequencing-by ligation, sequencing-by-hybridization, RNA-Seq, Digital Gene Expression, next generation sequencing (NGS), Single Molecule Sequencing by Synthesis (SMSS), massively-parallel sequencing, Clonal Single Molecule Array, shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia. primer walking, sequencing using PacBio, SOLiD, Ion Torrent, nanopore platforms, or SBX sequencing. The foregoing examples are illustrative and may include current or legacy sequencing technologies. Sequencing reactions can be performed in a variety of sample processing units, which may include multiple lanes, multiple channels, multiple wells or other means of processing multiple sample sets substantially simultaneously. Sample processing units can also include multiple sample chambers to enable the processing of multiple runs simultaneously.

[0154] The sequencing reactions can be performed on one more nucleic acid fragment types or cell-free nucleic acid samples known to contain markers of cancer or of other diseases. The sequencing reactions can also be performed on any nucleic acid fragment present in the sample. The sequence reactions may provide for sequence coverage of the genome of at least about 5%. 10%, 15%, 20%, 25%. 30%, 40%, 50%, 60%. 70%, 80%, 90%, 95%. 99%, 99.9% or 100% of the genome. In other cases, sequence coverage of the genome may be less than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.9% of the genome. Next Generation Sequencing (NGS)

[0155] Next-generation sequencing (NGS), also known as massively parallel sequencing sequences DNA or RNA at a high speed and low cost by sequencing many DNAstrands at the same time with low input sample and with high accuracy allowing for detection of variants with high sensitivity.

[0156] Simultaneous sequencing reactions may be performed using multiplex sequencing techniques. In some embodiments, cell-free polynucleotides are sequence with at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions (sequencing depth). In other embodiments, cell-free polynucleotides are sequenced with less than about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. Sequencing reactions are typically performed sequentially or simultaneously. Subsequent data analysis is generally performed on all or part of the sequencing reactions. In some embodiments, data analysis is performed on at least about 1000, 2000, 3000, 4000, 5000, 6000. 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. In other embodiments, data analysis may be performed on less than about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. An exemplary read depth is from about 1000 to about 50000 reads per locus (base position).

[0157] In some embodiments, a nucleic acid population is prepared for next generation sequencing by fragmenting DNA into short fragments of about 100-800 bp using a variety of methods (e.g., physical shearing, enzyme digestion and PCR based amplification). DNA fragments are then ligated to technology specific adaptor sequences forming a fragment library.

[0158] In some embodiments, DNA is cut enzymatically forming blunt-ends on double-stranded nucleic acids with single- stranded overhangs at one or both ends. In these embodiments, the population is typically treated with an enzyme having a 5 '->3' DNA polymerase activity and a 3 '->5' exonuclease activity in the presence of the nucleotides (e.g.. A, C, G and Tor U). Exemplary enzymes or catalytic fragments thereof that are optionally used include Klenow large fragment and T4 polymerase. At 5' overhangs, the enzyme typically extends the recessed 3' end on the opposing strand until it is flush with the 5' end to produce a blunt end. At 3' overhangs, the enzyme generally digests from the 3' end up to and sometimes beyond the 5' end of the opposing strand. If this digestion proceeds beyond the 5' end of the opposing strand, the gap can be filled in by an enzyme having the same polymerase activity that is used for 5' overhangs. The formation of blunt-ends on double-stranded nucleic acids facilitates, for example, the attachment of adapters and subsequent amplification.

[0159] In some embodiments, nucleic acid populations are subject to additional processing, such as the conversion of single- stranded nucleic acids to double-stranded and / or conversion of RNA to DNA.

[0160] With or without prior amplification, nucleic acids subject to the process of forming blunt-ends described above, and optionally other nucleic acids in a sample, can be sequenced to produce sequenced nucleic acids. Sequencing can be performed so as to provide sequence data of individual nucleic acid molecules in a sample either directly or indirectly from a consensus sequence of amplification products of an individual nucleic acid molecule in the sample.

[0161] In some embodiments, double-stranded nucleic acids with single- stranded overhangs in a sample after blunt-end formation are linked at both ends to adapters including barcodes, and the sequencing determines nucleic acid sequences as well as inline barcodes introduced by the adapters. The blunt-end DNA molecules are optionally ligated to a blunt end of an at least partially double- stranded adapter (e.g.. a Y shaped or bell- shaped adapter). Alternatively, blunt ends of sample nucleic acids and adapters can be tailed with complementary nucleotides to facilitate ligation (e.g., sticky end ligation).

[0162] In some embodiments, adaptors may also have a unique molecular “barcode,” so each sample can be tagged with a unique DNA sequence. This allows for multiple samples to be mixed together and sequenced simultaneously. For example, barcodes 1-20 can be used to individually label 20 samples and then analyze them in a single sequencing run by “pooling” or “multiplexing”, which saves time and cost, as pooled samples are processed together.

[0163] In addition to DNA fragment libraries, paired-end libraries and mate-pair libraries can be used. Paired-end libraries allow users to sequence the DNA fragment from both ends, instead of typical sequencing which occurs only in a single direction. Paired-end libraries are created like regular fragment libraries, but they have adaptor tags on both ends of the DNA insert that enable sequencing from two directions, which makes it easier to map reads and can be used to improve detection of genomic rearrangements, repetitive sequence elements, and RNA gene fusions or splice variants. However, improvements in modem library prep methods and analysis tools have made it possible to detect these features with single direction sequencing as well.

[0164] Mate-pair libraries involve much larger-sized DNA inserts (over 2 kb and up to 30 kb). Sequencing of mate-pair libraries generates two reads distal to each other and in theopposite orientation and subsequently using the physical information associated between the two sequencing reads, mate pair sequencing is useful for larger structural variant detection, and identification of complex genomic rearrangements.

[0165] The DNA library is attached to a solid surface and clonally amplified to increase the signal that can be detected from each target during sequencing. Each DNA molecule in the library is bound to the surface of a bead or a flow-cell and PCR amplified to create a set of identical clones and all the DNA is sequenced at the same time using a sequencing instrument where "sequencing by synthesis" is carried out, reading individual bases as they grow along a polymerized strand. DNA base synthesis on single stranded DNA is followed by detection of the incorporated base, and then subsequent removal of reactants to restart the cycle. Optical detection or electrical detection is used to determine nucleotide incorporation during DNA synthesis.

[0166] Raw signals from instrument detectors collected during each sequencing cycle are processed into digitized data or base calls which are assembled into sequencing reads (FASTQ files) and their associated quality scores (Phred quality score). Secondary analysis involves read filtering and trimming based on quality, followed by alignment of reads to a reference genome or assembly of reads for novel genomes, and finally by variant calling generating a BAM file containing aligned reads. TP 53 variant allele fraction levels from a baseline and a predetermined time-point after treatment are compared.Digital PCR

[0167] In some embodiments, the cfDNA is profiled by digital PCR (dPCR).

[0168] Digital PCR enables precise, highly sensitive quantification of nucleic acids without the need for standard curves. In digital PCR, a PCR sample is first partitioned into discrete subunits prior to PCR amplification, for example, into 20,000 droplets, each containing zero to one template molecules. Each partition is amplified and fluorescent probes are used to identify amplified DNA. After amplification, droplets containing target sequence are detected by fluorescence and scored as positive, and droplets without fluorescence are scored as negative. The ratio of positives to negatives in each sample is used to quantify, and Poisson statistical analysis of the numbers of positive and negative droplets yields absolute quantitation of the target sequence.

[0169] In some embodiments, dPCR provides high sensitivity and fine resolution when comparing between samples and detecting variants with low frequency (e.g., TP 53 variant alleles of the present disclosure).Methods of Determining Molecular Response and Adjusting Treatment

[0170] The assessment of the molecular response involves a measurement of the level of one or more mutant TP53 alleles or variants between cfDNA from tumors at a baseline prior to treatment and a predetermined early on-treatment timepoint (e.g., day 1 of treatment cycle 1, or day 1 of treatment cycle 2, etc.) to identify the therapeutic response and predicted patient outcomes. In some embodiments, wherein relapse monitoring is carried out, a comparison is between pretreatment or on-treatment time-points with a post-treatment timepoint.

[0171] By the present disclosure, the improved method of measuring molecular response is measured by converting a continuous TP53 variant allele fraction to a discrete variable. In some embodiments, wherein the patient has more than one TP53 variant, the median value of multiple TP53 variant allelic fractions (VAF) is used for comparison between at least two timepoints (such as baseline, prior to treatment initiation and predetermined timepoint during or post treatment).

[0172] In some embodiments, wherein two or more on-treatment samples are available from the patient, best MR (bMR) is a molecular response that is based on the higher molecular response between the samples being selected as the representative molecular response of the patient. In some embodiments, wherein two or more on-treatment samples are available from the patient, earliest MR (eMR) is a molecular response that is based on the earliest molecular response between the samples being selected as the representative molecular response of the patient.

[0173] In some embodiments, MR positivity (MR+) is a decrease in TP53 VAF greater than 50% between baseline and the available on-treatment sample(s). In some embodiments, Molecular Complete Response (mCR) is a decrease of TP53 VAF of -100%. In some embodiments, Molecular Partial Response (mPR) is a decrease of TP53 VAF of between -100% to -50%. In some embodiments, Molecular stable disease (mSD) is a decrease of TP53 VAF of greater than -50% and less than 0%, or same TP53 VAF. In some embodiments. Molecular progressive disease (mPD), is a change of TP53 VAF greater than 0%.

[0174] In some embodiments, wherein two on-treatment samples are available from the patient, confirmed MR positivity (cMR+) is defined as MR+ in both on-treatment samples. In some embodiments, unconfirmed MR positivity (uMR+) is defined as MR+ in either of two on-treatment samples. In some embodiments, confirmed MR negativity (cMR-) is defined as MR-in both on-treatment samples.

[0175] In some embodiments, provided herein is a predetermined threshold of 50% for measuring change in TP53 variant allele fraction levels as being predictive and indicative of clinical response and efficacy for treatment of HGSOC using azenosertib.

[0176] In some embodiments, the patient to be administered azenosertib that has less than 50% reduction in the level of TP53 variant allele fraction has less than 40%, 30%, 20% or 10% reduction. In some embodiments, the patient to be administered azenosertib that has less than 50% reduction in the level of TP53 variant allele fraction has less than 40% reduction. In some embodiments, the patient to be administered azenosertib that has less than 50% reduction in the level of TP53 variant allele fraction has less than 30% reduction. In some embodiments, the patient to be administered azenosertib that has less than 50% reduction in the level of TP53 variant allele fraction has less than 20% reduction. In some embodiments, the patient to be administered azenosertib that has less than 50% reduction in the level of TP53 variant allele fraction has less than 10% reduction.

[0177] In some embodiments, the patient to be administered azenosertib has substantially no reduction in the level of TP53 variant allele fraction.

[0178] In some embodiments, azenosertib is administered as a monotherapy.

[0179] In some embodiments, administration of azenosertib results in at least 50%, 60%, 70%, 80%, 90%, 99% or 100% reduction in the level of TP53 variant allele fraction. In some embodiments, administration of azenosertib results in at least 50% reduction in the level of TP53 variant allele fraction. In some embodiments, administration of azenosertib results in at least 60% reduction in the level of TP 53 variant allele fraction. In some embodiments, administration of azenosertib results in at least 70% reduction in the level of TP53 variant allele fraction. In some embodiments, administration of azenosertib results in at least 80% reduction in the level of TP53 variant allele fraction. In some embodiments, administration of azenosertib results in at least 90% reduction in the level of TP 53 variant allele fraction. In some embodiments, administration of azenosertib results in at least 99% reduction in the level of TP53 variant allele fraction. In some embodiments, administration of azenosertib results in 100% reduction in the level of TP53 variant allele fraction.Clinical Responsiveness

[0180] In some embodiments, administration of azenosertib in a patient with at least 50% reduction in the level of TP 53 variant allele fraction has tumor shrinkage by at least 20%. Insome embodiments, administration of azenosertib in a patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 30%. In some embodiments, administration of azenosertib in a patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 40%. In some embodiments, administration of azenosertib in a patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 50%.

[0181] In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP53 variant allele fraction have time to progression (TTP) of 4 months. In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP53 variant allele fraction have time to progression (TTP) of 5 months. In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP 53 variant allele fraction have time to progression (TTP) of 6 months.

[0182] In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP53 variant allele fraction have objective response rate (ORR) of at least 20%. In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP53 variant allele fraction have objective response rate (ORR) of at least 30%. In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP 53 variant allele fraction have objective response rate (ORR) of at least 40%. In some embodiments, administration of azenosertib in patients with at least 50% reduction in the level of TP53 variant allele fraction have objective response rate (ORR) of at least 50%.

[0183] In some embodiments, the treatment methods described herein results in a response rate at or greater than 10%, 15%, 20%, 25%, 30%, 35%. 40%, 45% or 50%. In some embodiments, the response rate is measured by complete response (CR), partial response (PR), or combination thereof. In some embodiments, response is determined based on progression free survival. In some embodiments, response is determined based on tumor response. In some embodiments, response is determined based on clinical benefit rate (CBR). In some embodiments, response is determined based on disease control rate (DCR). In some embodiments, response is determined based on overall survival (OS). In some embodiments, response rate is measured in a cohort of patients.

[0184] In some embodiments, the treatment results in progression-free survival (PFS) of 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,12 months or longer. Tn some embodiments, the treatment results in progression-free survival (PFS) of 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months. 14 months, 15 months. 16 months, 17 months. 18 months, 19 months. 20 months, 21 months, 22 months, 23 months, 24 months or longer. In some embodiments, the treatment results in progression-free survival (PFS) of 1 year, 1.5 years, 2 years, 2.5 years or longer. ). In some embodiments, PFS is measured in a cohort of patients.

[0185] As used herein, the term “progression” of tumor growth or a “progressive disease” (PD) as used herein in reference to cancer status indicates an increase in the sum of the diameters of the target tumors. Progression for the purposes of determining progression free survival may also be determined if at least one of the following criteria is met: 1) tumor assessment by CT / MRI unequivocally shows progressive disease according to RECIST 1.1 criteria; 2) additional diagnostic tests (e.g., histology / cytology, ultrasound techniques, endoscopy, positron emission tomography) identify new tumors or determine existing tumors qualify for unequivocal progressive disease and / or CA-125- progression according to Gynecologic Cancer Intergroup (GCIG)-criteria (see Rustin et al., “Definitions for response and progression in ovarian cancer clinical trials incorporating RECIST 1.1 and CA 125 agreed by the Gynecological Cancer Intergroup (GCIG)” Int J Gynecol Cancer (2011) 21:419-423, which is incorporated herein in its entirety); or 3) definitive clinical signs and symptoms of PD unrelated to non-malignant or iatrogenic causes ([i] intractable cancer-related pain; [ii] malignant bowel obstruction / worsening dysfunction; or [iii] unequivocal symptomatic worsening of ascites or pleural effusion) and / or CA-125 -progression according to GCIG-criteria.

[0186] As used herein, the term “partial response” or “PR” refers to a decrease in tumor progression in a subject as indicated by a decrease in the sum of the diameters of the target tumors, taking as reference the baseline sum diameters. In some embodiments, PR refers to at least a 30% decrease in the sum of diameters, taking as reference the baseline sum diameters. Exemplary methods for evaluating partial response are identified by RECIST guidelines. See Eisenhauer et al., “New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1.)” Eur. J. of Cancer (2009) 45:228-247.

[0187] As used herein, “stabilization” of tumor growth or a “stable disease” (SD) refers to neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD. In some embodiments, stabilization refers to a less than 30%, 25%, 20%, 15%, 10% or 5% change (increaseor decrease) in the sum of the diameters of the target tumors, taking as reference the baseline sum diameters. Exemplary methods for evaluating stabilization of tumor growth or a stable disease are identified by RECIST guidelines. See Eisenhauer et al., “New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1.)” Eur. J. of Cancer (2009) 45:228-247.

[0188] As used herein, the term “complete response” or “CR” is used to mean the disappearance of all or substantially all target lesions. In some embodiments, CR refers to an 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% decrease in the sum of the diameters of the target tumors (z.e., loss of tumors), taking as reference the baseline sum diameters. In some embodiments, CR indicates that less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the total lesion diameter remains after treatment. Exemplary methods for evaluating complete response are identified by RECIST guidelines. See Eisenhauer et al., “New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1.)” Eur. J. of Cancer (2009) 45:228-247.Methods of Treatment

[0189] The present disclosure provides methods of monitoring responsiveness and efficacy of treatment of cancer using the compound known as Azenosertib, or a pharmaceutically acceptable salt thereof.

[0190] Azenosertib is a WEE1 inhibitor of the formula:NIl■^N'Hi\ OHAzenosertibWO 2019 / 173082 and WO 2021 / 231653 describe the compound azenosertib, both of which are hereby incorporated by reference in their entireties. Azenosertib is also known as ZN-c3 and these terms are used interchangeably.

[0191] In some embodiments, a treatment method described herein results in a therapeutic effect (e.g., a desired pharmacologic and / or physiologic effect). A therapeutic effect can encompass partially or completely curing a disease, relieving one or more adverse symptoms attributable to the disease and / or delaying progression of the disease. By the present disclosure, the method comprises determining clinical response and administering or adjusting the dose of atherapeutically effective amount of a therapeutic agent (e.g., Azenosertib, or a pharmaceutically acceptable salt thereof, and / or a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof). A therapeutically effective amount can be an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., tumor growth inhibition, progression free survival, complete response, partial response etc.). A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding agent to elicit a desired response in the individual.Administration Routes

[0192] In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered orally, intravenously or subcutaneously. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered orally. One may also use alternative suitable techniques of administering the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, that are known to those skilled in the art including, but not limited to, oral, rectal, pulmonary topical, aerosol, injection, infusion and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections. In other embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, and / or a second therapeutic such as a chemotherapeutic, can be administered orally.

[0193] In some embodiments, the effective dose of azenosertib. or a pharmaceutically acceptable salt thereof, is administered orally, intravenously, subcutaneously, intrathecally, intramuscularly, intracavitary, intrapleural, intralesional, or intra-arterial. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered intrathecally, intramuscularly, intracavitary, intrapleural, intralesional, or intra-arterial.

[0194] In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered orally.Azenosertib Dosage and Schedule

[0195] In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered based on body weight of the subject. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is between 2 mg / kg and 20 mg / kg. In some embodiments, the effective dose azenosertib, or a pharmaceutically acceptable salt thereof, is between 2-18 mg / kg, 2-16 mg / kg, 2-14 mg / kg, 2-12 mg / kg, 2-10 mg / kg, 2-8 mg / kg, 2-6 mg / kg, 3-4 mg / kg, 3-5 mg / kg or 4-6 mg / kg. Tn some embodiments, the effective dose is at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least 8 mg / kg, at least 9 mg / kg, at least 10 mg / kg, at least 11 mg / kg, at least 12 mg / kg, at least 13 mg / kg, at least 14 mg / kg, at least 15 mg / kg, at least 16 mg / kg, at least 17 mg / kg, at least 18 mg / kg or at least 19 mg / kg.

[0196] In some embodiments, azenosertib may also be in the form of equivalent dose e.g., the compound in other salt forms). In some embodiments, the effective dose is a flat dose. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 200-800 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 200-600 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 300-600 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 400-600 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 400-800 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 50-350 mg, 50-290 mg, 100-290 mg, 100-250 mg, 150-250 mg or 180-220 mg once a day, or equivalents thereof. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, ranges from 50-400 mg, 100-400 mg, 150-400 mg, 200-400 mg, 200-375 mg, 200-350 mg, 200-300 mg, 200-400 mg or 400-600 mg, or equivalents thereof, once a day.

[0197] In some embodiments, the effective dose of azenosertib. or a pharmaceutically acceptable salt thereof, is about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg, or equivalents thereof, once a day.

[0198] In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 50 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 100 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 150 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable saltthereof, is about 200 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 300 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 350 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 400 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 450 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 500 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 600 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 700 mg, or equivalents thereof, once a day. In some embodiments, the effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 800 mg, or equivalents thereof, once a day.

[0199] In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at or greater than 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or an equivalent thereof. In some embodiments, the present disclosure provides administration of a high dose of azenosertib. or a pharmaceutically acceptable salt thereof, e.g., wherein the dose is or greater than 375 mg.

[0200] In some embodiments, provided herein is a method of treating ovarian cancer comprising administering to a subject in need thereof, a daily dose of azenosertib. or a pharmaceutically acceptable salt thereof, at or greater than 300 mg, or an equivalent thereof. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 300 mg. In some embodiments, the daily dose of azenosertib. or a pharmaceutically acceptable salt thereof, is about 350 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 400 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is about 450 mg.

[0201] Methods of the present disclosure include methods of adjusting the therapeutic regimen based on molecular response, wherein the standard therapy is administering azenosertib, or a pharmaceutically acceptable salt thereof, as a monotherapy or in combination with a secondchemotherapeutic agent, or a pharmaceutically acceptable salt thereof, in a suitable dosing schedule. For example, the azenosertib, or a pharmaceutically acceptable salt thereof, as a monotherapy or in combination with a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, described herein may be administered one or more times per day (for example once, twice or three times a day) for a certain number of days (continuous dosing) or for a certain number of days, followed by a period of days where no dose is given (intermittent dosing). This treatment cycle (including dosing days and no-dosing days) may then be repeated.Intermittent Dosing

[0202] In some embodiments, methods described herein comprises intermittent dosing. In some embodiments, methods described herein comprise continuous dosing, hi some embodiments, methods described herein comprise combination therapy comprises continuous dosing of one of the agents. In some embodiments, methods described herein comprise combination therapy comprises continuous dosing of one of the agents and intermittent dosing of azenosertib, or a pharmaceutically acceptable salt thereof.

[0203] In some embodiments, an intermittent dosing schedule comprising one or more dosing weeks with each dosing week comprising at least three consecutive dosing days and at least one day without dosing.

[0204] In some embodiments, the present disclosure provides administration of a high dose of azenosertib, or a pharmaceutically acceptable salt thereof, e.g., between about 300 mg to about 800 mg once daily, or between about 175 mg to about 400 mg twice daily at an intermittent dosing schedule, e.g., 5 days administration (“on” days) followed by 2 days break (“off’ days) i.e.5 / 2, 4 days administration followed by 3 days break i.e., 4 / 3, or 3 days administration followed by 4 days off, i.e., 3 / 4, or 6 days administration followed by 1 day off i.e., 6 / 1. Alternatively, the intermittent dosing schedule of azenosertib, or a pharmaceutically acceptable salt thereof, is also expressed as administering between about 350 mg to about 800 mg once daily, or between about 175 mg to about 400 mg twice daily at an intermittent frequency, e.g., 5 on / 2 off, 4 on / 2 off, 3 on / 4 off, among others.

[0205] In some embodiments, the one or more dosing weeks are separated by at least one week of break. In some embodiments, the intermittent dosing schedule described herein (for example, of 7 / 0, 6 / 1, 5 / 2, 4 / 3 or 3 / 4) is carried out for 2 weeks followed by one week of break, or one week followed by one week of break, thereby achieving a high efficacy while increasing safetyand tolerability in treating a cancer. Tn some embodiments, the intermittent dosing schedule described herein (for example, of 7 / 0, 5 / 2, 6 / 1, 4 / 3 or 3 / 4) is carried out for 3 weeks followed by one week of break, or one week followed by one week of break, thereby achieving a high efficacy while increasing safety and tolerability in treating a cancer. In some embodiments, the intermittent dosing schedule described herein (for example, of 7 / 0, 6 / 1, 5 / 2, 4 / 3 or 3 / 4) is carried out for greater than 3 weeks followed by one week of break, or one week followed by one week of break, thereby achieving a high efficacy while increasing safety and tolerability in treating a cancer.

[0206] In some embodiments, provided herein is a method of treating cancer comprising administering to a subject in need thereof, a daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, at or greater than 100 mg, or an equivalent thereof, in accordance with an intermittent dosing schedule, wherein the intermittent dosing schedule comprises one or more dosing weeks with each dosing week comprises at least three consecutive dosing days and at least one day without dosing, followed by at least one week of break. In some embodiments, the daily dose of azenosertib. or a pharmaceutically acceptable salt thereof, is at or greater than 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, or an equivalent thereof. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 225 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 250 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 275 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of greater than about 300 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib. or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 350 mg once daily in an intermittent dosing schedule.

[0207] In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at or greater than 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or anequivalent thereof. Tn some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at or greater than 375 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 400 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 425 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 450 mg. In some embodiments, the daily dose of azenosertib is at about 475 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 500 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 550 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 600 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 625 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 650 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 675 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 700 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 725 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 750 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 775 mg. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at about 800 mg, or an equivalent thereof.

[0208] In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered once per day.

[0209] In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is divided into twice per day.

[0210] In some embodiments, each dosing week comprises at least four, five or six consecutive dosing days.

[0211] In some embodiments, each dosing week comprises five consecutive dosing days and two days without dosing.

[0212] In some embodiments, each dosing week comprises four consecutive dosing days and three days without dosing. In some embodiments, each dosing week comprises threeconsecutive dosing days and four days without dosing. Tn some embodiments, each dosing week comprises seven consecutive dosing days and seven days without dosing.

[0213] In some embodiments, each intermittent dosing schedule comprises between about 7 days to about 10 consecutive dosing days. In some embodiments, each intermittent dosing schedule comprises between about 8 consecutive dosing days. In some embodiments, each intermittent dosing schedule comprises between about 9 consecutive dosing days. In some embodiments, each intermittent dosing schedule comprises between about 10 consecutive dosing days.

[0214] In some embodiments, the intermittent dosing schedule comprises twenty-one consecutive dosing days and seven days without dosing.

[0215] In some embodiments, the intermittent dosing schedule comprises two consecutive dosing weeks.

[0216] In some aspects, provided herein is a method of treating cancer comprising administering to a subject in need thereof, a daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, at or greater than 350 mg, or an equivalent thereof, in accordance with an intermittent dosing schedule, wherein the intermittent dosing schedule comprises at least two consecutive dosing days and at least one day without dosing.

[0217] In some embodiments, the intermittent dosing schedule comprises at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen consecutive dosing days. In some embodiments, the intermittent dosing schedule comprises greater than fourteen consecutive dosing days. In some embodiments, the intermittent dosing schedule comprises twenty-one consecutive dosing days. In some embodiments, the intermittent dosing schedule comprises twenty-eight consecutive dosing days. In some embodiments, the intermittent dosing schedule comprises thirty-two consecutive dosing days. In some embodiments, the intermittent dosing schedule comprises forty-two consecutive dosing days.

[0218] In some embodiments, the intermittent dosing schedule comprises at least one two. three, four, five, six, or seven days without dosing. In some embodiments, the intermittent dosing schedule comprises one day without dosing. In some embodiments, the intermittent dosing schedule comprises between about two to seven days without dosing. In some embodiments, the intermittent dosing schedule comprises two days without dosing. In some embodiments, the intermittent dosing schedule comprises three days without dosing. In some embodiments, theintermittent dosing schedule comprises four days without dosing. In some embodiments, the intermittent dosing schedule comprises five days without dosing. In some embodiments, the intermittent dosing schedule comprises six days without dosing. In some embodiments, the intermittent dosing schedule comprises seven days with dosing.

[0219] In some embodiments, the intermittent dosing schedule includes consecutive dosing days of between about two to seven days (“on” days), followed by a period of break of between about one to seven days (“off’ days).

[0220] In some embodiments, the intermittent dosing schedule comprises five consecutive dosing days and two days without dosing. In some embodiments, the intermittent dosing schedule comprises four consecutive dosing days and three days without dosing. In some embodiments, the intermittent dosing schedule comprises three consecutive dosing days and four days without dosing. In some embodiments, the intermittent dosing schedule comprises six consecutive dosing days and one day without dosing. In some embodiments, the intermittent dosing schedule comprises seven consecutive dosing days and seven days without dosing. In some embodiments, the intermittent dosing schedule comprises fourteen consecutive dosing days and seven days without dosing. In some embodiments, the intermittent dosing schedule comprises twenty-one consecutive dosing days and seven days without dosing.

[0221] In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is at or greater than 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or an equivalent thereof. In some embodiments, the present disclosure provides administration of a high dose of azenosertib, or a pharmaceutically acceptable salt thereof, e.g., wherein the dose is or greater than 300 mg In some embodiments, the present disclosure provides administration of a high dose of azenosertib, or a pharmaceutically acceptable salt thereof, e.g., wherein the dose is or greater than 325 mg In some embodiments, the present disclosure provides administration of a high dose of azenosertib, or a pharmaceutically acceptable salt thereof, e.g., wherein the dose is or greater than 350 mg In some embodiments, the present disclosure provides administration of a high dose of azenosertib, or a pharmaceutically acceptable salt thereof, e.g., wherein the dose is or greater than 375 mg. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 400 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceuticallyacceptable salt thereof, is administered at a dose of about 450 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 500 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 550 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 600 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of greater than about 600 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 650 mg once daily in an intermittent dosing schedule, hi some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 700 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 750 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 775 mg once daily in an intermittent dosing schedule. In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 800 mg once daily in an intermittent dosing schedule.

[0222] In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is administered once per day. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is divided equally into twice per day. In some embodiments, the daily dose of azenosertib. or a pharmaceutically acceptable salt thereof, is divided equally into three doses per day. In some embodiments, the daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, is divided equally into four doses per day.

[0223] In some embodiments, the twice per day of azenosertib. or a pharmaceutically acceptable salt thereof, is at or greater than 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg or an equivalent thereof.

[0224] In some aspects, provided herein is a method of treating ovarian cancer comprising administering to a subject in need thereof, a daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, at or greater than 400 mg, or an equivalent thereof, inaccordance with an intermittent dosing schedule, wherein the intermittent dosing schedule comprises five consecutive dosing days and two consecutive days without dosing.

[0225] In some aspects, provided herein is a method of treating cancer comprising administering to a subject in need thereof, a daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, at or greater than 450 mg, or an equivalent thereof, in accordance with an intermittent dosing schedule, wherein the intermittent dosing schedule comprises five consecutive dosing days and two consecutive days without dosing.

[0226] In some aspects, provided herein is a method of treating cancer comprising administering to a subject in need thereof, a daily dose of azenosertib, or a pharmaceutically acceptable salt thereof, at or greater than 400 mg, or an equivalent thereof, in accordance with an intermittent dosing schedule, wherein the intermittent dosing schedule comprises four consecutive dosing days and three consecutive days without dosing.

[0227] In some embodiments, provided herein is a method of treating ovarian cancer comprising administering to a subject in need thereof, a daily dose of azenosertib. or a pharmaceutically acceptable salt thereof, at or greater than 450 mg, or an equivalent thereof, in accordance with an intermittent dosing schedule, wherein the intermittent dosing schedule comprises four consecutive dosing days and three consecutive days without dosing.

[0228] In some embodiments, the intermittent dosing schedule is repeated.

[0229] In some embodiments, the method further comprises administering a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, during the intermittent dosing schedule. Without wishing to be bound by any particular theory, administration of azenosertib, or a pharmaceutically acceptable salt thereof, in combination with a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, renders responsive a subject initially resistant to treatment by the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, alone, or prevents or reduces toxicity by the agent, and / or improves efficacy of treatment as compared to monotherapy. Combination therapy using intermittent dosing schedule further benefits dosing by requiring, for example, a lower effective dose of the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, and / or azenosertib, or a pharmaceutically acceptable salt thereof.

[0230] In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered in combination with one or more second chemotherapeutic agents (including pharmaceutically acceptable salts thereof) in an intermittent dosing schedule.

[0231] In some embodiments, the second chemotherapeutic agent is selected from carboplatin, paclitaxel, gemcitabine and pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.Dosing for a Second Chemotherapeutic Agent

[0232] In some embodiments, the second chemotherapeutic agent is carboplatin, or a pharmaceutically acceptable salt thereof, wherein carboplatin, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 1-10 mg / mL*min for 15 minutes or longer once during the treatment cycle. In some embodiments, the second chemotherapeutic agent is carboplatin, or a pharmaceutically acceptable salt thereof, wherein carboplatin, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 3-6 mg / mL*min for 15 minutes or longer once during the treatment cycle. In some embodiments, the second chemotherapeutic agent is carboplatin, or a pharmaceutically acceptable salt thereof, wherein carboplatin, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 1-10 mg / mL*min, 2-10 mg / mL*min, 3-10 mg / mL*min, 4-10 mg / mL*min, 5-10 mg / mL*min, 6-10 mg / mL*min, 7-10 mg / mL*min, 8-10 mg / mL*min, 9-10 mg / mL*min, 2-8 mg / mL*min, 2-7 mg / mL*min, 3-7 mg / mL*min, 4-7 mg / mL*min, 5-7 mg / mL*min, 4-6 mg / mL*min, 2-6 mg / mL*min, 3-8 mg / mL*min, 9-10 mg / mL*min for 15 minutes or longer once during the treatment cycle.

[0233] In some embodiments, the second chemotherapeutic agent is PLD, or a pharmaceutically acceptable salt thereof, wherein PLD, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 10-100 mg / m2 over about 60 minutes once during the treatment cycle. In some embodiments, the second chemotherapeutic agent is PLD, or a pharmaceutically acceptable salt thereof, wherein PLD, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 5-50 mg / m2 over about 60 minutes once during the treatment cycle. In some embodiments, the second chemotherapeutic agent is PLD, or a pharmaceutically acceptable salt thereof, wherein PLD, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 10-40 mg / m2 over about 60 minutes once during the treatment cycle.

[0234] In some embodiments, the second chemotherapeutic agent is PLD, or a pharmaceutically acceptable salt thereof, wherein PLD, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 10-100 mg / m2, 10-90 mg / m2, 10-80 mg / m2, 10-70 mg / m2, 10-60 mg / m2, 10-50 mg / m2, 10-40 mg / m2, 10-30 mg / m2, 10-20 mg / m2, 20-90 mg / m2, 30-90 mg / m2, 40-90 mg / m2, 50-90 mg / m2, 60-90 mg / m2, 70-90 mg / m2, 20-80 mg / m2, 20-70 mg / m2, 20-60 mg / m2, 20-50 mg / m2, 20-40 mg / m2, or 30-40 mg / m2over about 60 minutes once during the treatment cycle.

[0235] In some embodiments, the second chemotherapeutic agent is paclitaxel, or a pharmaceutically acceptable salt thereof, wherein paclitaxel, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 10-120 mg / m2 over 60-70 minutes each time for three times during the treatment cycle. In some embodiments, the second chemotherapeutic agent is paclitaxel, or a pharmaceutically acceptable salt thereof, wherein paclitaxel, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 10-100 mg / m2, 20-100 mg / m2, 30-100 mg / m2, 40-100 mg / m2, 50-100 mg / m2, 60-100 mg / m2, 70-100 mg / m2, 80-100 mg / m2, 90-100 mg / m2, 10-90 mg / m2, 10-80 mg / m2, 10-70 mg / m2, 10-60 mg / m2, 10-50 mg / m2, 10-40 mg / m2, 10-30 mg / m2, 30-70 mg / m2, 40-70 mg / m2, 50-70 mg / m2, 60-70 mg / m2, 30-90 mg / m2, 30-80 mg / m2administered up to 3 hours each time for three times during the treatment cycle.

[0236] In some embodiments, the second chemotherapeutic agent is paclitaxel, or a pharmaceutically acceptable salt thereof, wherein paclitaxel, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 40-100 mg / m2administered up to 3 hours each time up to three times during treatment cycles.

[0237] In some embodiments, the second chemotherapeutic agent is gemcitabine, or a pharmaceutically acceptable salt thereof, wherein gemcitabine, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 500-1500 mg / m2over 15 minutes or longer once during the treatment cycle.

[0238] In some embodiments, the second chemotherapeutic agent is gemcitabine, or a pharmaceutically acceptable salt thereof, wherein gemcitabine, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 100 to 1000 mg / m2, 100 to 1000 mg / m2, 100 to 900 mg / m2, 100 to 800 mg / m2, 100 to 700 mg / m2, 100 to 600 mg / m2, 100 to 500 mg / m2, 100 to 400 mg / m2, 100 to 300 mg / m2, 100 to 200 mg / m2, 200 to 1000 mg / m2, 300 to 1000mg / m2, 400 to 1000 mg / m2, 500 to 1000 mg / m2, 600 to 1000 mg / m2, 700 to 1000 mg / m2, 800 to 1000 mg / m2, 200 to 800 mg / m2, 200 to 700 mg / m2, 200 to 600 mg / m2, 200 to 500 mg / m2, 300 to 900 mg / m2, 300 to 800 mg / m2, 400 to 700 mg / m2, 500 to 700 mg / m2, 500 to 800 mg / m2, 600 to 900 mg / m2over 15 minutes or longer each time for up to 3 times during the treatment cycle.

[0239] In some embodiments, the second chemotherapeutic agent is gemcitabine, or a pharmaceutically acceptable salt thereof, wherein gemcitabine, or a pharmaceutically acceptable salt thereof, is administered intravenously at a dose ranging from 100 to 1000 mg / m2over 15 minutes or longer each time for up to 3 times during the treatment cycle.EXAMPLES

[0240] While certain methods of the present disclosure have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the methods of the disclosure and are not intended to limit the same.Example 1. TP53 Based Molecular Response Predicted Azenosertib Efficacy in HGSOC Earlier than RECIST Outcome

[0241] This example outlines the methods of and response to an exemplary Weel inhibitor small molecule drug, azenosertib, in treatment of patients with high grade serous ovarian cancer.Study Design and Plasma cfDNA NGS Assay

[0242] Briefly, 139 high grade serous ovarian cancer (HGSOC) patients enrolled in single-agent azenosertib studies (NCT04158336. NCT05128825, NCT05198804) were eligible and included in this study based on criteria that the patients were 1) assigned to receive the same or higher than 300 mg QD of azenosertib on 5:2 intermittent dosing schedule (300 mg QD 19% (n = 24), 350 mg QD 2% (n = 3) and 400 mg QD 78% (n = 96)) and 2) plasma cfDNA were successfully profiled at baseline (Treatment Cycle 1 Day 1; C1D1) and at least one on-treatment timepoint (Treatment Cycle 2 Day 1; C2D1 (38% (n = 47)), Treatment Cycle 3 Day 1; C3D1 (9% (n = 11)), or C2D1 and C3D1 (53% (n = 65))). Each treatment cycle was given every 3 weeks, whereas radiological tumor assessments were every 6 weeks by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 (FIG. 1).

[0243] Time to progression (TTP) was defined as the interval from C1D1 to the date of disease progression. Patients with non-disease-related or unknown reason of death were censored at the date of death and included in TTP analysis. 123 out of 139 patients (88.5%) showedTP53 variants detectable at baseline and were considered evaluable for the validation analysis. Follow-up was censored on 27 Jan 2025, with a median follow-up of 8.6 months and 21% (24 / 123) objective response rate (ORR) in the evaluable cohort (n = 123).

[0244] Peripheral blood samples were collected from patients and centrifuged at 1600g for 10 min. Then plasma was centrifuged again at 16000g for 10 min followed by aliquoting into cryovials and storing at -80°C. 4 mL of plasma samples were used for cfDNA extraction and subsequent NGS analysis performed by Tempus using XF+ Liquid Biopsy Assay. Variant allelic fraction (VAF) of individual TP53 variants provided by Tempus were used to determine molecular response (MR).Molecular Response Calculation Method

[0245] If a patient had more than one TP53 variants, the median value of the multiple TP53 VAFs was used. MR positivity (MR+) was defined as the decrease of TP53 VAF greater than 50% between baseline and the first available on-treatment sample. For further stratification of the depth of MR, the definition of 4 MR stages were introduced. Molecular complete response (mCR), if the decrease of TP53 VAF was -100%; molecular partial response (mPR), if the decrease of TP53 VAF was higher than -100% and less than -50% (between -100% and -50%); molecular stable disease (mSD), if the decrease of TP53 VAF was same or greater than -50% and less than or equal to 0% (between -50% and 0); molecular progressive disease (mPD), if the change of TP53 VAF was greater than 0%. In the patients with both on-treatment samples available, confirmed MR positivity (cMR+) was defined as MR+ in both on-treatment samples, unconfirmed MR positivity (uMR+) as MR+ in either of two on-treatment samples, and confirmed MR negativity (cMR-) as MR- in both on-treatment samples.Results

[0246] The 123 patients of MR-evaluable cohort showed 57% (70 / 123) of molecular response rate (MRR), and 23% (28 / 123) of complete molecular response rate. To validate the correlation between molecular response and RECIST response, MRR was examined across different best overall response (BOR) groups (FIG. 2). Confirmed objective response (cOR) group showed the highest 80% MRR (20 / 25), stable disease (SD) group showed 56% MRR (40 / 71), and progressive disease (PD) group showed the lowest MRR of 35% (9 / 26). On the other hand, RECIST response rate was examined between MR+ and MR- groups (FIG. 3A and FIG. 3B).MR+ showed 29% (20 / 70) ORR, whereas MR- 9% (5 / 53) ORR (p = 0.012, Fisher’s exact test two-sided; odds ratio = 0.26). MR- also has a higher PD rate (32%) compared to MR+ (13%).

[0247] Molecular response also showed correlation with tumor size reduction at target lesions measured by RECIST scan (FIG. 4A and FIG. 4B). The four MR stages showed significantly differentiated tumor size reduction (p = 5.1e-5, Kruskal-Wallis test), including -30.6% median tumor shrinkage in mCR group and 5.7% median tumor burden increase in mPD group.

[0248] TTP analysis showed that MR predicted TTP in the entire evaluable cohort (TTP median 5.49 mo vs 2.69 mo, p = 3.8e-5, HR = 0.43) (FIG. 5A and FIG. 5B). Furthermore, MR also stratified TTP in the patients with stable disease at the first assessment (SD1A patients; TTP median 6.14 vs 3.56, p = 3.8e-3, HR = 0.44), indicating that MR can serve as an earlier predictor of TTP compared to RECIST scan. In the entire cohort, mCR and CMR+ were able to further stratify patients with longer TTP of 6.47 and 6.14 months, respectively, whereas mPD and cMR- stratified patients with shortest TTP of 2.69 and 2.76 months, respectively (FIG.6A and FIG.6B). mCR and cMR+ did not show significant overlap (P = 1, Fisher’s exact test), indicating the independence between the depth and duration of MR.

[0249] In a cohort of patients whose first RECIST assessment result was stable disease (SD1A patient cohort), it was examined if the first MR call can foresee the resultant RECIST outcome of each patient, and how earlier the MR-based anticipation could be than a conclusive RECIST assessment. In group 1 of FIG.7A and FIG. 7B, MR+ predicted latent clinical response a median of 14 weeks earlier than a confirmed RECIST response, whereas in group 4, MR-predicted disease progression without clinical response a median of 8 weeks earlier than a RECIST PD call (see FIG. 7A and FIG.7B).

[0250] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

1. CLAIMS1. A method of treating ovarian cancer, comprising:administering azenosertib to a patient at a daily dose of 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week,measuring a baseline level of TP53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point; and(a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or(b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

2. The method of claim 1, wherein the patient with less than 50% reduction in (b) shows Stable Disease (SD) in a RECIST 1.1 scan.

3. The method of claim 1, wherein the patient with less than 50% reduction in (b) shows Progressive Disease (PD) in a RECIST 1.1 scan.

4. The method of claim 1, wherein the treatment is continued at a daily dose of 400 mg.

5. The method of claim 1, wherein the treatment is continued at a daily dose of 350 mg.

6. The method of claim 1, wherein the treatment is continued at a daily dose of 300 mg.

7. The method of any one of claims 4-6, wherein the treatment is continued in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

8. A method of treating ovarian cancer, comprising:administering azenosertib to a patient at a therapeutically effective dose,measuring a baseline level of TP 53 variant allele fraction at or prior to treatment initiation, measuring a first on-treatment level of TP53 variant allele fraction at a pre-determined time point; and(a) continuing the treatment if the patient has at least 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level; or(b) discontinuing the treatment if the patient has less than 50% reduction in the first on-treatment level of TP53 variant allele fraction as compared to the baseline level.

9. The method of claim 8, wherein the therapeutically effective dose is a daily dose of 300 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

10. The method of claim 8, wherein the therapeutically effective dose is a daily dose of 350 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

11. The method of claim 8, wherein the therapeutically effective dose is a daily dose of 400 mg in accordance with an intermittent dosing schedule of five consecutive dosing days and two days without dosing (5:2) in a dosing week.

12. The method of claim 8, wherein treatment is continued at substantially the same therapeutically effective dose as on treatment initiation.

13. The method of claim 8, wherein the treatment is continued at a daily dose of 400 mg.

14. The method of claim 8, wherein the treatment is continued at a daily dose of 350 mg.

15. The method of claim 8, wherein the treatment is continued at a daily dose of 300 mg.

16. The method of any one of claims 1-15. wherein the baseline is measured at least two weeks prior to treatment initiation.

17. The method of claim 16, wherein the baseline is measured two weeks prior to treatment initiation.

18. The method of any one of claims 1-15, wherein the baseline is measured at treatment initiation.

19. The method of any one of the preceding claims, wherein the pre-determined time point is within 6 weeks of treatment initiation.

20. The method of claim 19, wherein the pre-determined time point is within 3 weeks of treatment initiation.

21. The method of any one of the preceding claims, wherein the pre-determined time point is day 1 of a treatment cycle.

22. The method of claim 21, wherein the treatment cycle is three weeks.

23. The method of claim 21, wherein the pre-determined time point is day 1 of cycle 2 (C2D1).

24. The method of claim 21, wherein the pre-determined time point is day 1 of cycle 3 (C3D1).

25. The method of any one of the preceding claims, wherein the TP 53 variant comprises a mutation selected from the group consisting of R273H, R248W, Y220C, R175H, R248Q, R273C, G266R, H179R, K132R, R282W, R337H, S241F and T256fs.

26. The method of any one of the preceding claims, wherein the level of TP53 variant allele fraction is measured by profiling plasma cell free DNA (cfDNA).

27. The method of claim 26, wherein the cfDNA is profiled by next-generation sequencing (NGS) assay or digital polymerase chain reaction (dPCR).

28. The method of claim 27, wherein the cfDNA is profiled by the next-generation sequencing (NGS) assay.

29. The method of any one of the preceding claims, wherein the patient with at least 50% reduction in the level of TP53 variant allele fraction has at least 60%, 70%, 80%, 90% or 99% reduction.

30. The method of claim 29, wherein the patient has substantially 100% reduction in the level of TP53 variant allele fraction.

31. The method of any one of the preceding claims, wherein the patient with less than 50% reduction in the level of TP53 variant allele fraction has less than 40%, 30%, 20% or 10% reduction.

32. The method of claim 31, wherein the patient has substantially no reduction in the level of TP53 variant allele fraction.

33. The method of any one of the preceding claims, wherein the patient with at least 50% reduction in the level of TP53 variant allele fraction has tumor shrinkage by at least 20%.

34. The method of any one of the preceding claims, wherein the patient with at least 50% reduction in the level of TP53 variant allele fraction has time to progression (TTP) of greater than 4 months.

35. The method of any one of the preceding claims, wherein the patient with at least 50% reduction in the level of TP53 variant allele fraction has progression free survival of at least 3 months.

36. The method of any one of the preceding claims, wherein the patient with at least 50% reduction in the level of TP53 variant allele fraction has an objective response rate (ORR) of at least 20%.

37. The method of any one of the preceding claims, wherein the ovarian cancer is highgrade serous ovarian cancer (HGSOC).

38. The method of any one of the preceding claims, wherein the azenosertib is administered as a monotherapy.

39. The method of any one of claims 1-37, wherein the azenosertib is administered in combination with at least one other therapeutic agent.

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